untitled ISSN 215 Prepara surface Khaldoun 1 Department of 2 College of Phar * Corresponding Tel.: +962.79.58 ARTICLE IN DOI: 10.5155/e Received: 29 Oc Received in rev Accepted: 29 No Published onlin Printed: 31 Mar KEYWORDS Surface Tension Chitosan Surfactant Particle size 1H NMR spectro 1. Introduct Chitin (N‐acetylgluc amino‐2‐deo extracted fro chitosan der with tailorin wastewater medicine as materials [7] Chitosan are believed and many availability positive char charged surf because of it good sorptiv complexation base and ins is soluble in convert the g rendering th 53‐2249 (Print) ation and active N‐ Abed Al‐So f Chemistry, Al Al‐B rmacy, Taif Univers g author at: Depart 861350. Fax: +962.2 FORMATION eurjchem.7.1.91‐96 ctober 2015 vised form: 16 Nov ovember 2015 ne: 31 March 2016 rch 2016 S oscopy tion (β(1→4)‐linked cosamine)) a oxy‐β‐D‐glucose om various pla rivatives has r ng of the polym treatment [3] wound dressin ] and drug deliv n exhibits a larg to be involved other substan of free amino rge and thus in faces/polymers ts high amino c ve abilities for n with the ami soluble in wate dilute aqueou glucosamine un he polysaccharid / ISSN 2153‐225 ht Europ character butyrated u’od 1,*, Moh Bayt University, Maf sity, Taif, 21974, Ki tment of Chemistry, 2.6297034. E‐mail a 6.1353 ember 2015 d‐2‐acetamido‐ nd chitosan e) (Figure 1) a ants and anima recently becom mer to specific ], the food in ngs [5], gel imp very systems [8 ge number of a in the interacti nces [10‐12]. groups in ch n turn reacts w s [13]. It was re content, has be r many heavy no groups [14] er and organic s us acidic solutio nits into a solub de a polyelectr European Journ Europ 57 (Online)  20 ttp://dx.doi.org pean Jo Journal web rization o d low mol hammad Mu fraq, 25113, Jordan ingdom of Saudi Ar y, Al Al‐Bayt Univers address: khaldoun@ ABSTRACT New classes o weight chitos new compoun and particle s 57% as confir with higher su found to be s breaking up o Cite this: Eur. 2‐deoxy‐β‐D‐gl (α‐(1→4)‐link are natural poly ls [1,2]. Synthe me of great int applications su ndustry [4] an plants [6], scaffo 8,9]. amine groups w ion between chi Due to the hitosan, it carr with many nega ported that chi een found to po metal ions, th ]. Chitosan is a solvents, althou on (pH < 6.5), w ble form R‐NH3+ olyte in acidic m al of Chemistry 7 pean Journal of C 016 Atlanta Pub /10.5155/eurjch ournal bpage: www. of lecular w ustafa Saade n rabia sity, Mafraq, 25113 @aabu.edu.jo (K. A of natural surfac ans (1.3, 6, 10, 1 nds were charac size were also d rmed by 1H NMR urface activity on soluble in all pH of chitosans inter . J. Chem. 2016, lucose ked‐2‐ ymers esis of terest, uch as nd in olding which itosan easy ries a atively tosan, ossess rough weak ugh, it which + [15], media [16 chit dea dea cos pro anti gro the hyd cou intr Sev acti mic the app biom sub con 7 (1) (2016) 91‐ Chemistry lishing House LL hem.7.1.91‐96.1 of Che .eurjchem.co weight chit elnour 1 and 3, Jordan. Al‐Sou’od). ctants have been 18 and 30 kDa) terized by 1H NM determined. The R spectra. These n comparison w H media in cont r‐strand hydroge 7(1), 91‐96 ]. The main pa tosan are its acetylation (D acetylated units There is a r metics concern operty to act imicrobial activ ups of chitosan surface of micr Chitosan by its drophobic segm uld improve su roducing hydro veral examples ivity have been celles and aggre entrapment o plications in t medical fields bstrates by elec nsiderable biom ‐96 LC ‐ All rights re 1353 emistry m tosan d Mayyas Mo n prepared throu with butyric an MR and FT‐IR sp degree of subs compounds pos with native chitos trast to native c en bonding upon arameters influ molecular we DD), represen s [17]. remarkable int ning the applic as natural pre vity on the hypo n at C‐2 interact roorganisms ca self have weak ments. Chemic uch surface a ophobic substit s of chitosan surveyed. The egates which h of water‐insolu the controlled s. Chitosan a ctrostatic and h medical applicat served ‐ Printed y ohammad Al ugh the reaction nhydride in aque pectroscopy. The titutions was in sess low aggreg san. The prepare chitosan. This m n substitutes. uencing the cha eight (MW) a nting the p terest in food cation of chito eservative. Chi othesis that pro t with anionic c using cell dama surface activity al modification ctivity. This is tuents in its glu derivatives w surface active have enormous uble drugs and d drug delive also interacts hydrophobic int ions [18]. d in the USA l‐Remawi 2 n of low molecul eous medium. Th eir surface tensio n the range of 4 ate size 12‐18 n ed compounds a may be due to th aracteristics of and degree of proportion of d science and san due to its itosan showed otonated amine constituents on age [18]. y since it has no ns of chitosan s achieved by ucosidic group. with surfactant polymers form importance in d consequently ry and many with several teractions with ar he on 4‐ m re he f f f d s d e n o n y . t m n y y l h 92 Al‐Sou’od et al. / European Journal of Chemistry 7 (1) (2016) 91‐96 (a) (b) Figure 1. The structure of chitin (a) and the structure of partially deacetylated chitosan (b). Figure 2. Synthesis of N‐butyrated chitosan. In this work, N‐butyrated chitosan (NBCh) from different low molecular weights chitosan (LMWC) were prepared by using butyric anhydride, and their structures were clarified by means of 1H NMR and FT‐IR techniques. Their self‐aggregation behaviors were examined by dynamic light scattering (DLS) and surface tension. 2. Experimental 2.1. Materials High molecular weight chitosan (HMWC) of a viscosity average molecular weight of 250 kDa was obtained from Hongo Chemical Company Ltd., China. The degree of deacetylation (DA) is 93%. Butyric anhydride (C8H14O3) obtained from Merck Schuchardt OHG Hohenbrunn, Germany. Water used to prepare the aqueous solutions was double distilled water. All reagents were of analytical grade and used without further purifications. 2.2. Methods 2.2.1. Depolymerization procedure of chitosan The high molecular weight chitosan (10 g, 250 kDa) were dissolved in 830 mL (0.1 M) HCl, and then 170 mL of concentrated HCl (37%) were added to dissolve chitosan. The dissolved chitosan was vigorously stirred (1000 rpm) with heating under reflux for the different depolymerization times. At the end of every reaction, each mixture was allowed to cool and around two liters of 96% ethanol were added to the depolymerized chitosan to enhance the precipitation of hydrochloric salt of chitosan oligomers. The hydrolysis reaction time for 1, 2, 3.5, 6 and 24 hours gives different molecular weights (30, 18, 10, 6 and 1.3 kDa) respectively. Finally the precipitated low molecular weight chitosan was filtered and centrifuged, the solid residue was washed several times using ethanol, and dried using the freeze dryer. Low molecular weight chitosans were stored as powder in glass vials at room temperature [19]. The viscosity average molecular weight of chitosan oligomers was calculated according to Mark‐Houwink equation (1) [12]. [η] = K×Ma (1) where [η] is the intrinsic viscosity, M is the average molecular weight. K and a are constants which had been determined by many authors. K = 1.38×10‐4 and a = 0.85 [20] and K = 2.14×10‐3 and a = 0.657 [21] another values were K = 1.4×10‐4 and a = 0.83 [22]. Another formula for liquid chitosan has been adapted [23] with K = 8.93×10‐4 and a = 0.71. 2.2.2. Synthesis of N‐butyrated chitosan The depolymerized chitosan (0.5 g) of 1.3, 6, 10, 18 and 30 kDa were dissolved in 50 mL of distilled water. The reaction mixture was adjusted to at pH (6.5‐7.0) with 0.2 M NaOH solution. Equivalent molar ratio (1:1) of butyric anhydride was added with mechanical stirrer to the chitosan solution, then, the solution was kept at room temperature overnight (25 °C). The resulted solution was dried in oven at temperature 40 °C for two days. The resulted films were crusted and kept in tightly closed vials. The synthetic N‐butyrated chitosan was prepared as shown in Figure 2. 2.3. Characterization LMWC and N‐butyrated LMWC were characterized by using different techniques, such as 1H NMR and FT‐IR spectroscopy, particle size, surface tension, and comparison between the native chitosan and N‐butyrated chitosan (NBCh). Table 1. Assign Sample NBCh 1.3 kDa 6 kDa 10 kDa 18 kDa 30 kDa 2.3.1. Viscos Viscosity wave Vibro Helsinki, Fin 2.3.2. 1H NM 1H NMR spectromete was dissolve (D2O) at tem deacetylated of substitutio 2.3.3. FT‐IR s Infrared composition instrument a IR spectrosc characterizat Infrared sp different typ frequencies. in the transm 2.3.4. Surfac Surface t Fisher Surfa method. Bef thoroughly distilled wat benzene flam carried out a is also cont water before tension of N (1.3 and 10 k 2.3.5. Partic The par concentratio Scattering (D UK). Sample and butyrate rations were μm and 0.2 repeated eig 3. Results an The poor high crysta hydrogen b substituents inter‐ or int crystallinity, However, a nments of chemica δ of H‐1 peak 5.294 5.150 5.259 4.908 5.263 5.238 sity measureme y measuremen o SV‐10/SV‐10 land). MR spectroscopy R spectra were er. About 5 g/L ed in 5 mm dia mperature 70 d chitosan and N on (DS) was cal spectroscopy spectroscopy of chitosan sa availability and copy is the one tion of chito ectroscopy of pes of interato The FT‐IR spe mission mode in ce tension mea tension measu ace Tensiomat fore each mea cleaned and ter, then with me for five m at 25±3 °C and trolled by the e each measure N‐butyrated sol kDa) was determ cle size measur rticle size of N ons was meas DLS) method u es of different ed) (1.3, 6, 10 e prepared. Eac μm syringe fi ht times. nd discussion r solubility of ch allinity and s onding. There into chitosan tra‐molecular h this favoring an excessive Al‐Sou’od et l shift (δ) for chito δ of (H3,4,5,6) p 3.61‐4.32 4.16‐4.63 4.12‐4.69 3.99‐4.68 4.15‐4.67 4.09‐4.21 ent ts were perfo 00 viscometer y e recorded on L of chitosan/N ameter tubes w °C. The peaks N‐acylated chit lculated from th can be used ample due to it independence e of the most s osan and N‐b ffers the poss omic bond vib ectra were mea n the range 400 asurements urements were t, which emp asurement, the rinsed three absolute etha minutes. The m the accuracy o surface tensio ment. After equ lutions of diffe mined. rements N‐butyrated ch sured using t sing a Malvern molecular wei and 15 kDa) a ch sample was f ilters and each hitosan in wate strong inter‐ fore, introduct backbone is l hydrogen bond g solvating of hydrophobic t al. / European J san and N‐butyrat peaks δ of H‐ 3.113 3.432 3.550 3.132 3.539 3.525 rmed using a (KSV Instrum n a Bruker AV N‐butyrated chi with deutrated for the comp tosan and the d he area peaks. to investigat ts simplicity, re of sample solu studied method butyrated chi sibility to me brations at dif asured in KBr p 00‐400 cm‐1. carried out us ploys the De platinum ring times with d anol and burne measurements of the measurem on measuremen uilibrium, the su erent concentra hitosan of dif the Dynamic n Zetasizer (Ma ght chitosan (n at different con filtered through h measuremen er is mainly due or intra‐mole tion of appro likely to disrup ding and weak chitosan in w substitution w Journal of Chemis ted chitosan (NBCh ‐2 peak δ of C ‐ 2.510 2.703 2.214 2.721 2.679 Sine‐ ments, V 300 itosan water pletely degree te the elative ubility. ds for tosan. easure fferent pellets sing a Nouy g was double ed on were ments nts of urface ations fferent Light alvern, native ncent‐ h 0.45 nt was e to its ecular priate pt the ken its water. would gen hyd asse 3.1. dete app app mol 9.85 3.2. from 30 refe but anh spe (LM in D The Equ %DS whe H‐1 F 3.3. fun chit pos gro mol from ran stry 7 (1) (2016) h). CH2 (c) peak δ ‐ 0 3 4 1 9 nerate water‐ drophobic inte embling” mode . Viscosity‐aver The average m ermined using plying equation plied our publ lecular weight 5, 17.6, 29.4 kD . 1H NMR spect In this work, m different mo kDa). We used er molecular w tyric anhydrid hydride/NH2 on ectra (Figures MWC) polymers D2O at 70 °C w e degree of sub uation (1) [25]. 3 H‐1 CH H‐1 I S 1‐ I I 3              ere IH‐1 and ICH3 1 of chitosan an igure 3. 1H NMR s . FT‐IR spectra Analysis of FT ctional group a tosan based p ssessing both h ups (substitut lecule, which a m reactions u dom substitutio ) 91‐96 δ of CH2(b) peak ‐ 1.948 2.040 1.896 2.073 2.011 ‐insoluble de eraction follow el [16]. rage molecula molecular weig intrinsic visco n (1) as mention lished procedu chitosan were Da. tra of chitosan N‐butyrated c olecular weight a prefixes 1.3, weight of N‐b de, the mono n repeating un 3‐5) of low s (1.3, 6, 10, 18 with peak assign bstitution can b 100% are integrals o d CH3 in butyl g spectra of chitosan a of chitosan an T‐IR spectra allo are present in t polymeric surf ydrophilic (‐NH ted butyl chain allows efficient under homoge on along the ma δ of CH3 (a) p ‐ 1.242 1.351 1.185 1.394 1.326 rivatives due wing a “hydr r weight deter ght of prepared osity measurem ned in experim ure [24]. Five obtained, nam and N‐butyrat chitosan (NBCh chitosans (1.3 , 6, 10, 18, and utyrated chito omolar ratio nit of chitosan) molecular w 8 and 30 kDa) w nments are sho be estimated ac of the signals co group, respectiv (NBCh) structure nd N‐butyrated ows insight int the sample. Th factants (CBPS H2 and ‐OH) an ns) on the ch t substitution o enous conditio ain chain of chi 93 peak DS % 0.000 55.05 44.55 57.69 50.33 49.83 e to strong rophobic self‐ rmination d chitosan was ment, and then mental part. We types of low mely 1.34, 5.92, ted chitosan h) is prepared 3, 6, 10, 18 and 30 in NBCh to osan. By using 1:1 (butyric ). The 1H NMR eight chitosan were dissolved own in Table 1. ccording to the (1) orresponding to vely. in D2O at 70 °C. d chitosan to what type of he specificity of Ss) consists of nd hydrophobic hitosan macro‐ of the product ns leading to tosan. g ‐ s n e w , d d o g c R n d . e o f f f c ‐ t o 94 Figure Figure Chitosan (‐NH2) at 16 asymmetrica broad band o stretching vi peaks at 100 saccharide st Compari can be seen a IR spectrum amide carbo N‐butyl ester in the range reaction occ which leadin 3.4. The surf When co samples, it chitosan (1.3 due to high measured su the critical shown in F respectively. concentratio = 0.67 g/10 weight chito 3.5. The par The part different con micelle conc different con NBCh and 10 were estima respectively, concentratio e 4. 1H NMR spectr e 5. 1H NMR spectr n exhibits main 622 and 1514 al and symmet observed at 340 ibration of the H 00‐1200 cm‐1 ar tructure of chit ng the spectra a significant sh m of chitosan to onyl group stre r groups is con e 1710‐1760 cm curs at ‐NH2 gr ng to the N‐buty face tension m omparing the s was found th 3 kDa) had a low her solubility urface tension i micelle concen Figures 6 and . The results on of the lower mL) was greate san (10 kDa = 0 rticle size meas ticle size of chi ncentrations w centration (CM ncentrations o 0 kDa NBCh). T ated to be 0.6 , which is s on which calcu Al ra of 1.3 kDa NBCh ra of 30 kDa NBCh characteristic p cm‐1; these b rical NH3+ ben 00‐3500 cm‐1 m H‐bonded N‐H re attributed to tosan. a of chitosan an hift of amine pea o a higher valu etching frequen firmed by the a m‐1. This suppo roups rather th yrated chitosan easurements surface tension hat the lowes wer surface ten in aqueous m s plotted again ntration (CMC 7 for chitosa showed that molecular weig er than that of t 0.32 g/100 mL) surements itosan and N‐b were used to de MC). The CMC w of N‐butyrated Their critical m 7 g/100 mL a similar to the ulated by surf l‐Sou’od et al. / E h in D2O at 70 °C. , in D2O at 70 °C. peaks of amine ands originate ding vibrations might be attribu and O‐H group C‐O stretching nd 1.3 kDa NB ak from 1622 c ue, 1649 cm‐1, d ncy. The presen absence of any orts the fact th han the ‐OH gr only. n of 1.3 and 10 t molecular w nsion, which mig medium. When st the concentr ) is determine an 1.3 and 10 the critical m ght chitosan (1. the higher mole ). utyrated chitos etermine the c was estimated chitosans (1.3 icelle concentra and 0.32 g/10 eir critical m face tension v European Journal group from s. The uted to s. The of the BCh, it cm‐1 of due to nce of peaks at the roups, 0 kDa weight ght be n the ration, ed, as 0 kDa, micelle .3 kDa ecular san at critical from 3 kDa ations 0 mL, micelle versus con follo Figu 25 ° Figu NBC Figu NBC (1.3 chit obs nati l of Chemistry 7 ( ncentrations. T owing Figures 8 ure 6. Surface tens C. Figure 7. Surface t ure 8. Particle siz Ch. ure 9. Particle siz Ch. The particle s 3, 6, 10, 18, 30 k tosan (1.3/B, served that the ive (Tables 2‐6) (1) (2016) 91‐96 These results c 8‐10. sion vs concentrati tension vs concent e vs concentration ze vs concentratio size of differen kDa), when com 6/B, 10/B, 18 particle size of ). 6 can be clearly on for chitosan an tration for 10 kDa N n for N‐butyrated n for N‐butyrated nt molecular w mpared with the 8/B, 30 kDA N‐butyrated is y seen in the nd 1.3 kDa NBCh at NBCh at 25 °C. chitosan 1.3 kDa d chitosan 10 kDa weight chitosan eir N‐butyrated NBCh), it was lower than the e t a a n d s e Table 2. Particl Chitosan conc 1.3 (0.5) 1.3 (0.5)/B 1.3 (0.4) 1.3 (0.4)/B 1.3 (0.3) 1.3 (0.3)/B 1.3 (0.2) 1.3 (0.2)/B Table 3. Particl Chitosan conc 6 (0.5) 6 (0.5)/B 6 (0.4) 6 (0.4)/B 6 (0.3) 6 (0.3)/B 6 (0.2) 6 (0.2)/B Table 4. Particl Chitosan conc 10 (0.5) 10 (0.5)/B 10 (0.4) 10 (0.4)/B 10 (0.3) 10 (0.3)/B 10 (0.2) 10 (0.2)/B Table 5. Particl Chitosan conc 18 (0.5) 18 (0.5)/B 18 (0.4) 18 (0.4)/B 18 (0.3) 18 (0.3)/B 18 (0.2) 18 (0.2)/B Table 6. Particl Chitosan conc 30 (0.5) 30 (0.5)/B 30 (0.4) 30 (0.4)/B 30 (0.3) 30 (0.3)/B 30 (0.2) 30 (0.2)/B Figure 10. Pa chitosan at diffe This due quasi‐globul le size of 1.3 kDa c entration (g/100 le size of 6 kDa chi entration (g/100 le size of 10 kDa an entration (g/100 le size of 18 kDa an entration (g/100 le size of 30 kDa an entration (g/100 article size of dif erent concentratio e to the chitosa ar conformatio Al‐Sou’od et chitosan and 1.3 kD 0 mL) itosan and 6 kDa N 0 mL) nd 10 kDa NBCh at 0 mL) nd 18 KDa NBCh at 0 mL) nd 30 kDa NBCh at 0 mL) fferent molecular ons (0.2‐0.5 g/100 an in solutions on stabilized by t al. / European J Da NBCh at differen Average particl 54.00 2.65 33.70 2.72 62.80 2.43 67.00 2.59 NBCh at different co Average particl 59.40 4.63 20.20 3.60 76.60 2.98 19.70 2.77 t different concent Average particle 73.50 7.44 70.20 4.10 109.00 4.44 119.00 4.06 t different concent Average particle 80.90 7.90 52.40 4.82 113.00 4.45 129.00 4.66 t different concent Average particle 73.10 8.37 52.40 5.25 121.00 4.85 128.00 6.15 weight of N‐bu mL). exists in the fo y extensive intra Journal of Chemis nt concentrations. le size Dp (n) [nm oncentrations. le size Dp (n) [nm trations. e size Dp (n) [nm trations. e size Dp (n) [nm trations. e size Dp (n) [nm tyrated orm of a‐ and inte cau syst bac hyd wel sma in T 4. C reac and new spe dete ran com stry 7 (1) (2016) m] m] ] ] ] er‐molecular h used chitosan tems. Therefor ckbone is likely drogen bonding ll, and thus the all molecules sy Tables 2‐6. Conclusion New natural s ction of differe d 30 kDa) with w compounds ectroscopy. The ermined. The ge 44‐57% a mpounds are h ) 91‐96 R 9 0 1 0 5 0 5 0 S 7 0 3 0 6 0 2 0 R 1 0 2 0 3 0 7 0 R 5 0 5 0 9 0 1 0 R 1 0 5 0 8 0 1 0 hydrogen bond to aggregate re, the introdu y to disrupt t g of chitosan an e N‐butyrated c ystems. The va surfactants hav nt molecular w butyric anhyd were characte ir surface tensi degree of sub s confirmed b highly water s Relative std. dev. 9.20 0.43 10.20 0.35 5.40 0.31 5.43 0.10 Std. dev. 7.27 0.46 3.30 0.23 6.40 0.35 2.05 0.40 Relative std. dev. 11.00 0.53 2.77 0.60 3.09 0.27 7.12 0.33 Relative std. dev. 5.87 0.70 5.20 0.52 9.50 0.44 11.20 0.67 Relative std. dev. 12.70 0.78 5.20 0.64 8.84 0.53 12.90 0.56 ding. The hydr into large s uction of N‐but the inter‐ or i nd weaken its chitosan tends t lues of particle ve been prepare weight chitosans ride in aqueou rized by 1H N ion and particle bstitutions (DS by 1H NMR s oluble in cont 95 rogen bonding supramolecular tyl in chitosan ntra‐molecular crystallinity as to aggregate in e size are listed ed through the s (1.3, 6, 10, 18 s medium. The MR and FT‐IR e size were also S) were in the spectra. These trast to native g r n r s n d e 8 e R o e e e 96 Al‐Sou’od et al. / European Journal of Chemistry 7 (1) (2016) 91‐96 chitosan, this is may be due to breaking up of chitosans inter‐ strand hydrogen bonding. The new surfactants have a reduced particle size. Acknowledgements The authors express their thanks to Al Al‐Bayt University for financial support. References [1]. Jayakumar, R.; New, N.; Tokura, S.; Tamura, H. Int. J. Biolog. Macromol. 2006, 40(3), 175‐181. [2]. Guibal, E. Prog. Polym. Sci. 2005, 30, 71‐109. [3]. Crini, G. Prog. Polym. 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