1-7 s.k. saha r c o s t n s.k. saha / bibechana 11(1) (2014) 1-7 (online publication: march, 2014) p.1 bibechana a multidisciplinary journal of science, technology and mathematics issn 2091-0762 (online) journal homepage: http://nepjol.info/index.php/bibechana weak interactions-strong effects: key role of hydrophobic interaction in the formation of stimuli-responsive viscoelastic gel of worm-like micelles swapan k. saha*1 department of chemistry, university of north bengal, darjeeling, 734 013, india e-mail: ssahanbu@hotmail.com accepted for publication: february 02, 2014 abstract among all the weak interactions, which are operative in the domain of chemistry, hydrophobic effect is one that leads to new structural motifs like molecular self-assemblies viz., micelles, vesicles etc. worm-like micelles are polymer like aggregates which have been formed in aqueous surfactant solutions in the presence of additives. these are relatively new materials with exciting material properties. these stimuli-responsive viscoelastic materials are interesting from both fundamental understanding as well as application points of view. the physico-chemical properties of worm-like micelles are studied and the morphological transitions to vesicle as functions of various parameters have been investigated. © 2014 rcost: all rights reserved. keywords: hydrophobic effect; vesicles; worm-like micelles; viscoelastic materials. 1. introduction from the days of alchemists, the primary goal of chemistry has been to understand the behavior of molecules and their construction from constituent atoms. in recent years chemists have extended their goal beyond the atomic and molecular chemistry into the realm of supramolecular chemistry controlled by weak interactions. in chemistry, weak interactions are the intermolecular forces mainly governed by (i) vander waals interaction (0.5-2 kcal/mol) (ii) hydrophobic interactions (1-2 kcal/mole) (iii) π—π interaction (2-3 kcal/mole) and (iv) hydrogen bonding (12-16 kcal/mole). among all the weak interactions, which are operative in the domain of chemistry, hydrophobic effect is one that leads to new structural motifs like molecular self-assemblies viz., micelles, vesicles etc. in aqueous media. the unfavorable contact between water and the apolar part of surfactant molecules lead to their congregation into well organized entities, viz., micelles, vesicles, fibers, discs and tubes [1-3]. although micelles are usually spherical in shape, under certain conditions, e.g., concentration, salinity or in the presence of hydrophobic counter ions, etc., they may undergo uniaxial growth. this subsequently results in the formation of significantly long (100 – 500 µ m) yet highly flexible aggregates referred to as 1* prof. swapan k. saha, keynote speaker international conference on emerging trends in science and technology (march 22-23, 2014) biratnagar, nepal s.k. saha / bibechana 11(1) (2014) 1-7 (online publication: march, 2014) p.2 “worm-like micelles (wlm)” [4-6]. the research of wlm has drawn considerable interest as its rheology is very challenging due to the presence of multiple pertinent length scales and stress relaxation mechanisms. this relatively new material has many applications including that of fractured fluids in oil fields, efficient drag reducing agent in hydrodynamic engineering and home care, personal care and cosmetic products [7-8]. viscoelastic wlms are formed in various surfactant systems in the presence of different additives [9-12]. most extensively studied system is the cetyltrimethylammonium bromide (ctab) micelles in presence of hydrotrope, sodium salicylate (ss). unlike simple halides, salicylate promotes sphere to wormlike micellar transition at very low concentrations, viz., near the normal critical micelle concentration (cmc, ~1 mm) of ctab. the flexible and elongated wormlike micelles under dilute conditions show complex and unusual rheological phenomena, which include strong viscoelasticity and shear-induced structure (sis) formation. it is particularly interesting that, while a wide variety of wormlike ionic micellar solutions display identical rheological responses, a common element in most of these systems was the presence of salt anions such as ss. in view of the importance of an efficient wlm promoter, which might be effective for various applications at low surfactant concentrations and in the presence of metal ion impurities, organic πconjugated molecules with h-bonding functionality, viz., naphthols are highly promising [13-16]. microstructural transitions of micellar aggregates, especially the nature of transition from ordinary micelles to long worm-like giant micelles and the vesicles, mediated by organic π-electron systems are of fundamental scientific interest and have been reported in several papers only recently [17-18]. moreover, synthetic vesicular systems are interesting from a number of standpoints, not the least being their structural similarity with the constituent of the biomembrane, viz., phospholipid. they offer a convenient way to probe interactions involving membrane systems. vesicle aggregation or adhesion is the primary step for the fusion of the vesicles in membrane. therefore, the elucidation of the molecular mechanism of vesicular aggregation would greatly contribute to a better understanding of these biological phenomena. further, it may be anticipated that a simple and effective route to design a ph responsive microstructure could well be based on the neutral naphthol dopants, which form salts only at high ph (pka ~ 9.2). as a function of ph, ionization of the oh group of naphthol molecules may switch the onset of charge screening, paving the way to effect further morphological transitions (viz., vesicle formation). one of the objectives of the present work is, therefore, to design a simple effective route of ph-responsive morphological transition for the aqueous molecular aggregates of single chain cationic surfactant, viz., ctab from micelles to long wormlike micelle to unilamelar vesicles. 2. historical overview since the pioneering work of prof. h. hoffmann [9] and his group of bayreut university, germany on the unusual rheological property of wlm in the year 1988, quite a large number of physical chemists, physicists and chemical engineers became interested in this fascinating area and have contributed immensely to the advancement of wlm research. among others this list includes prof. s. candau and m. cates [19-20] of louis pasteur university, france; prof. h.t. davis [21], university of minnesota, u.s.a.; prof. d.j. pine [22] and prof. s.l. keller [23], both from the university of california, u.s.a.; prof. e.w. kaler [24], university of delware, u.s.a. and prof. t. imae [25,26] of nagoya university of japan. the coevolution of strong theoretical framework alongside application of a range of sophisticated experimental tools engendered rapid advancement in the study of wlm. presently the main focus of research in this field has been shifted to the development of new surfactant systems and new protocol by which wlm formation could be triggered. prof. s.r. raghavan [27] and his group of university of maryland have developed a series of new shear, temperature and light responsive wlm systems, which are of high technological interests. a mixture of ctab and trans orthomethoxy cinnamic acid (omca) (photo-rheological fluid) form long wormlike micelles leading to strong viscoelasticity under the s.k. saha / bibechana 11(1) (2014) 1-7 (online publication: march, 2014) p.3 influence of uv light. this trans omca is converted to cis isomer of omca, which interacts only weakly with ctab and eventually viscoelasticity disappears. prof. raghavan’s group has also studied wlm to vesicle transition as influenced by heat. prof. a. bose [28] of university of rhode island and prof. v.t. john [28] of tulane university, usa has investigated surfactant solubilization and direct encapsulation of interfacially active phenols in mesoporous silicas. template synthesis of mesoscopic tube silicates using aqueous mixtures of naphthalene diol and ammonium surfactant has been carried out by prof. m. isayama [29] of kyushu university, japan. dr. r. g. shrestha [30] and her groups, also from japan, are involved in extensive research on wlm with the aid of saxs, rheometer, cryo-tem etc. on some novel and interesting systems including poly (oxyethylene) cholesteryl ether. in china, the researchers in the field of wlm include dr. g-z. li [31] of shardong university, dr. s. liu [32] of science and technology university and prof. j. huang [33] of peking university. dr. l. zheng [34] of key laboratory of colloid and interface chemistry has been engaged in wlm research involving surface active ionic liquids (sail). in view of the fascinating rheology and technological applications of wlm, a tunable and efficient method of preparation of wlm is required. in spite of remarkable interests among the scientists on this exciting area of research, there is a major gap in the method of preparation and triggering of the wlm formation from a wide range of surfactants by metal ion inert neutral promoters (hydroxyl aromatic compounds). unless this limitation is addressed properly a complete understanding of the phenomenon, its application and control may prove to be far from complete success. 3. results and discussion despite high promise shown by neutral hydrotropes like naphthols in triggering the formation of wlm from cationic surfactants, surprisingly these hydroxyl aromatic compounds have been used only scarcely. aqueous ctab (2-10 mm) and 1or 2-naphthol (2-10 mm in 2-5% methanol, naphthols being sparingly soluble in water) solutions show viscosities similar to those of water. but as soon as these solutions are mixed together at room temperature, a thick gel-type fluid with high viscoelasticity is developed. from the initial visual observation it was found that the viscosity of the gel was very much dependent on the concentration of ctab and the additives. therefore, we first determined the ctab: dopant mole ratio at which the gel shows maximum viscosity [14]. much like the ctab-nasal system, it was found that ctab-naphthols also display maximum viscoelasticity at a 1:1 molar ratio of surfactant and the promoter (fig. 1). on the other hand the effect of the dihydroxynaphthalenes (2,3-dhn and 2,7-dhn) on micelles of ctab are completely different. equimolar mixtures of only ctab and 2,3-dhn gives a highly viscous gel in aqueous solutions but 2,7-dhn/ctab does not. therefore, further attempts were not made to study the viscosity of 2,7-dhn/ctab system [15]. the argument that an excess or deficiency of charge on the micelles due to adsorption of hydrotrope anions (e.g., nasal) would shorten the micellar life time and size is not apparently true for the present system because under the present experimental condition of solution ph (~6.5), the naphthols and the dihydroxynaphthalenes are mostly protonated, i.e., uncharged (pka’s > 9.0) [16]. therefore, it seems apparent that the symmetrical distribution of surfactant and the promoter molecules leading to highly compact spherical micelles facilitates an optimum surface curvature to attain in presence of h bonding (discussed later), and this results in the sphere to rod transition easily. for further experiments, dopant to surfactant ratio was chosen to produce strongest viscoelasticity, i.e., 1:1 mole ratio. further, the rheological responses for a viscoelastic system (viz., an aqueous ctab-1-naphthol system as a function of concentration (1:1 mol ratio) at 250c (ph ~5.0)) shows a shear thinning property up to a shear rate of 25 s-1 and then the shear thickening phenomenon starts to occur at low concentrations (~2 mm), but above a shear rate of 60, the fluid shows a newtonian type behavior [17]. however, an overall non-newtonian nature is apparent as the concentration of the ctab and naphthol (1:1) system is raised above 2.0 mm. at still higher concentrations (~5.0 mm), the nature of the rheological response s.k. saha / bibechana 11(1) (2014) 1-7 (online publication: march, 2014) p.4 changes dramatically and the system starts displaying an unusual rheology as a function of shear rate. up to a shear rate of 60 s-1, the fluid shear thins. an onset of viscosity rise is observed at the shear rate of 60 s-1, and the system again shear thins, passing through a maximum at 70 s-1. figure 1: variation of viscosity as a function mole ratio of ctab and the dopants (2-naphthol, 1-naphthol and 2,3-dihydroxynaphthelene) that form viscoelastic gels. at further higher concentrations (8.5 mm), the viscosity-shear rate profile again changes feature; the initial shear thinning characteristics disappear. the overall behavior is consistent with building up of long worm-like micellar bundles at relatively high concentrations. spectral modification of micelle-embedded dopants: in view of the differences in the viscoelastic responses and the morphological transitions of ctab micelles induced by neutral naphthols and the methoxynaphthalenes, uv absorption spectra of these dopants may be interesting to study in micellar media. to understand the kind of interactions which are operative in the micelle-dopant systems, the key element of the present study is to compare the spectral characteristics of naphthols (hns which contain oh) with those of methoxy naphthalenes (mns, which do not contain oh) under various conditions in order to visualize a consistent molecular picture eliminating the untenable suggestions. aromatic compounds, e.g., naphthalene, in general, have two strongly overlapped bands in the near uv region, viz., the longitudinally polarized 1la ← 1a band and the transversely polarized 1lb ← 1a band. while the vibrational structure of these bands appears differently in different substituted compounds, effects of extending conjugation in 1 and 2 positions by oh or ch3o groups in naphthol and methoxynaphthalene molecules, respectively, are interesting. both in 1-naphthol and 1-methoxy, naphthalene conjugation is extended in the transverse direction and, therefore, it affects the transverse polarized 1la band. in 2-naphthol and 2-methoxy naphthalene, on the other hand, conjugation is primarily extended in the longitudinal direction, affecting both the intensity and the frequency of the longitudinally polarized 1lb band compared to the unsubstituted naphthalene. it is well-known that the near uv spectra of aromatic compounds are affected by specific interactions like hydrogen bonding. noncovalent interactions like π-π and cation-π also cause shifts in the electron distributions of the molecule. the near uv absorption of 1-naphthol which arises from two strongly overlapped π-π* transitions remains unaffected in the presence of submicellar aqueous ctab solution, indicating the absence of any appreciable interaction. the effect of ctab micelles on the uv spectra of 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 4 8 12 16 20 24 28 32 v is co si ty ( cp ) ctab:dopant 2,3-dhn 1-naphthol 2-naphthol s.k. saha / bibechana 11(1) (2014) 1-7 (online publication: march, 2014) p.5 2-naphthol is also similar (fig. 2). however, interestingly, significant red-shift starts to occur (6.4 nm at λmax ~293 nm) in the presence of ctab just above its cmc (0.96 mm) with a well-defined isobastic point at 296 nm. such shifting of λmax continues until most of the naphthol molecules are partitioned in the micellar phase at high surfactant/ naphthol ratio (80:1). figure 2: absorption spectra of 2-naphthol (0.25 mm) in water at varying concentrations of ctab at 25 °c. [ctab]: (1) 0.0, (2) 0.65, (3) 0.79, (4) 0.94, (5) 1.13, (6) 1.36, (7) 1.63, (8) 1.96, (9) 2.35, (10) 2.82, (11) 3.39, (12) 5.08, (13) 7.63, (14) 11.44, (15) 17.16, (16) 20.60 mm the result suggests that the protruded oh groups of micelle-embedded naphthols form a h-bond with interfacially located (deff ~45) water molecules and act as a h-donor. it may also be argued that at a mole ratio of 1:1 of naphthol and the ctab, at which maximum viscoelastic response is observed under shear due to the presence of entangled worm-like micelles, not all of the naphthol molecules are embedded in the micelles, but some are located in the palisade layer. these naphthols may, however, be involved in hbond network formation with embedded molecules via interfacial water. the relatively less polar and less mobile water molecules compared to bulk water form a strong h-bond with the oh group of embedded naphthols, which act as h-donors and result in an optimum orientation of aromatic π-electron systems in the micelles to shield the surfactant headgroup charges efficiently; may be via cation-π interaction; i.e., the cation charge of surfactant head groups interacts with the quadrupole moment of the aromatic πsystem of naphthols. on the other hand, as the h atom of oh is replaced by a ch3 group (viz., the methoxynaphthalene molecule), the ability of intermolecular h-bond formation disappears. instead, the h-accepting tendency from a potential donor is enhanced. the nature of changes encountered in the uv spectra of methoxynaphthalenes on the addition of ctab above its cmc indicates the permeation of the dopant molecules in the micelles. the small red-shift compared to that in naphthols indicates a weaker noncovalent interaction takes place. the large drop in intensity on first addition of 0.33 mm ctab is the signature of breaking of a h-bond with bulk water molecules. due to their directionality and spatial arrangement, complementary multiple h-bonding interactions at the micellar interface lead to engineering well-defined supramolecular structure via micellar headgroup charge shielding by π-electron systems of naphthols. shear-induced viscosity and ph: the role of neutral hydroxyaromatic dopants, viz., 1and 2naphthol, stimulates the idea of designing a route for ph-responsive vesicle formation. this idea stems from the fact that the dopants, which under neutral conditions activate the formation of worm-like micelles at ph ~5.0, may on partial ionization of the oh group increase the packing parameter further via charge screening. while the viscosity of both, 2as well as 1-hn-ctab systems, is quite high due to s.k. saha / bibechana 11(1) (2014) 1-7 (online publication: march, 2014) p.6 formation of long worm-like micelles at low ph, the viscosity of the former system falls initially, indicating formation of shorter micelles with ph until ph ~5.0 is reached. on the other hand, for 1-hnctab, the onset of viscosity rise as a function of ph is found to occur from very low ph (ph ~2.0). for 2-naphthol-ctab, the onset of viscosity rise is observed at higher ph (~5.0) and the viscosity-ph profile passes through a maximum at ph ~8.5. further increase in ph results in the ionization of oh group further, and the packing parameter probably exceeds the critical value of 1/2 via enhanced charge screening, leading to vesicle formation (for naphthols, pka ~9, which means 50% ionization of the oh group at ph ~9.0). this results in the fall of viscosity of the system. since 1-naphthol could modulate the micellar surface curvature more efficiently, a little dissociation of the oh group (at low ph range) leads to an appreciable decrease of surface curvature via charge screening and promotes long worm-like micelle formation. in fact, for the 1-hn-ctab system, vesicles start to form even at slightly higher than ph ~5.0. a simple and effective route to design ph responsive viscoelastic worm-like micelles and less viscous globular vesicles based on naphthol dopants may be tuned by controlling the degree of charge screening of ctab micelles via controlled ionization of naphtholic oh groups. the result of ph-responsive morphology modification is further investigated by means of cryo-tem. cryogenic transmission electron microscopic study: cryo-tem images of the ctab-2naphthol system at high ph’s is shown in fig. 3. at high ph (ph~9.4), the system contains very long wormlike micelles, which coexist with large unilamellar vesicles. this is undoubtedly due to enhanced charge screening of micelles by naphtholate anions. the field is seen to populate mainly by large vesicles of diameter ~30 nm along with thinly populated smaller vesicles. it is also seen that the long worm-like micelles are highly entangled. sometimes they are found to elongate linearly under shear flow. the solutions are completely transparent. the direct imaging by cryo-tem supports the rheological observation as a function of ph. at low ph, the worm-like micelles are formed via headgroup charge shielding by aromatic π electrons, whereas, at high ph, ionization of oh groups takes place and the packing parameter exceeds the critical value of 1/2 via enhanced charge screening by naphtholate ion. this leads to unilamellar vesicle formation along with long worm-like micelles. figure 3: cryo-tem image of ctab-2-naphthol at a ph of ~ 9.4. 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[34] dong, j. zhang, l. zheng, s. wang, x. li, t. inoue, j. colloid interface sci., 319 (2008) 338. microsoft word mahesh chandra _6-13_.doc mahesh chandra and r.c. meena / bibechana 7 (2010) 6-13 : bmhss 6 bibechana a multidisciplinary journal of science, technology and mathematics issn 2091-0762 (online) journal homepage: http://nepjol.info/index.php/bibichana role of reductant for generation of solar energy in photogalvanic cell mahesh chandra a∗∗∗∗ , r.c. meena b a department of chemistry,deshbandhu college,new dehli-110019 b department of chemistry, jai narain vyas university, jodhpur (rajasthan)-342 005 e-mail: rcmeena007@rediffmail.com article history: received 15 october 2010;revised 29 october 2010; accepted 1 november 2010 a b s t r a c t the photo voltages and photocurrents in photo galvanic cell containing a dye rose bengal and oxalic acid as reducing agent have been determined. the photo-outputs with oxalic acid are higher than rose bengal and mannitol system. the efficiency of the rose bengal –oxalic acid photo galvanic cell has been estimated to be 0.981 % .the photo potentials and photocurrent garneted, conversion efficiency, power of the cell and cell performance of the photo galvanic cells were determined. the effects of different parameters on electrical output of the cell were observed .a mechanism has also been proposed for the generation of the photocurrent in photo galvanic cell. keywords: photo galvanic cell; photopotential; rose bengal; oxalic acid,; mannitol; power point 1. introduction today, global warming and the rapid decrease in energy resources caused by the largescale consumption of fossil fuels have become serious. accordingly renewable energy resources are attracting a great deal of attention, and solar energy is one of the most promising future energy resources. the photo effects in electrochemical systems were first reported by becquerel [1, 2] in his investigation on the solar illumination of metal electrodes long back. surash and hercules [3] proved that only negative photo potential should be obtained with carbonyl compounds. alonso et al. [4] reported the use of electrodeposited cdse0-5 te0-5 electrode for solar energy conversion. jana and bhowmik [5] reported enhancement in the power output of a solar cell consisting of mixed dyes. hara et al. [6] investigated design of new coumarin dyes having thiophene moieties for highly efficient organic dye-sensitized solar cells. ameta et al. [7] reported use of toluidine blue nitroloacetic acid (tb-nta) system in photo galvanic cell for solar energy conversion. they also reported the use of micelles in photo galvanic cell for solar energy conversion and storage in azur a-glucose system [8], bromophenol-edta system [9] and fluoroscein-edta system [10]. bohrmann-linde and tausch [11] reported photo galvanic cells ∗ corresponding author: dr. mahesh chandra, asstt. prof., department of chemistry, deshbandhu college, new dehli-110019 [india]. mob.no. +91-09968347362, e-mail: drmahesh100@gmail.com mahesh chandra and r.c. meena / bibechana 7 (2010) 6-13 : bmhss 7 for classroom investigation, and monat and mccusker [12] reported femto-second excited state dynamics of an iron (ii) polypyridyl solar cell. schwarzhurg and willig [13] explored the origin of photo voltage and photocurrent in nanoporous, dye-sensitized, photo electrochemical solar cell. the sensitization of nanoporous films of tio2 with santaline (red sandal wood pigment) and the construction of a dye–sensitized solid-state photovoltaic cell was attempted by tennakone and kumara [14]. yadav et al. [15] reported use of bismarck brown-ascorbic acid (bb-aa) system in photogalvanic cell for solar energy conversion. photo galvanic cells containing reductants and photo sensitizers [16-33] were reported. the research in the field of photogalvanic cells is still in its infancy with respect to its viability and practical applicability and, therefore, requires thorough exploration to increase the conversion efficiency and storage capacity by selecting a suitable redox couple and photo sensitizers. a detailed survey of literature reveals that no attention has been paid to electrochemical behaviour of rose bengal-oxalic acid and mannitol has also been investigated at platinum electrode in presence of these reducing agents; therefore, the present work has been undertaken 2. experimental rose bengal (loba), oxalic acid (loba), mannitol (loba) and sodium hydroxide (s.d. fine) were used without further purification in present work. all the solutions were prepared in doubly distilled water and were kept in amber colored containers to protect them from sunlight. the whole system was set up systematically for photo galvanic studies consists of two thin foilles of electrochemically treated platinum as electrodes and saturated calomel electrodes as a reference electrode the distance between the illuminated and dark electrode is approx 45 mm .an ordinary tungsten lamp of 200 as light source. water filter was used to cut off ir radiations. the photopotetial was obtained as the difference between the initial potential of the system in dark and the equilibrium potential the system attains under constant illumination. the potential was first measured in dark and the change in potential on illumination was measured as a function of time. all the solution was bubbled with prepurified nitrogen gas for nearly twenty minutes to remove dissolved oxygen. solutions of dye, reductant and sodium hydroxide were taken in an htube glass tube. a platinum electrode (1.0x1.0 cm 2 ) was immersed into one arm of h-tube and a saturated calomel electrode (sce) was kept in the other. the whole system was first placed in dark till a stable potential was obtained, then the arm containing the sce was kept in the dark and the platinum electrode was exposed to a 200 w tungsten lamp. a water-filter was used to cut off infrared radiations. the photochemical bleaching of rose bengal was studied potentiometrically. a digital ph meter (systronices) and a micro-ammeter (osaw, india) were used to measure the potential and current generated by the system, respectively. the i-v characteristics of the cells were studied using an external load (log470k) in the circuit. the effect of variation of different parameters has also been observed. the rate of change in potential after removing the source of illumination was 1.20 min in rose bengal-oxalic acid; therefore, the system may be used in photo galvanic cell more successfully than the rose bengal and mannitol system. 3. results and discussion 3.1 rose bengal-oxalic acid systems the photo potential of rose bengal-oxalic acid system was measured at different ph values and maximum photo potential was found at ph 12.6 all the subsequent measurements were made at this ph value. the variation of photo potential with time for this system is shown in figure 1. as can he has seen from the figure, the photo potential increases upon illumination to a mahesh chandra and r.c. meena / bibechana 7 (2010) 6-13 : bmhss 8 value of 1239.0 mv in about 260.0 min. and remains constant on further illumination. when the light is switched-off, the system regains its original potential immediately, thereby showing that the system is perfectly reversible. 3.2 rose bengal-mannitol systems the photo potential of rose bengal-mannitol system was measured at different ph values and maximum photo potential was found at ph 11.0. all the subsequent measurements were made at this ph value. the variation of photo potential with time for this system is shown in figure 2. as can he seen from the figure, the photo potential increases upon illumination to a value of 1080 .0 mv in about 315.0 min. and remains constant on further illumination. when the light is switched-off, the system regains its original potential immediately, thereby showing that the system is perfectly reversible. we have also examined the photocurrent of rose bengal in presence of mannitol. the observed photo potentials and currents are comparable less than that of rose bengal-oxalic acid system (table 1). the photoionduced short circuit currents of rose bengal-oxalic acid and rose bengal-mannitol in photo galvanic cells are shown in fig.3. on illumination maximum photocurrents are obtained 190.0 in 315.0 min in rose bengal – oxalic acid system and 180.0 in 260.0 min in rose bengal-mannitol system. the rose bengalmannitol system takes much smaller time than that of rose bengal-oxalic acid system. the trend in short circuit photocurrents of rose bengal-oxalic acid is much better than rose bengalmannitol system (table 1). 3.3 power conversion efficiency of photo galvanic cell one of the important characteristics of any electrochemical cell is its power conversion efficiency. the i-v characteristics of rose bengaloxalic acid and rose bengal-mannitol photo galvanic cell have been investigated to estimate the power conversion efficiency of the cell. the typical i-v curve shown in figure 3. the possible power output from the cell can be obtained from the rectangle of maximum area which can be drawn under i-v curve. 100 200 300 400 500 600 700 1000 800 900 0 p h o t o p o t e n t ia l ( m v ) 1100 1200 1300 1400 1500 1600 50 100 150 200 2500 time (min) figure 1: variation of photo potential with time in rose bengal-oxalic acid in photo galvanic cell mahesh chandra and r.c. meena / bibechana 7 (2010) 6-13 : bmhss 9 the power point (a point on the curve where the product of potential and current was maximum) in i-v curves were determined and their fill factors were also calculated. these data are summarized in table 2. the efficiency of the rose bengal-oxalic acid photo galvanic cell has been calculated to be 0.981 percent, comparable to that of and rose bengal-mannitol photo galvanic cell has been calculated to be 0.744 percent table 1 observations rose bengaloxalic acid system rose bengal-mannitol system open circuit voltage (voc) 1239.0 mv 1080.0 mv photo potential (v) 1137.0 mv 968.0 mv equilibrium photocurrent (ieq) 160.0 µa 160.0 µa maximum photocurrent (imax) 190.0 µa 180.0 µa short circuit current (isc) 175.0 µa 160.0 µa current at power point (ipp) 110.0 µa 100.0 µa potential at power point (vpp) 1022.0 µa 704.0 µa power at power point 64.0 µa/min 62.8 µa/min rate of generation 56.8 µa/min 56.0 µa/min conversion efficiency 0.981% 0.744% charging time 180.0 min 260.0 min t1/2 90.0 min 60.0 min fill factor (n) 0.51 0.44 mahesh chandra and r.c. meena / bibechana 7 (2010) 6-13 : bmhss 10 0 35 70 105 140 175 210 245 280 315 100 200 300 400 500 600 700 1000 800 900 0 1100 p h o t o p o t e n t ia l ( m v ) time (min) figure 2: variation of photo potential with time in rose bengal-mannitol in photo galvanic cell 100 0 200 300 0 35 70 105 140 175 210 245 280 315 time (min) p h o t o c u r r e n t ( a ) µ i ii figure 3 : variation of photo potential with time in (i) rose bengal-oxalic acid and (ii) rose bengalmannitol photo galvanic cell mahesh chandra and r.c. meena / bibechana 7 (2010) 6-13 : bmhss 11 table 2: i-v characteristics of the photogalvanic cells systems voc (mv) isc (µ a) vpp (mv) ipp (µ a) n rose bengal-oxalic acid 1239 175.0 1022.0 110.0 0.51 rose bengal-mannitol 1080 160.0 704.0 100.0 0.44 the conversion efficiency and sunlight conversion data for these two systems are reported in table 3. table 3 : conversion efficiency and sunlight conversion data sunlight conversion data system fill factor (n) conversion efficiency (%) photo potential (mv) photocurrent ( µ a) rose bengal-oxalic acid 0.51 0.98 1239 190 rose bengal mannitol 0.44 0.74 1080 180 on the basis of these observations, the highest conversion efficiency was found in rose bengaloxalic acid system. 3.3.1 performance of the cell the results obtained have been given in the tables 4. all the two systems were studied by applying the desired external load to have the potential and current corresponding to power point. the time t1/2 was determined after removing the source of light. it is the time taken in reaching half the value of power. the performance of cells was studied and comparative values are summarized in the table 4. table 4: performance of the photogalvanic cells in dark system power ( µ w ) t1/2 (min) rose bengal-oxalic acid 64.0 90.0 rose bengal-mannitol 62.8 60.0 on the basis of the observed data, the rose bengal-oxalic acid is the most efficient from power point of view (electrochemical studies of photo sensitizers). mahesh chandra and r.c. meena / bibechana 7 (2010) 6-13 : bmhss 12 3.3.2 mechanism on the basis of these observations, a mechanism is suggested for the generation of photocurrent in the photo galvanic cell as: illuminated chamber dye dye * (1) dye * + r dye (semi or leuco) + r + (2) at platinum electrode dye dye + e (3) dark chamber dye + e dye (semi or leuco) (4) dye + r + dye + r (5) where dye, dye*, dye , r and r+ are the excited form of dye, semi or leuco dye, reductant and oxidized form of the reductant, respectively. 4. conclusion photo galvanic cells are low cost due to the use of a dye, which are cheap and used in minute quantities reductant like ascorbic acid, is also not that very expensive. so overall working with a photo galvanic cell has lot of scope for its development. on the basis of observation in the whole study it is concluded that photo galvanic cells are better option for solar energy conversion and storage. also this system with better electrical output good performance and storage capacity may be used in near future. according to observed photo galvanic effect in all these two systems, rose bengal-oxalic acid system was the most efficient in all the ways. acknowledgement the authors are grateful to principal, deshbandhu college, kalkaji new delhi for providing the necessary laboratory facilities. references [1] e.becquerel, c .r. acad. sci. paris., 9 (1839a) 145. [2] e.becquerel, c. r. acad. sci. paris, 9 (1839a) 561. [3] j.j.surash , d.m.hercules, j. phys. chem., 66 (1962)1602. [4] a.v.alanso, m. belay, p.chartier, v.ern, rev. phys. appl.,16 (1981)5. [5] a.k.jana , b.b.bhowmik, j. photochem. photobiol., 122a , 53(1999) 53. [6] k.hara ,m.kurashige,y.dan-oh,c.kasada, a .shinpo ,s. suga , k.sayama , h. arakawa , new j. chem., 27 (2003) 783. mahesh chandra and r.c. meena / bibechana 7 (2010) 6-13 : bmhss 13 [7] s .c.ameta, r. ameta, s. seth, t.d.dubey, afinidad, xlv (1988)264. [8] s .c.ameta, s. khamesare, r. ameta , m. bala , int. j. energy res., 14 (1999)163. [9] s.c. ameta, p.b. punjabi, j.vardia, s .madhwani, s.chaudhary,j.power sources, 159 (2006)747. [10] s.madhwani, r. ameta, j .vardia, p.b.punjabi, v.k. sharma, energy sources, 29 (2007 )721. [11] c .bohrmann-linde , m.w. tausch, j. chem. educ., 80 (2003) 1471. [12] j.e. monat , j. k. mccusker, j. amer. chem. soc., 122 (2000) 4092. [13] k.schwarzburg, f. willig, j. phys. chem., 103b (1999) 5743. [14] k.tennakone, grr.a.kumara, j. photochem. photobiol, 117a (1998)137. [15] sushil yadav, r. d. yadav , gautam singh , int. j. chem. sci., 6(4) (2008) 1960. [16] r.c. meena , r.s.sindal, int. j. chem. sci., 2(3) (2004) 321. [17] suresh c. ameta, sadhana khamesra, anil k. chittoro , k.m.gangotri.,.int.j.energy res., 13 (1989) 643. [18] k.m.gongotri, r.c. meena , rajni meena., j. photochem and photobiol. a: chem; 123 (1999) 93. [19] k.m.gangotri , chhagan lal., int. j. energy res., 24 (2000)365. [20] a.c. fisher, l.m. peter, e.a. ponomareve, a.b. walker , k.g.u. wijayantha., j. phys. chem. b, 104(5) (2000) 949. [21] zhi-ying zhang, chun-yanliu., j. photochem. and photobio. a: chemistry, 130 (2000)139. [22] michael gratzel, j. photochem.and photobiol. a: chemistry, 164 (2004) 3. [23] a.hameed , m.a.gondal, j. molecular catalysis a: chemical, 219 (2004) 109. [24] coralie houarner-rassin, errol blart, pierrick buvat , fabriceodobel, j. photochem.and photobiol a: chemistry, 186 (2007)135. [25] j.bandara, u.w. pradeep , r.g.s.j. bandara., solar cells , 170 (2005) 273. [26] r.s.sindal, r.k.gunsaria,mahesh chandra , r.c. meena, the arabian journal for science and engineering, 31(2a) (2006)177. [27] minna toivola, lauripeltokorpi, janne halme, peter lund, solar energy materials and solar cells, 9 1 (2007) 1733. [28] mural kenisarin, khamid mahkamov, renewable and sustainable energy reviews, 11 (2007) 1913. [29] r.s.sindal,mahesh chandra , r.c.meena , j.ind.council chem ,25(2) (2008)131. [30] sonal sirvi, vijay kumar meena, r. c.meena, j. indian chem.soc., 85 (2008) 825. [31] r.c.meena, j.indian chem. soc., 85 (2008) 280. [32] r.s.sindal, m. chandra, v.kumar, r.c. meena , energy sources, 31(16) (2009) 1. [33] manju kumari, ram babu pachwarya, r.c. meena, int j. energy sources, 31(11) (2009) 1. microsoft word mani raj bhattrai _82-86_.doc mani raj bhattrai / bibechana 7 (2011) 83-86: bmhss 83 bibechana a multidisciplinary journal of science, technology and mathematics issn 2091-0762 (online) journal homepage: http://nepjol.info/index.php/bibichana riemann integral and its relation with lebesgue integral mani raj bhattrai ∗∗∗∗ dept. of mathematics, bhanu m.h.s. school, biratnagar, nepal article history: received 29 october 2010; revised 25 november 2010; accepted 5 december 2010 abstract this review explains the limitations of the riemann integral and focuses on the more general concept. it has been suggested that a better route is to abandon the riemann integral for lebesgue integral and explained the importance of lebesgue integral in real analysis and other fields of mathematical science. keywords: riemann integral; lebesgue integral; fourier series 1. introduction as part of a general movement toward rigor in mathematics in the nineteenth century, attempts were made to put the integral calculus on a firm foundation. the riemann integral, proposed by bernhard riemann (1826–1866) [1], is a broadly successful attempt to provide such a foundation. riemann's definition starts with the construction of a sequence of easily-calculated areas which converge to the integral of a given function. this definition is successful in the sense that it gives the expected answer for many already-solved problems, and gives useful results for many other problems. in the branch of mathematics called real analysis, the riemann integral was the first definition of the integral of a function on an interval, created by bernhard riemann,. while the riemann integral is unsuitable for many theoretical purposes, it is one of the easiest integrals to define. some of these technical deficiencies can be remedied by the riemann–satieties integral, and most of them disappear in the lebesgue integral [2]. bernhard riemann,no doubt acquired his interest in problems connected with trigonometric series through contact with dirichlet when he spent a year in berlin. he almost certainly attended dirichlet's lectures. for his habilitationsschrift (1854) riemann under-took to study the representation of functions by trigonometric functions. he concluded that continuous functions are represented by fourier series. he also concluded that functions not covered by dirichlet do not exist in nature. but there were new applications of trigonometric series to number theory and other places in pure mathematics. this provided impetus to pursue these foundational questions. riemann began with the question: when is a function integrable? by that he meant, when do the cauchy sums converge? ∗ corresponding author: mani raj bhattrai, dept. of mathematics, bhanu m.h.s. school, biratnagar, nepal, email: manirb08@yahoo.co.in mani raj bhattrai / bibechana 7 (2011) 83-86: bmhss 84 however, riemann integration does not interact well with taking limits of sequences of functions, making such limiting processes difficult to analyze. this is of prime importance, for instance, in the study of fourier series, fourier transforms and other topics. the lebesgue integral is better able to describe how and when it is possible to take limits under the integral sign. the lebesgue definition considers a different class of easily-calculated areas than the riemann definition, which is the main reason the lebesgue integral is better behaved. the lebesgue definition also makes it possible to calculate integrals for a broader class of functions. in mathematics, lebesgue integration refers to both the general theory of integration of a function with respect to a general measure, and to the specific case of integration of a function defined on a sub-domain of the real line or a higher dimensional euclidean space with respect to the lebesgue measure. 2. limitations of the riemann integral here we discuss the limitations of the riemann integral and the greater scope offered by the lebesgue integral. we presume a working understanding of the riemann integral. with the advent of fourier series, many analytical problems involving integrals came up whose satisfactory solution required interchanging limit processes and integral signs. however, the conditions under which the integrals ( ) ( )k k k k f x dx and f x dx       ∑ ∑∫ ∫ are equally proved quite elusive in the riemann framework. there are some other technical difficulties with the riemann integral. these are linked with the limit taking difficulty discussed above. 2.1 unsuitability for unbounded intervals. the riemann integral can only integrate functions on a bounded interval. it can however be extended to unbounded intervals by taking limits, so long as this doesn't yield an answer such as ∞÷∞ . 3. domain of integration a technical issue in lebesgue integration is that the domain of integration is defined as a set (a subset of a measure space), with no notion of orientation. in elementary calculus, one defines integration with respect to an orientation: ∫ ∫−= a b b a ff . generalizing this to higher dimensions yields integration of differential forms. by contrast, lebesgue integration provides an alternative generalization, integrating over subsets with respect to a measure; this can be notated as ∫ ∫ µ=µa ]b,a[ fdfd to indicate integration over a subset a. for details on the relation between these generalizations, see differential form: relation with measures. 4. intuitive interpretation to get some intuition about the different approaches to integration, let us imagine that it is desired to find a mountain's volume (above sea level). mani raj bhattrai / bibechana 7 (2011) 83-86: bmhss 85 4.1 the riemann-darboux approach divide the base of the mountain into a grid of 1 meter squares. measure the altitude of the mountain at the center of each square. the volume on a single grid square is approximately 1x1(altitude), so the total volume is the sum of the altitudes. 4.2 the lebesgue approach draw a contour map of the mountain, where each contour is 1 meter of altitude apart. the volume of earth contained in a single contour is approximately that contour's area times its height. so the total volume is the sum of these volumes. folland [1] summarizes the difference between the riemann and lebesgue approaches thus: "to compute the riemann integral of f, one partitions the domain [a, b] into subintervals", while in the lebesgue integral, "one is in effect partitioning the range of f". the riemann integral is a linear transformation; that is, if f and g are riemann-integrable on [a,b] and α and β are constants, then ( ) ( ) ( ) b b b a a a f g d x f x d x g x d xα + β =α + β∫ ∫ ∫ because the riemann integral of a function is a number, this makes the riemann integral a linear functional on the vector space of riemann-integrable functions. it can be shown that a real-valued function f on [a,b] is riemann-integrable if and only if it is bounded and continuous almost everywhere in the sense of lebesgue measure. if a real-valued function on [a,b] is riemann-integrable, it is lebesgue-integrable. if function is a uniformly convergent sequence on [a,b] with limit f, then riemann integrability of all function implies riemann integrability of f, and b b b n n n n a a a f d x lim f d x lim f d x → ∞ → ∞ = =∫ ∫ ∫ if a real-valued function is monotone on the interval [a,b], it is riemann-integrable, since its set of discontinuities is denumerable, and therefore of lebesgue measure zero. 5. alternative formulations it is possible to develop the integral with respect to the lebesgue measure without relying on the full machinery of measure theory. one such approach is provided by daniell integral [3]. there is also an alternative approach to developing the theory of integration via methods of functional analysis. the riemann integral exists for any continuous function f of compact support defined on rn (or a fixed open subset). integrals of more general functions can be built starting from these integrals. let cc be the space of all real-valued compactly supported continuous functions of r. define a norm on cc by ( ) .= ∫f f x d x then cc is a normed vector space (and in particular, it is a metric space.) all metric spaces have hausdorff completions, so let l 1 be its completion. this space is isomorphic to the space of lebesgue integrable functions modulo the subspace of functions with integral zero. furthermore, the riemann integral ∫ is a uniformly continuous functional with respect to the norm on cc, which is dense in l 1 . hence ∫ has a unique extension to all of l1. this integral is precisely the lebesgue integral. mani raj bhattrai / bibechana 7 (2011) 83-86: bmhss 86 this approach can be generalized to build the theory of integration with respect to radon measures on locally compact spaces. it is the approach adopted by bourbaki [4] for more details see radon measures on locally compact spaces. references [1] w.k.clifford, “on the hypotheses which lie at the foundation of geometry”, nature, 8 (1873) 183. [2] h.j. wilcox and d.l. myers, an introduction to lebesgue integration and fourier series, dover publications, inc., new york, (1978). [3] p. daniell, "a general form of integral" ann. of math. , 19 (1917) 279. [4] n. bourbaki, elements of mathematics, springer, (2004). 403 forbidden forbidden you don't have permission to access this resource. apache/2.4.54 (ubuntu) server at www.nepjol.info port 443 microsoft word ram p. koirala _14-17_.doc ram p. koirala / bibechana 7 (2011) 14-17 : bmhss 14 bibechana a multidisciplinary journal of science, technology and mathematics issn 2091-0762 (online) journal homepage: http://nepjol.info/index.php/bibichana temperature regulation in animals ram prasad koirala ∗∗∗∗ dept. of physics, m.m.a.m. campus, biratnagar, nepal article history: received 5 september 2010; revised 22 october 2010; accepted 5 november 2010 a b s t r a c t we have studied the rate of evaporation of sweat for the body of human of two different surface areas at different temperatures. we have also studied the rate of evaporation of sweat at different values of heat produced by chemical metabolism. it is found that a thin man perspires at lower temperature than a fat man when they both produce the same heat by chemical metabolism. keywords: chemical metabolism; evaporation of sweat; normal breathing 1. introduction most biological processes are temperature dependent. so the body temperature must be kept within a narrow range. the chemical metabolism of food is the source of body heat. depending on the air temperature and clothing worn, the heat generated may be needed to overcome convective and radiative loss and radiation from the skin and by the evaporation of sweat from the skin and of water from the lungs [1, 2]. the evaporation of sweat is primary cooling mechanism used by the body. the temperature – control mechanisms of many warm blooded animals make use of heat of vaporization, removing heat from the body using it to evaporate from the tongue (panting) or from the skin (sweating). evaporation by perspiration is an important mechanism for temperature control of warm blooded animals evaporating cooling enables humans to maintain normal body temperature in hot, dry desert climates where the air temperature may reach 55 o c (about 130 o f). the skin temperature may be as much as 30 o c cooler than the surrounding air. under these conditions a normal person may perspire several liters per day, and this lost water must be replaced [3]. if the interior temperature begins to increase, the body first increases blood flow near the skin surface to increase convective and radiative losses and then if necessary, uses evaporative loss mechanism. human sweats from glands located over much of the body and thus benefits from evaporation over a large surface area. as much as 1.5 kg sweat per hour can be evaporated from the human body under many conditions. evaporation by perspiration is primary cooling mechanism for temperature control of human body. this mechanism can also be observed in ∗ corresponding author: ram pd. koirala, dept. of physics m.m.a.m. campus, biratnagar, nepal, email:sijuwa_3@yahoo.co.in ram p. koirala / bibechana 7 (2011) 14-17 : bmhss 15 other warm-blooded animals. in this paper we intend to study about the rate of sweat from human body in different conditions in light of physics. 2. theory the various contributions to the rate of heat loss and production of heat for a typical human adult are approximately as follows [4]: if the area a is given in square meter, the skin temperature ts and air temperature ta are given in degree celsius, and the rate r of sweating is in units of kilogram per hour, then =mh rate of heat generated by metabolism ( w1600to80 ; average hm = 300 w) hr = rate of heat lost by radiation = )tt(ad asr − hc = rate of heat lost by convection = )tt(ad asc − (still air) hl = rate of heat lost by evaporation from the lungs = dl hs = rate of heat lost by evaporation of sweat = ds r the d’s are constants whose values are given by 12 c kwm1.7d −− = 12 r kmw5.6d −− = 1 s kghw674d − = w5.10d l = for normal breathing. the value of hl is given for normal breathing; it increases to the proportion of rate of breathing. as the variation is very small, we neglect the variation. for the body temperature to be held constant, the heat loss are related as hm = hs + hr+ h c+ hl lrcms hhhhh −−−= 3. results and discussion for a given value of heat produced by chemical metabolism in a man, the rate of sweating has been found to increase with increase in air temperature. evaporation begins at different temperature depending upon the amount of heat produced by metabolism. with a given surface area, as heat produced by metabolism is increased, sweating is found to begin at lower temperature and the rate of sweat production is higher for larger value of heat of metabolism, hm. for a typical human of a=1.0 m 2 , sweating begins around air temperature of 32 0 c (r =0.0022 kgh -1 ), 16 0 c (r =0.0057 kgh -1 ), 2 0 c(r = 0.0200 kgh -1 ) and below 0 0 c for hm=80w, hm = 300w, hm = 500w and hm = 1000w respectively. for a human of a=1.2m 2 , sweating begins around 33 0 c (r=0.0063 kgh -1 ), 20 0 c (r=0.0179 kgh -1 ), 8 0 c (r=0.02406 kgh -1 ) and far below for hm=80w, 300w, 500w and 1000w respectively. ram p. koirala / bibechana 7 (2011) 14-17 : bmhss 16 figure 1, 2 and 3: rate of evaporation of sweat (r) verses temperature, ( ) for a human of surface area 1.0 square metre and (……..) for human of surface area 1.2 square metre. thus rate of perspiration varies with the heat produced by the chemical metabolism and with the temperature of air. the evaporation of sweat forms an important mechanism for the temperature control of human body. the constant body temperature of other warm blooded animals can also be explained on the principle of evaporation by perspiration. ram p. koirala / bibechana 7 (2011) 14-17 : bmhss 17 4. conclusions a thin man perspires at lower temperature than a fat man when they both produce the same heat by chemical metabolism. at higher temperature rates of sweating are nearly the same for fat and thin man. acknowledgement i gratefully acknowledge associate professor devendra adhikari, presently in t.m. bhagalpur university, for his valuable suggestions and inspiring discussions. references [1] t.c. ruch and h.d. patton; physiology and biophysics vol. 3, w.b. saunders co. philadelphia, chap.5 ( 1973 ). [2] h. bornd, scientific american ( 1981) 146. [3] f.w.sears, zemnasky et.al., university physics, 11th ed. pearson education , delhi, (2004). [5] j .w. kane and m.m. sterntheim, physics, john willey and sons 2nd ed. ( 1948 ) p. 260. further readings j.r. cameron and j. g. skofronick, medical physics, john willey and sons inc. new york ( 1978). microsoft word ram chandra adhikari _21-25_.doc ram chandra adhikari / bibechana 7 (2011) 21-25 : bmhss 21 bibechana a multidisciplinary journal of science, technology and mathematics issn 2091-0762 (online) journal homepage: http://nepjol.info/index.php/bibichana household solid waste production and its management in biratnagar sub-metropolitan city ram chandra adhikari ∗∗∗∗ department of zoology, tribhuvan university, central campus of science and technology, hattisar, dharan article history: received 2 october 2010; revised 5 november 2010; accepted 10 november 2010 a b s t r a c t the study was conducted from march to may month of 2010 a d in biratnagar regarding the waste production and management. total amount of waste produced was recorded about 46.877 tonnes per day. the highest amount was from urban rural area (44.637 tonnes) and the lowest was from main bazaar (0.689 tonnes). about 69.19 percent households collect waste separately . 45.90 percent households dump degradable waste. 80 percentages of all households sell non degradable waste and only 33.67 percentage people of this town have scientific knowledge of waste management. keywords: solid waste; biodegradable waste; improper settlements 1. introduction biratnagar sub-metropolitan city (26º 28ú n, 86º19ú e and 72 m altitude) is one of four submetropolitan cities of nepal lying in koshi zone, morang district . taking the position as second largest town it locates with the embanked by singia river in east and keshlia in west. it enjoys on sub-tropical and monsoon type of climate. this town covers an area about 59.9 square kilometers. a total of 26,699 households and 1, 80,138 people reside in this town. due to availability of facilities belonging to education, healthcare, employment etc. a lot people are migrating to this town every year. improper settlements, rapidly growing population, industrialization and lack of sufficient awareness result over production of solid waste and that create many problems. this town has been facing growing solid waste problems. any unwanted and discarded materials from residential, commercial, industrial, mining and agricultural activities that cause environmental problems may termed as solid waste. on the other words solid waste can be defined as those wastes which have been rejected for further use and can neither be transported by water into streams nor readily escape into atmosphere . according to rao [1] the waste arising from human and animal actives that normally solid and described as useless or unwanted. solid waste is a byproduct of human activities which tends to increase with rapid urbanization, improved living standards and changing consumption patterns. ∗ corresponding author: ram chandra adhikari, department of zoology, tribhuvan university, central campus of science and technology, hattisar , dharan email: ramchandra.adhikari@gmail.com ram chandra adhikari / bibechana 7 (2011) 21-25 : bmhss 22 management of increasing amounts of solid waste has become a major challenge in many cities in developing countries. the amount of waste generated from every person is increasing day per day as a result of social, economical and technological changes. according to who [2], it varies from 0.2 to 3 kg per person per day. unicef [3] reported that waste production depends on income of people. people with per capita income below 360 us dollar produce about 0.5 kg solid waste. similarly those with per capita income 360 to 3500 dollar produce 1.5 kg and income above 3500 dollar produce up to 4 kg of solid waste per day. on average 58 municipalities in the country are spending about 13 percent of their total budget on waste management related activities but in most cases this amount is not being spent in an efficient manner [4]. according to cbs 1997 [5] only 17 percent of urban households have their waste collected by waste collectors. furthermore, in low-income households, indicated by houses having no toilets, only 2 percent of the households have their waste collected. open waste piles are a common site and the work of municipalities is often limited to sweeping the streets and dumping the waste in the nearest river or vacant land. solid waste can be classified into two categories. they are bio degradable waste and non degradable waste. papers, garbage, dead bodies and compost -able waste are the examples of degradable solid waste and plastics, metals, ceramics etc. are non degradable ones. the waste should be managed properly but in the case of biratnagar there is not suitable dumping site or sanitary landfill site. so problems on the management of solid waste have been rising. waste management comprises of purposeful and systematic control of the generation, storage, collection, transportation, separation, processing, recycling, recovery and disposal of waste [6]. objectives the main objectives of present study were: -to measure the total quantity of waste production -to study management practice of household waste 2. materials and methodology the following important materials, tools or equipments were used for designing experiment scientifically. the materials used in the research are as following :collecting drums, spring balance, shovel, thermometer, gloves, field book, forceps, mask, questionnaires, calculator, camera, tongs, plastic bags, spade etc. firstly biratnagar town was divided into three areas namely main-bazaar ( ward number 8,9,10 and 14), semi-urban ( 3,4,12 and 13) and urban-rural (1,2,5,6,7,11,15,16,17,18,19,20,21 and 22 ). it was done according to municipality profile. after finding total number of households, 10 percent (about 300 households) of total number was taken for the study. special attention was given to include the households of all toles. fifteen individuals were trained as observers and used in the field with researcher. from each sample house, waste production was weighed with the help of spring balance twice a week and amount of average production was calculated. during the study period the waste management methods were also observed. by giving a questionnaire to fill up to the public and direct observation of existing management practice was studied. the period of study was three months from march to may 2010. ram chandra adhikari / bibechana 7 (2011) 21-25 : bmhss 23 3. result and discussion 3.1 waste production the average of solid waste generation from urban-rural region is tabulated in the table 1. table 1 s.n. month types of waste average daily production ( in kg) 1 march bio degredable + non degrdable+mixed 17540+6440+20000=4398 0 2 april bio degredable+non degredable+ mixed 18702+7845+21033=4758 3 3 may bio degredable+ non degredable +mixed 16980+5488+19882=4235 0 average 44637.667 from the above table in march biodegradable waste was produced 17540 kg, non degradable waste 6440 kg and non-separated was 20000 kg. similarly 18702 kg of biodegradable,7845 kg of non degradable and 21033 mixed type of waste was produced and total amount was 47583 kg in april. in the case of may month biodegradable 16980 kg, non degradable 5488 kg mixed 19882 kg and total 42350 kg was generated per day. the sum of total waste generation was found to be 44637.667 kg. the average of solid waste solid waste generation from semi urban region is tabulated in table 2. table 2 s.n. month types of waste average daily production ( in kg) 1 march bio degredable + non degrdable+mixed 540+192+732=1464 2 april bio degredable+non degredable+ mixed 555+243+679=1477 3 may bio degredable+ non degredable +mixed 675+272+765= 1712 average 1551 above table shows that from semi urban area in march 540 kg of bio degradable waste, 192 kg of non degradable and 732 kg of non-separated waste was excreted out per day. the total amount of solid waste produced was about 1464 kg daily. in the month of april 555 kg biodegradable, 243 kg non degradable, 679 kg mixed and total 1477 kg of non separated waste was found to come out from the houses. 675 kg bio degradable, 272 kg non degradable and 765 kg mixed waste and a total of 1712 kg was excreted out daily. in average 1551 kg waste was found to be generated everyday from this area. the average of solid waste solid waste generation from main bazaar region is furnished in table 3. ram chandra adhikari / bibechana 7 (2011) 21-25 : bmhss 24 table 3 s.n. month types of waste average daily production ( in kg) 1 march bio degredable + non degrdable+mixed 240+100+331=671 2 april bio degredable+non degredable+ mixed 385+123+ 342=850 3 may bio degredable+ non degredable +mixed 299+112+235= 546 average 689 above table tells that from main bazaar area in march 240 kg of bio degradable waste, 100 kg of non degradable and 331 kg of non-separated waste was excreted out per day. the total amount is about 671 kg daily. in the month of april 385 kg biodegradable, 123 kg, non degradable 342 kg mixed and total 850 kg of waste comes out from the houses. 299 kg biodegradable, 112 kg non degradable and 546 kg mixed waste and a total of 546 kg was excreted out daily. 689 kg was the average daily waste production. total waste generation from whole biratnagar sub-metropolitan city is displayed in table 4. table 4 s. n. area waste production (in kg per day ) 1 urban rural 44637.667 2 semi urban 1551 3 main bazaar 689 total 46877.667 according to the above table total amount of waste production from this town was found to be about 466877.667 kg or 46.6877 metric tonnes per day . it was made by 44637.667 kg from urban rural as maximum and 689 kg from main bazaar as minimum. 3.2 waste management practice during the study the following practices were observed in whole sub-metropolitan city. data were taken region wise and total of the town is presented here in all the topics. • 69.19 percent households collect biodegradable and non degradable waste separately. • about 45.90 percent households dump degradable waste in the pit in house compound. rest of them throws the solid waste randomly. • 80 percentages of all households sell non degradable waste. • only 33.67 percentage people have scientific knowledge of waste management. 4. conclusion biratnagar sub-metropolitan city is in trouble of solid waste problem. a huge amount of solid waste (46.687 tonnes) has daily been produced. there are not sanitary landfill site, proper ram chandra adhikari / bibechana 7 (2011) 21-25 : bmhss 25 awareness and cultural practices of waste management. some people (69.19 percent) collect waste separately but large population throws them everywhere. about 80 percentages of households sell plastics and metal born waste as they don't have sufficient practice of reuse. according to the study 33.67 percentage people know the method of waste management. this town is seeking for a good guideline, enough budget, proper awareness, skilled human resource etc. for waste management. acknowledgements the author is thankful to sagaramatha community development center (scdc) for financial support to this study. the author is also grateful to dr. damodar thapa for the valuable suggestions and inspiration to conduct the project. references [1] c.s. rao, study of solid waste management for environment pollution control engineering, wiley eastern limited, delhi, (1985). [2] who, solid waste disposal and control, public health papers 38, geneva , (1971). [3] unicef, the state of children and environment, new york, (1990). [4] swmrmc, a d i a g n o s t i c r e p o r t o n s t a t e o f s o l i d w a s t e m a n a g e me n t i n municipalities of nepal, solid waste management and resource mobilization centre, lalitpur, (2004). [5] cbs, urban population survey 1996, central bureau of statistics, his majesty's government of nepal, kathmandu, (1997). [6] s.s. dara, a text book of environmental chemistry and pollution control, s. chand and company ltd, new delhi, ( 1998) . further readings [7] a.k deo, environmental chemistry, new age international limited, new delhi, (2001). [8] r.c. adhikari, study of solid waste management, a thesis submitted to p.g. campus biratnagar,(2oo5) . 403 forbidden forbidden you don't have permission to access this resource. apache/2.4.54 (ubuntu) server at www.nepjol.info port 443 microsoft word mohd yusuf yasin.doc mohd yusuf yasin / bibechana 8 (2012) 31-36 : bmhss, p. 31 bibechana a multidisciplinary journal of science, technology and mathematics issn 2091-0762 (online) journal homepage: http://nepjol.info/index.php/bibechana scientific calculators and the skill of efficient computation mohd yusuf yasin ece department, integral university, lucknow, up, india e-mail: mmyasin@rediffmail.com article history: received 12 may 2011; accepted 21 june 2011 abstract calculators are part and parcel of modern education. involvement of science and engineering in different fields of knowledge is increasing with each bit of time is passed by, and they are playing a role in description and characterization of the delicate phenomena of nature arising day by day. these fields of knowledge and mathematics in particular, are influencing even those distant branches of knowledge, which were so far imagined to be free of mathematics. even art is not free of mathematics and there exists mathematical art. computations are getting lengthy and complex specially in design and analysis of engineering systems. scientific calculators are handy tools. but an efficient computation is a skill that can be developed. keywords: scientific calculators; calculator techniques; efficient computation; efficient use of calculators, computation skills; numerical techniques 1. introduction today’s world is of science and technology. science has pervaded in other branches of knowledge, it has become difficult to think a subject thoroughly explained without inviting science to help. more insight into the natural phenomena are required and as a way, they demand computations. subjects specifically related to engineering and technology, are heavily computational. fortunately, powerful computing devices are readily available and in every body’s reach. it has been observed that usually students buy scientific calculators. but it is a pity that most of these students usually imagine the operations of multiplying and dividing, and computations of functions like sine and cosine is the all a scientific calculator can do. today’s scientific calculators are highly powerful and if their operation is properly understood, a little innovative imagination can do wonders. long calculations are a normal part of design and analysis, where students spend time like anything, and conclude the computational procedure in distaste. in this short paper efforts are made to point out that this attitude needs a rethinking on the process afresh to settle down the matters favorably. mohd yusuf yasin / bibechana 8 (2012) 31-36 : bmhss, p. 32 2. preliminaries a little knowledge is required before one should start working with a calculator. every calculator is complemented with a manual, which delineates the calculator capabilities. it needs proper attention to go through the manual. scientific calculators are highly capable and therefore are complex entities. their functioning is grouped. therefore a calculator needs to be prepared for a proper functioning. a very common mistake usually observed in this context is the computation performed on functions containing ‘circular functions’ without a care of the proper mode of their arguments or computations on functions containing a ‘log’ without an idea of the natural log or log on base 10. such improper modes lead to wrong results and may cause a failure to maintain interest in the process of design and analysis both. 3. calculator techniques almost all scientific calculators posses a temporary register under the key ans, which automatically modifies after each computation, and holds the current result. in design or analysis, this result is usually needed by the next step. still there are examples where recursive computations are necessary to ascertain the final results. normally, calculators have a few more variable like a, b, c, ;, but presently a description is restricted on some simple tricks based upon the ans key only. in fig.1 the general scheme adopted here is presented. figure 1: general flow graph of the computational procedure mohd yusuf yasin / bibechana 8 (2012) 31-36 : bmhss, p. 33 in the following sections, two examples are considered for the purpose of description on how the calculator tricks are applicable to ease the process of approaching final accurate result recursively. in both these examples, due to their simplicity and excellent bidirectional approach to the final accurate solution, the simple newton – raphson method is employed. 3.1 calculators referred in this article, scientific calculators referred to as the casio make only, like fx-82ms, fx-991ms or later versions. all these calculators have a distinct feature of displaying full scientific equations before and after the calculation is done. a ‘scientific calculator’ afterwards refers to a calculator possessing these specific features. other brands of calculators with similar features can also be used for the purpose of this article, possibly with a little or no modification in the computational scheme depending upon their operating principles [1,2]. 3.2 example – 1: square root of a given number in the first example, computation of the square root of a given number ‘a‘ is considered. let x be the square root of the number a thus mathematically this can be represented as ax = 2 , and the corresponding functional equation is axxf −= 2 )( . according to the ordinary newton – raphson method, the square root is approached by the equation )( )( '1 n n nn xf xf xx −=+ , where n x is the first approximation, 1+nx is the new improved approximation and )( ' n xf is the first derivative of the function )( n xf . when applied to the required square root of the given number, this approximating equation can be expressed as [3]:       +=+ n nn x a xx 2 1 1 (1) this method is particularly important as it is quite fast in approaching the final reasonable result in just a few iterations. further, the final result is possible even when an absurd initial approximation is adopted, of course, in such a case, total number of iterations may be increased. for applying a scientific calculator, the above root approximating equation is considered for determining a square root of the given number 3, with an initial approximation, say, 1. the procedure can be as follows. 1. type the initial approximation, (say, ‘1’ for this example) and press ‘=’ (enter through the key ‘=’ in casio calculators, refer, fx-82ms, fx-991ms). this step stores the ‘1’ in the register associated with the key ‘ans’. as every result is automatically stored under the key ‘ans’, therefore this key can be used as a variable. hence, the square root determining equation can be re written with ‘ans’ as variable,       += ans a ansans 2 1 (2) here in equation (2), the ‘ans’ inside the braces refers to n x and contain the last result, whereas the ‘ans’ outside refers to 1+nx and automatically modifies to the latest result. as mohd yusuf yasin / bibechana 8 (2012) 31-36 : bmhss, p. 34 per the data handling scheme of the calculators, the equation (2) is read as       +← ans a ansans 2 1 , where the calculation results on the right hand side are transferred to the register attached to the ‘ans’ key, 2. second step is to type the equation in accordance with the right hand side of the above equation assuming the variable key ans and do the required computation by just pressing the ‘=’ key as many number of times as required. the sequence of keys pressed on a calculator may be as follows: 1 =; ( ans + a / ans ) / 2 = = = (3) in this construct, each key is separated by multiple spaces for the sake of clarity only, the spaces are not a part of the equation. as is evident from the calculation of the examples below, for three iterations, the total number of key presses are just 14 in each case. convergence of 3 to it’s final numerical value is as follows: ans ← 1.0 ans ← 2.0 ans ← 1.75 ans ← 1.732142857 ans ← 1.73205081 ans ← 1.732050808. (4) the value calculated from the calculator is 732050808.13 = , thereby showing no error in the two results. however, 732.13 = is normally the accepted approximate value, and is available within third iteration. 3.3 root of an equation again, consider to find the intersection of the curves [4] xy cos= and xy = . (5) it is a single root problem. a solution exists as shown in fig. 2. analytically, the solution is obtained as follows. applying the ordinary newton raphson method, equation for solution is obtained as xxxf cos)( −= from eq. (5) above. the corresponding approximation equation to it’s root is n nn nn x xx xx sin1 cos 1 + − −=+ n nnn x xxx sin1 cossin + + = . (6) mohd yusuf yasin / bibechana 8 (2012) 31-36 : bmhss, p. 35 the computational equation for a calculator is written as given in equation (7) ans ansansans ans sin1 cossin + + = (7) convergence pattern of the root of the above equation with 1.0 as initial approximation, and for ‘radians’ as the mode of calculation, is figure 2: graphical solution of the equations of section 3.3 [4] ans ← 1 ans ← 0.750363867 ans ← 0.73911289 ans ← 0.739085133 ans ← 0.739085133 (8) a root of third decimal place accuracy is again achieved just within three iterations. it is important to consider that the concerned equations contain circular function, and therefore the selection of the mode radian becomes important. the results presented above are obtained in radian mode. purpose of the problem in equation (5) is to bring out the importance of consideration as mentioned earlier. in the solution of equation (6), ‘deg’ mode selection will produce a look alike root, 0.999847784, which unfortunately is not a root but merely an approximation of the equation (5) for the argument of the circular function tending 0 ( 0;cos →xx ). this situation arises due to an inappropriate mode selection. 4. discussions the above examples are common yet simple and are readily available in text books on advanced mathematics for engineers and scientists, numerical techniques etc. here the point of emphasis is how to make the computation effectively. the effective computational process for example in section 3.2 for a single digit number as per the flow graph, needs, all in all, 14 judicial key presses mohd yusuf yasin / bibechana 8 (2012) 31-36 : bmhss, p. 36 on an already on calculator. however, the engineering students have often been observed haggling for hours over solving such problems and with absurd or at least, inaccurate come outs. 5. conclusion for fields like numerical methods, where usually computations are huge but are iterative in nature, it is important to optimize computational efforts. such efforts are based on computational skills. these are some of the calculator tricks presented here which can be found quite useful. scientific calculators are quite powerful, but their real power remains untapped. a little practice can help develop the capability to perform computations in technical manner. references [1] users’ guide, fx-82ms/83ms/85ms/270ms/300ms/350ms, http://support.casio.com [2] users’ guide, fx-95ms/100ms/115ms/(912ms)/570ms/991ms, http://support.casio.com [3] erwin kreyszig, advanced engineering mathematics, 5 th edition, wiley eastern limited, 1989, pp 764 [4] http://www.sosmath.com/calculus/diff/der07/der07.html; graphical solution for the equations given in eq. (5) microsoft word main.doc 22 narendra kumar chaudhary food additives narendra kumar chaudhary m.m.a.m. campus, biratnagar, tribhuvan university, nepal abstract different types of food additives have been discussed. i have discussed the role of food additives to preserve nutritional values and to maintain desired consistency of the product. the antioxidants and antimicrobials food preservatives have also been studied. keywords: food additives; preservatives; colorants; sweeteners introduction various chemical substances added to foods to produce specific desirable effects are called food additives. additives such as salt, spices, and sulfites have been used since ancient times to preserve foods and make them more palatable. with the increased processing of foods in the 20th century, there came a need for both the greater use of and new types of food additives. many modern products, such as low-calorie, snack, and ready-to-eat convenience foods, would not be possible without food additives. food additives and their metabolites are subjected to rigorous toxicological analysis prior to their approval for use in the industry. feeding studies are carried out using animal species (e.g., rats, mice, dogs) in order to determine the possible acute, short-term and long-term toxic effects of these chemicals. the lowest level of additive producing no toxicological effects is termed the no-effect level (noel). there are four general categories of food additives: nutritional additives, processing agents, preservatives, and sensory agents. nutritional additives nutritional additives are utilized for the purpose of restoring nutrients lost or degraded during production, fortifying or enriching certain foods in order to correct dietary deficiencies, or adding nutrients to food substitutes. the fortification of foods began in 1924 when iodine was added to table salt for the prevention of goitre. vitamins are commonly added to many foods in order to enrich their nutritional value. for example, vitamins a and d are added to dairy and cereal products, several of the b vitamins are added to flour, cereals, baked goods, and pasta, and vitamin c is added to fruit beverages, cereals, dairy products, and confectioneries. other nutritional additives include the essential fatty acid linoleic acid, minerals such as calcium and iron, and dietary fibre. processing agents a number of agents are added to foods in order to aid in processing or to maintain the desired consistency of the product. several of these agents are discussed in more detail below. emulsifiers are used to maintain a uniform dispersion of one liquid in another, such as oil in water. the basic structure of an emulsifying agent includes a hydrophobic portion, usually a long-chain fatty acid, and a hydrophilic portion that may be either charged or uncharged. the hydrophobic portion of the emulsifier dissolves in the oil phase and the hydrophilic portion dissolves in the aqueous phase, forming a dispersion of small oil droplets. emulsifiers thus form and stabilize oil-in 23 bibechana vol. 6, march 2010 water emulsions (e.g., mayonnaise), uniformly disperse oil-soluble flavour compounds throughout a product, prevent large ice crystal formation in frozen products (e.g., ice cream), and improve the volume, uniformity, and fineness of baked products. stabilizers and thickeners have many functions in foods. most stabilizing and thickening agents are polysaccharides, such as starches or gums, or proteins, such as gelatin. the primary function of these compounds is to act as thickening or gelling agents that increase the viscosity of the final product. these agents stabilize emulsions, either by adsorbing to the outer surface of oil droplets or by increasing the viscosity of the water phase. thus, they prevent the coalescence of the oil droplets, promoting the separation of the oil phase from the aqueous phase (i.e., creaming). chelating, or sequestering, agents protect food products from many enzymatic reactions that promote deterioration during processing and storage. preservatives food preservatives are classified into two main groups: antioxidants and antimicrobials. antioxidants are compounds that delay or prevent the deterioration of foods by oxidative mechanisms. antimicrobial agents inhibit the growth of spoilage and pathogenic microorganisms in food. antioxidants the oxidation of food products involves the addition of an oxygen atom to or the removal of a hydrogen atom from the different chemical molecules found in food. two principal types of oxidation that contribute to food deterioration are autoxidation of unsaturated fatty acids and enzyme catalyzed oxidation. the autoxidation of unsaturated fatty acids involves a reaction between the carbon-carbon double bonds and molecular oxygen (o2). the products of autoxidation, called free radicals, are highly reactive, producing compounds that cause the off-flavours and off-odours characteristic of oxidative rancidity. antioxidants that react with the free radicals (called free radical scavengers) can slow the rate of autoxidation. these antioxidants include the naturally occurring tocopherols (vitamin e derivatives) and the synthetic compounds butylated hydroxyanisole (bha), butylated hydroxytoluene (bht), and tertiary butylhydroquinone (tbhq). specific enzymes may also carry out the oxidation of many food molecules. the products of these oxidation reactions may lead to quality changes in the food. for example, enzymes called phenolases catalyze the oxidation of certain molecules (e.g., the amino acid tyrosine) when fruits and vegetables, such as apples, bananas, and potatoes, are cut or bruised. the product of these oxidation reactions, collectively known as enzymatic browning, is a dark pigment called melanin. antioxidants that inhibit enzyme-catalyzed oxidation include agents that bind free oxygen (i.e., reducing agents), such as ascorbic acid (vitamin c), and agents that inactivate the enzymes, such as citric acid and sulfites. antimicrobials antimicrobials are most often used with other preservation techniques, such as refrigeration, in order to inhibit the growth of spoilage and pathogenic microorganisms. sodium chloride (nacl), is probably the oldest known antimicrobial agent. organic acids, including acetic, benzoic, propionic, and sorbic acids, are used against microorganisms in products with a low ph. nitrates and nitrites are used to inhibit the bacterium clostridium botulinum in cured meat products. sulfur dioxide and sulfites are used to control the growth of spoilage microorganisms in dried fruits, fruit juices, and 24 narendra kumar chaudhary wines. nisin and natamycin are preservatives produced by microorganisms. nisin inhibits the growth of some bacteria while natamycin is active against molds and yeasts. sensory agents colorants colour is an extremely important sensory characteristic of foods; it directly influences the perception of both the flavour and quality of a product. the processing of food can cause degradation or loss of natural pigments in the raw materials. in addition, some formulated products, such as soft drinks, confections, ice cream, and snack foods, require the addition of colouring agents. colorants are often necessary to produce a uniform product from raw materials that vary in colour intensity. colorants used as food additives are classified as natural or synthetic. natural colorants most natural colorants are extracts derived from plant tissues. the use of these extracts in the food industry has certain problems associated with it, including the lack of consistent colour intensities, instability upon exposure to light and heat, variability of supply, reactivity with other food components, and addition of secondary flavours and odours. in addition, many are insoluble in water and therefore must be added with an emulsifier in order to achieve an even distribution throughout the food product. synthetic colorants synthetic colorants are water-soluble and are available commercially as powders, pastes, granules, or solutions. special preparations called lakes are formulated by treating the colorants with aluminum hydroxide. they contain approximately 10 to 40 percent of the synthetic dye and are insoluble in water and organic solvents. lakes are ideal for use in dry and oil-based products. the stability of synthetic colorants is affected by light, heat, ph, and reducing agents. a number of dyes have been chemically synthesized and approved for usage in various countries. all synthetic colorants have undergone extensive toxicological analysis. brilliant blue fcf, indigo carmine, fast green fcf, and erythrosine are poorly absorbed and show little toxicity. extremely high concentrations (greater than 10 percent) of allura red ac cause psychotoxicity, and tartrazine induces hypersensitive reactions in some persons. flavourings the flavour of food results from the stimulation of the chemical senses of taste and smell by specific food molecules. taste reception is carried out in specialized cells located in the taste buds. the four basic taste sensations—sweet, salty, bitter, and sour—are detected in separate regions of the tongue, mouth, and throat because the taste cells in each region are specific for certain flavour molecules. in addition to the four basic tastes, the flavouring molecules in food stimulate specific olfactory (smell) cells in the nasal cavity. these cells can detect more than 10,000 different stimuli, thus fine-tuning the flavour sensation of a food. a flavour additive is a single chemical or blend of chemicals of natural or synthetic origin that provides all or part of the flavour impact of a particular food. these chemicals are added in order to 25 bibechana vol. 6, march 2010 replace flavour lost in processing and to develop new products. flavourings are the largest group of food additives, with more than 1,200 compounds available for commercial use. natural flavourings are derived or extracted from plants, spices, herbs, animals, or microbial fermentations. artificial flavourings are mixtures of synthetic compounds that may be chemically identical to natural flavourings. artificial flavourings are often used in food products because of the high cost, lack of availability, or insufficient potency of natural flavourings. flavour enhancers are compounds that are added to a food in order to supplement or enhance its own natural flavour. the concept of flavour enhancement originated in asia, where cooks added seaweed to soup stocks in order to provide a richer flavour to certain foods. the flavour-enhancing component of seaweed was identified as the amino acid l-glutamate, and monosodium glutamate (msg) became the first flavour enhancer to be used commercially. the rich flavour associated with l-glutamate was called umami. umami is often considered the fifth basic taste because it is distinctly different from the other basic tastes (sweet, salty, sour, and bitter) and it is believed to activate a separate set of taste receptors. other compounds that are used as flavour enhancers include the 5′-ribonucleotides, inosine monophosphate (imp), guanosine monophosphate (gmp), yeast extract, and hydrolyzed vegetable protein. flavour enhancers may be used in soups, broths, sauces, gravies, flavouring and spice blends, canned and frozen vegetables, and meats. sweeteners sucrose or table sugar is the standard on which the relative sweetness of all other sweeteners is based. because sucrose provides energy in the form of carbohydrates, it is considered a nutritive sweetener. other nutritive sweeteners include glucose, fructose, corn syrup, high fructose corn syrup, and sugar alcohols (e.g., sorbitol, mannitol, and xylitol). efforts to chemically synthesize sweeteners began in the late 1800s with the discovery of saccharin. since then, a number of synthetic compounds have been developed that provide few or no calories or nutrients in the diet and are termed nonnutritive sweeteners. these sweeteners have significantly greater sweetening power than sucrose, and therefore a relatively low concentration may be used in food products. in addition to saccharin, the most commonly used nonnutritive sweeteners are cyclamates, aspartame, and acesulfame k. the sensation of sweetness is transmitted through specific protein molecules, called receptors, located on the surface of specialized taste cells. all sweeteners function by binding to these receptors on the outside of the cells. the increased sweetness of the nonnutritive sweeteners relative to sucrose may be due to either tighter or longer binding of these synthetic compounds to the receptors. nonnutritive sweeteners are primarily used for the production of low-calorie products including baked goods, confectioneries, dairy products, desserts, preserves, soft drinks, and tabletop sweeteners. they are also used as a carbohydrate replacement for persons with diabetes and in chewing gum and candies to prevent dental caries (i.e., tooth decay). unlike nutritive sweeteners, nonnutritive sweeteners do not provide viscosity or texture to products, so that bulking agents such as polydextrose are often required for manufacture. toxicological analysis of the nonnutritive sweeteners has produced variable results. high concentrations of saccharin and cyclamates in the diets of rats have been shown to induce the development of bladder tumours in the animals. because of these results, the use of cyclamates has been banned in several countries and the use of saccharin must include a qualifying statement regarding its potential health risks. however, no evidence of human bladder cancer has been reported with the consumption of these sweeteners. both aspartame and acesulfame k are relatively safe, with no evidence of carcinogenic potential in animal studies. 26 narendra kumar chaudhary references 1. r. macrae, r.k. robinson and m.j. sadler (eds.), encyclopaedia of food science, food technology, and nutrition, vol. 8 (1993); 2. y.h. hui (ed.), encyclopedia of food science and technology, vol. 4 (1992), 3 p. fellows, food processing technology: principles and practices (1988), 4. a. larry branen, p. michael davidson, and seppo salminen (eds.), food additives (1990), 5. t. e. furia (ed.), crc handbook of food additives, 2nd ed., 2 vol. (1972, reissued 1980), 6. j. a. maga and t. anthony tu (eds.), food additive toxicology (1995), 7. t. smith (ed.), food additive user's handbook (1991), 403 forbidden forbidden you don't have permission to access this resource. apache/2.4.54 (ubuntu) server at www.nepjol.info port 443 microsoft word mohan k. shrestha _70-75_.doc mohan k. shrestha / bibechana 7 (2011) 70-75: bmhss 70 bibechana a multidisciplinary journal of science, technology and mathematics issn 2091-0762 (online) journal homepage: http://nepjol.info/index.php/bibichana effect of sewage and industrial effluents on flowering behavior and yield of pea (pisum sativum l. var. arkel) mohan kumar shrestha ∗∗∗∗ dept. of biology, bhanu memorial higher secondary school, biratnagar, nepal article history: received 25 november 2010; accepted 5 december 2010 abstract municipality sewage and effluents from shah udyog and dairy farming were taken to assess the flowering behavior and yield of pea. the effluents from dairy farming initiated flowering earlier than the other effluents while effluents of shah udyog retarded comparatively. the same trend was followed for anthesis, withering and pod appearance. duration of anthesis was found increasing in the order: dairy farming, municipality sewage, control and shah udyog respectively. on the other hand, number of seed per plant and dry weight per seed was found in decreasing order from dairy farming to shah udyog through municipality sewage. key words: effluents; flowering; yield; inhibition and promotion 1. introduction discharge of waste unscientifically is a major problem in the large cities and industrial area like biratnagar (lat n 26 0 29’, long e 87 0 16’, altitude 72m).the industries viz. textile , sugar, leather, soap, plastic, iron rod, stainless steel, liquors, biscuits, dairy, oil seed extraction mills etc. discharge their untreated waste directly into the natural environments thereby polluting them [1-5]. the liquid waste of this city is discharged mainly to the singhia river through municipal sewage. the industrial effluents are discharged directly into the neighbouring water bodies or into the agricultural land. the polluted water thus produced is being used by the farmers to irrigate their crop land that adds plant nutrients to some extent and increases fertility of soil but also includes toxicants that change soil fertility and micro-flora [6]. as it needs to investigate the effects of effluents on the different parameters before using them in irrigation, an attempt has been made to find out effect of effluents from municipality sewage, dairy farming and shah udyog on flowering behavior and yield of pea (pisum sativum l. var. arkel). 2. materials and methods the seeds of pea were obtained from siwani seeds selling centre, biratnagar and were stored in air tight plastic bottle in dark room under ambient conditions. the municipality sewage ∗ corresponding author: mohan k. shrestha, dept. of biology, bhanu memorial higher secondary school, biratnagar, email:stha.mohan@yahoo.com mohan k. shrestha / bibechana 7 (2011) 70-75: bmhss 71 was collected from the discharge point of the sewer of biratnagar municipality, located at the bank of singhia river. the effluents were collected directly from the outlet of the factories and were stored inside the air tight polythene containers in dark room under ambient conditions. the colour (visual appearance), ph (ph meter method), dissolved oxygen (winkler’s method), solute particles [7]and organic matter [8] were recorded within three days of effluent collection(table 1). the concrete pots (diameter 44.5cm, depth 23.4cm) were filled with mixture of garden soil, compost manure and sand in the ratio of 2:1:1 by volume. ¾ parts of them were buried in the field. the seeds were sown in the pots and were irrigated daily with tap water. each treatment was carried out in triplicates. the effluent treatment was started when the seedlings were 30 days old. each pot for particular effluent/control was irrigated with 500ml of 100% effluent/tap water at the interval of 10 days. the seedlings were irrigated with tap water daily except the day of treatment. when the plants entered in reproductive phase, flowering behavior was observed daily for each treatment/control, the number of plants having floral buds, number of flowers, withered flowers and pods were recorded separately. the former two observations were recorded daily and the rest were recorded at the intervals of twelve hours. for the observation of anthesis, withering of flowers and pod appearance, 40 plants from each treatment were tagged randomly. for each treatment/control, the smallest visible bud was tagged and the time taken for anthesis was recorded. three independent observations were carried out to calculate the mean value. after harvesting, the number of pod(s) per plant and number of seeds per pod were recorded separately. the seeds from each treatment/control were oven dried at 80 0 c for 24 hours and the dry weight per seed was calculated. the experiments were conducted in 1999. 3. results and discussion the observation revealed that dairy farming and municipality sewage effluents initiated reproductive phase earlier (fig.1). the rate of increase in number of flowering plants was initially high in municipality sewage effluent but was leaded by dairy farming effluent in final days. the shah udyog effluent delayed the initiation of reproductive phase and the rate was also low. comparatively, the dairy farming and municipality sewage effluents correlated in other flowering and yield parameters i.e.; anthesis (fig.2, table 2), withering (fig.3 ), pod appearance (fig.4 ), number of pods, seed set and dry weight of seed (table 3 ). the shah udyog effluent on the other hand delayed all the parameters than the other effluents. the municipality sewage receives the discharge of liquid waste of colleges, hospitals, hotels, nursing homes and large number of household by-products that may contain high amount of organic and inorganic toxicants and stimulants for plant growth and development. the dairy farming effluent on the other hand may have less amount of inorganic toxicants but more organic ones as it involves more biological action on the milk and its products. similarly, shah udyog effluent contains different classes of dyes including naphthol, sulphur, chromate, basic and direct dyes used in cotton fabric production. some of these contain toxic chemicals e. g; chromium [9]. shrestha and niroula [10] reported that municipality sewage and dairy farming effluents are inhibitory for seed germination while shah udyog effluent stimulated this parameter. the inhibitory effect of the former two effluents on seed germination and stimulatory effect in flowering and yield suggests that these effluents are not suitable for the earlier developmental stages of pea but can be used to increase the yield after some chemical treatment. the shah udyog effluent on the other hand, though stimulatory in earlier stages of development, proved that it is not useful to get good yield of pea plant as it not only delayed the yield but the seeds set inside the pods were not fully developed. plants show different degree of responses with effluents at different stages and also the magnitude of toxicity depends upon species [2,5,11]. the present study also has shown that the mohan k. shrestha / bibechana 7 (2011) 70-75: bmhss 72 inhibitory effect of the effluents on the same plant in earlier stages of development has changed to stimulatory effect. to understand the exact nature and causes of different effluents/ sewage on the tested plants, it still awaits detail physico-chemical analysis and further research in different growth parameters. though the industrial and municipal sewage effluents may contain some useful organic and inorganic nutrients required for plants, their more toxic nature than useful property suggests not bringing them in use for irrigation without proper treatment. it is also indispensable to treat the effluent chemically before discharge to the surrounding water bodies to minimize water pollution and to save the quality of soil and surrounding air. table 1: physico-chemical characteristics of effluents and tap water (control) s.n. effluents /tap water parameters color solute (g/l) d.o.(mg/l) organic matter ph 1. dairy farming silvery white 1.4 3 only small amount 7.4 2. municipality sewage blackish turbid 1.5 2.5 high 7.1 3. shah udyog violet 1.7 3.6 moderate 7.1 4. tap water ---------0.4 4.15 negligible 6.9 mohan k. shrestha / bibechana 7 (2011) 70-75: bmhss 73 mohan k. shrestha / bibechana 7 (2011) 70-75: bmhss 74 table 2. effect of effluents on duration of anthesis of p. sativum. s.n. effluents/tap water time (in hours) 1. control 74.5 2. dairy farming 73.4 3. municipality sewage 73.6 4. shah udyog 76.1 table 3. effect of effluents on yield of p. sativum. seed s.n. effluents/tap water pod number / plant number/pod dry weight seed (mg) 1. control 1 1.2 66.7 2. dairy farming 1.2 1.4 87.1 3. municipality sewage 1.2 1.4 76.9 4. shah udyog 1.1 1.1 65.0 acknowledgement author is grateful to p. g. campus, biratnagar, nepal for providing laboratory facilities. mohan k. shrestha / bibechana 7 (2011) 70-75: bmhss 75 references [1] a. p. sharma and a. rijal, impact of industrial effluent on pollution of river and adjoining land, project report, national committee for man and biosphere (mab), kathmandu, nepal, (1988). [2] b. niroula, evaluation of some effluents on germination and growth of oryza sativa l.. cv. “chaite 4” and vigna mungo (linn) hepper. m. sc. thesis, dept. of botany, p. g. campus, biratnagar, nepal, (1996). [3] m. dahal, m. d. kafle and d. rijal, environmental impact assessment of rani, biratnagar, ( fieldwork report), department of botany, post graduate campus, t. u., biratnagar, nepal, (1997). [4] s. jha and b. niroula, t.u. journal, xxi(1)(1998) 63. [5] m. shrestha, effect of industrial and municipality sewage effluents on germination, growth and yield of pea (pisum sativum l. var. arkel ), m.sc. thesis, department of botany, post graduate campus, t. u., biratnagar, nepal, (2000). [6] p. k. goel and s. m. kulkarni, effects of sugar factory waste on germination of gram seed ( cicer arietinum l.) j. of environ. pollution, 1(1994) 35. [7] d. b. zobel, p. k. jha, m. j. benan and u. k. r. yadav, a practical manual for ecology, ratna book distributor, kathmandu ,(1987). [8] p. michael, ecological methods for field and laboratory investigations, tata mc graw hill publishing co. ltd., new delhi ,(1984). [9] npc/ iucn implementation project, balaju industrial district pollution control study, jeevan printing press, kathmandu, nepal, (1992). [10] m. shrestha and b. niroula, our nature, 1(2003) 33. [11] n. chapagain, physiological impact of dhobikhola (katmandu) water pollution on persicaria perfoliata l. leaves and germination of some vegetable seeds, m. sc. thesis, central department of botany, t.u., kathmandu, nepal, (1991). . microsoft word sitnshu s. chaudhary _18-20_.doc shitanshu shekhar choudhary and raju ram thapa / bibechana 7 (2011) 18-20 : bmhss 18 bibechana a multidisciplinary journal of science, technology and mathematics issn 2091-0762 (online) journal homepage: http://nepjol.info/index.php/bibichana locally compact space and continuity shitanshu shekhar choudhary , raju ram thapa ∗∗∗∗ p.g. campus, biratnagar, tribhuvan university article history: received 27 september 2010; revised 1 november 2010; accepted 7 november 2010 abstract topological spaces for being t0, t1, t2 and regular space have been discussed. the conditions for a topological space to be locally compact have also been studied. we have found that a continuous function preserves locally compactness. keywords: topological spaces; compactness; regular space 1. introduction it has been seen that any product of compact spaces is compact. it has been also seen that most of the spaces turn out to be closed subspaces of products of compact spaces and such spaces are necessarily compact. the n-dimensional euclidean space r n is the most important type of topological space which has a great importance in modern analysis. a topological space is locally compact if each of it's point has a neighborhood with compact closure. as a result, r n is locally compact for any open sphere centred on any point, is the neighborhood of the point whose closure, being a closed and bounded subspace of r n is compact. it's application is in the field of geometry and analysis. 2. definitions 2.1 t0space a topological space (x, j) is called t0 space or kolmogorff space iff given any pair of points xy,x ∈ (distinct) there exists an open set containing one of them but not the other. 2.2 t1space a topological space (x, j) is called t1space or frechet space ∗ corresponding author: raju ram thapa, dept. of mathematics, post graduate campus, biratnagar, tribhuvan university, email: thaparajuram@yahoo.com shitanshu shekhar choudhary and raju ram thapa / bibechana 7 (2011) 18-20 : bmhss 19 iff given any pair of points xy,x ∈ there exist two open set, one containing x not y & other containing y not x. 2.3 t2space a topological space (x, j) is said to be t2-space or hausdroff space iff given any pair of points xy,x ∈ there exits two disjoint open sets one containing x and other y. regular space : a topological space x is called regular if for each closed subset f of x and xx ∈ such that fx ∉ , there exist disjoint open sets g and h such gf ⊆ and hx ∈ . 2.4 t3 space a regular t1-space is called t3-space. 2.5 locally compact space a top. space (x, j) is said to locally compact if given xx ∈ and any nbd. u of x, there is a compact set a such that .uaax 0 ⊂⊂∈ 0 a : it is union of all open sets contained in a called interior of a, obviously aa 0 ⊂ . 3. formalism proposition: let (x, j) be a t2-space then x is locally compact iff given xx ∈ , there is a compact set a such that 0 ax ∈ . proposition: any compact t2-space is locally compact. proposition: any locally compact t2-space x is t3. proposition: if a space x is t2 and locally compact then every open and closed subspace is also t2 space and locally compact. proposition: a substance y of a locally compact t2-space x is locally compact iff it is the intersection of an open set and a closed set. proposition: let f be a continuous and open function from one topological space (x, j) to another topological space (y, j') then x is locally compact y⇒ is locally compact. proof: let (x, j) and (y, j') be two topological spaces and f be a continuous and open mapping from x onto y. let x is locally compact. to show that y is also locally compact, let yy ∈ and n be the neighborhood of y then )x(fy = for some xx ∈ . since f is continuous so there exists a neighborhood v of x such that f(v) ⊂ n. since x is locally compact so there exists a compact set b such that 0 bx∈ nb ⊂⊂ . then .n)b(f)b(fy)x(f 0 ⊂⊂⊂= but )b(f 0 is open, being f is open mapping and also compactness is invariant under continuous mapping so f(b) is compact. thus n)b(f)b(fx 0 ⊂⊂∈ . which shows that y is locally compact. shitanshu shekhar choudhary and raju ram thapa / bibechana 7 (2011) 18-20 : bmhss 20 proposition: let iii }j,x{ ∈ be a countable families of non-empty spaces and ix∏ be the product spaces. then ix∏ is locally compact iff each component spaces is locally compact and all of the component spaces except atmost finitely many are compact. proof: let ip be the projection mapping i ii ii xx:p →∏ ∈ which is continuous onto and open so each ix is locally compact. let a be any compact subset of ix∏ such that some point y of ∏ ∈ii ix is in 0 a . then there is a basis neighborhood ∏ ∈ii iv of y such that ii xv = for all but at most finitely many i and ∏ ∈ ⊂ ii 0 i av . thus ii x)a(p = for all but at most finitely many i, since ip is continuous and a is compact so ix is compact for all but at most finitely many i. now we assume that each ix is locally compact and all but finitely many of ix are compact. let ∏ ∈ ∈ ii ixy,x and let iy be the i th co-ordinate of y. if u is any neighborhood of y then u contains a basis neighborhood of y of the form ∏ ∈ii iv , where iv is open in ix for each i and ii xv = for all ii ∈ , except for at most finitely many say n21 i.,.........i,i . since each ix is locally compact so for each ii ∈ there is a compact subset ia of ix . such that ii 0 ii vaay ⊂⊂∈ . there are at most finitely many more ii ∈ , other than n21 i.,.........i,i say m2n1n i,.........i,i ++ such that m2n1n xi.........,,xi,xi ++ are not compact. for any i not in n21 i.,.........i,i , m2n1n i,.........i,i ++ we may let ii xa = then ∏∏ ∏∏ ∈∈ ∈∈ ⊂⊂⊂∈ ii i ii ii i 0 i ii i 0 va)a(ay . but ∏ ∈ii ia is product of compact sets and is therefore compact. hence ∏ ∈ii ix is locally compact. 4. conclusion locally compactness is not invariant under continuous mapping but in under certain assumptions which are openness, a continuous function preserves local compactness. references [1] g. a. quarw, point countable open covering in countable compact space; academic press, new york (1967). [2] r. arers, remark on concept of compactness; port maths (1950). [3] n. dykes, pac.j. maths(1970). [4] m. henrikse and j.r., isbell duke maths j. (1958). [5] h. wickle & j.m. worrel, proc. aurer, maths society (1976). devendra sir cover page copy.jpg bibechana 17 (2020) 20-27 20 bibechana issn 2091-0762 (print), 2382-5340 (online) journal homepage: http://nepjol.info/index.php/bibechana publisher: department of physics, mahendra morang a.m. campus, tu, biratnagar, nepal structure-activity relationship and mm2 energy minimized conformational analysis of quercetin and its derivatives in the dpph• radical scavenging capacity gan b. bajracharya 1* , mohan paudel 2 , rajendra k. c. 2 , sajan l. shyaula 1 1 faculty of science, nepal academy of science and technology (nast), khumaltar, lalitpur, nepal 2 department of chemistry, tri-chandra multiple campus, tribhuvan university, kathmandu, nepal *email: ganbajracharya@yahoo.com; gan.bajracharya@nast.gov.np article information: received: august 13, 2019 accepted: september 7, 2019 keywords: antioxidant 1,1-diphenyl-2-picrylhydrazyl flavonoid free radical ic50 sar abstract antioxidant activity of quercetin (1) and its derivatives (2-15) was evaluated by using dpph assay and ic50 values were calculated. dihedral angles α of c3-c2c1’-c6’ chain and β of o1-c2-c-1’-c2’ chain between ac and b rings of these flavones were determined by using mm2 energy minimized structures. structureactivity relationship study revealed that quercetin (1), quercetin-5-methyl ether (2), quercetin-3’-methyl ether (3) and quercetin-3’,5-dimethyl ether (4) displaying a high antioxidant activity (ic50 = 47.20-119.27 μm) possess similar dihedral angles (α 11.1-11.5º and β 6.3-6.6º). monoand/or di-methoxy substituent(s) at 3’ and 5 positions of the flavone are most suitable for the preservation of the antioxidant capacity while retaining conformational geometry. 1. introduction reactive oxygen species (ros) such as hydrogen peroxide (h2o2), singlet oxygen ( 1 o2), superoxide (o2•–), hydroxyl radicals (oh•), etc. are generated during oxygen metabolism in biological systems. imbalance between generation and elimination of ros leads cellular aging, mutagenesis, carcinogenesis, immunodeficiency syndrome, diabetes, coronary heart diseases, neurodegenerative diseases, etc. [1-3]. dietary flavonoids are considered as powerful antioxidants since they act as radical scavengers, metal chelators and enzyme inhibitors, and produce the beneficial health effects [4-7]. in several publications, the antioxidant activity of flavonoids have been evaluated using 1,1-diphenyl2-picrylhydrazyl radical (dpph•) [8], hydroxyl radical (ho•) [9], superoxide (o2• ˗ ) [10], peroxyl radical (roo•) [11], and hypochlorite (hocl/ocl ⁻ ) [12], and structure-activity relationships (sar) of flavonoids in antioxidant activity have been documented [1, 11-27]. hydrogen or electrondonating properties of flavonoids are considered to be the basis of their antioxidant activity [1, 25]. this work is licensed under the creative commons cc by-nc license. https://creativecommons.org/licenses/by-nc/4.0/ doi: https://doi.org/10.3126/bibechana.v17i0.25208 http://nepjol.info/index.php/bibechana mailto:ganbajracharya@yahoo.com mailto:gan.bajracharya@nast.gov.np https://creativecommons.org/licenses/by-nc/4.0/ https://doi.org/10.3126/bibechana.v17i0.25208 gan b. bajracharya, mohan paudel et al / bibechana 17 (2020) 20-27 21 structural requirement in flavonoids for the hydrogen-donation by single-electron transfer includes ortho-dihydroxyl substituent in the b ring and c2-c3 double bond in conjugation with c-4 carbonyl group in the c ring, which expands electron delocalization for radical stabilization and determines the co-planarity of the hetero ring. quercetin, a pentahydroxy flavone (1), seems to be a paradox which satisfies these structural variations and so efficiently captures free radicals thereby exhibiting high antioxidant activity. in case of absence of a catecholic structure in b ring, the antioxidant activity is compensated by 3and/or 5hydroxyl substituent(s). blocking or removing the 3-oh group decreases antioxidant property. the dihedral angle of the b ring with respect to remaining structure in flavones is considered for strong influence on radical scavenging activity [18, 27]. however, the roles of chemical structure and underlying molecular phenomena have stayed elusive [23, 28]. the nature of rapid conjugation with glucuronosyltransferases and sulfotransferases present in the small intestine halts quercetin (1) to reach the malignant organs through gastrointestinal tract in oral therapy [29]. when the hydroxyl groups in polyphenols are methylated, the resulting compounds are much less prone to glucuronidation and sulphation, and hence are more metabolically stable increasing their bioavailablity. these underlying facts promoted us to commence a research program to study the sar of quercetin derivatives for possible enhancement of antioxidant capacity. 2. experimental chemicals and equipments quercetin (1), dpph and gallic acid were purchased from sigma-aldrich and were used as received. quercetin derivatives (2-15) used in this work were previously synthesized in our laboratory [30]. spectrophotometric analysis was performed with a cary 60 uv-visible spectrophotometer (agilent technologies). dpph radical scavenging assay the dpph assay was performed according to the procedure described by brand-williams et al. with a slight modification [31]. to generate free dpph• radical, 11.7 mg of dpph in methanol (300 ml) was stirred overnight at 0 °c and used immediately. sample solutions of the compounds 1-15 of different concentrations (5, 25, 50, 100, 250 and 500 μm in acetone) were prepared. each sample solution (0.5 ml) was mixed with freshly prepared dpph• solution (2.5 ml). a control solution was prepared by mixing acetone (0.5 ml) and dpph• solution (2.5 ml). the content was shaken well, kept in dark at room temperature for 30 min and then absorbance was measured at 517 nm against the blank solution consisting acetone (0.5 ml) and meoh (2.5 ml). the percentage of inhibition was calculated by the equation: i (%) = (1 – asample / acontrol) × 100 where, asample and acontrol are the absorbance values of the reaction mixture with and without sample, respectively. thus obtained data of % inhibitions at different concentrations were computed to calculate ic50 values by employing the equation: ic50 = (50 μm c) / m where, ic50 = concentration causing 50% inhibition of absorbance, c = intercept, m = slope of a linear curve describing dependence of % inhibition with concentration. computational analysis energy minimized structure of quercetin (1) and synthesized quercetin derivatives (2-15) were produced by using molecular mechanics part 2 (mm2) calculation, cambridgesoft’s chem3d pro 12.0.2.1076. thus optimized geometry of the structures were studied for determining the dihedral angles between the planes of the ac-ring and the b-ring employing the angels of c3-c2-c1’-c6’ (α) and o1-c2-c1’-c2’ (β). gan b. bajracharya, mohan paudel et al / bibechana 17 (2020) 20-27 22 3. results and discussion dpph assay measures the antioxidant capacity of a compound in terms of its ability to donate hydrogen atom to the free dpph• radical. the dpph• radical, which shows absorption at 517 nm, is reduced to the corresponding hydrazine when it reacts with hydrogen donors. dpph assay is considered a valid and easy assay to evaluate the sar of antioxidants [32]. some of the compounds used in this study were not completely soluble in meoh; therefore, standard procedure was slightly modified by using acetone as a dissolving solvent to prepare the sample solutions. in the dpph assay, six different concentrations of each compound were used in order to obtain ic50 value. linear regression curve of concentration versus percentage of inhibition for each compound was plotted where r 2 values were found nearly equal to 1. the slope and intercept values of linear regression curve were used to obtain the ic50 value and the result is presented in table 1. the ic50 value of quercetin (1) in dpph assay was reported by several groups and it ranged from 1.82 to 119.60 μm [26, 33-39]. in this study, compared to the parent quercetin 1 (ic50 = 47.20 µm), its monomethylated derivatives 2 (ic50 = 52.24 µm) and 3 (ic50 = 52.45 µm) have retained the antioxidant capacity in the dpph assay (table 1). the dimethylated derivative 4 also displayed antioxidant property with ic50 value of 119.27 µm. the antioxidant capacity was gradually reduced as the number of substituents other than hydroxyl group is increased. although 3,5-dioh and/or 3’,4’-dioh groups are important for scavenging of dpph• radical; however, this study showed that one (or two) of these hydroxyl group(s) can be replaced with methoxy group(s). this conclusion is also supported by moalin et al., who have reported that substitutions of methoxy group at 5 and/or 7 position(s) in ring a of quercetin (1) retain the antioxidant capacity in abts assay [27]. acetylation of free hydroxyl groups reduced the antioxidant capacity; however, quercetin 3,3’,4’,5-tetraacetate (5) and quercetin 3,3’,4’,5,7pentaacetate (6) derivatives have exhibited a mild antioxidant activity with ic50 values of 516.26 and 790.57 µm, respectively. these results also indicated that a free 7-oh group in a flavone has a crucial role. remaining quercetin derivatives (7-15) were found to be lost their dpph• radical scavenging capacity displaying ic50 value >2000 µm. olejniczak and potrzebowski have reported that a small perturbation of geometry has a great influence on bond orders in ring b in density functional theory (dft) calculation of quercetin (1) [40]. and, the change in conformers may significantly change the susceptibility for the formation of radicals and consequently alters the biological property. although molecular structure of quercetin (1) (and its derivatives as well) can be represented by exact conformation; however, the presence of many flexible hydroxyl groups and hydrogen bonding networks in the system leads a wrong conclusion in the crystal lattice provided by x-ray diffractometry (xrd). later, filip et al. have performed the conformational calculation of quercetin (1) by employing the molecular mechanics (mm) level of theory that combined with previous xrd and solid state nmr data [41, 42]. it was concluded that small changes of conformation and hydrogen bonding pattern greatly influence the bond order parameters of quercetin (1). the authors have further mentioned that no matter how precisely the conformational analysis is performed, the most important is finding of the most probable molecular conformation. the mm2 energy minimization calculation can identify the more stable conformation [43]. therefore in the present study, the mm2 calculations of the compounds 1-15 were performed. in the case of quercetin (1), the orientation of the hydroxyl groups at 3 and 3’ carbon atoms in a planar arrangement of a, b and c rings produces anti and syn conformers. herein, energy minimized structures of compounds 1-15 were computed by keeping 3 and 3’ substituents at anti fashion. the optimal conformations of the compounds obtained are depicted in figure 1 and their dihedral angles are given in table 1. gan b. bajracharya, mohan paudel et al / bibechana 17 (2020) 20-27 23 table 1: ic50 values of quercetin scaffolds (1-15) in dpph• free radical scavenging activity. the dihedral angles α = 11.5º and β = 6.6º were found in mm2 energy minimization calculation of quercetin (1), which exhibited a high antioxidant property (ic50 = 47.20 µm) (table 1, figure 1). interestingly, in comparison with quercetin (1), quercetin-5-methyl ether (2) (α 11.2º and β 6.4º), quercetin-3’-methyl ether (3) (α 11.1º and β 6.3º) and quercetin-3’,5-dimethyl ether (4) (α 11.2º and β 6.3º) showed similar dihedral angels and they were also highly efficient in scavenging of dpph• radical with the ic50 values of 52.24, 52.45 and 119.27 µm, respectively. the decrease in dihedral angles α and β decreased the antioxidant property as shown by quercetin-3,3’,4’,5-tertaacetate (5) (α = 9.2º, β = 4.6º, ic50 = 516.26 µm) and quercetin3,3’,4’,5,7pentaacetate (6) (α = 7.3º, β = 3.9º, ic50 = 790.57 µm). the mm2 calculation of remaining compounds (7-15) showed that they exist in more or less planar conformation. therefore, a slightly twisted conformation between rings b and c with dihedral angles α 11.5-7.3º and β 6.6-3.9º plays a pivotal role in preserving the antioxidant capacity of parent compound quercetin (1) and perfect planarity is merely not necessary [27]. while modifying the structure of a flavone in search of the molecule with enhanced antioxidant activity, preservation of its planarity is therefore a must. 4. conclusion in conclusion, we have evaluated the dpph• free radical scavenging capacity of quercetin (1) and its derivatives (2-15). quercetin (1) and its methoxyl derivatives 2, 3 and 4 were found as highly efficient antioxidants. the acetyl derivatives 5 and 6 exhibited a mild antioxidant capacity while other remaining derivatives (7-15) were found to be inefficient. dihedral angles α of c3-c2-c1’-c6’ chain (11.5-7.3º) and β of o1-c2-c-1’-c2’ chain compound substituents dihedral angles ic50 (μm) r 1 r 2 r 3 r 4 r 5 α° β° quercetin (1) oh oh oh oh oh 11.5 6.6 47.20 quercetin 5-methyl ether (2) oh ome oh oh oh 11.2 6.4 52.24 quercetin 3’-methyl ether (3) oh oh oh ome oh 11.1 6.3 52.45 quercetin 3’,5-dimethyl ether (4) oh ome oh ome oh 11.2 6.3 119.27 quercetin 3,3’,4’,5-tetraacetate (5) oac oac oh oac oac 9.2 4.6 516.26 quercetin 3,3’,4’,5,7-pentaacetate (6) oac oac oac oac oac 7.3 3.9 790.57 quercetin 3,3’,4’,7-tetraacetate (7) oac oh oac oac oac 7.3 1.7 >2000 quercetin 3,3’,4’,7-tetrabenzyl-5-methyl ether (8) obn ome obn obn obn 3.4 1.3 >2000 quercetin 3,4’,7-tribenzyl ether (9) obn oh obn oh obn 2.8 0.9 >2000 quercetin 3,3’,4’,7-tetrabenzyl ether (10) obn oh obn obn obn 2.7 1.1 >2000 quercetin 3,4’,7-tribenzyl-3’,5-dimethyl ether (11) obn ome obn ome obn 2.6 0.9 >2000 quercetin 3,4’,7-tribenzyl-3’-methyl ether (12) obn oh obn ome obn 2.3 0.8 >2000 quercetin 3,3’,4’,5,7-pentamethyl ether (13) ome ome ome ome ome 1.7 0.5 >2000 quercetin 3,3’,4’,7-tetramethyl ether (14) ome oh ome ome ome 1.5 0.4 >2000 quercetin 3,4’,7-trimethyl ether (15) ome oh ome oh ome 1.4 0.3 >2000 gan b. bajracharya, mohan paudel et al / bibechana 17 (2020) 20-27 24 (6.6-3.9º) between ac and b rings as can be found in quercetin (1) are suitable for the preservation of antioxidant capacity. monoand/or di-methoxy substituent(s) at 3’ and 5 positions of the flavone are most suitable for the preservation of the antioxidant capacity. free 7-oh group of a flavone also plays a crucial role in preserving of the antioxidant capacity. fig. 1: three dimensional energy minimized structure of quercetin (1) and its derivatives (2-15) with their dihedral angles α and β. gan b. bajracharya, mohan paudel et al / bibechana 17 (2020) 20-27 25 acknowledgements the world academy of sciences (twas) is gratefully acknowledged for supporting our research program by purchasing precious instruments (process station personal synthesizer pps-ctrl 1 and microwave synthesis reactor monowave 300) through the research grant no. 13198 rg/che/as_g. references [1] k. e. heim, a. r. tagliaferro, d. j. bobilya, flavonoid antioxidants: chemistry, metabolism and structure-activity relationships, j. nutr. biochem. 13 (2002) 572–584. https://doi.org/10.1016/s0955-2863(02)00208-5. 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extremely high corrosion resistance properties of the spontaneously passivated sputter–deposited w–xta alloys was investigated using a non−destructive angle resolved x−ray photoelectron spectroscopy (angle resolved xps) techniques in this study. in−depth surface analyses of the thin passive films formed on the spontaneously passivated amorphous/nanocrystalline w–xta alloys using angle resolved xps analyses revealed that the high corrosion resistance of the alloys is mostly due to the formation of homogeneous passive double oxyhydroxide films consisting of w ox and ta 4+ cations with a small concentration gradients in–depth particularly after immersion between 20– 168 h in 12 m hcl solution open to air at 30°c. consequently, tantalum metal acts synergistically with tungsten in enhancing the spontaneous passivity as well as the high corrosion resistance of the sputter–deposited binary w–xta alloys in 12 m hcl solution. keywords: sputter deposition; w–xta alloys; 12 m hcl; take–off angle 1. introduction an angle resolved xps proves as one of the promising in−depth compositional analyses of thin films to investigate chemical compositions, oxidation states, chemical shifts, binding energies and electronic structures of the surface films [1−7]. for this purpose the non−destructive angle resolved xps technique is widely used by a number of researchers to gain about the in−depth concentration profiles of the passive films. in principle, the in−depth information of the passive film depends on the effective escape depth of the ejected photoelectrons, which increases with an increase in the take−off angle of photoelectrons relative to the surface of the sample specimen [1−8]. therefore, the surface sensitivity is varied by changing the take−off angle of photoelectrons (θ) as explain by equation (1). in general, the surface sensitivity is varied by changing the take−off angle of photoelectrons. at a lower take−off angle of photoelectrons, the intensity signals (i) from the sample species located in the exterior part of the surface films are enhanced. therefore, the apparent compositions of the surface films are changed with the take−off angle of photoelectrons in the angle resolved xps measurements. jagadeesh bhattarai / bibechana 8 (2012) 8-16 : bmhss, p. 9 i = k exp(−z/λ sinθ) (1) where, i is the intensity of the ejected photoelectrons, k is constant, z is the depth of the surface and θ is the take−off angle of the ejected photoelectrons. in recent years, angle resolved xps (arxps) technique is one of the very useful in−depth surface characterization tools for corrosion scientists to study the mechanism of high corrosion resistance of materials during last four decades after a valuable contribution of kai siegbahn and his research groups [9]. it allows us to examine the formation of homogeneous or heterogeneous passive surface films formed on the corrosion−resistant alloys [8,10−19]. tungsten and tantalum are effective elements in enhancing the corrosion resistance of alloys in aggressive environments. several surface studies have been carried out for a better understanding of the role of tungsten in the corrosion resistance properties of the sputter−deposited binary [12,14−16,18,20−37] and ternary [19,38−43] tungsten−based alloys in aggressive environments. it is reported that the addition of tungsten to amorphous fe−p−c alloys with and without chromium [44,45] and to the amorphous ni−p alloys [46] is effective in improving the corrosion resistance in aggressive hydrochloric acid solutions. similarly, tantalum is widely known for its superior corrosion resistance in aggressive acidic media. amorphous nickel−base alloys containing certain amounts of tantalum exhibit very high corrosion resistance in boiling concentrated acids [47,48]. lee et al. reported a beneficial effect of tantalum to improve the corrosion resistance of nickel base alloys in 12 m hcl [48,49]. a series of sputter−deposited binary [11,13,20,50,51] and ternary [52,53] tantalum−containing alloys showed high corrosion resistance due to spontaneous passivation in aggressive media. in particular, the sputter-deposited w-xta alloys were passivated spontaneously and showed significantly high corrosion resistance in 12 m hcl solution. their corrosion rates were found about two orders of magnitude lower than that of sputter-deposited tungsten and even lower than that of tantalum. the conventional xps analyses confirmed that the formation of the spontaneously passivated films formed on the alloys composed of double oxyhydroxide of tungsten and tantalum ions was found to be responsible for their higher corrosion resistance those of alloying elements of the wxta alloys in 12 m hcl solution. in this context, the present work is aimed to clarify the mechanism of the higher corrosion resistance of the sputter–deposited nanocrystalline w–23ta and w–60ta alloys than those of tungsten and tantalum in 12 m hcl solution open to air at 30°c using angel resolved xps techniques. 2. materials and experimental methods the sputter–deposited w–23ta and w–60ta alloys were characterized as nanocrystalline single–phase solid solution having an apparent grain size of 10–20 nm from xrd patterns using scherrer’s formula [54]. the compositions of the alloys hereafter are all denoted in atomic percentage (at%). prior to the corrosion tests and angle resolved xps analyses, the sputter– deposited w–xta alloy specimens were mechanically polished with a silicon carbide paper up to grit number 1500 in cyclohexane, degreased by acetone and dried in air. the average corrosion rate of the alloys was estimated from weight loss after immersion for 168 h in 12 m hcl solution open to air at 30°c. the composition of the spontaneously passivated films formed on the w–xta alloys was analyzed by angle–resolved xps. before and after immersion in 12 m hcl solution, xps spectra were measured by a shimadzu esca–850 photoelectron spectrometer with mg k (hν = 1253.6 ev) radiation for surface analyses including the in–depth surface compositions of the alloys. xps spectra for both the nanocrystalline w–23ta and w–60ta alloys over a wide binding energy region (that is, 0–1000 ev) exhibited peaks of tungsten, tantalum, carbon, oxygen and chlorine. jagadeesh bhattarai / bibechana 8 (2012) 8-16 : bmhss, p. 10 the most intense peaks of the w 4f, ta 4f, c 1s, o 1s and cl 2p electrons were measured in the binding energy range of 20 ev for all spectra except for w 4f and ta 4f electrons. the most intense peaks of the w 4f and ta 4f spectra are located very close to each other and they are partially overlapped as discussed elsewhere [20,55]. hence, the w 4f and ta 4f electrons were measured in the binding energy range of 30 ev [20,55]. the peak shift (chemical shift) caused by the charging effect was corrected using the difference the measured binding energy of the contaminant c 1s peak and assumed value of 285.0 ev. for the alloy specimens after immersed in 12 m hcl solution, a very weak cl 2p spectrum was detected at about 199.0 ev. however, the concentration of the chloride ion was not considered for the quantitative analyses of the surface films in this study because the intensity of the chloride ion is very low for quantitative analyses. the o 1s spectrum was composed of two peaks; the lower binding energy peak at 530.4 ev was assigned to om oxygen, and the higher binding energy peak at 532.3 ev was assigned to oh oxygen [56]. the spectra from the w–xta alloys constituents indicated the presence of the oxidized and metallic species; the former comes from the surface film and the latter from the underlying alloy surface. the measured spectrum of w 4f electron was separated into w ox 4f and w m 4f and the measured spectrum of ta 4f electron was separated into ta 4+ 4f and ta m 4f for the sputter–deposited w–xta alloys. furthermore, the w m 4f spectrum was consisted of w 4+ 4f, w 5+ 4f and w 6+ 4f spectra. the integrated intensities of the w 0 4f, w 4+ 4f, w 5+ 4f, w 6+ 4f, ta m 4f, and ta 4+ 4f were obtained by the same method as those described elsewhere [20,55]. after the integrated intensities of these all spectra observed from the surfaces of the sputter−deposited w−xti alloys were obtained, the compositions and thickness of the passive films and the compositions of the underlying alloy surfaces were quantitatively determined using three layers model proposed by asami et al. [56,57]. the diagrammatic sketch of the three layers model is shown in fig. 1. for angle–resolved xps, the angle between the alloy specimen surface and the direction of photoelectron to the detector (take–off angle of photoelectrons) was changed by using tilted–specimen holders at 30°, 45°, 60° and 90° as discussed elsewhere [55]. the photo–ionization cross–section of the w 4f and t a 4f electrons relative to the o 1s electrons used were 2.97 [58] and 2.617 [59], respectively. fig. 1: a diagrammatic sketch of the three layers model for the quantitative xps analyses of the surface films formed on the sputter–deposited w–xta alloys. jagadeesh bhattarai / bibechana 8 (2012) 8-16 : bmhss, p. 11 3. results and discussion it was reported that the synergistic effect of tungsten and tantalum in forming the double oxyhydroxide was responsible for the higher corrosion resistance of the sputter−deposited amorphous/nanocrystalline w−xta alloys than those of the alloy−constituting elements in 12 m hcl solution open to air at 30°c from the conventional xps analyses. however, the experimental results of the angle resolved xps measurements are presented here to clarify the mechanism of showing the higher corrosion resistance behavior of the sputter−deposited binary w−xta alloys than those of alloy−constituting elements in 12 m hcl. because, the in−depth surface analyses of the spontaneously passivated double oxyhydroxide films formed on the w−xta alloys using angle resolved xps measurements give very important information for a better understanding of the effects of tungsten and tantalum additions in the corrosion−resistant behavior of the alloys. an information about in−depth composition changes of the spontaneously passivated films formed on the w−23ta and w−60ta alloys were obtained by a non−destructive depth profiling technique of the angle resolved xps to know whether the spontaneously passivated films formed on the alloys are homogeneous or not. the spectra from the w–xta alloys constituents indicated the presence of the oxidized and metallic species; the former comes from the surface film and the latter from the underlying alloy surface. the measured spectrum of ta 4f electrons was separated into ta 4+ 4f and ta m 4f spectra as shown in fig. 1 and the measured spectrum of w 4f electrons was separated into w m 4f and w ox 4f as shown in fig. 1 for the sputter–deposited w–45ta alloy in the given conditions [55]. the ta 4+ 4f and ta m 4f spectra were consisted of lower and higher binding energy peaks corresponding to ta 4+ 4f7/2 and ta 4+ 4f5/2 electrons, and ta m 4f7/2 and ta m 4f5/2 electrons, respectively, as shown in fig. 1. furthermore, the w m 4f spectrum was consisted of lower and higher binding energy peaks corresponding to w m 4f7/2 and w m 4f5/2 electrons. similarly, the w ox 4f spectrum was also composed of three doublets of the overlapped spectra of three oxidized species, that is, doublets of w 4+ 4f7/2 and w 4+ 4f5/2 ; w 5+ 4f7/2 and w 5+ 4f5/2 ; and w 6+ 4f7/2 and w 6+ 4f5/2 electrons as shown in fig. 1. the quantitative result of the in−depth compositional change in the surface films formed on the sputter−deposited w−xta alloys using angle resolved xps analysis is shown in fig. 2. figures 2 (a) and 2 (b) show the changes in apparent cationic fractions in the spontaneously passivated films and the apparent atomic fractions in the underlying alloy surfaces of the w−23ta and w−60ta alloys, respectively, after immersion for different periods of time in 12 m hcl solution open to air at 30°c, respectively, as a function of take−off angle of photoelectrons. it is clearly raveled that tantalum is concentrated in the spontaneously passivated films, while tantalum is deficient in the underlying alloy surfaces. the concentration of tantalum ion is slightly higher in the exterior part (at take−off angle of 30°) of the passive films formed on both the sputter−deposited w−23ta and w−60ta alloys by immersion for 20 hours or more in 12 m hcl, while there is also small concentration gradient of cations in−depth of the underlying alloys. on the other hand, the concentration of tungsten is slightly higher in the exterior part (at take−off angle of 90°) of the underlying alloy surfaces and the tungsten concentration decreases with take−off angles after immersion in 12 m hcl solution. jagadeesh bhattarai / bibechana 8 (2012) 8-16 : bmhss, p. 12 fig. 2: changes in the apparent cationic fractions in the passive film and the atomic fractions in the underlying alloy surface for the (a) w−23ta and (b) w−60ta alloy as a function of take−off angle of photoelectrons. for a better understanding of the role of tungsten ions in the passive films formed on the alloys, the changes in the ratios of w 4+ , w 5+ and w 6+ ions in the surface films were determined quantitatively. figure 3 shows the concentration ratios of w 4+ , w 5+ and w 6+ ions to the total tungsten ions in the spontaneously passivated films formed on the w−23ta and w−60ta alloys after immersion for 20 h in 12 m hcl solution open to air at 30°c, as a function of take−off angle of photoelectrons. after immersion for 20 h in 12 m hcl solution, the ratio of w 6+ ions in the passive films formed on both the w−23ta and w−60ta alloys are significantly higher than those of w 4+ and w 5+ ions in the spontaneously passivated films. w 6+ ion decreases with increasing take−off angle of photoelectrons, while w 4+ ion increases with take−off angles. the ratio of w 5+ ion remains almost constant with take−off angle of photoelectrons. accordingly, w 4+ ion is particularly concentrated in the interior (at take−off angle of 90°) of the spontaneously passivated film formed on the sputter−deposited w−xta alloys, while the concentration of w 6+ ion is higher in jagadeesh bhattarai / bibechana 8 (2012) 8-16 : bmhss, p. 13 the exterior (at take−off angles of 30 to 60°) of the spontaneously passivated films. it is generally known that air exposure of the specimen during transfer from the electrolyte to the xps analyzing chamber give rise to oxidation of w 4+ to w 6+ ion. accordingly the relative ratio of w 6+ ion is higher in the exterior of the surface films and decreases with the depth of the passive film. fig. 3: changes in the apparent cationic fractions in the passive film and the atomic fractions in the underlying alloy surface for the (a) w−23ta and (b) w−60ta alloys, as a function of take−off angle of photoelectrons. in general, one of the characteristic of the thin passive films is the concentration of oxygen species in the spontaneously passivated films formed on the corrosion−resistant alloys. figure 4 shows the changes in the ratios of [o 2− ]/[cations] and [oh]/[cations] in the spontaneously passivated films formed on the sputter−deposited amorphous w−23ta and w−60ta alloys after immersion for 20 h in 12 m hcl solution open to air at 30°c, as a function of take−off angle of photoelectrons. the ratios of [o 2− ]/[cations] slightly increase with the take−off angles and the ratios of [oh]/[cations] significantly decrease with take−off angles in the films formed on the w−xta alloys. consequently, the interior part of the passive films formed on the sputter−deposited w−xta alloys is rather dry and well developed by m−o−m bridging, while the exterior part of the passive films formed on the alloys is slightly wet with oh − ion and h2o. jagadeesh bhattarai / bibechana 8 (2012) 8-16 : bmhss, p. 14 fig. 4: changes in the ratios of [o 2− ]/[cations and [oh − ]/[cations] in the surface films formed on the w−23ta and w−60ta alloys in the given conditions, as a function of take−off angle of photoelectrons. 4. conclusion the mechanism of the synergistic effect of tungsten and tantalum metals enhancing the higher corrosion resistance of the sputter–deposited nanocrystalline w–23ta and w–60ta alloys than those of alloy–constituting elements has been studied using angle resolved xps measurements in 12 m hcl solution open to air at 30°c. the following conclusions are drawn: 1. the angle–resolved xps measurement revealed that all cations of the binary w−xta alloys are distributed homogeneously in the spontaneously passivated films formed on the alloys. the higher corrosion resistance of the alloys than those of the alloying elements is mostly due to the formation of the homogeneous passive films composed of both w 4+ and ti 4+ ions with a small concentration gradient in–depth. 2. the w 4+ ion is particularly concentrated in the interior of the spontaneously passivated films formed on both the w−23ta and w−60ta alloys, while the concentration of w 6+ ion is higher in the exterior of the passive films. 3. the interior part of the passive films formed on the w−xta alloys is rather dry and well developed by m−o−m bridging, while the exterior part of the passive films formed on the alloys is slightly wet with oh − ion and bound h2o molecule. acknowledgements the author is very thankful to professors emeritus; dr. koji 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[59] j. h. kim, h. yoshioka, h. habazaki, a. kawashima, k. asami and k. hashimoto, corros. sci. 33 (1992) 1507. bibechana a multidisciplinary journal of science, technology and mathematics issn 2091-0762 (print), 2382-5340 (0nline) journal homepage: http://nepjol.info/index.php/bibechana publisher: research council of science and technology, biratnagar, nepal physical properties of dust particles around white dwarf in far infrared sky at 12.80 galactic latitude b. b. sapkota1*, b. aryal2, r. weinberger3 1mahendra ratna campus, tahachal, tribhuvan university, kathmandu, nepal. 2center department of physics, tribhuvan university, kirtipur, nepal. 3 institute of astro-particle physics, insbruck university, austria. *email: bhanusapkota45@gmail.com article history: received 15 october, 2017; accepted 12 november, 2017 doi: http://dx.doi.org/10.3126/bibechana.v15i0.18488 this work is licensed under the creative commons cc by-nc license. https://creativecommons.org/licenses/by-nc/4.0/ abstract we study the active region in the interstellar medium in which the process of cavity formation is expected. the interaction between wind and its surroundings in the interstellar medium (ism) provides a laboratory to study the behavior of dust particles. the information obtained from the dust colour temperature and dust mass of wd2116+675 will be presented. it is found that the dust colour temperature and dust mass lie in the range 20 k to 22.3 k and 2.1×1023 kg to 2.9×1023 kg respectively. with the help of number density, flux density, dust colour temperature and dust mass maps, the variation of temperature and mass in different position of dust will be presented and discussed. keywords: white dwarfs; flux density; cavity. . 1. introduction white dwarf are hot (~10,000k) low luminosity stars composed mostly of carbon, oxygen and helium. their luminosity is low because their surface is small. the electrons in the white dwarf are degenerate. the largest possible white dwarf is 1.4 of msun. this is called chandrasekhar limit, and is the most mass that the electron degenerate core can support without collapsing under its own gravity. no white dwarf with masses less than 0.6msun have been observed yet. the matter and radiation that exists in the space between the star system in galaxy is called interstellar medium (ism). the ism is the dust and gas between the stars. 99%of ism is gas, and only 1% of the mass is dust. about 20% of the galaxies mass is ism [1]. the interstellar dust consists of particles of silicates or carbon. the gas is mostly hydrogen gas, atomic and molecular gas. dust emits in the infrared but blocks visible light. the reduction in amount of light or other radiation received from star as a result of absorption and scattering of the radiation by intervening dust grains in space is called extinction. the extinction decreases with increase of wavelength of the radiation and increases with the path length through the absorbing medium and .b. b sapkota et al. / bibechana 15 (2018) 43-49: rcost p.43 http://nepjol.info/index.php/bibechana mailto:bhanusapkota45@gmail.com http://dx.doi.org/10.3126/bibechana.v15i0.18488 https://creativecommons.org/licenses/by-nc/4.0/ with the density of the medium. blue light is scattered and absorbed more than red in interstellar medium. this effect is called reddening of light. in other words, the extinction of blue light by dust is greater than red light [2]. in the present work we intend to study a new cavitylike structure around white dwarf. 2. region of interest and methods we have compiled a database of 1978 number of white dwarfs which are listed in the catalogue of holberg et. al.[3]. out of which we have studied wd2116+675 here. we have carried out a systematic search of iras maps available in the sky view virtual observatory (http://skyview.gsfc.nasa.gov). this sample white dwarf has the right ascension of 20h 17m 17s and declination of +670 44m 44s in equatorial coordinate system. the following input parameters were used for the search: (1) coordinate: j2000, (2) projection: gnomonic (tan), (3) image size (pixel): 500×500 (4) image size (degrees) : 0.50×0.50 (5) brightness scaling: histogram equilization (histeq) (6) colour table: stern special. we have downloaded flexible image transport system (fits) v3 image of the selected region. we selected fits format of 0.50×0.50 at 60 and 100µm for the image processing. using aladin v2.5 software the fits image carries the information concerning the flux density, position, etc for each pixels. aladin v2.5 is an interactive sky atlas developed and maintained by the center de donne's astronomiques de strasbourg (cds) for the identification of astronomical sources through visual analysis of reference sky images. aladin v2.5 allows the user to visualize digitized images of any part of the sky, to superimpose entries from the cds astronomical catalogues and tables, and to interactively access related data and information from simbad, ned or other archives of all known objects in the field. in ordered to separate the region of minimum flux density region, contours are drawn at 60 and 100 μm respectively. because we are interested to study temperature and mass profile of the region. we intend to study the cavity like structure at 60 µm and 100 µm. measured flux density is subtracted with the background values. background flux is the flux emitted by other sources lying nearby the region of interest (not from the region of interest). the average value of the background flux is obtained by noting and summing up of the minimum flux densities around the region of interest and dividing the sum by total number of pixel with this minimum flux density. when this background flux is subtracted from the obtained flux density of each pixels in the region of interest, it is said to be background flux density. 3. dust color temperature estimation we use data base from the iras 60 µm and 100 µm flux densities is similar to that of schnee et. al.[4]. by knowing the flux densities at 60µm and 100 µm, the temperature contribution due to dust color can be calculated. the dust temperature 𝑇𝑑 in each pixel of a fits image can be obtained by assuming that the dust in a single beam is isothermal and that the observed ratio of 60 µm to 100 µm emission is due to black body radiation from dust grains at 𝑇𝑑, modified by a power law of spectral emissivity index. the flux density of emission at awavelength 𝜆𝑖is given by 𝐹𝑖 = [ 2ℎ𝑐 𝜆𝑖 3(𝑒 ℎ𝑐 𝑘𝑇𝑑 −1) ] 𝑁𝑑 𝛼𝜆𝑖 −𝛽 𝛺𝑖) (1) b. b sapkota et al. / bibechana 15 (2018) 43-49: rcost p.44 where, 𝑁𝑑 is the column density of dust grains, is a constant which relates the flux withthe optical depth of the dust, β is the spectral emissivity index, and 𝛺𝑖 is the solid angle subtended at 𝜆𝑖 by the detector. following [6], we use the equation 𝛽 = 1 𝛿+𝜔𝑇𝑑 (2) to describe the observed inverse relationship between temperature and emissivity spectral index. here, 𝛿 and 𝜔 are free parameters found that the temperature dependence of the emissivity index fits very well with the hyperbolic approximating function. considering temperature as an independent variable, the best fit gives 𝛿 = 0.40 ± 0.02 and 𝜔= 0.0079 ±0.0005 𝐾−1, with the 𝜒2/degree of freedom = 120/120. with the assumptions that the dust emission is optically thin at 60 𝜇m and 100 𝜇m and that 𝛺𝜔 ≅ 𝛺100 (true for iras image), we can write the ratio “r” of the flux densities at 60 𝜇m and 100 𝜇m as 𝑅 = 0.6−(3+ 𝛽) 𝑒 144 𝑇𝑑 – 1 𝑒 240 𝑇𝑑 – 1 ) (3) the value of 𝛽 depends on dust grain properties as composition, size, and compactness. for reference, a pure blackbody would have 𝛽 = 0, the amorphous layer-lattice matter has 𝛽~1, and the metals and crystalline dielectrics have 𝛽~ 2. for a smaller value of 𝑇𝑑, 1 can be dropped from both numerator and denominator of equation and it takes the form 𝑅 = 0.6−(3+ 𝛽) 𝑒 144 𝑇𝑑 𝑒 240 𝑇𝑑 (4) taking natural logarithm on both sides of equation (4) we find the expression for the temperature as 𝑇𝑑 = −96 1 𝑙𝑛{𝑅×0.6(3+𝛽)} (5) where r is given by r = f(60 𝜇m) f(100 𝜇m) (6) f(60 𝜇m)and f(100 𝜇m) are the flux densities at 60 𝜇m and 100 𝜇m, respectively. in this way we can use equation (5) for the determination of the dust grain temperature [6]. 4. mass estimation since the longer wavelength measurements give us more precise dust masses due to the characteristics of the planck curve, the far infrared emission which is used for the derivation of the dust mass is measured from the 100 𝜇𝑚 iras images. the dust masses are estimated from the ir flux densities. in order to estimate the dust masses from the infrared flux densities at 100 𝜇𝑚, followingthe calculation of young et. al.[7]. we need the background correction of flux and convert the relative flux into absolute flux. the background correction is done by subtracting the average flux emitted by the external sources other than the object of interest. the blackbody intensity can be calculated using the basic expression as given in equation (2). the resulting dust mass depends on the physical and chemical properties of the dust grains, the adopted dust temperature td and the distance d to the object. 𝑀𝑑𝑢𝑠𝑡 = 4 3 𝑎𝜌 𝑄𝜈 [ 𝑆𝜈𝐷 2 𝐵(𝜈,𝑇) ] (7) where a, ρ,qvand sv represent weighted grain size, grain density ,grain emissivity and flux density of the region of interest, respectively. here, sν = f × 5.288 × 10 −9 mjy s the distance (d) to the cavity is 205 pc, known from odenwald et .al.[5]. the planck’s function is given by, b(ν, t) = 2hν3 c2 [ 1 e hν kt−1 ] (8) b. b sapkota et al. / bibechana 15 (2018) 43-49: rcost p.45 where h,c,ν ,and t represent planck's constant, velocity, frequency of light, and average temperature of region respectively. by using, a=0.1 µm [5], ρ=3000 kgm-3, and qν =0.0010 for 100 µm and 0.0046 for 60 µm respectively [6] ,the expression (7) takes the form : mdust=0.4 [ sνd 2 b(ν,t) ] (9) we use the equation (9) for the calculation of the dust mass it is clear from the expression (8) that the value of planck function 𝐵 (𝜈, 𝑇) for longer wavelength is higher than that of the shorter wavelength. consequently, the range of 𝐵 (𝜈, 𝑇) for fixed temperature (say∆t) goes narrower if wave-length of the images increases. 5. results and discussion fig. 1: 0.5 0 ×0.50 image of the region centered at r.a.(j2000)=21h 17m 17s,dec. (j2000)=+67 044'44" at 60µm (left) and 100µm (right) of wd 2116+675. fig 2 fig.3 60micron 60micron 100micron b. b sapkota et al. / bibechana 15 (2018) 43-49: rcost p.46 fig. 2: cavity with contour and major diameter (ab) and minor diameter (cd). the size of image is 0.50×0.50 at 100 µm iras maps. fig. 3:showing different flux density inside the different contour level outside whole region of wd2116+675 centered at r.a.= 21h 17 m 17 s and dec. = +67 044' 44". + sign represents position of white dwarf and a, b, c, and d represents four contour level drawn at 2, 40, 69, and 255 respectively. figure (1) represents 60 and 100 µm iras image of the region of interest. the region of minimum and maximum flux is represented by black and white colors respectively. diameters are drawn passing through minimum flux inside the cavity as shown in figure (2). figure (3) shows four contour level a, b, c, and d that surround flux density 23.86,25.75, 25.02 and 30.57 mjysr-1 respectively. contour a encloses minimum flux density and b encloses maximum flux density inside the cavity. similarly, contour level c , and d encloses minimum and maximum flux density including whole region of the region. the flux density obtained inside the contour level c helps to determine background count. similarly, the flux density of other required region can be obtained with the help of aladin software. by comparing figures (1), (2) ,and (3), we get same result. fig. 4 fig. 5 b. b sapkota et al. / bibechana 15 (2018) 43-49: rcost p.47 fig 4 : contour map of mass distribution. the map is centered at r.a.(j2000)=21h 17m 17s, dec. (j2000)=+67 044'44". fig. 5: dust colour temperature contour plot. the map is centered at r.a.(j2000)=21h 17m 17s, dec. (j2000)=+67 044'44".the contour levels are shown. in figure (4), different colour counter represents the different range of mass. from above plot mass is higher at centre and decreases across the outward region. figure (5) shows the different colour map of temperature. from above plot temperature is lower at centre and increases across the outward region. by comparing figure (4) and figure (5) we get those region at which mass is minimum, greater the value of temperature of dust. fig. 6 fig. 7 fig. 6: the flux density at 60m versus 100m plot. the best fit can be seen. fig. 7: dust mass distribution. the gaussian fit and the ±1σ statistical error bars can be seen. here σ = √n. figure (6) is the plot of relative flux density of 60 µm versus 100 µm. the solid line represents linear fit. the slope of the line has been used to find average dust color temperature of the region of interest. figure(7) shows the gaussian plot of mass distribution. the solid curve represents gaussian fit of mass with error bar. the dust mass is very well fitted. the gaussian parameter i.e. gaussian centre is 2.37×1023 kg and offset mass is -0.11 kg. fig. 8: distribution of dust color temperature. the gaussian fit and the ±1σ statistical error bars can be seen. here σ = √n. b. b sapkota et al. / bibechana 15 (2018) 43-49: rcost p.48 the maximum and minimum dust color temperature of structure wd2116+675 is found to be 22.34k and 20.02k respectively. total mass of dust structure is found to be 7.42×1024kg. the dust mass and dust colour temperature is found to be well fitted by gaussian distribution. the size of cavity is 0.78 pc × 0.329 pc. the cavity is found to be formed due to presence of nearby white dwarf. the white dwarf evolves from the planetary nebula stage by ejecting its outer envelopes. acknowledgements we thank editor and referee for the constructive comments and suggestion. one of the authors (bbs) thanks university grants commission of nepal for providing research fund and acknowledge central department of physics, tribhuvan university , nepal for various kinds of support during ph.d. in addition, we acknowledge sky view virtual observatory ( http://skyview.gsfc.nasa.gov ) simbad ( http://skyview.gsfc.nasa.gov ) for providing database and software. references [1] s. palen, schaum's outlines astronomy, mc graw hill, usa, 2004. [2] h. karttunen, p. kroeoger, h. oja, m. poutanen, k.j. donner, fundamental astronomy, springer berlin heidelberg, fifth edition. usa, 2007.doi.org/10.1007/978-3-540-34144-4. [3] j. b. holberg, terry d. oswalt,and e. m. sion., a determination of the local density of white dwarf stars astrophysical journal 571 (2002) 512-518. doi.org/10.1086/339842. [4] s. l.schnee, n. a. ridge, n.a., goodman, a.a, jason, g. l. , a complete look at the use of iras emission maps to estimate extinction and dust temperature, astrophysical journal 634 (2005) 442450. doi.org/10.1086/491729. [5] s. f. odenwald and l. j. richard, hydrodynamical process in the draco, astrophysical journal 318 (1987) 702-711. [6] x. dupac, j.p. bernard, n boudet, m. giard, j.m. lamarre, c meny, f. pajot, i. ristorcell., g. serra, b. stepnik, j.p. torre, inverse temperature dependence of the dust submillimeter spectral index astronomy & astrophysics 404 (2000) l11-l15. doi.org/10.1051/0004-6361:20030575. [7] k. young., t.g. philip., & g.r. knapp, circumstellar shells resolved in iras survey data. ii – analysis, astrophysical journal, 409, (1993) 725-738. doi.org/10.1086/172702 b. b sapkota et al. / bibechana 15 (2018) 43-49: rcost p.49 figure(8) shows the gaussian plot of dust color temperature the solid curve represents gaussian distribution. the dust color temperature is well fitted as gaussian distribution. the offset temperature is 0.28k and gaussian centre is 20.77k. this suggest that the cavity is in thermal equilibrium and not effected by external cause. there is less deviation in temperature. 6. conclusions http://skyview.gsfc.nasa.gov/ http://skyview.gsfc.nasa.gov/ https://doi.org/10.1007/978-3-540-34144-4 https://doi.org/10.1086/339842 https://doi.org/10.1086/491729 https://doi.org/10.1051/0004-6361:20030575 https://doi.org/10.1086/172702 403 forbidden forbidden you don't have permission to access this resource. apache/2.4.54 (ubuntu) server at www.nepjol.info port 443 microsoft word bibechana _final_.doc 1 bibechana vol. 6, march 2010 regular associated solution model for the estimation of heat of mixing of binary liquid alloys d. adhikari a , b.p. singh a , i.s. jha b a univ. dept. of physics, t.m.bhag. university, bhagalpur, bihar, india b dept. of physics, m.m.a.m. campus (tribhuvan university), biratnagar, nepal abstract we have determined the equilibrium constants and pairwise interaction energies between the species and the complexes of liquid cusn, agal and fesi, alloys on the basis of regular associated solution model. these parameters are then used to estimate the heat of mixing of each alloy. the observed asymmetry in the heat of mixing of each alloy with respect to concentration is well explained. key words: free energy of mixing; asymmetry; binary alloys, interaction energy 1. introduction several models have been proposed to solve the difficulties and complexities of obtaining thermodynamic parameters. one of the models successfully used for accounts of the thermodynamic characteristics of binary liquid alloy systems, is the model of regular associated solution model. in regular associated solution model, it is assumed that strong associations among the constituent species exist in the liquid phase close to the melting temperature. these associations are given different names such as 'complexes', 'pseudomolecules', 'clusters', 'associations' etc. this assumption has been used by several researchers [1-12] to explain the asymmetry of the properties of mixing for binary alloys. thus binary alloys in a liquid phase can be considered as a ternary mixture of unassociated atoms of components and complexes, all in chemical equilibrium. jordan [3] proposed that activity of unassociated atoms and the complexes can be estimated by treating the mixture as a ternary system and termed this mixture as regular associated solution. jordan [3] applied this idea in the congruently melting semiconductors zn-te and cd-te and determined thermodynamic equations for liquidus curve (the melting temperature against concentration curve) of these alloys. this model is further extended and applied by other researchers [4, 5, 12] for the determination of thermodynamics and microscopic parameters of different alloy systems in molten state. in present paper, we intend to apply regular associated solution model to obtain the heat of mixing of liquid cusn, agal and fesi alloys. for this, we have assumed cu3sn ,ag3al and 2 d. adhikari et al. fe2si complexes in liquid cusn, agal and fesi alloys respectively by studying their corresponding phase diagram in solid state[13]. theoretical formalism is given in section 2, section 3 deals with the numerical result and discussion. conclusion is provided in section 4. 2. theory suppose there be three species in equilibrium in the liquid alloy, namely, monomers a, b and apb molecules in the respective concentrations ,n,n ba and apbn moles. considering a solution of 1n atoms of a and 2n atoms of b, the formation of apbn complex requires apba1 pnnn += and apbb2 nnn += for conservation of mass in the partially associated solution. when there is association, the thermodynamic behaviour of complexes a and b components is governed by their true mole fractions ax , bx and apbx rather than their gross mole fraction 1x and 2x , where )nn/(nx 2111 += etc. and )nnn/(nx apbbaaa ++= etc. using above relations, the two sets of mole fractions are related to each other by the relations apb21a xpxxx −= , apb22b x)px1(xx −−= (1) in regular associated solution solutions, the gross chemical potentials of components 1 and 2 are equal to the chemical potentials of the monomeric species a and b [14]. the activity coefficients aγ , bγ and apbγ of monomers and complex can be expressed in terms of pairwise interaction energies through [3] )ωωω(xxωxωxγlnrt 132312apbb13 2 apb12 2 ba +−++= (2a) )ωωω(xxωxωxγlnrt 121323apba12 2 a23 2 apbb +−++= (2b) )ωωω(xxωxωxγlnrt 231213apbb23 2 b13 2 apba +−++= (2c) where 12ω , 13ω and 23ω are interaction energies for the species a, b ; a, apb and b, apb respectively, t the temperature and r stands for the universal gas constant. the equilibrium constant in a regular associated can be obtained [6] as ]x)px1(x[ rt ]x)x1(px[ rt ]x)x1(px[ rtx xx lnkln bbapb 23 aaapb 13 abb 12 apb b p a −− ω +−− ω ++− ω +         = (3) 3 bibechana vol. 6, march 2010 now using the equations listed above the integral excess free energy xsg∆ is given by klnrt )px1( x )xlnxxlnx(rt)xlnxxlnxxlnx( )px1( rt )xxxxxx( )px1( 1 g apb apb 2211apbapbbbaa apb 23apbb13apba12ba apb xs + ++−++ × + +ω+ω+ω + =∆ (4) once the expressions for g∆ )]xlnxxlnx(rtg[ 2211 xs ++∆= is obtained, heat of mixing can be found using standard thermodynamic relation p,t t g∆ tg∆h∆         ∂ ∂ −= (4a) the pairwise interaction energies and equilibrium constant are determined by the following method: in a regular associated solution aa11 γxγx = and bb22 γxγx = , where 1γ and 2γ are respective gross activity coefficients of components 1 and 2. thus 1 a a1 x x lnlnln +γ=γ (5a) and 2 b b2 x x lnlnln +γ=γ (5b) the pairwise interaction energies, the equilibrium constants and the activity coefficients at infinite dilution can be written as [9] rt ln 12 0 1 ω =γ (6a) o 2 o 1 o 2 o 1 13 γγ γγ )rt/ωexp(k − = (6b) where oγ1 and o γ 2 are activity coefficients of component a and that of b at zero concentrations. solving equations (2a) and (2b) we obtain 2 apb 12 bb a 1 b b 2 b 13 x rt )x1(x x a ln)x1( x a lnx rt ω −−        −+        = ω (7) 2 apb 12 aa b 2 a a 1 a 23 x rt )x1(x x a ln)x1( x a lnx rt ω −−        −+        = ω (8) using equations (6), (16) and (17), we can derive         +         −        +                + =+ apb 2 p 112 b 2 apb b a 1 apb a13 x aa ln rt ω x a ln x x x a ln x x1 rt ω kln (9) 4 d. adhikari et al. 3. results and discussion to find heat of mixing of binary alloys, we have found the complex concentration in a regular associated solution of respective alloys by using equations (6), (9) and observed data of corresponding activities [13]. the complex concentration of liquid alloys in molten state are presented in table 1. we have found the equilibrium constants and the pairwise interaction energies between the species and the complexes using equations (3), (4), (6), (7), (8), and observed data of corresponding integral excess free energies of mixing. the equilibrium constants and pairwise interaction energies of different binary alloys are listed in table 2. 3.1 cusn alloys at 1400k it is found from the analysis that the heat of mixing is negative at all concentration. our theoretical calculation shows that the minimum value of the heat of mixing is -4.95 kj at cux = 0.8 which exactly matches with the experimental value [1]. further it is observed that the concentration dependence of asymmetry in h∆ can be explained only when one considers the temperature dependence of the pairwise interaction energies. the agreement between the calculated and experimental values is also good. the calculated and observed values of heat of mixing are compared in figure 1. figure-1: free energy of mixing (∆hrt) versus xcu of liquid cusn solution (1400k) ;(–––) theory, (○○○) experiment [13] 5 bibechana vol. 6, march 2010 3.2 agal alloys at 1273k it is found from the analysis that the enthalpy of mixing is negative at all concentration, being minimum around stoichiometric composition ( h∆ = -0.611rt at xag = 0.72). further it is observed that the concentration dependence of asymmetry in h∆ can be explained only when one considers the temperature dependence of the pairwise interaction energies. the calculated and observed values heat of mixing is compared in figure 2. figure-2: free energy of mixing (∆h/rt) versus xag of liquid agal solution (1273k); (––––) theory, (○○○) experiment [1] 3.3 fesi alloys at 1873k it is found from the analysis that the heat of mixing is negative at all concentration. our theoretical calculation shows that the minimum value of the heat of mixing is -2.585 kj at fex = 0.55. further it is observed that the concentration dependence of asymmetry in h∆ can be explained only when one considers the temperature dependent of the 6 d. adhikari et al. pairwise interaction energies. figure 3 show the comparison between the experimental and calculated values of heat of mixing and entropy of mixing. figure-3: free energy of mixing (∆g/rt) versus xfe of liquid fesi solution (1873k); (––––) theory, (○○○) experiment [13] table 1 xcu/ xag/ xfe sncux 3 alagx 3 sifex 2 0.1 0.001095 0.008257 0.01635 0.2 0.008378 0.03075 0.05294 0.3 0.02658 0.0656 0.1086 0.4 0.05146 0.1138 0.1869 0.5 0.08541 0.1803 0.2903 0.6 0.1240 0.2517 0.3337 0.7 0.1525 0.3075 0.3720 0.8 0.1650 0.2355 0.2968 0.9 0.1192 0.1056 0.1098 -3 -2.5 -2 -1.5 -1 -0.5 0 0.2 0.4 0.6 0.8 1 r t / h ∆ fex 7 bibechana vol. 6, march 2010 table 2 liquid alloy systems parameter cusn at 1400k agal at 1273k fesi at 1873k k 0.1652 0.0494 0.004139 12ω (jmol -1 ) -13500 -11382 -64170 13ω (jmol -1 ) -24900 -617 -14720 23ω (jmol -1 ) -16500 -28064 -45500 4. conclusion the regular associated solution model is found to be suitable to estimate the heat of mixing for both weakly and strongly interacting binary liquid alloys. the observed asymmetries in the heat of mixing of binary liquid alloys with respect to the concentrations are well explained on the basis of regular associated solution models. acknowledgement one of the authors (d. adhikari) is thankful to university grant commission (ugc), nepal, for providing financial support to pursue the research. references [1] bhatia, a.b. and hargoove, w.h. 1974.phys. rev. b-10:316 . [2] singh, r.n. 1987. can j. phys. 65: 309. [3] jordan, a.s. 1970. metall. trans. 1:239. [4] lele, s.and ramchandra rao, p. 1981. metall. trans. 12 b: 659. [5] osmura, k. and predel , b. 1977. tans. j. phys. inst. met. 18 :765 . [6] k. hoshino and w.h. young, j. of phys. f: met. phys. 10,1365 (1980). [7] mcalister, s.p. and crozier, e.d. 1974. j. of phys. c-7:3509. 8 d. adhikari et al. [8] jha, i.s. ,singh, r. n. shrivastava ,p.l. and mitra, n.r. 1990.phil. mag. 61:8445. [9] singh, r. n. , jha, i.s. and pandey, d.k. 1993.j. condens. matter 5: 2469 . [10] bhatia,a.b. and singh, r. n. 1980.phys. letters a-78:460 . [11] gerling, u. pool ,m. j. and predel, 1983.b. z. metallkde 74:616 . [12] adhikari, d., jha, i.s. and singh, b. p. 2010.physica b405 :1861 . [13] hultgren, r. desai, p. d.,hawkins, d.t., gleiser, m. and kelley, k.k. (asm,metal park,1973).selected values of the thermodynamic properties of binary alloys [14] prigogine, i. and defay, r. ,( longmans green and co.london, 1974) chem. thermodynamicsp.257. microsoft word main.doc 15 bibechana vol. 6, march 2010 diversity of herpetofauna in and around the koshi tappu wildlife reserve damodar thapa chhetry p.g. campus, biratnagar, tribhuvan university, nepal abstract the paper deals with the herpetofauna of koshi tappu wildlife reserve and its surroundings. a total of 23 species of herpetofauna belonging to 19 genera and 13 families were recorded. of these 8 species belonging to 6 genera and 3 families were amphibians, and 15 species belonging to 13 genera and 10 families were reptiles. keywords: herpetofauna.; gavialis gangeticus; koshi tappu 1. introduction the koshi tappu wildlife reserve is the first ramsar site in nepal. it extends between 86°55'-87°05’e longitude and 26°34'-26°45'n latitudes on the alluvial flood plain of the sapta koshi river which is fed by seven tributaries, the indrawati , bhote koshi , tama koshi , dudh koshi , liku , arun and tamor rivers. the reserve covers part of sunsari, saptari and udayapur districts of the eastern development region. it is touched by twelve village development committees. eastern and western embankments of 5-7 m high were constructed by the koshi dam project to control flood. in and around of koshi tappu wildlife reserve are many notable wetlands like rivers, floodplain, oxbow lakes and riverine marshes, fresh water marshes and ponds, seasonally flooded grassland, swamp forest, reservoir, paddy fields etc. these wetlands are the suitable habitat for the herpetofauna. several herpetologist have provided literature on herpetofauna of nepal (swan and levition,1962; dubois,1974; nonhoe and ouboter, 1987; shah and giri, 1992; schleich, 1993; shah, 1995; schleich and kaestle, 2002). however, very few literature (suwal,1993; wmi/iucn,1994;) are available on herpetofauna of koshi tappu wildlife reserve and its surroundings. 2. materials and methods the herpetofauna was studied from july, 2002 to june, 2004, once in every month at regular interval from koshi tappu wildlife and its surroundings. the amphibians were collected by the help of local people and fisher men. they used different materials like rubber glove, long iron tongue, different nets (cast net, scoop net etc.). the collected specimens were preserved in plastic containers in 10 % formalin for further study. in case of reptiles, long iron tongue, rubber glove, scoop net, plastic container etc. were mainly used for collection. some of the collected specimens were preserved in 10 % formalin for further study. some reptiles were released after taking photographs. informations about snakes were collected from local people. reports about the presence of gavialis gangeticus and crocodylus palustris were also collected from local people. the collected specimens of amphibia 16 damodar thapa chhetry and reptile were identified with standard literature (stebbins, 1966; cochran and goin, 1970; shrestha, 1981; daniel, 1983; conant and collins, 1991; das, 1991; schleich, 1993; murthy, 1995; shrestha, 2001; and schleich and kaestle, 2002). 3. results and discussion the present study recorded altogether 23 species of herpetofauna belonging to 19 genera and 13 families. of these 8 species belonging to 6 genera and 3 families were amphibians, and 15 species belonging to 13 genera and 10 families were reptiles (table 1). among the amphibians, 4 species were found commonly during the whole study period and 4 species were found rarely. five species belonging to the ranidae family was observed. among the ranidae species the sphaerotheca breviceps was found rarely. but the family rhacophoridae and bufonidae included all rarely found species. among the reptiles, 5 species found commonly during the whole study period, 4 species found rarely and 6 species found rarely. according to the local informations, the abundance of herpetofauna are in decreasing trend due to loss of habitat, poaching, killing and cosuming by people. specially, the turtle species: aspideretes hurum, lissemys punctata, pangshura flaviventer kill by local people for meat. similarly, python molurus molurus poach for leather. naja naja is highly poisonous and the people kill this animal when they see. references 1. d.m. cochran and c.j. goin, the new field book of reptiles and amphibians, putnam nature field book, new york (1970). 2. r. conant and j. t. collins, field guide to the reptiles and amphibians (1991). 3. j.c. daniel, the book of indian reptiles. bombay natural history society, india (1983). 4. i. das, colour guide to the turtles and tortoise of the indian sub continent. .a. publishing ltd., england (1991).5. a. dubois, diagnoses de troisespeces nouvelles d’ nepal. bull. zool. soc. france 98(1974) 495. 6. eastern and central north america, 3rd edition. houghton mifflin co., boston, mass. 7. t.s.n. murthy, reptiles of india, r.r. publishing corporation, delhi. (1995). 8. l.m.r. nanhoe and p.e. ouboter, the distribution of reptiles and amphibians in the annapurnadhaulagiri region (nepal). zool. verhandel. leiden. netherland 229(1987) 1. 9. h.h. schleich, contribution to the systematics and to a bibliography of the amphibians and reptiles from nepal . j. nepal res. centre 9(1993) 141. 10. h.h. schleich and w. kaestle, amphibians and reptiles of nepal. koeltz scientific books, koenigstein (2002) 11. k.b. shah, enumeration of the amphibians and reptiles of nepal. publication no. 2. department of national parks and wildlife conservation, ministry of forest and soil conservation, nepal (1995). 12. k.b. shah and m. k. giri, some amphibians and their local uses in arun basin. j. nat. hist. mus. t.u., nepal 12(1992) 9. 13. t.k. shrestha, distribution of amphibians of nepal and their conservation. in: introduction to fauna and wildlife of nepal. (ed. majupuria, t.c.) (1981) 148160, lashkar, kathmandu. 14. t.k. shrestha, herpetology of nepal. mrs. bimala shrestha, kathmandu, nepal (2001). 15. r.c. stebbins, a field guide to western reptiles and amphibians, houghton mifflin co., boston, mass (1966) . 16. r. n. suwal, koshi tappu wildlife reserve: conservation issues and management measures. a survey report submitted to iucn, nepal (1993). 17. l.w. swan and a.e. levition, the herpetology of nepal: a history checklist and zoogeographical analysis of the herpetofauna. proc. calif. acad. sci., san francisco, usa 32(1962) 103. 17 bibechana vol. 6, march 2010 18. wmi/ iucnnepal biodiversity of koshi tappu wildlife reserve and its adjacent area. applied database for integrated biodiversity conservation in nepal. woodland mountain institute / iucn – nepal( 1994). herpetofauna in and around the koshi tappu wildlife reserve. family scientific name common name local name local status ranidae hoplobatrachus crassus (jerdon, 1853) jerdon’s bull frog sandhe bhyaguto commonly found ranidae hoplobatrachus tigerinus (daudin,1802) indian bull frog singare baghe bhyaguto ’’ ranidae euphlyctis cyanophlyctis (schneider, 1799) skittering frog ahale bhyaguto ’’ ranidae limnonectes teraiensis (dubois, 1984) terai cricket frog madhese kithre bhyaguto. ’’ ranidae sphaerotheca breviceps (schneider,1799) indian burrowing frog. bharati khopilte bhyaguto. rarely found rhacophoridae polypedates taeniatus (boulenger, 1906) terai tree frog madhese singare rukh bhyaguto ’’ bufonidae bufo melanostictus (schneider, 1799) common asian toad khasre bhyaguto ’’ bufonidae bufo stomaticus (lutken, 1862) marbled toad khasre bhyguto ’’ agamidae calotes versicolor (daudin,1802) common garden lizard bagaiche chheparo commonly found varanidae varanus bengalensis (daudin,1802) bengal monitor bhaise gohoro very rarely found varanidae varanus flavescens ( hardwicke and gray, 1827) yellow monitor sun gohoro ’’ colubridae xenochrophis piscator (schneider, 1799) checkered keel back water snake kothe dhodia sarpa commonly found colubridae amphiesma stolatum (linnaeus,1758) buff-striped keelback harara sarpa ’’ elapidae bungarus fasciatus (schneider, 1801) banded krait pate gana guwali sarpa ’’ elapidae bungarus caeruleus (schneider,1801) common indian krait karet ’’ elapidae naja naja (linnaeus,1758) spectacled cobra goman very rarely found boidae python molurus molurus (linnaeus,1758) asiatic rock python ajingar ’’ testudinidae indotestudo elongata (blyth , 1853) elongated tortoise kubadi kachhuwa rarely found gavialidae gavialis gangeticus (gmelin, 1789) gharial lamo thutune gohi. very rarely found crocodylidae crocodylus palustris (lesson,1831) mugger crocodile magar gohi ’’ trionychidae aspideretes hurum (gray,1831) indian peacock soft shell turtle naram khabate kachhuwa rarely found trionychidae lissemys punctata (webb,1980) indian flapshell turtle putali kachhuwa ’’ bataguridae pangshura flaviventer (gunther,1864) yellow-bellied tent turtle. pahelo bhude dhuri kachhuwa ’’ microsoft word sujit kumar shah et al.doc sujit kumar shah et al. / bibechana 8 (2012) 37-45 : bmhss, p.37 bibechana a multidisciplinary journal of science, technology and mathematics issn 2091-0762 (online) journal homepage: http://nepjol.info/index.php/bibechana a comparative study of critical micelle concentration (cmc) and free energy of micellization (∆g o m) of cationic surfactant (dodecyltrimethylammonium bromide, dtab) and anionic surfactant (sodiumdodecyl sulphate, sds) in different composition of methanol-water mixed solvent media by conductometric method at 308.15 k sujit kumar shah, tulasi prasad niraula, ajaya bhattarai, sujeet kumar chatterjee* department of chemistry, mahendra morang adarsh multiple campus (tribhuvan university), biratnagar, nepal *corresponding author: sujeetkumarchatterjee@yahoo.com article history: received 14 june, 2011; accepted 10 july, 2011 abstract precise measurements on the specific conductivity of cationic surfactant (dodecyltrimethylammonium bromide) and anionic surfactant (sodiumdodecyl sulphate) in methanol-water mixed solvent media containing 0.1, 0.2 and 0.3 volume fractions of methanol are reported at 308.15 k. specific conductivities of dodecyltrimethylammonium bromide and sodiumdodecyl sulphate increase with increase in concentration and decrease with increase in the volume fractions of methanol. critical micelle concentration (cmc) increases with increase in volume fraction of methanol in case of both surfactants. free energy of micellization (∆gºm) has been calculated. increase in cmc with increase in volume fractions of methanol has been explained in terms of breaking of structure of water due to co-solvent effect. keywords: critical micelle concentration; methanol-water mixed solvent media; specific conductivity; dodecyltrimethylammonium bromide; sodiumdodecyl sulphate 1. introduction surfactants have various applications to different daily use products and in research fields [1]. zana and coworkers [2-6] have reported extensive studies on the effect of the linear alcohols ethanol to hexanol on the critical micelle concentration (cmc), micelle molecular weight and degree of ionisation. the behaviour of sodiumdodecyl sulphate micelles in the presence of n-alcohols has been extensively investigated [714]. according to rubio et al. [13] moderately hydrophobic alcohols in low concentration promote micellization probably by residing at the micellar surface and reducing unfavourable water hydrocarbon contacts. however, at higher concentrations these alcohols destabilize micelles by displacing water from the surface, therefore decreasing its effective dielectric constant, increasing head group repulsions, and disrupting surfactant packing. since water-alcoholsurfactant systems are frequently used as media in the studies of chemical equilibria and reaction rates, it is essential to investigate the effect of the nature of the alkyl groups in the alcohol on the cmc of the surfactants. addition of alcohols to aqueous solutions of sujit kumar shah et al. / bibechana 8 (2012) 37-45 : bmhss, p.38 surfactants has allowed the investigation of the effect of hydrophobic interactions on the micellar structure [15]. almgren et al. [16] investigated on the effect of formamide and other solvents, including dimethylsulfoxide and dimethylacetamide on the micelle formation of sodiumdodecyl sulphate. they observed the reduction of both critical micelle concentration (cmc) and mean aggregation number of sodiumdodecyl sulphate micelles upon the addition of formamide. the gradual replacement of water with other organic polar solvent allows one to explore a wide bulk phase polarity range and its influence on micellization. addition of small amount of an organic solvent has been known to produce marked changes, in the critical micelle concentration (cmc) of ionic surfactants due to tendency of added organic solvent either to break or make the water structure through solvation of hydrophobic tail of the surfactant by the hydrocarbon part of the organic solvent [17-18]. akbas et al. [19] carried out an investigation on the effect of ethanol and ethylene glycol on the cmc of cetyltrimethylammonium bromide. they observed that cmc decreases upon the addition of ethanol and ethylene glycol. the aim of the present work is to investigate the effect of methanol on the micellar behaviour of dodecyltrimethylammonium bromide and sodiumdodecyl sulphate at 308.15 k by conductance measurement. 2. experimental section methanol (merck, india) was distilled with phosphorous pentoxide and then redistilled over calcium hydride. the purified solvent had a density of 0.77723 ± 0.00004 g.cm -3 and a co-efficient of viscosity of 0.47424 ± 0.00005 mpa.s at 308.15 k; these values are in good agreement with the literature values [20]. triply distilled water with a specific conductance less than 10 -6 s.cm -1 at 308.15 k was used for the preparation of the mixed solvents. the physical properties of methanol-water mixed solvents used in this study at 308.15 k are shown in table 1 and those values are matched with the published works [21-23]. the relative permittivity of methanol-water mixtures at the experimental temperatures were obtained by regressing the relative permittivity data as function of solvent composition from the literature [24]. dodecyltrimethylammonium bromide (98% pure) and sodiumdodecyl sulphate (98% pure) were obtained from loba chemi, india and were dried in oven for one hour before use. solutions were prepared of dodecyltrimethylammonium bromide and sodiumdodecyl sulphate by weighing appropriate amount in an electronic balance, (afcoset-er120a) with a precision of 0.0001g. conductivities were measured using digital conductivity meter (306) of systronics which is a microcontroller based instrument for measuring specific conductivity of solutions. the accuracy in conductance measurements is ± 1%. the cell was calibrated by the method of lind and co-workers [25] using aqueous potassium chloride solution. measurements were carried out in a jacket containing conductivity cell of cell constant 1.002 cm –1 . water was circulated in the jacket from thermostat and the temperature was maintained within ± 0.1ºc. the details of the experimental procedure have been described earlier [26-27]. several independent solutions were prepared and runs were performed to ensure the reproducibility of the results. due correction was made for the specific conductance of the solvent by subtracting the specific conductance of the relevant solvent medium from those of the electrolyte solutions. in order to avoid moisture pickup, all solutions were prepared in a dehumidified room with utmost care. in all cases, the experiments were performed in three replicates. 3. result and discussion conductivity measurements are carried out in 0.1, 0.2 and 0.3 volume fractions of methanol in methanolwater mixed solvent media for dodecyltrimethylammonium bromide and sodiumdodecyl sulphate at 308.15 k in order to evaluate the cmc. the plots between specific conductance and concentration of surfactants solution in methanol-water mixed solvent media at 308.15 k is shown in fig. 1 to fig. 6. the critical micellar concentrations (cmc) of dodecyltrimethylammonium bromide and sodiumdodecyl sulphate are found very easily from conductometry. these are obtained from the inflections in the plots of 0 1 2 3 0 0.02 0.04 0.06 [s d s ] / mol l -1 κ / m s c m -1 sujit kumar shah et al. / bibechana 8 (2012) 37-45 : bmhss, p.39 specific conductivity versus surfactant concentration. the data points above and below the inflection are fitted to two linear equations, and the cmcs are obtained from the common intersection. this method is found to be reliable and convenient for the present system because of the significant variations of specific conductivity with surfactant concentration in the preand postmicellar regions which allowed us to draw two unambiguous straight lines above and below the cmc. temperature dependence values of cmc and α (degree of ionisation) can be used to obtain information about thermodynamics of micellization. degree of ionisations are calculated by taking ratio of slope of line after micelle and the slope of line before micelle. the standard gibbs free energy of micellization for ionic surfactant (∆gºm) are calculated from the relation derived for the charged phase separation model of micellization[28-29]. this relation is given by ∆gºm = (2-α) r t ln(χcmc) (1) χcmc represents cmc in mole fraction. the values of cmc and standard free energy of micellization of dodecyltrimethylammonium bromide and sodiumdodecyl sulphate are listed in table 2. with the increase in the volume fraction of methanol, values of cmc for both surfactants increases. this increase in values of cmc is also explained by the decrease in negative values of standard free energy change. fig. 7 shows the effect of volume fraction of methanol on the free energy change for the micellization of dodecyltrimethylammonium bromide at 308.15 k. it can be observed that the volume fraction of methanol in water increases the negative values of free energy of micellization (∆gºm) decreases thus making less favourable for the micelle to form. similar behaviour can be seen in case of sodiumdodecyl sulphate (fig. 8). it is well known that addition of solvents, which act as water structure breakers decrease the hydrophobic effect resulting into an increase in the cmc of ionic surfactants [30]. breaking of water structure by methanol would facilitate interactions between the hydrophobic tail of the surfactant molecules and the hydrophobic part (methyl group) of the methanol and consequently, the local concentration of methanol molecules around the surfactant monomers becomes larger than the average of the bulk. solvation of the surfactant molecules by the hydrophobic part of the organic solvent would, therefore, lead to delaying the aggregation of the surfactant monomers to form micelles and hence the increase in the cmc of the surfactant. fig. 1: variation of specific conductance with molar concentration of sodiumdodecyl sulphate (sds) in methanol water mixed solvent media containing 0.1 volume fraction of methanol at 308.15 k 0 1 2 3 0 0.02 0.04 0.06 [ s d s ] / mol l -1 κ / m s c m -1 0 1 2 3 0 0.02 0.04 0.06 [ s d s ] / mol l -1 κ / m s c m -1 sujit kumar shah et al. / bibechana 8 (2012) 37-45 : bmhss, p.40 fig. 2: variation of specific conductance with molar concentration of sodiumdodecyl sulphate (sds) in methanol water mixed solvent media containing 0.2 volume fraction of methanol at 308.15 k. fig. 3: variation of specific conductance with molar concentration of sodiumdodecyl sulphate (sds) in methanol water mixed solvent media containing 0.3 volume fraction of methanol at 308.15 k. 0 0.5 1.0 1.5 2.0 0 0.01 0.02 0.03 0.04 0.05 [dtab] / mol l -1 κ / m s c m -1 0 0.5 1.0 1.5 2.0 0 0.01 0.02 0.03 0.04 0.05 [dtab] / mol l -1 κ / m s c m -1 sujit kumar shah et al. / bibechana 8 (2012) 37-45 : bmhss, p.41 fig. 4: variation of specific conductance with molar concentration of dodecyltrimethylammonium bromide (dtab) in methanol water mixed solvent media containing 0.1 volume fraction of methanol at 308.15 k. fig. 5: variation of specific conductance with molar concentration of dodecyltrimethylammonium bromide (dtab) in methanol water mixed solvent media containing 0.2 volume fraction of methanol at 308.15 k. 0 0.5 1.0 1.5 2.0 0 0.01 0.02 0.03 0.04 0.05 [dtab] / mol l -1 κ / m s c m -1 15 20 25 30 35 40 0 0.1 0.2 0.3 0.4 volume fraction of methanol in water -∆ g 0 m / ( k j m o l1 ) sujit kumar shah et al. / bibechana 8 (2012) 37-45 : bmhss, p.42 fig. 6: variation of specific conductance with molar concentration of dodecyltrimethylammonium bromide (dtab) in methanol water mixed solvent media containing 0.3 volume fraction of methanol at 308.15 k. fig. 7: variation of standard free energy change of micellization of dodecyltrimethylammonium bromide (dtab) with volume fractions of methanol in water. 15 20 25 30 35 40 0 0.1 0.2 0.3 0.4 volume fraction of methanol in water -∆ g o m / ( k j m o l1 ) sujit kumar shah et al. / bibechana 8 (2012) 37-45 : bmhss, p.43 fig. 8: variation of standard free energy change of micellization of sodiumdodecyl sulphate (sds) with volume fraction of methanol in water. table 1: properties of methanol-water mixtures containing 0.1, 0.2, and 0.3 volume fractions of methanol at 308.15 k. t/k 0 ρ /g.cm -3 0 η / mpa.s d 0.1 volume fraction of methanol 308.15 0.9797 0.8665 71.57 0.2 volume fraction of methanol 308.15 0.9663 1.0217 68.14 0.3 volume fraction of methanol 308.15 0.9516 1.1418 64.25 sujit kumar shah et al. / bibechana 8 (2012) 37-45 : bmhss, p.44 table 2: values of cmc and free energy of micellization of sodiumdodecyl sulphate (sds) and dodecyltrimethylammonium bromide (dtab) in methanol water mixed solvent media containing 0.1, 0.2 and 0.3 volume fractions of methanol at 308.15 k volume fractions of methanol cmc (mm) degree of ionisation (α) -∆gºm ( kj mol -1 ) sds 0.0 8.7 * 0.1 9.31 0.493 33.28 0.2 10.04 0.473 32.97 0.3 10.75 0.466 32.83 dtab 0.0 15.6 ** 0.1 18.75 0.263 35.29 0.2 21.62 0.237 34.90 0.3 28.86 0.233 33.38 ref. no. [31] *, [32] ** 4. conclusion , effects of concentration and solvent composition on dodecyltrimethylammonium bromide and sodiumdodecyl sulphate in methanol–water mixed solvent media have been studied by measuring specific conductance through conductometric method. the following conclusions have been drawn from the above results and discussion. the conductance decreases with increase of alcohol content for the studied methanol-water mixed solvent system. the presence of methanol reduces the dielectric constant of the solvent phase and makes easier for the formation of ion-pairs in the solution phase. the cmc increases with the increase of methanol for dodecyltrimethylammonium bromide and sodiumdodecyl sulphate but dodecyltrimethylammonium bromide has higher cmc values in comparison with sodiumdodecyl sulphate in the measured ranges of methanol-water mixed solvent media. furthermore, the negative value of free energy of micellization decreases with the increase of methanol for dodecyltrimethylammonium bromide and sodiumdodecyl sulphate but dodecyltrimethylammonium bromide has higher values in comparison with sodiumdodecyl sulphate in the measured ranges of methanol-water mixed solvent media. acknowledgements the authors are grateful to mr. g. shrivastav (head of the department of chemistry), mahendra morang adarsh multiple campus for providing research facilities. one of the authors (s.k. shah) is thankful to university grants commission (ugc), nepal, for providing financial support to pursue the research. sujit kumar shah et al. / bibechana 8 (2012) 37-45 : bmhss, p.45 references [1] s. k. shah, a. bhattarai and s. k. chatterjee, bibechana 7(2011) 61. 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[32] d. lópez-díaz and m. m. velázquez, chem. educator 12 (2007) 5. 403 forbidden forbidden you don't have permission to access this resource. apache/2.4.54 (ubuntu) server at www.nepjol.info port 443 microsoft word shobha kanta lamichhane-final 59-66.doc shobha kanta lamichhane / bibechana 8 (2012) 59-66 : bmhss, p.59 bibechana a multidisciplinary journal of science, technology and mathematics issn 2091-0762 (online) journal homepage: http://nepjol.info/index.php/bibechana experimental investigation on anisotropic surface properties of crystalline silicon shobha kanta lamichhane department of physics, prithvi narayan campus, pokhara, nepal e-mail: sklamichhane@hotmail.com article history: received 31 may, 2011; accepted 25 july, 2011 abstract anisotropic etching of silicon has been studied by wet potassium hydroxide (koh) etchant with its variation of temperature and concentration. results presented here are temperature dependent etch rate along the crystallographic orientations. the etching rate of the (111) surface family is of prime importance for microfabrication. however, the experimental values of the corresponding etch rate are often scattered and the etching mechanism of (111) remains unclear. etching and activation energy are found to be consistently favorable with the thermal agitation for a given crystal plane. study demonstrate that the contribution of microscopic activation energy that effectively controls the etching process. such a strong anisotropy in koh allows us a precious control of lateral dimensions of the silicon microstructure. keywords: microfabrication; activation energy; concentration; anisotropy; crystal plane 1. introduction anisotropic etching of silicon is used to obtain varied microstructures [1–3], and anisotropic etchants extensively used are koh, edp (ethylene diamine, pyrocatechol), tmh (tetryl methylammonium hydroxide), etc. the mechanisms of anisotropic etching of silicon and the etching processes that occur in the crystalline surfaces are not well known. many authors have suggested a different sort of mechanism for the reactions to explain the anisotropic behaviour of etching of silicon in alkaline solutions [4, 5]. seidel et al [6] has proposed an electro-chemical model which involves the transfer of the electrons from ions in the solution to the crystal surface. in this model, the rate determining step is the transfer of electrons from the surface state, which lies in the forbidden gap, to the conduction band. this process is thermally activated. seidel states that the difference in energy between final and initial state contributes significantly to the macroscopic activation energy. anisotropy can be accounted for by the fact that the energy level of the surface state depends on the surface orientation. thus the (111) surface which exhibits the highest activation energy when etched with koh, has the lowest level energy state. the (110) and (100) planes present roughly the same level below that corresponding to (111) surface state. in other words, they have approximately the same activation energy. on the other hand, elwenspoek [7] puts forward a model inspired from the crystal growth theories in which the etch rate should be governed by a nucleation barrier of one atomic layer deep cavities and the activation energy depends on the step-free energy and under saturation. shobha kanta lamichhane / bibechana 8 (2012) 59-65 : bmhss, p.60 micro-electro mechanical structure (mems) devices are the output of either an etching process (or micromachining) from larger structures or are built up atom by atom through material deposition processes. maintaining dimensional control and retaining consistent mechanical property is considerably difficult because feature sizes are in the micrometer range. silicon mems offer these challenges. formation of a functional mems structure on a substrate, it is necessary to etch the thin films previously deposited or the substrate itself. in general there are two type of etching process: wet etching, where the material is dissolved when immersed in a chemical solution and, dry etching, where the material is sputtered or dissolved using reactive ions. in the following we briefly discuss the most popular technology, wet etching, is a simplest etching technology. all it requires is a container with a liquid solution that will dissolve the material. unfortunately, there are complications since a mask is desired to selectively etch the material. one must find a mask that will not dissolve or at least etch much slower then the material to be patterned. anisotropic is contrast to isotropic etching means different etch rates in different plane surfaces. the classic example of this is the (111) crystal plane sidewalls that appears when etching a hole in (100) silicon wafer in a chemical such as koh. the result is a pyramid shaped whole instead of a hole with round sidewalls with an isotropic etchant. etching technologies in thin film processes play an important role in the semiconductor industry. wet chemical etching techniques are used extensively in semiconductor processing because of their low cost, high throughput, and excellent selectivity. important progress in the fabrication of microelectrical structure with integrated circuits has been achieved. though detailed kinetic and electrochemical measurements have not been made, koh solutions are extensively used for the etching of single crystal silicon [8, 9]. fabrication of umos transistors on silicon (111) wafers for high power and high current densities has been achieved by applying koh anisotropic wet etching to the silicon substrate. other applications of koh wet etching include the fabrication of vmosfets, radio frequency amplifiers, power supplies, and microcomputers [10]. using its selective and anisotropic etching properties, koh wet etching has also been applied to yield such devices as field emission devices, optical waveguides, pressure sensors and ink nozzles [11]. koh wet etching is one of the old anisotropic orientation dependent wet etching techniques. etching of (100) oriented silicon using aqueous koh creates v-shaped grooves with (111) planes at an angle of 54.74° from the (100) surface. koh solution can also be used to produce mesa structures. there are several models proposed for the silicon etching mechanism in aqueous koh. bean and runyan found that the slow etch rate in the (111) direction is a consequence of the diamond lattice structure because the (111) plane is a double layer bound together by more atomic bonds than that of other planes. glembocki et al. proposed an electrochemical reaction mechanism that exists in the rate determining step for koh etching. a compromise model was proposed by seidel et al. in 1990, assuming that electrochemical reactions dominate during etching and that anisotropic etching was caused by the orientation-dependent number of dangling bonds available per surface area. the (100) silicon surface etches faster than the (111) because the free surface of (100) silicon is attacked by one oh per silicon atom, whereas the (111) silicon surface is attacked by two oh per silicon atoms [12]. although the mechanisms are not clear and need to be answer all these questions. the approach followed so as to determine the significant factor in a given electrochemical process and their quantification are prime importance from technological point of view. such proposed quires are being addressed in this work. if we have answer of all these quires in hand, then there is no doubt of etching silicon in aqueous koh is of both technological and fundamental importance. etching of (100) oriented silicon using aqueous koh creates v-shaped grooves with (111) planes at an angle of 54.74° from the (100) surface. koh solution can also be used to produce mesa structures. there are several models proposed for the silicon etching mechanism in aqueous koh. bean and runyan found that the slow etch rate in the (111) direction is a consequence of shobha kanta lamichhane / bibechana 8 (2012) 59-65 : bmhss, p.61 the diamond lattice structure because the (111) plane is a double layer bound together by more atomic bonds than that of other planes [10]. a compromise model was proposed by assuming electrochemical reactions dominate during etching and that anisotropic etching was caused by the orientation-dependent number of dangling bonds available per surface area. the (100) si surface etches faster than the (111) because the free surface of (100) si is attacked by one oh per si atom, whereas the (111) si surface is attacked by two oh per si atoms. although the mechanisms are not clear, it is known that electrochemical reactions play an important role in this process. 2. methodology the design of the present work was lab based experimental work. the procedure followed during this study was mentioned herewith sequentially. the wafers used in this study were thermally bonded silicon on insulator (soi) wafers with (100) orientation. the thickness of the top silicon, buried sio2 layer and bottom silicon were 2 and 450 mm respectively. the wafers were of p-type silicon with a resistivity of 4 – 631022 v-cm for the top silicon and 12–15 v cm for the bottom silicon. only the top silicon layer was etched by the koh solution. first, the soi wafers were prepared with standard rca cleaning. a 450 å layer of si3n4, which acts as a koh etching mask, was deposited on each wafer using low pressure chemical vapor deposition (lpcvd). oxide can be used as an etch mask for short periods in the koh solution, but for long periods, nitride is a better etch mask as it etches more slowly. the soi wafer was then cut into 10 mm×10 mm pieces. with the diced pieces held on a vacuum chuck, positive photoresist, ocg 825 [sutter et al, 2003; pp 4] was used to pattern the si3n4 for the koh etching mask. the nitride was etched with cf4 plasma and then the exposed portion of the top silicon on each soi wafer was etched in koh solutions of varying temperature and concentration. to assure the samples were free of particulate and other airborne contaminants. the experiments were conducted in a clean room environment. 3. results and discussion koh etch is a bulk silicon etch whose etch rate is depends on the orientation of the crystal plane can be exploited to create specific geometries (for example, v-grooves sown below). formation of such geometrical structure on silicon crystal depends on various factors: temperature, concentration, activation energy, surface finish and its cleaning process etc. we have performed on crystalline silicon with aqueous koh for the temperature ranging from 70°c to 90°c and its concentration was 30%. our results shows that the optimum etch rate with the minimum surface roughness, indicates low activation energy at 80 0 c. with this one can easily understand that a single result is the outcome of various factors including koh concentration. the etch rate versus temperature as well as etch rate versus koh concentration are displayed in figure 2. figure 2 (a) and (b) shows that etch rate increases with increasing reaction temperature, regardless about koh concentration. within the working range investigated, the etch rate exhibits a maximum at approximately 20% koh and 90°c. below 70°c, little dependence of etch rate with the koh concentration is observed. without considering the effect of etching, we conclude that the highest etch rate can be achieved with a high temperature reaction. figure 2 (d) demonstrate the effect of changing koh concentration with the etch rate. as we conclude from figure. 2 that er depends on concentration are less significant than reaction temperature. dimensional control of a microstructure is strongly related to mems performance. etching technique allows control of the dimension. working in micromachining we need to both add and shobha kanta lamichhane / bibechana 8 (2012) 59-65 : bmhss, p.62 remove the material in a controlled way. adding material on a silicon wafer can be done by chemical vapor deposition (cvd). removing material is done almost by chemical etching, where a material is removed atom by atom [13]. this can be done either in a liquid (wet etching) or in plasma (dry etching). still the most simple and versatile anisotropic wet chemical etching for silicon is in koh. generally, etch anisotropy is given by, ea= 1(erl/erv), where erl and erv are the lateral and vertical etch rates. a process is said to be purely anisotropic (ea=1) if lateral etch rate is zero. on the other hand, if ea=0, lateral and vertical etch rates are identical and etching is said to be isotropic. the geometry what we got is shown in fig.1 below. fig.1: schematic showing anisotropic etches of silicon the final shape of the etched wafer depends highly on relative etching speed along crystallographic planes. the (111) plane is comparatively inert to alkaline etchants, while the relative etching speed of other planes depends on nature of etchant, temperature, concentration etc. in our experiment, we have taken 33 % koh by weight as an etchant for silicon (111) and (100) planes ambient conditions, unless otherwise specified. 3.1 surface finish of etched silicon contaminants are attached to the wafer surface physically or chemically. chemical cleaning is necessary in many steps during fabrication. the process that is widely used is so called “rca cleaning” consist of two consecutive cleaning solutions including h2o-h2o2-nh4oh (standard clean1, i.e. sc1 clean) and h2o-h2o2-hcl (standard clean 2, i.e. sc2). the sc1 cleaning with volume ratios typically 5:1:1 is to remove organic contaminants. the sc1 can remove particles physically attached to a wafer surface by etching with nh4oh that detaches the particles from the wafer surface and prevents re-deposition. the sc2 clean, with typical volume ratios of 6:1:1 removes metals from wafer surface and prevents re-deposition [13]. a modified rca clean (i.e., dilute hf after sc1) is designed to eliminate the thin oxide layer grown on silicon surface. modification of rca clean exists on the volume ratios. as for example, in order to reduce silicon surface roughness one needs to greatly reduce the volume fraction of nh4oh in sc1 from the original composition. surface taken by afm is depicted in fig. 3 below. other cleaning processes, such as piranha (h2so4 -h2o2-h2o), ozonized water etc. are also effective. however, rca clean have been most popular cleaning process, is adopted in this work. shobha kanta lamichhane / bibechana 8 (2012) 59-65 : bmhss, p.63 70 75 80 85 90 0.0 0.1 0.2 0.3 0.4 e tc h r a te ( µµ µµ m /m in ) temperature ( 0 c) 20 40 60 80 100 0 1 2 3 4 5 6 e tc h r a te ( µµ µµ m /m in ) temperature ( 0 c) 100 110 111 (a) (b) 0.0027 0.0030 0.0033 -24 -22 -20 -18 -16 ln (e r m /s ) 1/t (k -1 ) 100 110 111 10 20 30 40 50 60 70 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 e tc h r a te ( m ic ro n /m in ) koh concentration (wt % ) 100 110 111 (c) (d) fig. 2: behaviors of etching of crystalline silicon: (a) variation of er along (111) plane with temperature (b) variation of er along (111, 110, 100) plane with temperature (c) arrhenius plot of er for primary crystal planes (d) variation of er along (111, 110, 100) plane with koh concentration 3.2 association between etch rate and activation energy the macroscopic activation energy of er is explained by the sum of two terms. one corresponds to the average of the microscopic activation energies and other accounts for the existence of fluctuation in the fraction of particles at a fixed temperature. er depends on pi and t. in order to understand this point, we define er as ∑ ∆ ∆ = ∆ ∆ = i iimc mc pz tt z er ... .. )( 1 (1) where, ..mc z∆ is the distance traveled by the center of mass of the surface, pi, is the removal probability of the surface atom i, and is given by tk e ii b ia epp − = 0 (2) shobha kanta lamichhane / bibechana 8 (2012) 59-65 : bmhss, p.64 where t is the temperature, kb is the boltzmann constant, eai is the microscopic activation energy for removal of site i and poi is a prefactor. hence, macroscopic and microscopic activation energy and temperature are interrelated. thus, er is the distance traveled by the moving surface per unit time. when the etching process has reached steady state, er is simply the ratio of the distance traveled by the center of mass (cm) of the surface (zcm) to the period of time elapsed (t). i.e. er = zcm/t. during a modeling, zcm can be determined as the sum of all individual shifts (zcm)i of the surface following each successful event, i.e. the sum over all successful particle removal is occurring during ‘t’. although (zcm)i is typically positive, occasionally it may be negative if the removal of site i involves a reduction in the total number of surface sites [14]. in particular, certain sitetypes of our formula for wet chemical etching, typically contribute to the motion of the cm with a negative shift on average. alternatively, we may consider the sum over all events, independently of whether the event is a successful removal or not. note that the positive/negative shift of the cm of the surface due to the removal of atom i, (zcm)i, may be calculated independently, whether the atom is removed or not. equation suggests that the etch rate is composed of two factors: one purely geometrical (the cm-shifts) and one purely numerical. indeed, er is proportional to the average number of surface atoms removed from the surface per unit time. here, proportionality constant (z) is precisely a measure of the average shifts in the cm of the surface per removed atom. z is an exclusively geometrical feature of the er. in particular, it is independent of temperature. although z may take different values for different surface orientations, z does not depend on temperature for a specified orientation. this is an important observation because the appearance of negative cm-shifts (zcm)i may be accepted as the interpretation of the relative importance (weight) of the different particle types for the calculation of an average. actually, the interpretation becomes meaningless if some of the weights are negative. however, the use of i is free of these artifacts and allows unambiguous interpretation, as shown in this study. there are three alternate ways to determine the rate of removal of particles [15]: (i) as in the case of the er, of the removal of particles can be determined using only successful events. note that the averages of the number of surface atoms n are not constant. (ii) the rate of removal of particles can be written in terms of a sum over the different types of surface sites. the rate of removal of particles may also be expressed in terms of the average fraction of particles removed per unit time. here we have defined as the average fraction of particles. activation energy, in relation to the er, the activation energy (ea) is, by definition, the slope of the curve. 3.3 etched crystallographic planes it is essential to monitor the evolution of the surface configuration and roughness during etching. although we are dealing with ideal conditions, this gives us an idea of the difference in etching between the different planes. it is thought that all faces that are rough in the atomic scale etch in the same manner. this is supported by the afm micrograph representation of one plane viz. (111). this is a characteristic feature of rough faces at the atomic level when etched with an alkaline solution. shobha kanta lamichhane / bibechana 8 (2012) 59-65 : bmhss, p.65 fig.3: structure of (111) silicon plane as seen from the top and its afm micrograph. seidel et al [6] has carried out precise experiments to determine the values of the er of the three silicon orientations (100), (110) and (111). in their experiments, the concentration of koh was also modified. the result shows that the anisotropic ratio (110)/(100) has a small dependence on the concentrations (from 10–60%), and the value is on the order of 1.55 at 80 0 c. the macroscopic activation energies were found to be similar for (100) and (110) planes [16], being approximately 0.6 ev. we found an anisotropic ratio (110)/(100) of the same order as above (1.5). for activation energies, we found approximately 0.6 ev for (100) and (110) planes. 4. conclusion the etching mechanism is ‘layer-by-layer’ removal. this mechanism leads to a particular evolution of the roughness which shows an alternation of peaks. anisotropic etching of silicon with koh is a well-known method of forming grooves in the silicon-surface. etching of (100) silicon on silicon on insulator wafers was carried out over a wide range of temperatures and concentrations. using statistical methods, we show that the factors important in the silicon er are reaction temperature, koh concentration, and interaction between them. the activation energy belongs to etching at different koh concentration was calculated from arrhenius plots. in addition, in this work we report a qualitative study of the surface roughness formed in koh etching. the smoothness increases with temperature to a critical point and then decreases at very high temperature and concentration. results presented here are temperature dependent er along the crystallographic orientations. based on this work, temperature is a crucial factor, which control er. activation and cohesive energies along the crystallographic planes are thus microscopic quantities that determine er. er is slow for (111) face and fast in (110). strong er anisotropy allows us a precious control of lateral dimension of the microstructure bound by the (111) surface and activation energy depends on the step-free energy and undersaturation. references [1] k. e. petersen ieee proc. 70 (1982) 420. [2] d. l. kendall, annual rev. mater. sci. vol 9, ed r a huggins (1979) 373. [3] h. namatsu, m. nagase, k. kurihara, k. iwadate and k.murase, microelectron. eng. 27 (1995) 7. shobha kanta lamichhane / bibechana 8 (2012) 59-65 : bmhss, p.66 [4] o. j. glembocki, e d palik, g r de guel and d l kendall, j. electrochem. soc. 138 (1991) 1055. [5] e. d. palik, v m bermudez and o j glembocki, j. electrochem. soc. 132 (1985) 871. [6] h. seidel, l. csepregi, a heuberger and h baumgartel, j. electrochem. soc 137 (1990) 3612. [7] e. vazonyi and t. szkufcik, journal of micromechanics and microengineering 13 (2003) 165. [8] n. p. krivorotov, y. g. svinolupov, t. i izaak, v. v. bychkov, sensors and actuatiors a 113(2004) 350. [9] k.. mylvaganam and c. l. zhang, key engineering materials 233 (2003) 615. [10] p. sutter, e. sutter and m. g. lagally, surface science 30 (2003)1. [11] p. waish, r. omeitchenko, r. k. kalla, a. nakano and p. vashishta, applied physics letters 82 (2003) 118. [12] x. yan, t. xu, g. chen, s. yang, and huiwenliu, journal of physics d: applied physics 37 (2004) 907. [13] p. j.french and a. g. r. evans, solid state electronics 32 (1989) 1. [14] p. j french. and a.g.r evans, journal of physics e: scientific instruments 19 (1986) 1055. [15] j. duck lee, b. chang shim, h. soo and b. gook, electron device letters 20 (1999) 215. [16] k. e. bean and w. r. runyan, semiconductor integrated circuit processing technology, addisionwesley, new york 1990. microsoft word ujjol.doc ujjal alok et al. / bibechana 8 (2012) 67-72 : bmhss, p.67 bibechana a multidisciplinary journal of science, technology and mathematics issn 2091-0762 (online) journal homepage: http://nepjol.info/index.php/bibechana acoustoelectric effect in semiconductor superlattice ujjwal alok 1* , manoj kumar 1 , b.k.singh 1 , praveen kumar 2 1 univ. dept. of physics, t.m.b. university, bhagalpur (bihar) india 2 research scholar, s.k.m. university, dumka (jharkhand) india ∗ corresponding author, e-mail : okujjwal@rediffmail.com article history: received 8 july, 2011; accepted 7 august, 2011 abstract acoustoelectric effect in semiconductor superlattice (sl) in the region ql >>1 has been studied. the equation of motion of the lattice has been transformed into a simple form which becomes identical with the dynamic equation for the wave amplitude in the theory of plasma turbulence. a dispersion relation has been derived from the acoustoelectric current j on the constant electric field e. it is noted that when the electric field is negative the current j rises, reaches a peak and falls off. on the other hand, when the electric field is positive the current decreases, reaches a peak and then rises. a similar observation has been noted for an acoustoelectric interaction in a multilayered structure resulting from the analysis of the si/sio2 structure. keywords: acoustoelectric current; acoustomagnetothermal effect; quantum transparency 1. introduction the propagation of acoustic wave through a semiconductor has been discussed from many angles. blotekjaer and quate [1] have used the coupled mode approach in which one regards the acoustic wave as the lattice mode which are coupled piezo – electrically to the space charge modes of the electron distribution. in [2] thakur et al. observed the effect of a dc current on the drift of optically generated carriers in a quantum well. another interesting mechanism based on the transfer of energy and momentum is the interaction of acoustic phonons with carrier charges in semiconductor materials. this mechanism occurs not only during the scattering of quasimomentum carriers with lattice vibrations but also when acoustic waves propagate through the material. among the effects observed are the absorption ( amplification ) of acoustic waves [3-5], acoustoelectric effect (ae) [6-10], acoustomagnetoelectric effect (ame) [11-12], acoustothermal effect [13], and acoustomagnetothermal effect [13]. these phenomena have, however, received very little attention in (sl) even though they have immense device applications. acoustoelectric effect is the transfer of momentum from acoustic waves to the conduction electrons as a result of which may give rise to a current usually called the acoustoelectric current j or in the case of an open circuit, a constant electric field e. the study of this effect is vital because of the complimentary role it may play in the understanding of the properties of the sl which we believe should find an important place in the acoustoelectric devices. experimental evidence of the dependence of the acoustoelectric effect on the ujjal alok et al. / bibechana 8 (2012) 6772: bmhss, p.68 parameters of sl has been reported in [14]. in [15] experimental work on the acoustoelectric interaction of saw in gaasin gaas superlattice has been reported. a theoretical model for measuring transverse acoustoelectric voltage in multilayered structure resulting from the analysis of the si/sio2 has also been reported in [16]. 2. distribution function and threshold field assume the sound wave as collimated monochromatic phonons with all exactly in phase with one another, we write distribution function, as. f(k) = δ(k q) (1) where ‘k’ is the current phonon wave vector, in many semiconductors, such as gaas the conduction band has a minimum at zone centre and an indirect gap with higher energy at another minimum, at a critical point on the zone boundary. the sl has above other minimum on zone boundary. if the gaas quantum well width is small enough, the confined state of the zone centre can be pushed higher than the other zone boundary. in this case, electrons will fall from the zone centre into other minimum. the valence band holes will remain confined in this layers, however. this is called a type -ii sl. a structure in which both the electrons and the holes are in the same layer is called a type –i sl. sl have been used to demonstrate the basic effect of bloch oscillations [17] in which a d c electric field generates. it is assumed that the sound wave and the applied constant electric field e propagates along the z – axis of the sl. the problem will be solved in the quasi – classical case, i.e. 2∆ >>τ -1 (ћ = 1), eed << 2∆ ( d is the period of the sl, 2∆ is the width of the lowest energy miniband and e is the electron charge ). the current density associated with an acoustic wave may be obtained from the expression [18]. j = (2) where, u = (3) ‘ ’is the sound flux density, ωq and are the frequency and the group velocity of sound wave with the vector and is the solution of the boltzmann kinetic equation in the absence of a magnetic field. if we introduce a new term = p-q in the second term of the integrals in eq. (3) and take = . we can express eq. (3) in the form j = (4) where the vector as indicated in [19] is the mean free path li(p). thus the acoustoelectric current in eq. (4) in the direction of sl axis becomes jz= (5) where f( ) is the distribution function, p is the momentum of electrons, g(p2, ) is the matrix element of the electron-phonon interaction and for qd<<1 it is given as, ujjal alok et al. / bibechana 8 (2012) 6772: bmhss, p.69 (6) where c is the deformation potential constant. is the density of the sl in the τ approximation and further when τ is taken to be constant, lz = τ sz (7) sz = (8) inserting eqs. (6) and (7) into eq. (5) we obtain jz = (9) for superlattices the dispersion law is given by (10) where pt and pl are the transverse and longitudinal ( relative to the sl axis ) components of the quasi momentum respectively, ∆υ is the half width of the υth allowed miniband (11) are the sizequantized levels in an isolated conduction film, d = d0+d1 ( d0 is the width of the rectangular potential wells and d1 is the barrier width a non-zero quantum transparency) is the sl period. the distribution function in the presence of the applied constant field e is obtained by solving the boltzmann equation in the τ approximation, given by f(p) = (12) here, (13) where n is the electron density, t is the temperature in energy units and io(x) is the modified bessel function. we assume that electrons are confined to the lowest conduction miniband (υ=1) and omit the miniband indices. this is to say that the field does not induce transitions between the filled and empty minibands. the electron velocity is given by (14) we further assume that and write eq. (10) in the usual form as (15) substituting eqs. (12),(14) and (15) into eq. (4) and solving for a non-degenerate electron gas. ujjal alok et al. / bibechana 8 (2012) 6772: bmhss, p.70 jz= (16) where is the heaviside step function. b = x = sinh , and = we shall solve eq. (16) for two particular case, (i) in the absence of the applied constant field (e=0), from eq. (16) we obtain jz = (17) if , jz =0 i.e; there appears a transparency window. this is a consequence of the conservation law. (ii) in a weak constant electric field, eed , , from eq. (16) we obtain jz= (18) from eq. (18) it is observed that at e > e0 = ωq the acoustoelectric current changes sign. the value e0 can be interpreted as a threshold field. e0 is a function of the sl parameters d and , and temperature t, frequency and the wave number q. for example, at , , d= cm, , and . for these values we obtain the threshold field e0 = 8.65vcm -1 which is small and can be observed. 3. results and discussion the general solution of eq. (16) cannot be obtained analytically. we, therefore, obtained it numerically and the graph of jz on e have been plotted. it is noted that the acoustoelectric current has a peak at some values e. these peaks decrease with a corresponding decrease of . more interesting is the nature of the acoustoelectric current. it is observed that when the electric field is negative the current rises and reaches a maximum and then falls of in a manner similar to that ujjal alok et al. / bibechana 8 (2012) 6772: bmhss, p.71 observed during a negative differential conductivity. on the other hand, when the electric field is positive the current decreases and reaches a minimum then increases. this can be attributed to the bragg reflection at the band edge. it is further observed that the ratio of the height of the peak corresponding to absorption to that corresponding to amplification differ from one. this value also decreases with a decrease in . the threshold field e0 also increases with the decrease of . it is noteworthy to show that a similar relation was obtained for a transverse acoustoelectric voltage experiment on si/sio2 and this result agrees quite well with our result [15] fig : dependence of jz/jo on ------= 0.09 -------= 0.07 evv..... = 0.05 ev references [1] k. blotekjaer and c.f.quate, proc. ieee 52 (1964) 360 [2] r. y. thakur, b. k. singh. and anjula singh, semicond.sci. technol. uk 5 (1990) 405 [3] k.b.tolpygo and z.i.uritskii, zh. eksp. teor. fiz 30 (1956) 929 [4] g. weinreich, phys. rev. 104 (1956) 321 [5] a.r.hatson, j.h mcfee and d.l.white, phys. rev. lett. 7 (1961) 237 [6] r.h.parmenter, phys. rev. b 89 (1953) 990 [7] m. rotter, a.v.kalameitsev, a.o.govorov, w. ruile and a. wixforth, phys. rev. lett. 82 (1999) 2171 [8] j. m. shilton, d.r.mace, v.i. talyanskii, yu. galperin, m.y.simmons, m.pepper and d.a.ritchie, j.condens. matter 8 (1996) l 337 [9] v.l.gurevich and v.i. kozub, phys. rev. b 58 (1998) 13088 [10] v.v.afonin, yu. m. gal’perin, semiconductor 27 (1993) 61 [11] m.i.kaganov, sh. t. mevlyut and i.m. suslev, sov. phys jetp 51 (1980) 189 [12] a.d. margulis and v.a.margulis j.condens. matter 6 (1994)6139 [13] yu. v. gulyaev and e.m.epshlein sov. phys. solid state 9 (1967) 674 [14] v.a vyun, yu.o. kanter, s.m.kikkarin, v.v.pnev, a.a.fedorov, and yakovkin 1988, academy of science ussr 11 th all union conference on physics of semiconductors ( kishinev, 1988) vol 3 (kishinev academy) p118 ujjal alok et al. / bibechana 8 (2012) 6772: bmhss, p.72 [15] v.a vyun, yu.o.kanter, s.m. kikkarin, v.v.pnev, a. a.fedorov, and i.b.yakovkin, solid state communications 78 (1991) 823 [16] f. palma, j.appl. phys. 66 (1989) 292 [17] c. waschke, physical review letters 70, (1993) 3319 [18] m.i. kaganov. sh.t. mevlyut, and i.m.suslev, zh. eksp. teor. fiz 78 (1980) 376 [19] s.v. krychkov and n.p. mikheev, fiz. tekh. poluprov. 16 (1982) 169 403 forbidden forbidden you don't have permission to access this resource. apache/2.4.54 (ubuntu) server at www.nepjol.info port 443 403 forbidden forbidden you don't have permission to access this resource. apache/2.4.54 (ubuntu) server at www.nepjol.info port 443 403 forbidden forbidden you don't have permission to access this resource. apache/2.4.54 (ubuntu) server at www.nepjol.info port 443 403 forbidden forbidden you don't have permission to access this resource. apache/2.4.54 (ubuntu) server at www.nepjol.info port 443 403 forbidden forbidden you don't have permission to access this resource. apache/2.4.54 (ubuntu) server at www.nepjol.info port 443 403 forbidden forbidden you don't have permission to access this resource. apache/2.4.54 (ubuntu) server at www.nepjol.info port 443 403 forbidden forbidden you don't have permission to access this resource. apache/2.4.54 (ubuntu) server at www.nepjol.info port 443 403 forbidden forbidden you don't have permission to access this resource. apache/2.4.54 (ubuntu) server at www.nepjol.info port 443 microsoft word article bharat subba-corrected.doc bharat raj subba and satya narayan meheta / bibechana 8 (2012) 96-104 : bmhss, p.96 bibechana a multidisciplinary journal of science, technology and mathematics issn 2091-0762 (online) journal homepage: http://nepjol.info/index.php/bibechana ovarian histomorphology and gonadial cycle of freshwater garfish xenentodon cancila (hemiltonbuchanan) (beloniformes:belonidae) bharat raj subba and satya narayan meheta department of zoology,post graduate campus (tu), biratnagar, nepal email:subbabharat@yahoo.com article history: received 5 december, 2011; accepted 26 december, 2011 abstract the histomorphology and gonadial cycle of a freshwater garfish xenentoton cancila revealed that the ovaries of the fish pass through resting, early maturing advanced maturing ,pre-spawning, spawning and spent phases within one year.the oogenetic activity starts in november and continues upto june(gsi=10.19)-july(gsi=11.38),when ovaries are full of yolky eggs.only young oogonia and oocytes l appeared in september to october.the oocytes of l,ll,lll,lv and v stages were present during november to february.the oocytes vl and vll were seen dominated by ripe oocytes during june to late july when spawning takes place.asychronism mode of oocyte development was recorded in x.cancila. keywords: xenentodon cancila; oocyte development; reproductive cycle; spawning 1. introduction the studies on fish biology encompassing habitat ecology,ovarian morphology,spawning,physiology and reproductive habits have been indespensible for the fish culture development and safeguard of fish species.pisciculture has been an important adjunct to aquaculture on one hand and the conservation of fish has been a global attention because of climate change and population explosion on the other.conserving fish is only possible when the life cycle is fully understood. the study of spawning habit of a fish is carried out in the natural habitat but as fish are aquatic ,it is not possible to observe all the steps taking place during spawning and before that too.therefore, considering the above facts, the study of ovarian histomorphology of the freshwater garfish xenentodon cancila has been made to understand the reproductive potential of the species in the natural habitat. several workers have studied the gonads of fishes. among many, most important contributions on the histological and seasonal changes in the ovary of fishes were made[ 1-11]. shrestha and khanna[12] have worked on structure and histological changes in the ovary of the nepalese snow trout schizothorax. plagiostomus. . rita and nair [13] made studies on oogenesis in a tropical loach, lepidocephalus thermalis and hillstream loach noemachelilus triangular. toshimori, and fumioki [14] studied gap junctions between microvilli of an oocyte and follicle cell in teleosts, plecoglossus attivelis. chakrabarti and mandal [15] studied seasonal histological changes in the vary of puntius japanieus. in spite of large contributions noted above, the study in ovarian structure of freshwater garfish (kauwa) is meager indeed., taking this point in view, subba [16] has carried out an investigation on ovarian histomorphology and gonadial cycle of a hill-stream fish lepidocephalichthys guntea. bharat raj subba and satya narayan meheta / bibechana 8 (2012) 96-104 : bmhss, p.97 2. materials and methods live specimens of xenentodon cancila were collected every month from muriyadhar which runs parallel to east koshi dam in sunsari. the weight of the specimens and their ovaries were recorded in situ.the ovaries taken out were washed in distilled water properly so as to remove blood stains. fresh pieces from anterior, middle and posterior regions of the ovary were taken and fixed separately in bouin's fixative. serial sections were cut at 5 to 6µ and stained in iron haematoxylin using eosion as a counter stain. mallory triple stain was also used to study connective tissues. the gonadosomatic index (gsi) was calculated for every month as below. weight of the ovary gsi = × 100 total weigh of the fish 3. results 3.1 ovarian morphology the ovaries of xenentodon cancila are paired and elongated sac like structures which lie in the abdominal cavity ventral to the kidney. they are attached to the body wall by means of the mesovarium. the anterior portions of the ovary are free, broad but posterior ends are united into one to form oviduct (fig 1). the ovary is of typical teleostean type consisting of an ovarian wall and ovocoel. the ovary is covered with a thin peritoneal covering or serosa. beneath this is the elastic and muscular connective tissue, the connective tissue fibers bear the blood vessels, project into the ovarian lumen, ovocoel to form finger like folds known as the ovigerous lamellae, which encloses numerous ova in different stages of development and growth. the ovaries are of light cream colour during early stages of maturation but become yellow in the breeding season, ripe ova project out of the surface and gonads become semitransparent. as maturity advances the weight of the ovaries also increases gradually and reaches maximum in the mouths of jun-july showing highest gsi value was 11.3772. the weight became minimum in the months of oct-november with gsi 0.9416. after spawning the ovaries became, thin flaccid, and were considerably reduced in size. 3.2. histology the wall of the ovary is composed of an outer thin layer, peritoneal membrane, which is the outermost layer, overlying the tunica albuginea. the tunica albuginea is made of fibrous connective tissue and blood capillaries. the inner most layer is the germinal epithelium which projects into the ovocoel in the form of lamellae. these ovigerous lamellae are the seat for the development of oogonia, which are visible in various stages of development. oogonia of various stages are arranged on either side of these lamellae. these oogonia mature into oocytes. 4. developing oocytes an oogonium has a large nucleus and a thin layer of ooplasm which is chromophobic. each oogonium undergoes successive maturation divisions and form new generation of oocytes. before ripening an ovum, a series of cytonuclear changes take place in the oocyte. bharat raj subba and satya narayan meheta / bibechana 8 (2012) 96-104 : bmhss, p.98 stage oocyte (plate-i a,plate-2 g) :the earliest stage of germ cell in the ovary is the oogonium, which arises from the residual oogonia, occurring in the year. an oogonium has a large nucleus which is single thin layer of ooplasm which does not take basic stain. this stage is also known as chromatin nucleus stage. each oogonium passess through seven different stages called the oocytes, before it becomes a ripe ovum. the following stages of oocytes have been identified in the ovary of this fish. the mean diameter of an oogonium is 0.035 mm. stage i oocyte (plate i a,b plate-2 ): in this stage, the oogonia which acquire larger amount of cytoplasm are called oocytes. an oocyte can be distinguished from an oognium because of a prominent round central nucleus and two or three nucleoli. in this stage, oocyte is encircled by an incomplete layer of simple squamous cells. this is the beginning of the follicular layer. the cytoplasm is basophilic. the oocyte in this stage measured 0.1 mm in diameter. stage ii oocyte (plate i b,d plate-3): with an increased quantity of cytoplasm, increases strong affinity for basic dyes. in this stage many oocytes have a yolk nucleus situated close to the nuclear membrane in the cytoplasm. the yolk nucleolus ,first appears near the nuclear membrane but moves towards the periphery in the later stage. several nucleoli of various sizes are arranged along the periphery of nuclear membrane. this stage is conventionally known as perinucleolus stage. the oocyte measured 0.15 mm. stage iii oocyte (plate ic, plate-2 i) :further development of the oocytes is marked by the formation of a thin layer of follicular cells around the cytoplasm. owing to the differential affinity to the basic dyes, the zonation is exhibited in ooplasm. the oocytes of this stage show an intensely stained inner zone and lightly stained outer portion. a few nucleoli extrude out of the nuclear membrane into the cytoplasm, some of them can be seen emerging through nuclear membrane while others are found in cytoplasm. yolk nucleous is seen frequently at this stage. the oocyte, at this stage was of 0.24 mm in diameter. stage iv oocyte (plate 1 b,): this stage is characterized by the appearance of minute vaculoes in the ooplasm. the vacuoles appear as if they are empty because they don't take basic dyes. the ocyte is now known to have reached the yolk vesicles stage. as oocytes grow further, more yolk vesicles bharat raj subba and satya narayan meheta / bibechana 8 (2012) 96-104 : bmhss, p.99 appear at the periphery and proceed inwards towards the nucleous and fill the entire cytoplasm. at this stage a few of the nucleoli can be seen to pass out of the undulating nuclear membrane. in this stage a vitelline membrane or zona radiata is also recognizable between ooplasm and follicular layer. the oocyte in this stage measured 0.29 mm in diameter. stage v oocyte (plate 1 d) : after the cytoplasm has become full of yolk vesicles, yolk begins to appear in the form of small granules in the extra vesicular ooplasm. most of them are present near the periphery of the oocyte and accumulate in high number. the yolk granules then proceed centripetally to the entire cytoplasm and become impregnated with them. the smaller granules fuse each other in later stage to produce large yolk globules. the process of nuclear extrusion contiunes in some of the oocytes. the zona radiata and follicular layer become more thicker in this stage. the oocytes measured 0.33 mm in diameter at this stage. stage vi oocyte (plate 1 e) :this stage is characterized by the appearance of the yolk globules. the nucleus disappears. the yolk starts depositing in the form of large globules in the extravesicular ooplasm. the entire cytoplasm becomes impregnated with yolk globules or granules proceeded in words. some yolk vesicles of smaller size collect along the periphery of the oocyte and form individual so called cortical alveoli. outside the follicular layer, a thin layer of fibroblast develop. the oocytes measured 0.44 mm in diameter. stage vii oocyte (plate 1f) :at this stage nucleus becomes irregular in outline and smaller in size. the nucleus can be observed gradually migrating towards the periphery of the oocyte. so this stage is known as migratory nucleus stage. the nuclear membrane becomes indistinct and probably after that the contents of the nucleus mingle with cytoplams. the oocyte of this stage measured 0.48 mm in diameter. ripe oocyte: the ripe oocyte is recognized by its largest size, yellowish colour and translucent. large amount of yolk globules and yolk vesicles may lie scattered in it. it is covered over by three distinguishable layers namely theca an external layer, followed by the follicular epithelium (zona granulosa), and the innermost layer ,zona radiata. zona granulosa is made up of follicular epithelium. the ripe oocyte is full of yolk and nucleus is not visible. the ripe, oocyte measured 0.56 mm in diameter. this is the largest size oocyte and the ovary contains several ripe eggs. spawing takes place at this stage. a ripe egg is full of large amount of noncontiguous yolk and numerous yolk vesicles remain scattered in it. 4. reproduction cycle on the basis of histomorphological changes, the ovarian cycle of xenentodon cancila has been divided into the following phases. resting phase ( october to november ) :it extends from october to november. in this phase ovaries are thin translucent, pale and dirty brown in colour, with less vascular supply. histologically, the ovary shows ovigerous lamellae, packed with oogonia. the oogonia are budded off from the germinal epithelium and are arranged in nests. early maturing phase ( december to january ) plate 1 a,b : this phase extends from december to january. during this phase the ovaries become slightly thicker, opaque, yellowish in colour. vascularisation is feeble. the ovigerous lamellae are greatly swollen and laden with oocytes of different stages. tunica albuginea becomes slightly thinner than in previous months. there is an increase in the weight to the ovary. advanced maturing phase (february to march) plate 1 e,f : this phase extends from february to march. in this phase, colour of the ovaries changes into deep yellow. the blood capillaries become inconspicuous because of profuse vascular supply. number of immature oocytes decreases. the ova are tightly held and the ovary can not be striped by applying gentle pressure. the tunica albuginea becomes extremely thin. due to the presence of large number of fully mature ova. the ovocoel is greatly reduced. migratory nucleus reduced in size is seen towards the periphery. the yolk globules become enlarged because of their fusion with one another. there is further increase in the weight and volume of the ovaries. bharat raj subba and satya narayan meheta / bibechana 8 (2012) 96-104 : bmhss, p.100 pre-spawning phase (april to may) plate i e,f : this stage extends from april to may. during this phase the ovaries become deep yellow in colour. vascularisation is extensively developed. both transparent and opaque ova are present and the ovaries attain maximum weight. the fish abdomen seems bulging due to the presence of ripe ova inside. in this phase nuclear extrusion is seen. histological condition is the same as that of previous months. spawning phase (early june to late july) plate 1f,plate-3m : this phase begins in the early june and ends in the late july. during this period the ovaries are yellowish and turgid due to the presence of a large number of translucent eggs. ripe eggs are present in the oviduct to be discharged out side. the fish spawns number of times during this period. vascularisation of the ovary reaches its peak and the ovaries are said to be in running phase. in this phase ova ooze out in the oviduct with slight pressure in the abdomen. tunica albuginea becomes extremely thin and inter follicular space is greatly reduced. the ovigerous lamellae are inconspicuous. histological section of the ovaries in june and july shows a number of discharged follicles, such follicles can be detected up to the end of september. spent phase (august to september) :this phase starts in august and lasts up to september. the ovaries become thin, flaccid, delicate, slender and dull in colour. there is a decrease in the volume and weight of the ovary. vascularisation is reduced. in this phase, oogonia are seen budding from the germinal epithelium. the tunica albuginea again becomes thicker. histologically, the ovary shows some residual oocytes as well as discharged follicles. the nest of oogonia are seen among the ovigerous lamellae. 5. discussion xenentodon cancila (ham.) does not show sexual diamorphism, but during the breeding period, they exhibit notable sexual difference by the presence of relatively enlarged belly in female fishes. in this fish, the ovary is of cystovarian type, because lumen of the ovary is continuous with the oviduct. 5.1 origin of oogonia different authors have expressed different views regarding the origin of the new oogonia, yamamoto [18] believed that the new oogonia originate from the follicular epithelial cells of spent follicles but according to, [,19,20] the new crop of the oogonia are produced by the germinal epithelium.unlikely to the above suggestion some [7-9] suggest that in schizothorax richardsonii the new crop of oogonia arises from the residual oogonia. as observed by rita and nair [13] the new crop of oogonia originates from ovarian lamellae formed by germinal epithelium. shrestha and khanna [12] showed that the crop of oogonia arises from the residual oogonia. in this fish, cancila the crop of oogonia are derived from the residual oogonia. this finding supports the finding of belsare [4,8,12]. 5.2 yolk nucleus the investigators differ in their views regarding the origin and function of the yolk nucleus. according to yamamoto [21] the yolk nucleus is of nuclear in origin, while chopra [22] believed it to be cytoplasm in origin. wallace [17] suggested that the yolk nucleus has the capacity to form yolk in fishes, whereas wheeler [23] suggested that the yolk nucleus remains inactive and takes no visible part in the formation of yolk. khanna [24] has suggested that the yolk nucleus is the initial centre of growth and dispersal of all important cellular inclusions. bara [20] and nayar [25]. reported some relationship between the yolk nucleus, golgi bodies and mitochondrial elements according to rita and nair [13] the yolk nucleus is entirely wanting in any stage of oocyte maturation of lepidocephalus thermalis. in x. cancila the yolk nucleus is present in the periphery of young stage iii oocytes. as it gradually disappears in the maturing oocytes, it may have some relationship with the process of vitellogenesis.a similar view has been expessed by khanna, and sanwal [8] in channa gachua and bisht and joshi [4] in schizothorax richardsonii bharat raj subba and satya narayan meheta / bibechana 8 (2012) 96-104 : bmhss, p.101 bharat raj subba and satya narayan meheta / bibechana 8 (2012) 96-104 : bmhss, p.102 plate -1 a. photomicrograph of t.s. ovary of xenentodon cancila showing oogonium,ooc l, x 40 b. general observation of t.s. ovary of x.cancila showing stage 11 and iv,ooc l x 100. c. general observation of t.s. ovary of x.cancila showing stage iii and yolk nucleus x 200 d. t.s. ovary of x.cancila showing appearance of yolk vesicles in the periphery in stage v&lll oocytes x 200. e. t.s. ovary of xenentodon cancila showing stage vl x 200 f. t.s. ovary of xenentodon cancila showing stage vll x 200 (ge = germinal epithelium, 10 = immature oocyte, mn= migratory nucleus, ns = nest, nu = nucleus, nm = nuclear membrane, 0 = oogonium, ooc = oocyte, vm= vitelline membrane, yv = yolk vesicle, yg = yolk granule) plate -2 g. t.s. mature oocyte of xenentodon cancila. showing stage lv x 50. h. showing fusion of yolk globules x 100 i. stage vii showing migratory nucleus x 100. j. plate of a ripe ovum x 200. k. showing both mature and immature oocytes. l t.s. ovary of x.cancila showing immature oocytes. ovocoel. (fl = follicular layer, 10 = immature oocyte. mn = migratory nucleus, mo = mature oocyte, nu = nucleus,. th = thica, vm = vitelline membrane, yg = yolk globules, yv = yolk vesicles; zr = zona radiata.) plate-3 m. t.s. ovary of xenentodon cancila showing stage vll x 200 yg=yolk globules,mn=migrating nucleus,yv=yolk vesicles , n. ts showing of ovary of x.cancila atretic egg o. ts of ovay of x.cancila showing p= peritoneum,bc-blood capillary,ge=germinal epithelium,io=immature oocyte, ac = atretic egg 5.3 nuclear extrusion there are several views regarding the origin, extrusion and functions of the nucleoli in teleosts. rai [6] believed that large number of nucleoli are produced by division and fragmentation of a single nucleus of an oogonium, contrary to the above view, yamamoto et.al. [8,13,18,20,] did not observe any fragmentation in the fishes examined by them. according to their views, the fusion of minute fulgen positive particles present in peripheral nucleoplasm form peripheral nucleoli. according to bisht [5,9 23] in schizothorax richardsonii the nucleoli are produced by the division and fragmentation of the nucleolus. bharat raj subba and satya narayan meheta / bibechana 8 (2012) 96-104 : bmhss, p.103 in x.cancila many nucleoli of different sizes were observed in the early stages of oocyte, increasing in number by division, but the number diminishes as oocyte advances to maturity. this result corroborates closely with the finding of khanna et. al.[5,8,23, ]. extraction of nucleoli has been studied by several investigators in the past, but there is a great controversy in the origin of extrusion of nucleoli. according to dixit [3] the nuclear membrane does not allow the nucleoli to pass through it. rai et. al [6 ,8] have also observed extrusion of nucleoli into the ooplasm. extrusion of nucleoli is seen in x. cancila. several nucleoli of various sizes are seen in the early stage of oocyte. as oocytes reach maturity the nucleoli are seen increasing in number and their size goes on diminishing. this result agrees with the finding of khanna [5]. as the extrusion of nucleoli was seen more in number during the formation of yolk. it appeared to be associated with the formation of yolk. 5.4 yolk formation according to the mode of yolk formation the fish eggs have been classified into two types by yamamoto [26] uamamoto namely (i) eggs having a continuous mass of yolk, as in oryzia [18] and (ii) eggs having a noncontinuous mass of yolk, as in clupea [27]. in x.cancila yolk appears at the periphery of oocytes, in the form of the globular, later the whole ooplasm is filled with yolk globules. this process is similar to clupea [21] tot tor [6] channa gachua [8], puntius sophore [28] similarly results were obtained in x.cancila, where the yolk vesicles and yolk globules are independent of each other and the former give rise to so called cortical alveoli. and the yolk vesicles as such seen to play no role in the formation of yolk globules. nayar et. al [21,25] have described breaking down the vesicles of the inner side to take place in the formation of yolk globules in cyprinus and gobio which contradicts the above findings. 5.5 spawning there are three modes of oocytes development, according to prabhu [29] in some fishes eg. oncorhynchus masou [20] all the oocytes develop together at the same time in the ovary, such fishes spawn only once in a life time. this phenomenon is called total synchromism, there are some species which spawn once or twice in a season, developing two group of oocytes side by side (group synchronism), as in speckled trout, viadykov [30] still other species spawn several times in a breeding season. in these species large number of oocytes are present at various stages of their development in the same ovary asynchronism) as in channa gachua [8] and puntious sophore [28]. the spawning season of x.cancila extends from early june to late july. since various stages of developing oocytes are found in the same ovary and the fish spawns several times during a breeding season. x. cancila comes under the third group asynchronism. references [1] a .l. craig-bennett,linn. philos. trans. b 219 (1930)197. [2] c.f. hickling, z.j. mar. biol, ass. y.k. 20 (1936)443. [3] r.k. dixit, proc. nat. acad, sci, india b. 30 (1956) 214. [4] d.k.belsare, indian j.fish. 9 (1962)140. [5] s.s. khanna, and pant, m.c. copeil (1967) 83. [6] b.p.rai,acta.zool. 48 ( 1967)289. [7] g.k. lehri, acta anat. 69 (1968) 105. [8] s.s. khanna, and r. sanwal, zool. geitr, floge, 17 (1971)311. [9] j.s.bisht and m.l.joshi, acta, anat. 93 (1975)512. [10] c.p kapoor,acta. anat, 96 (1976)1-8. [11] c.p. kapoor, zekrosk. anat, forach. (leipz) 90 (1978)1049. [12] t.k. shrestha and s.s. khanna, matsya, 5 (1979)23. [13] s.d. kumari rita and n.b. nair, acad. sci. sect.b. 88 (1979)45. bharat raj subba and satya narayan meheta / bibechana 8 (2012) 96-104 : bmhss, p.104 [14] k. toshimori, and y. fumioki, forsch (lepiz) 93 (1979) 458. [15] p.chakrabarti and d.k. mandal,journal of freshwater bil.7 (1995)195. [16] b.r subba,j. freshwater biol.10 (1998)103. [17] w. wallace, quart. j. micr. sci. 47 (1904)161. [18] k.yamamoto,ann.zool.jap.28 (1955)233. [19] v.sriramalu and m. rajalaxmi, z. mikrosk. anat, forsch., 75 (1966)65. [20] g.bara, rev.fac,sci, univ. 25 (1960)50. [21] k.yamamoto, h.kal and r.ished,20 (1959)109. [22] h.c. chopra, experimentia. 17 (1961)120. [23] j.f.c. wheeler, quart. j. micr. sci. 68 (1925)641. [24] h.s. chaudhary, z.zellforosch. 37 (1952)455. [25] r.p.nayar, quart. j. micr, sci. 105 (1964)353. [26] k.yamamoto, j.fac.sci.hokkaido univ.13 (1957)346. [27] k.yamamoto,bull.fac.fish.hokkaido,univ.8 (1958)270. [28] r.k.dixit and n.agrawal,acta.anat.90 (1974)133. [29] m.s.prabhu, indian j.fish 3 (1956)59. [30] v.d.viadykov, j. fish. res. canada 13 (1956)799. microsoft word n.a. rathar_28-32_.doc n. a. rather and faroz ahmad / bibechana 9 (2013) 28-32 : bmhss, p.28 (online publication: nov., 2012) bibechana a multidisciplinary journal of science, technology and mathematics issn 2091-0762 (online) journal homepage: http://nepjol.info/index.php/bibechana on the critical points of a polynomial n a rather, faroz ahmad * department of mathematics, university of kashmir * corresponding author: email: farozmaths1080@gmail.com article history: received 25 march, 2012; accepted 13 august, 2012 abstract let denote the set of all polynomials of the form with and , . in this paper, we show that in for all polynomials . for , this reduces to a result due to aziz and zargar. keywords: polynomial, critical point; sendove conjecture; walish coincidence theorem. 1. introduction the gauss-lucas theorem states that if s is the set of zeros of a polynomial , then every zero of the derivative is contained in the smallest convex set that contains s. this is best possible, in the sense that, if has all its zeros in , then no proper subset of can be guaranteed to contain even one zero of , (as is shown by the polynomial of the form , since has zeros only at , which can lie anywhere in ). gauss-lucas theorem has been rather thoroughly investigated [8] and sharped in several ways. however, there is one related question that deserves attention, namely given one specific zero of , what can be said about a neighborhood of that will always contain a zero of . n. a. rather and faroz ahmad / bibechana 9 (2013) 28-32: bmhss, p.29 (online publication: nov., 2012) the following conjecture was made by bulgarian mathematician b l sendov in 1962 but became later known as ilief’s conjecture (see [4, problem 4.5] or [6, p.795]) . conjecture 1. let be a polynomial of degree having all its zeros in the unit disk . if is any one of these zeros, then has at least one zero in the disk . since in 1962, when it is first became known, conjecture 1 has been the subject of more than thirty articles. however, it was fully verified only for polynomials of degree (see [13]). a variety of special cases have been dealt with over the years (see [2, 7, 11] for references), among which we mention that of a polynomial with at most five distinct zeros [5], as well as miller’s qualitative result [10], according to which those zeros of lying sufficiently close to the unit circle satisfy an even stronger condition that the one stated in sendov’s conjecture (see also [12]). another stronger conjecture than that of ilief was made in 1969 by goodman, rahman and ratti [3]. conjecture 2. let be a polynomial of degree having all its zeros in the unit disk . if is any one of these zeros, then has at least one zero in the disk conjecture 2 has been proved when [3], but some counter examples have been devised for case by m. j. miller [10]. recently aziz and zargar [2] have proved the following result . theorem a. if is a polynomial of degree with , , then does not vanish in . in this paper we establish a generalized form of above theorem. in fact we prove the following interesting result which extracts that portion of complex plane in which the above polynomial does not vanish. theorem 1. let be a polynomial of degree with and , , then does not vanish in the disk n. a. rather and faroz ahmad / bibechana 9 (2013) 28-32: bmhss, p.30 (online publication: nov., 2012) . the result is best possible as shown by the polynomial , further taking we get theorem a. by using a similar argument, we can prove the following more general result. theorem 2. let be a polynomial of degree with and , where then has fold zero at and remaining zeros of lie in the region . the result is best possible as shown by the polynomial , . for the proof of this theorem we need the following lemma which is the coincidence theorem of walish [8, p.62] (see also [1]). lemma. let be a symmetric -linear form of total degree in and let c be a circular region containing the points , then there exists at least one point belonging to c such that . proof of theorem 2. by hypothesis, where and let , then is a polynomial of degree , having all its zeros in and we have . this implies . n. a. rather and faroz ahmad / bibechana 9 (2013) 28-32: bmhss, p.31 (online publication: nov., 2012) if now is any zero of , then from , we get (2) this is an equation which is linear and symmetric in the zeros of that is, in . hence an application of the above lemma with circular region shows that will also satisfy the equation obtained by substituting into the equation (2) , where is suitably chosen point in the circular region . that is satisfies the equation or equivalently . thus has the values or , where is suitably chosen point in . if , then using the fact that , it follows that if then clearly thus in any case since is an arbitrary zero of , it follows that every zero of lie in the disk this completes the proof of theorem 2. corollary . if we take in theorem 2, we get theorem 1. n. a. rather and faroz ahmad / bibechana 9 (2013) 28-32: bmhss, p.32 (online publication: nov., 2012) references [1] a. aziz, pacific j. math., 118 (1985) 17-26. [2] aziz and zargar, aus. math. soc., 57 (1998) 173-174. [3] a. w. goodman, q. i. rahman and j. s. ratti, proc. amer. math . soc., 21 (1969) 273-274. [4] w. k. hayman, research problems in function theory, athlone press london (1967). [5] s. kumar and b. g. shenoy, j. math. anal. appl., 171 (1992) 595-600. [6] m. marden, much ado about nothing, amer. math. monthly, 83 (1976)788-789. [7] m. marden, amer. math. monthly, 90 (1983) 267-276. [8] m. marden, geometry of polynomials, iind edition math. surveys, 3. amer .math. soc., providence, rhode island (1966). [9] a. meir and a. sharma, pacific j. math., 31 (1969) 459-467. [10] m. j. miller, j. math. anal. appl., 175 (1993) 632-639. [11] g. schmeisser, math. z., 156 (1977) 165-173. [12] v.vajaitu and z. zaharesen, london. math. soc., 25 (1993) 49-54. [13] jonny e. brown and guangping xiang, proof of the sendov conjecture for polynomials of degree at most eight (1965) [14] z. rubinstien, pacific j. math., 26 (1968) 159-161. microsoft word a bhattarai _63-68_.doc a. bhattarai et al. / bibechana 9 (2013) 63-68: bmhss, p. 63 (online publication: nov., 2012) bibechana a multidisciplinary journal of science, technology and mathematics issn 2091-0762 (online) journal homepage: http://nepjol.info/index.php/bibechana effect of solvent composition on the critical micelle concentration of sodium deoxycholate in ethanol-water mixed solvent media ajaya bhattarai*, sujit kumar shah, ashok kumar yadav, janak adhikari department of chemistry, mahendra morang adarsh multiple campus, tribhuvan university, biratnagar, nepal * corresponding author: e mail: bkajaya@yahoo.com article history: received 18 october, 2012; accepted 3 november, 2012 abstract the precise measurement of the specific conductivity of sodium deoxycholate in pure water and ethanolwater mixed solvent media containing 0.10 and 0.20 volume fraction of ethanol at 303.15 k are reported. the concentration were varied from ~ 0.01 mol l -1 to ~ 0.0002 mol l -1 .the conductivity of sodium deoxycholate decreases with the increase in the volume fraction of ethanol. the critical micelle concentration of sodium deoxycholate increases with the increase in the volume fraction of ethanol. keywords: sodium deoxycholate; conductivity; critical micelle concentration; ethanol-water mixed solvent media 1. introduction surfactants are organic substances, which significantly decrease the surface tension of water at relatively low concentrations, are at least partially water soluble. surfactants are amphiphilic materials containing both a polar long-chain hydrocarbon “tail” and polar, usually ionic, “head” groups. in polar solvents, for example water, this dual character of the amphiphile leads to self-association or micellization, the surfactant molecules arrange themselves into organized molecular assemblies known as micelles. the hydro-phobic part of the aggregate forms the core of the micelle, while the polar head groups are located at the micelle–water interface in contact with and hydrated by a number of water molecules. depending on the chemical structure of the surfactant, its micelle can be cationic, anionic, ampholitic (zwitterionic), or nonionic. when dissolving them, after they reach a certain value of concentration, molecules or ions of surfactants begin to associate and to organize themselves into micelles. in colloidal and surface chemistry, the critical micelle concentration (cmc) is defined as the concentration of surfactants above which micelles form and almost all additional surfactants added to the system go to micelles [1]. critical micelle concentration values are important indicators when considering which surfactant will provide optimal performance benefits. the determination of cmc is generally based on the localization of the position of a breaking point in the concentration dependencies a. bhattarai et al. / bibechana 9 (2013) 63-68: bmhss, p. 64 (online publication: nov., 2012) of selected physical or chemical properties of surfactant solutions. because of the surface activity of these substances, measurements of the surface tension of surfactant solutions represent the principal method of cmc determination. however, it is rather tedious and time-consuming procedure. in the case of ionic surfactants, the utilization of electrochemical measurement is much more convenient, especially the measurements of the electrical conductivity of their solutions with varying concentration. the conductometric method is based on the finding of a breaking point on the curves, which describe the concentration dependence of conductivity [2]. it is well-known, that the conductivity of any solution is directly proportional to the concentration of its ions. the point, where the micelle formation starts, is indicated on the concentration dependence of specific conductivity (κ) as a breaking point. it is easy to find the breaking point, because it marks a significant change of the linear slope of the dependence κ =f(c). the requested value of cmc is the intercept of two linear functions with mutually different slopes. sodium deoxycholate (fig.1), the sodium salt of deoxycholic acid, is a water-soluble, bile-acid, ionic detergent commonly used in protein methods. it is most frequently used as a component of cell lysis buffers (e.g., ripa buffer), but also has been used for liposome preparation, isolation of membrane proteins and lipids, preventing nonspecific binding in affinity chromatography and a cell culture media supplement. deoxycholic acid has been used since its discovery in various fields of human medicine. in the human body deoxycholic acid is used in the emulsification of fats for the absorption in the intestine. it has, in some countries (including switzerland) been licensed as an emulsifier in food industry [3]. fig.1: structure of sodium deoxycholate. our aim of this paper is to determine the cmc of sdc in pure water and ethanol-water mixed solvent media containing 0.10 and 0.20 volume fraction of ethanol at 303.15 k by conductometric method. 2. experimental methods ethanol (e. merck, india) was used for the experimental works. the purified solvent had a density of 0.78097 g.cm -3 and a co-efficient of viscosity of 0.9490 mpa.s at 303.15 k; these values are in good agreement with the literature values [4]. distilled water with a specific conductance less than 10 -6 s.cm -1 at 303.15 k was used for the preparation of the mixed solvents. the physical properties of ethanol-water mixed solvents used in this study at 303.15k were taken from the published works [5-8]. a. bhattarai et al. / bibechana 9 (2013) 63-68: bmhss, p. 65 (online publication: nov., 2012) conductance measurements were carried out on a pye-unicam pw 9509 conductivity meter at a frequency of 2000 hz using a dip-type cell with a cell constant of 1.15 cm -1 and having an uncertainty of 0.01%. the cell was calibrated by the method of lind and co-workers in 1959[9], using aqueous potassium chloride solution. the measurements were made at 303.15 k in a jacket containing conductivity cell of cell constant 1.002 cm –1 . water was circulated in the jacket from thermostat and the temperature was maintained within ± 0.1 o c. the details of the experimental procedure have been described earlier [10-11]. several independent solutions were prepared and runs were performed to ensure the reproducibility of the results. due correction was made for the specific conductance of the solvent by subtracting the specific conductance of the relevant solvent medium from those of the electrolyte solutions. in order to avoid moisture pickup, all solutions were prepared in a dehumidified room with utmost care. in all cases, the experiments were performed in three replicates. 3. result and discussions conductivity measurements were performed in pure water and ethanol -water mixed solvent media at 303.15 k in order to evaluate the cmc of sodium deoxycholate. the cmc of sodium deoxycholate found at different volume fraction of ethanol –water mixed solvent media are listed in table 1.the cmc of sodium deoxycholate in pure water was found to be 2.04 mm by fluorescence measurements at 298.15 k[12] which is almost matching with static light scattering measurements[13]. as the temperature increases of the solution of sodium deoxycholate in water,the cmc also increases[13-14] and this is the best evidence for our conductivity measurements of sodium deoxycholate in pure water. the experimental specific conductivities of sodium deoxycholate as a function of the its molar concentration at 303.15 k of pure water and two different ethanol-water mixtures (containing 0.10 and 0.20 volume fractions of ethanol) are depicted in fig. 2 to 4. it was observed that cmc of sodium deoxycholate increases with the increase in the volume fraction of ethanol. this trend is due to decrease in the relative permittivity of the medium. the relative permittivity of the medium decreased at a given temperature with increasing in the ethanol content and similar trends have been observed in the previous works [6-7]. the presence of ethanol reduces the dielectric constant of the solvent phase and makes easier for the formation of ion-pairs in the solution phase. it is also well known that addition of solvent which acts as water structure breakers decrease the hydrophobic effect result in to the increase in the cmc value of ionic surfactants [15]. breaking of water molecules by ethanol molecules would facilitate interation between the hydrophobic tail of the surfactant molecule and the hydrophobic part of the ethanol and consequently the local concentration of ethanol molecules around the surfactants monomers becomes larger than the bulk solvation of the surfactant molecules by the hydrophobic part of the organic solvent would therefore lead to the delay aggregation of the surfactant monomers to form micelle and hence the increase in the cmc of the surfactant [16]. table 1: the critical micellar concentratiom (cmc) obtained from conductometry of sdc in pure water and ethanol-water mixed solvent media containing 0.10, 0.20 volume fraction of ethanol at 303.15 k t (k) water cmc (mm) 0.10 volume fraction of ethanol cmc (mm) 0.20 volume fraction of ethanol cmc (mm) 303.15 2.91 3.74 4.15 a. bhattarai et al. / bibechana 9 (2013) 63-68 : bmhss, p. 66 (online publication: nov., 2012) 0 0.15 0.30 0.45 0.60 0.75 0 0.002 0.004 0.006 0.008 0.010 cmc= 2.91 mm ↑ [sdc] (mol l -1 ) κ ( s c m -1 ) fig. 2: specific conductivities of sdc as a function of the surfactant concentration in 303.15 k: opened circles represent pure water. 0 0.1 0.2 0.3 0.4 0.5 0 0.002 0.004 0.006 0.008 0.010 ↑ cmc= 3.74mm [sdc] (mol l -1 ) κ ( s c m -1 ) fig. 3: specific conductivities of sdc as a function of the surfactant concentration in 303.15 k: opened circles represent 0.10 volume fractions of ethanol in the solvent mixture. a. bhattarai et al. / bibechana 9 (2013) 63-68: bmhss, p. 67 (online publication: nov., 2012) 0 0.1 0.2 0.3 0.4 0.5 0 0.0025 0.0050 0.0075 0.0100 ↑ cmc= 4.15mm [sdc] (mol l -1 ) κ ( s c m -1 ) fig. 4: specific conductivities of sdc as a function of the surfactant concentration in 303.15 k: opened circles represent 0.20 volume fractions of ethanol in the solvent mixture. 4. conclusions the following conclusions have been drawn from the above results and discussion. experimental result for the conductivity of sdc solution in pure water and ethanolwater mixed solvent media has been presented as a function of salt concentration and different percentage composition of ethanol-water mixed solvent media. the cmc of sdc are found to increase with the increase in the volume fraction of ethanol in ethanol-water mixed solvent media. acknowledgements the authors are thankful to professor dr. sujeet kumar chatterjee at the department of chemistry, mahendra morang adarsh multiple campus, tribhuvan university, biratnagar, nepal for the valuable suggestions and discussions. the authors are also thankful to associate professor ghanashyam shrivastav, the head of department of chemistry, mahendra morang adarsh multiple campus,tribhuvan university, biratnagar, nepal for providing us the research facilities to conduct this research work. references [1] a. d. mcnaught and a. wilkinson, compendium of chemical terminology, 2nd ed. blackwell scientific publications, oxford, iupac (1997). [2] a. bhattarai, s.k. shah and a. k. yadav, nepal j. sci. and tech.,13 (2012) 89. [3] h. streuli, et. al, slmb schweizer lebensmittelbuch, chapter 58, 4/3, (1992). [4] n. a. lange and j. a. dean, lange's handbook of chemistry, 10th ed. mcgraw-hill, new york, (1967). [5] j .j. lee and m. d. lee, korean j. chem. eng., 5 (1988) 5. a. bhattarai et al. / bibechana 9 (2013) 63-68: bmhss, p. 68 (online publication: nov., 2012) [6] a. bhattarai and s. k. sah, res. j. chem. sci., 1 (2011) 1. [7] a. bhattarai and j. raut, j. alpine chemistry, 2 (2011) 44. [8] h. s. harned and b. b. owen, the physical chemistry of electrolytic solutions, reinhold, new york, (1957) 234. [9] j. e., jr.lind, j. j. zwolenik and r.m. fuoss, j. am. chem. soc., 81 (1959) 1557. [10] b. das and d. k. hazra, bull. chem. soc. jpn., 65 (1992) 3470. [11] b. das and d. k. hazra, j. phys. chem., 99 (1995) 269. [12] s. das, u. thapa and k. ismail, bull. chem. soc. jpn., 83 (2010) 1352. [13] k. matsuoka and y. moroi, biochimica et biophysica acta, 1580 (2002) 189. [14] l. r. bogdanovaa, o. i. gnezdilovb, b. z. idiyatullina, r. kh. kurbanova, yu. f. zueva, and o. g. usyarovc, colloid j., 74 (2012) 1. [15] k. gracie, d. turner and r. palepu, can. j. chem., 74 (1996) 1616. [16] s.k.shah, t.p. niraula, a.bhattarai and s.k. chatterjee, bibechana, 8 (2012) 37. microsoft word arun kumar khan _88-91_.doc a. k. khan / bibechana 9 (2013) 88-91 : bmhss, p.88 (online publication: nov., 2012) bibechana a multidisciplinary journal of science, technology and mathematics issn 2091-0762 (online) journal homepage: http://nepjol.info/index.php/bibechana compressibility and moelwyn-hughes parameter of nai crystals under high pressure, as the function of thermal properties arun kumar khan department of physics, r.m college, saharsa, india e-mail : arunkumarkhan@gmail.com article history: received 8 october, 2012; accepted 7 november, 2012 abstract the credit for the commencement of inter ionic potential function goes to born and lande, one of whose milestone has been their expression into inverse function. similarly, born and mayer also proposed the exponential function of the potential for the repulsive component. however none of them could get the desired success. subsequently, many more prominent scientists devoted their effort towards these thermal properties of the crystals, among which the endeavour of jha and thakur cannot be overlooked, who proposed a new interionic function to observe the properties of ionic crystal under high pressure. during its formulation they included both an inverse power function due to born and lande and an exponential function due to to born and mayer. appreciably, jha and khan used it for studying the hyped thermal properties of nai and csi crystal under high pressure up to 100 kilo bar. keywords: moelwyn-hughes parameter; isothermal compressibility; nai crystals; debye temperature 1. introduction solid state physics went through a roller coaster ride in the begining of the last century. riding on the same momentum, born model of ionic solids [1] provided a substantial method to calculate the elastic and thermal properties of ionic crystal under high pressure, whose calculations are based on an inter ionic potential function. repulsive term being an important component of the potential function, has been described by born and lande by an inverse power function, whereas, the same has been described as an exponential function by born and mayer. in contrary, roberts and smith [2] found none of these functions to be successful, if the compression of sodium halide crystals are concerned. 2. formalism the new inter ionic potential function as proposed by jha and thakur [3] has included both an inverse power function due to born and lande and an exponential function due to born and mayer as it repulsive component. after which, jha [4] used it for studying the thermal properties of nai and csi crystal under a. k. khan / bibechana 9 (2013) 88-91 : bmhss, p. 89 (online publication: nov., 2012) high pressure. to our happiness, we disclose the thunderous success of this potential to describe the elastic properties of nai crystals under high pressure up to 100 kbar. using reduced units of equilibrium inter ionic distance r0 for distance and e2/r0 for energy, the new interionic potential for the crystal under study can be written as [3] where r is the inter ionic distance, a , b , m & n are the potential parameters which are constants for a given cnrystal , m is the madelung constant , c & d are van der walls costants for dipole-dipole and dipole-quadruple interactions , respectively and e is the electronic charge.crystal properties have also been calculated using born-lande’ and born mayer potential for the sake of comparison. these can be written as: 3. results and discussion jha and thakur [3] have described a procedure, using which the parameters of the new potential have been calculated. m obtains only integral values, whereas n has been varied in steps of 0.1. the data for input crystal has been taken from tosi [1] and ashcroft and mermin [5]. potential parameters of the born – mayer potentials have been calculated in the usual manner. values of potential parameters for the crystal under study have been listed in table 1. table 1: potential parameters of nai crystals in reduced (dimensionless) units parameter value parameter value c 0.0595 born lande’ potential d 0.0075 8.3678 new potential 0.25868 m 4 born mayer potential n 2.9 1/ee 9.3678 a 0.213852 2704.6774 b 2.110957 at a given compression, the pressure has been calculated with the help of debye’s equation of state [6], p = -( ðeo/ ðv) + γ ed/v (4) here eo is the internal energy of the volume, p pressure at 0 kelvin, ed the debye energy of the crystalat the temperature at which the crystal properties are being studied, and γ the gruneisen parameter. here we a. k. khan / bibechana 9 (2013) 88-91 : bmhss, p.90 (online publication: nov., 2012) have taken in to account the variation of ed and γ with pressure, but their temperature has been ignored. ed has been calculated by calculating the debye temperature. jha and thakur[3] explained the calculation fig. 1. variation of isothermal compressibility with pressure of gruneisen parameter and debye temperature. the isothermal compressibility has been calculated with the relation kt = 1/v (ðv/ðp)t (5) the moelwyn-hughes parameters[7,8] is the pressure derivative of the isothermal bulk modelus bt. it is defined by c1 = (ðbt/ðp)t (6) the computed results have been displayed in fig. 1 and 2. fig. 1 shows the variation of kt with pressure at room temperature (298 k) . fig. 2 shows the variation of c1 with pressure at room temperature. curves marked l have been drawn using the born-lande’ potential, curves marked m using the born-mayer potential and curves marked n using the new potential. the open circles are estimates of kt and c1 obtained from the compression data of bridgman [9] using a modified murnaghan equation [10, 11]. this equation helps us to express bt as a parabolic function of p, bt= b0+b1p+1/2b2p 2 (7) it follows from the definition of the moelwyn-hughes parameter that c1=b1+b2p (8) values of b0, b1 & b2 have been mentioned in figure 1. a. k. khan / bibechana 9 (2013) 88-91 : bmhss, p.91 (online publication: nov., 2012) fig. 2. variation of moelwyn-hughes parameter with pressure 4. conclusion however, the observation cannot be overlooked that the inverse power law of born lande’ is not apposite for predicting the elastic properties of nai crystal under high pressure, whereas the exponential laws is better for studying the elastic properties of crystal under high pressure. the new potential is most successful in explaining mechanical and thermal properties of nai crystal under high pressure i.e, from 100 kilo bar study. references [1] m.p. tos, solid state physics, ed. f. seitz and d. turnbull, academic press, 16 (1964) 1. [2] r. w. roberts and c. s. smith, j. phys. chem. solids, 31(1970) 619. [3] b. n. jha and k. p. thakur, physics, 173b (1991) 408. [4] b. n. jha, physica, 192b (1993) 253. [5] n. w. ashcroft and n. d. mermin, solid state physics, holt, rinehart and winston, new york (1976) 459. [6] c. kittel, introduction to solid state physics, john wiley and sons, inc., new york (1996) 182. [7] k. p. thakur, acta cryst., a32 (1976) 363. [8] k. p. thakur, physics of the earth and planetary interiors, 27 (1982) 235. [9] p. w. bridgman, proc. amer. acad. arts sci., 76 (1945) 1. [10] f. d. murnaghan, proc. nat. acad. sci. usa, 30 (1944) 244. [11] f. d. murnaghan, finite deformation of an elastic solid, john wiley and sons, inc., new york (1951). further readings [12] zhang hong tangjin and cheng xin-lu, chinese physics letters (2008). [13] ayu.. kuznetsov, l. dubrovimin sky, a kurnosov, m. m. lucchese, w. crichton and c.a. achete, advances in physical chemistry, (2009)article id 180784. [14] alapana tiwari, n. k. gaur and p singh, journal of physics (2010). [15] a.k. khan, bibechana, 7 (2011) 49. bibechana vol. 20, no. 1, april 2023, 76-85 issn 2091-0762 (print), 2382-5340 (online) journal homepage: http://nepjol.info/index.php/bibechana publisher:dept. of phys., mahendra morang a. m. campus (tribhuvan university)biratnagar biosorption of hexavalent chromium (cr(vi)) from contaminated water using charred tea waste puspa lal homagai, miksha bardewa, anup subedee hari bhakta oli, ram lal shrestha, deval prasad bhattarai∗ department of chemistry, amrit campus, tribhuvan university, kathmandu, nepal ∗corresponding author. email: deval.bhattarai@ac.tu.edu.np abstract the goal of this study is to develop charred tea waste (ctw) via chemical process for the removal of cr(vi) from contaminated water. batch adsorption experiments were conducted as a function of ph, initial concentration, contact time and adsorbent dosage. characterization of the adsorbent was analyzed by ft-ir and xrd. maximum adsorption capacity (qm) of the ctw was found to be 85.32 mg/g at optimum ph 2 in 120 minutes. the adsorption on ctw was well fitted to the langmuir isotherm and the kinetic data is consistent with the pseudo-second order kinetic model. the findings suggest that ctw could be an efficient and promising adsorbent for the removing cr(vi) from aqueous solution. keywords hexavalent chromium, charred tea waste, langmuir isotherm, pseudo-second order. article information manuscript received: march 14„ 2023; accepted: march 31, 2023 doi https://doi.org/10.3126/bibechana.v20i1.53458 this work is licensed under the creative commons cc by-nc license. https://creativecommons. org/licenses/by-nc/4.0/ 1 introduction access to safe drinking water is internationally recognized human right. presence of organic pollutants, inorganic pollutants and microbes are causing water pollution. presence of metals or metal ions such as cadmium, mercury, lead, arsenic, chromium, nickel, etc. in environment is causing heavy metal ions pollution which brings about an adverse effect on human health as well as aquatic flora and fauna [1]. discharge of heavy metal ions such as chromium rich effluent from tanning, leather, metallurgy and electroplating industries into water body is being a serious issue these days. its prevalence is increasing due to industrial production of dyes, leather, textiles, electroplating materials, and chemical manufacturing and some similar channels [2]. different oxidation states of chromium are present, and they all affect people differently. cr(vi) is toxic and highly carcinogenic, despite the fact that trace amounts of chromium (iii) have been reported to be necessary for proper lipid, insulin, and glucose metabolism [3]. world health organization (who) has assigned its upper permissible level in drinking water as 50 ppb [4]. chromium gets exposed to the environment via 76 http://nepjol.info/index.php/bibechana deval.bhattarai@ac.tu.edu.np https://doi.org/10.3126/bibechana.v20i1.53458 https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ puspa lal homagai et al./ bibechana 20 (2023) 76-85 77 natural as well as anthropogenic routes [5]. many chromium-based materials are based on hexavalent chromium which exhibits its strong oxidizing power. hexavalent chromium either directly enters into the workers through dermal contact or enter into the human via water resource. industrial wastewater and human activities are polluting the environment more than a bearable level. studies have been undertaken using various types of adsorbents for its efficient and selective removal due to its extreme toxicity and pollution concerns. over use of heavy metals pollutes the water. the use of heavy metals has health, environmental and aquatic effects [6]. in connection to the increasing amount of cr(vi), various absorbents have been synthesized for the effective and selective removal of such heavy metal ions. among the different types of adsorbents, the biomass-based adsorbents are getting interest these days due to their non-toxic nature towards environment, low-cost and tunable functionality. in order to remove chromium from industrial effluents, varieties of methods and materials are used for the effective and efficient removal of heavy metal ions from the sources. some of the commonly used approaches are chemical precipitation, ion-exchange methods, electrochemical precipitation, chemical reduction, solvent extraction, membrane filtration or separation and so on [7]. however, many of these methods are less sensitive for adsorptive removal of heavy metal ions below the tolerance level due to lack of sensitivity or selectivity. removal of heavy metal ions by adsorption methods is the most promising for overall treatment. though different types of nanoparticles, polymers/conductive polymers, especially heteroatom containing conductive polymers [8, 9], layered compounds [10] are being used for the removal of heavy metal ions, biomass-based low-cost materials are getting interest for the effective removal of toxic metal ions form water. these adsorbents are easily available, eco-friendly, and cost effective with higher capacity of adsorption. in addition, such types of biomass-based materials can be chemically processed to increase its effectiveness. commonly, activated carbons are being widely used as adsorptive materials for waste water treatment [11, 12]. commonly used waste materials used to prepare biomass-based adsorbent are banana peels, plum kernels, oil palm shells, tea leaves, saw dust, wheat bran, coconut husks, coffee grounds, rice husks, jackfruit peels and many others. in this context, most commonly used bioadsorbents are fruit gum, wheat bran, cork powder, sugar beet, rice bran, rice husk, nut shells, tea leaves and so on. each of the mentioned adsorbent capacitates to adsorb chromium form aqueous solution [5]. activated carbon is considered as a potent adsorbent with high surface area, microporosity and surface chemistry [13]. one of the biomass-based precursors for the preparation of activated carbon could be tea waste which is a common material left after the preparation of tea. nepal is rich in tea production. with the processing and refining into tea powder, tea leaves remain as residue. such waste materials can be employed for the preparation of activated carbon and further chemical treatment. furthermore, the composition of tea leaves may be different as a function of geography and climate. tea waste contains cellulose, hemicellulose, lignin and a lot of pectin in the cell walls. pectin is polysaccharides in which pectic acid and acidic polysaccharides, mainly composed of dgalacturonic acid and methyl ester, are present. tea leaves contain various polyphenols including epigallocatechin gallate (usually known as eggcg), flavonoids and other catechins [14]. large-scale hot water extraction of tea leaves is employed for the instant production of canned or bottled teas and producers have to struggle to dispose the discarded tea-leaves wastes. therefore, the use of such waste materials is highly preferable for the synthesis of activated carbon [15]. in this context, tea waste can be regarded as a potential precursor which can be used for the removals of cr(vi) ions from aqueous media. this method is benefitted by the facile mode of processing, easily available, cost-effective and environmentally friendly features [16]. the synthesis of charred carbon materials from tea waste of nepal origin has not been reported for the adsorptive removal of chromium (vi) listed in the comparison table 1. in this work, activated carbon has been prepared from the tea waste followed by charring with concentrated sulphuric acid. the objective of the present study is to find out the maximum uptake capacity of prepared charred tea waste for the removal of cr(vi) from contaminated water. batch adsorption experiments would be carried out changing the ph of solution, adsorbent dosage and contact time. the biosorbent ctw is ecofriendly and it can be incinerated with zero emission due to its natural constituents. on the other hand, the adsorbent is cost-effective since it is prepared from agricultural byproduct of tea. it could be an efficient and promising biosorbent for the treatment of cr(vi) from contaminated water. 2 materials and methods 2.1 preparation of chromium (vi) solution stock solution of cr(vi) of 1000 ppm concentration was prepared by dissolving 1.414 g of potassium dichromate in water followed by the addition of 100 ml solution of 2 n hno3. then the volume puspa lal homagai et al./ bibechana 20 (2023) 76-85 78 of the solution was made exactly 500 ml by adding distilled water. all the chemicals used in this study were of analytical grades. 2.2 preparation of charred tea waste waste tea leaves were collected from the tea shop, lazimpat, kathmandu. the tea waste was washed with distilled water several times, dried and ground. for charring process, 250 grams of ground tea waste were placed in a beaker of 1 liter capacity followed by the addition of concentrated sulphuric acid at an installment with constant stirring until the content turns to the black. in this step, ring opening of cellulose unit occurs. the reaction was left for 24 h for completion. the carbon was washed with deionized water several times to bring up to neutralization. then the sample was dried in vacuum at 50 °c for 9 h. the dried charcoal was ground in an agate mortar using pestle and sieved through 200 microns. finally, a sample was completely dried keeping in desiccator which is known as charred tea waste (ctw) and stored in an airtight bottle. 2.3 instrument used uv-visible double beam spectrophotometer (labtronics, lt-2802, india), ftir-spectrometer (perkin elmer 10.6.2, usa), digital ph meter (hana, hi2002-01 edge), four digit weighing machine (phoenix, ph2204c), hot air oven (elite oven), magnetic stirrer (labinco l34) and sieve (new dehli-110055). 2.4 sorption study stock solution of cr(vi) was used for the study of cr(vi) adsorption by charred tea waste. for this purpose, the stock solution of cr(vi) was diluted to prepare a series of solution form 10-300 ppm. the effect of dosage, effect of initial concentration, ph of the solution and contact time of the adsorption process was investigated. the ph of solution was maintained by using 0.5 n naoh and 0.5 n hcl solution where necessary. 20 mg of prepared adsorbents was taken in a stopper bottle and 25ml of chromium solution were agitated in a shaker at room temperature for 5-6 hours. then, the solution was filtered and the filtrate solution was taken. the absorbance of metal ion solution before and after adsorption was determined by using double beam uv-visible spectrophotometer. the amount of metal ion adsorbed onto adsorbent is calculated using the following relation given in equation (1). q = ( ci − ce w ) × v (1) here, q (mg/g) is the quantity of adsorbate adsorbed per unit mass of adsorbent. in the equation, v is the volume of solution in liter. w(g) is the mass of adsorbent, ci and ce are concentration of cr(vi) solution, respectively. the percentage adsorption capacity (%a) of the metal ion can be used by the relation of equation (2): percentage adsorption(%a) = ci − ce ci ×100 (2) 3 results and discussion 3.1 x-ray diffraction (xrd) analysis of adsorbents x-ray diffraction (xrd) study was carried out to study the crystalline/amorphous nature of the adsorbent. the xrd pattern of the raw tea waste (rtw) and charred tea waste (ctw) exhibit a broad peak in the 2θ range of 14-23° indicating a predominantly an amorphous nature. before and after modification, the xrd patterns of rtw and ctw are not so significantly different shown in figure 1. the amorphous biosorbent allows the cr(vi) ions adsorption by penetrating the surface rapidly, hence increase in adsorption capacity [17]. 3.2 fourier transform infra-red spectra (ftir) analysis the functional group study for the rtw and ctw materials were carried out in terms of ftir analysis in the department of chemistry, amrit campus. the ftir spectra of rtw and ctw are shown in figure 2. the broad absorption at 3200-3500 cm−1 corresponds to the –oh group of the adsorbent and small absorption band at 2917 cm−1 corresponds to the ch stretching in methylene group. the absorption band around 1704 cm−1 and1610 cm−1 correspond to the c=o and c=c group, respectively [18]. the c-oh stretching band at 1032 cm−1 of rtw got shifted and slightly deformed in ctw which could be associated with the charring process of the material [19]. the ftir spectra reveal the intensity and frequency change in the transformation of rtw to ctw indicating the involvement of chemical reaction. this happens due to breakdown of chemical bonds during the process of charring. puspa lal homagai et al./ bibechana 20 (2023) 76-85 79 figure 1: xray diffraction of rtw and ctw. figure 2: ftir spectra of rtw and ctw. 3.3 effect of ph on hexavalent chromium adsorption the ph of the adsorbate solution has been reported to be one of the variable for cr(vi) adsorption. for this study, 50 ppm of chromium(vi) solution was taken and its ph was maintained using ph meter in different reagent bottles from ph 1 to ph 5 keeping 0.02g of ctw in each bottle. from ph studies it was found that the % removal of chromium by ctw increased from 48.77% to 81.42% then again decreased up to 40.91%. the maximum percent cr(vi) removal was found to be 81.42% by which we can conclude that the optimum ph found to be 2 as shown in figure 3. this finding is in quite agreement with the reported literature [6]. below the ph 2, some of the chromium (vi) exists as h2cro4 which is hardly adsorb onto the adsorbent. from ph 0 to 6.5, chromium(vi) exists as hcro4− (hydrogen chromate) while above this ph range chromium(vi) exists as chromate ions (cro42−) predominantly. at the optimum ph value, chromium exists predominantly as hcro4− anion in aqueous media. with increasing the ph value the complexation process of cr(vi) goes on decreasing resulting into the low uptake of cr(vi). 3.4 effect of dose adsorption experiment was carried out at lab temperature (25°c) taking different weight of adsorbent to find the optimize amount of adsorbent dosage for the removal of cr(vi) from its 50 ppm aqueous solution. the upper tolerance level of chromium in drinking water is also 50 ppb. adsorption capacity of adsorbents increases upto a certain weight then it remains constant keeping in cr(vi) solution. with the increase of adsorbent dosages from 5 mg to 100 mg under the experimental conditions like ph, temperature and adsorbate concentration constant, the puspa lal homagai et al./ bibechana 20 (2023) 76-85 80 figure 3: effect of ph for adsorption of cr(vi) onto ctw. figure 4: effect of ctw dose on cr(vi) adsorption. adsorption study was carried out. low mass of adsorbent corresponds to the low active sites and high mass of adsorbent corresponds to the high active sites. with the increase of active sites, the percentage removal of cr(vi) got increased. the result shows that the percentage removal of cr(vi) increases with up to 20 mg adsorbent doses and remains almost constant for the studied adsorbent doses as shown in figure 4. the highest adsorption for 50 ppm adsorbate of cr(vi) is found to be 20 mg. 3.5 effect of concentration of cr(vi) solution the adsorption capacity of ctw can be evaluated with the help of an adsorption isotherm for the adsorption of cr(vi). as the concentration of cr(vi) ions increases, the adsorption of metal ions increases. at low cr(vi) ions concentration, the concentration of cr(vi) ions are limiting and get sufficient sites for their adsorption whereas at high concentration, the active sites on adsorbent are limiting compared to the cr(vi) concentration and hence become saturated. the absorbance of the supernatant clear liquid is measured spectrophotometrically at the maximum wavelength of after 4h equilibrium interval. a graph was plotted and used to calculate the amount of cr(vi) adsorbed. the maximum adsorption capacities of experimental results shown in fig.5 found to be 85.32 mg/g onto ctw at optimum ph2. puspa lal homagai et al./ bibechana 20 (2023) 76-85 81 figure 5: effect of concentration for adsorption of cr(vi) onto ctw. figure 6: langmuir isotherm plot for biosorption of cr (vi) onto ctw. 3.6 adsorption isotherm the correlation coefficient (goodness of fit) values for freundlich and langmuir isotherms were used to determine the applicability of the isotherms. langmuir and freundlich’s relation, equation (3) and (4), respectively, were used to calculate adsorption isotherms. ce qe = 1 qmb + ce qm (3) log qe = log kf + 1 n × log ce (4) in these equations, ce(ppm) is the equilibrium concertation of adsorbate, qe (mg/g) is the amount of cr(vi) ions adsorbed per gram of adsorbent at equilibrium, qm (mg/g) is the maximum adsorption capacity, b (l/mg) is the constant. by plotting a graph of ce/qe versus ce, the value of qm and b can be evaluated. the kf (l/mg) is adsorption capacity and 1/n is adsorption intensity which indicates the relative distribution of energy and adsorption heterogeneity site. the result shows that the maximum adsorption capacity is found to be 85.32 mg/g onto ctw at the ph of 2. higher initial concentration can increase the higher adsorption of metal ions. with the increase of concentration, the adsorption process get saturated. the higher value of langmuir correlation coefficient (r2 = 0.9974) indicates the better fitness of langmuir model compared to the freundlich model. it shows that the metal ion adsorption is predominantly a unimolecular. additionally, in langmuir’s isotherm, the equilibrium parameter, the dimensionless equilibrium puspa lal homagai et al./ bibechana 20 (2023) 76-85 82 figure 7: time on adsorption of cr(vi) onto ctw. figure 8: pseudo second order adsorption kinetics of cr(vi) onto ctw. constant (kl), given by equation (5): kl = 1 1 + bci (5) where b is the langmuir constant and ci is the initial metal ion concentration. in this research work, the value of kl is between 0 and 1 indicating the nature of favorable langmuir model of adsorption isotherm. 3.7 effect of contact time for the study of effect of contact time in the adsorption process, 20 mg of adsorbent was put into the 20 ml of 50 ppm of cr(vi) solution. the ph of the solution was maintained 2 for the study. after shaking the solution for the 24 h, the content was filtered and the clear solution was processed for its concentration measurement using double beam uv-vis spectrophotometer. adsorption of cr(vi) was initially increased with increase of the contact time and remain constant. figure 7 shows that the amount of adsorption increased from 5 minutes up to 120 minutes, and then remained constant thereafter. 3.8 adsorption kinetics the experimental adsorption kinetic data can be evaluated following integrated form of pseudosecond-order kinetics model explained by ho and mckay [20] as given in equation (6). t qt = 1 k2q2e + t qe (6) puspa lal homagai et al./ bibechana 20 (2023) 76-85 83 here, qt (mg g−1) is the amount of the adsorption at time (min), k2 (gmg−1min−1) is the rate constant of pseudo-second order kinetic of adsorption. using the plot of t/qt versus time, we can calculate the values of k2 and qe. pseudo-second order kinetics plot gives the perfect straight line for the adsorption of cr(vi) onto ctw shown in fig.8. correlation coefficient r2 value is 0.992 which indicates the pseudo second order kinetic model for the adsorption. the correlation coefficient value is about unity i.e. 0.992 confirms that metal ions, cr(vi) are chemisorbed onto ctw. 3.9 biosorption mechanism the high value of qm could be associated with high surface area resulted from ring opening of cellulose, hemicellulose or lignin mass due to acid treatment in charring process. the ring opening process of cellulose increased the active surface sites during the charring process. in the hexavalent chromium having ionization process at ph2, the concentration of hcro−4 ions are maximum. furthermore, at a low ph condition, protonation is possible due to high concentration of protons. as a result, hcro4 is electrostatically attracted to the protonated surfaces of the biosorbents, which helps it to bind to the surface of ctw (figure is not given). similarly, cr(vi) get adsorbed onto the protonated sites within the ctw by an ion-exchange mechanism [20, 21]. table 1: maximum cr(vi) biosorption capacities (qm) of ctw with other reported biosorbents. sn biosorbent ph qm (mg/g) reference 1 arthrobacter viscous biomass 2.0 20.377 [21] 2 waste newspaper 3.0 59.88 [22] 3 coconut husk 2.0 29.00 [23] 4 cacktus fruit (opuntia) 2.0 18.50 [24] 5 carbon slurry 2.0 15.24 [25] 6 nutshell powder 2.0 72.12 [26] 7 coconut coir pith 2.0 76.3 [27] 8 banana peel 1.5 10.42 [28] 9 charred tea waste 2.0 85.32 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al./ bibechana 20 (2023) 76-85 85 [27] c. namasivayam et al. removal of chromium (vi) from water and wastewater using surfactant modified coconut coir pith as a biosorbent. bioresource technology, 99(7):2218–2225, 2008. 10.1016/j.biortech.2007.05.023 [28] s. parlayici. comparative study of cr (vi) removal by bio-waste adsorbents: equilibrium, kinetics, and thermodynamic. journal of analytical science and technology, 10(1):1–8, 2019. 10.1186/s40543-019-0175-3 https://doi.org/110.1016/j.biortech.2007.05.023 https://doi.org/110.1186/s40543-019-0175-3 introduction materials and methods preparation of chromium (vi) solution preparation of charred tea waste instrument used sorption study results and discussion x-ray diffraction (xrd) analysis of adsorbents fourier transform infra-red spectra (ftir) analysis effect of ph on hexavalent chromium adsorption effect of dose effect of concentration of cr(vi) solution adsorption isotherm effect of contact time adsorption kinetics biosorption mechanism conclusion bibechana vol. 20, no. 2, august 2023, 175–182 issn 2091-0762 (print), 2382-5340 (online) journal homepage: http://nepjol.info/index.php/bibechana publisher:dept. of phys., mahendra morang a. m. campus (tribhuvan university)biratnagar in-vitro dissolution study of gallstone with medicinal plant extracts bijaya b.k.1, achyut adhikari1,∗ gobinda gyawali2 1central dept. of chemistry, tribhuvan university, kirtipur 44618, kathmandu 2department of fusion science and technology, sun moon university tangjeong myeon, asan si, chungnam 31460, republic of korea ∗corresponding author. email: achyutraj05@gmail.com abstract background: gallstone disease poses a substantial economic burden on healthcare systems globally, necessitating safer alternatives to current treatments like dissolution therapy and cholecystectomy. natural compounds from plants offer a potential solution, but research on their cholelitholytic activity is limited. in vitro dissolution studies are crucial for identifying effective plant-based therapies. objective: this study aims to investigate the in vitro cholelitholytic activity of six plants and ayurvedic medicines, selected based on ethnopharmacological knowledge and folk medicinal practices. methods: gallstone samples were categorized as combined cholesterol gallstones (ccgs) or black pigment gallstones based on external morphology and cross-sectional analysis. in vitro dissolution studies were conducted using extracts from bergenia ciliata, berberis asiatica, cuscuta europaea, kalanchoe pinnata, teraxacum officinale, macrotyloma uniflorum, and ayurvedic medicines (cystone®, gokshuradi, and calcury). the samples were immersed in the extracts and controls separately and incubated in a shaking water bath. the gallstone dissolution capacity was assessed by recording the dry weight of the samples at multiple time points. results: t. officinale was highly effective in dissolving black pigment gallstones, while b. asiatica exhibited superior efficacy for ccgs. m. uniflorum and c. europaea also demonstrated significant dissolution activity against black pigment gallstones. however, k. pinnata was less effective for both gallstone types. b. ciliata and c. europaea exhibited equal effectiveness against both types. ayurvedic medicine extracts were less effective compared to plant extracts. conclusion: this in vitro study showed the plants can dissolve gs effectively. however, the effectiveness of the plant to dissolve gs depends on the type of the stone. the findings from this study serve as a basis for further in vivo research. keywords gallstone, in vitro dissolution, plant extracts, combined cholesterol gallstones, black pigment gallstones. article information manuscript received: june 4 2023; accepted: june 17, 2023 doi https://doi.org/10.3126/bibechana.v20i2.55865 this work is licensed under the creative commons cc by-nc license. https://creativecommons. org/licenses/by-nc/4.0/ 1 introduction gallstones (gs), which are solid deposits formed inside the gallbladder, are mainly composed of cholesterol and bilirubin [1]. the formation of gs involves supersaturation, nucleation, crystallization, and aggregation of the insoluble components of the bile like cholesterol and bilirubin [2]. when the 175 http://nepjol.info/index.php/bibechana achyutraj05@gmail.com https://doi.org/10.3126/bibechana.v20i2.55865 https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ bijaya b.k. et al./ bibechana 20 (2023) 175-182 176 concentration of these insoluble components exceeds their solubility or there is a decrease in bile acid concentration or the presence of foreign substances, gallstones form [3]. gallstone disease is a prevalent health problem that affects 10-20% of the global population [4] and is a significant contributor to morbidity and mortality [5], with an estimated global cost of $6.5 billion annually [6]. its prevalence in nepal is 4.87% with females being more affected than males [7]. therefore, gallstone disease imposes a significant economic burden on healthcare systems worldwide [8]. the treatment of gallstone disease can be done through surgery or non-surgical methods. nonsurgical methods include taking bile acids like chenodeoxycholic acid (cdca) and ursodeoxycholic acid (udca) orally (oral dissolution therapy) [9] or installing litholytic solvent like methyl tert-butyl ether (mtbe) [10], 2-methoxy-6-methylpyridine (mmp) [11], or ethylenediaminetetraacetic acid (edta) [12], directly into the gallbladder through a percutaneous transhepatic catheter (contact dissolution therapy) [13]. however, these methods have limitations due to side effects, toxicity, low efficiency [14], incomplete dissolution [15], and gallstone reoccurrence is common [16]. laparoscopic cholecystectomy, the surgical removal of the gallbladder, is considered the best option [17] but has postoperative complications such as bile leakage, bile duct injury [18], persistent pain [19], and fat intolerance [20]. due to the limitations and potential risks of current treatment options, natural compounds from plants could be a safer alternative. it is believed that herbal medicines have lesser or no side effects. while extensive studies have been conducted on the cholelitholytic activity of organic solvents, research on plants is limited, and in vitro dissolution studies are necessary to identify potential plant-based therapies. therefore, in this study, we investigated the in vitro cholelitholytic activity of six plants (selected based on ethnopharmacological knowledge and folk medicinal practices) and ayurvedic medicines available in the market. the results of this in vitro study will provide a basis for further research in vivo. 2 materials and methods 2.1 collection and pre-treatment of gallstones dr. barun kumar shah, norvic international hospital, thapathali, kathmandu, nepal has provided the gallstone samples. cholecystectomy was performed to extract gallstones from patients. altogether, 12 ccgs (from two patients) and 15 black pgs (from one patient) were collected. the collected gallstones were thrown away materials with no human tissues or genetic material. the gallstone samples were washed with deionized water and dried at 60 ℃ in an incubator until the constant weight [21]. 2.2 macroscopic classification of gallstones based on the external morphology and internal cross-sectional analysis, we categorized the gallstones into two groups: combined cholesterol gallstone (ccgs) and black pigment gallstone (black pgs). photographs of the gallstones were taken, their morphological features like shape, size, color, and internal cross-section were studied. the outer dimensions of the stone were measured with a digital vernier caliper. one gallstone from each patient was cut into equal halves using jeweler’s saw and the internal cross-section was studied. the remaining gallstones were stored on a desiccator filled with calcium chloride in a dark cabinet, and later on, they were used for in vitro dissolution with different extracts of plant and herbal medicines. 2.3 collection of plant and ayurvedic medicines the rhizomes of bergenia ciliata were collected from central nepal, parbat district, panchase. the root of berberis asiatica and tendrils of cuscuta europaea were collected from central nepal, parbat district, modi rural municipality-7, ranpu. the leaves of kalanchoe pinnata were collected from central nepal, kaski district, pokhara-15, nayagaun. leaves of teraxacum officinale were collected from central nepal, kathmandu district, kirtipur. the legumes of macrotyloma uniflorum were purchased from the local market of kirtipur, nayabazar. herbarium samples of each plant were prepared and the plants were identified from national herbarium and plant laboratories, godawari, lalitpur. ayurvedic medicines (cystone®, gokshuradi, and calcury) were purchased from arogyadham homoeopathic hospital, pokhara with the suggestion of homeopathic physician dr. bishnu prasad chapagain. the plant materials were washed with tap water, cut down into small pieces, and shed dried for two weeks. the shed dried plant samples were crushed into powder with the help of a grinding mill at the central department of chemistry. the powder samples were put into an airtight glass jar and stored at ambient temperature. 2.4 in vitro dissolution study of gallstones the dried and powdered plant samples were extracted with ethanol using soxhlet apparatus. to bijaya b.k. et al./ bibechana 20 (2023) 175-182 177 obtain crude extracts, 50 g sample powder was refluxed with 500 ml of ethanol at 40 °c until a clear solution was obtained in the thimble. the dilute extract was evaporated under reduced pressure using a rotatory evaporator at 40 °c. the semisolid crude extract obtained from rotavapor was dried at ambient temperature for several days to get the dry crude extract. the dried crude extracts were collected into 15 ml flat-bottom borosil culture tubes, labeled properly, and stored at 4 °c in a refrigerator. 50 mg/ml extract solution was prepared by dissolving 5 g of the solid crude extract in 100 ml of distilled water. the solution was slightly warmed and sonicated for half an hour, let to settle down to get a clear stock solution. the clear solution was collected into a 100 ml volumetric flask, the flask was stoppered, labeled, and stored in a refrigerator at 4 °c. two tablets of each ayurvedic medicine were powdered in a mortar and pestle and the powder was taken into the centrifuge tube with a screw cap. distilled water (10 ml) was added and the content was kept in a water bath for a night at 37 °c, centrifuged at 25 °c in a high-speed centrifuge at 8000 rpm to get clear supernatant. the clear supernatant was collected into a 100 ml volumetric flask, the flask was labeled and stored at 4 °c in a refrigerator [22]. 2.5 dissolution of gallstones with plant and medicine extracts for the in vitro dissolution study, the protocol used by igimi et al. [23] was followed with slight modification. the gallstone samples that were oven-dried (at 60 ℃) and stored in a desiccator in a dark cabinet at least for 15 days were taken for dissolution. the gallstone samples were taken, weighed, and immersed in vitro into 10 ml of the extract, positive control, and negative control separately. borosil culture tubes (15 ml volume) with round bottom and screw cap were used for the purpose. the tubes with content were put in a test tube rack and kept in a shaking water bath at 37 ℃ with gentle and constant shaking (60 rpm). the plant extracts were changed every day to minimize any effect due to saturation of the solution by dissolved components of the stone [23]. the stones were taken out of the extract by filtration through filter paper (whatman no1), washed with distilled water, transferred into a pre-weighed crucible, dried at 60 °c in an incubator for two days to get constant weight, and the weight reduced was noted [11,24]. to determine the gallstone dissolution capacity of each extract and solvent, we recorded the dry weight of the stone samples at three different time points (4, 94, and 190 h) after they were directly immersed in the solution. photographs of the stones were also taken after each time interval. the stone samples with comparable weight and size were taken for dissolution study. the average weight of different ccgs samples was 14.48±0.21 mg and that of black pgs samples was 21.46±0.22 mg. in the present research, 2% edta solution maintained at ph 9.5 adjusted with hcl and 95% ethanol was used as a positive control whereas distilled water was used as a negative control. it was found that the higher the ph of the edta solution higher is its litholytic activity [25]. the ph of 8.5 is within the harmless range to use to the human body [25], but we maintained the ph 9.5 because studies have found that this is the most effective ph at which edta shows the maximum dissolution of gs [26]. first of all, the weight dissolved by negative control at every time point was subtracted from the weight reduction value recorded for each of the preparations at respective time points to calculate the actual weight dissolved by the preparations. then the percentage dissolution was calculated by using the following formula [11, 24]: %dissolution(w/w) = actual wt. dissolved initial wt. of the stone ×100 (1) 3 results 3.1 general observation and macroscopic classification of gallstones the photographs showing the external morphology and internal cross-section of the gallstone samples are given in figure 1. the gallstone sample, which is multifaceted, whitish-brown with a hard and smooth outer surface [27, 28], with a distinct inner pigmented yellow core and a white external shell (figure 1a, b) [28, 29] was classified as a combination cholesterol gallstone (ccgs). the gallstone sample, which was irregular with a rough and thin yellowish surface [27, 28], amorphous in cross-section [28, 29] with a black inner part and outer thin yellowish layer (figure 1c, d) was classified as black pigment gallstone (black pgs) [28]. furthermore, we conducted a separate study to support this macroscopic classification via uv-vis and sem-eds analysis [28]. 3.2 in vitro dissolution study of gallstones the final data obtained for cumulative dissolution of gallstone samples in different extract and solvent preparations is presented in table 1. the observation tables for recorded weight (initial, after 4 h, 94 h, and 190 h), dissolution and cumulative dissolution are included in supplementary material bijaya b.k. et al./ bibechana 20 (2023) 175-182 178 figure 1: photographs of the gallstone samples; (a) ccgs, (b) internal cross-section of ccgs, (c) black pgs, and (d) internal cross-section of black pgs (annexure 1). photographs of gallstones at different time are also given in supplementary material (annexure 2). after 190 hours, 14.3 mg of the ccgs and 8.6 mg of the black pgs was dissolved in etoh, whereas in edta, 11.5 mg of the black pgs and 5.1 mg of ccgs was dissolved. therefore, it can be inferred that the ccgs was more soluble in etoh and black pgs was more soluble in edta (table 1). figure 2 illustrates the comparative study of the final dissolution of gallstone samples in different extract and solvent preparations. it shows that e1 (m. uniflorum), e4 (c. europaea), e5 (t. officinale), and edta dissolved black pgs more effectively than ccgs, while the m2 (calcury) showed almost similar dissolution for both the stones. in contrast, e2 (b. asiatica), e3 (b. ciliata), m1 (cystone), m3 (gokshuradi), and etoh showed better dissolution for ccgs than for black pgs, while in e6 (k. pinnata), dissolution of both the gallstones was comparable. table 1: comparative study of the final dissolution of ccgs and black pgs in different extract and solvent preparations. extract or solvent weight dissolved after 190 hours (mg) ccgs black pgs e1 (m. uniflorum) 5.6 9.3 e2 (b. asiatica) 10.8 5.4 e3 (b. ciliata) 4.7 3.7 e4 (c. europaea) 7.3 8.5 e5 (t. officinale) 3.0 9.6 e6 (k. pinnata) 2.2 2.1 m1 (cystone®) 3.1 1.8 m2 (calcury) 3.9 4.0 m3 (gokshuradi) 5.8 3.5 edta (+ve control) 5.1 11.5 etoh (+ve control) 14.3 8.6 note: edta and etoh are used as positive controls and distilled water as a negative control. the data for each experiment is expressed after subtracting the data obtained for the negative control. 4 discussion close interpretation of the graph in figure 2 shows that, in e1 (m. uniflorum), e4 (c. europaea) and e5 (t. officinale), black pgs was more soluble than ccgs. among different extracts preparations (both plant and medicine), e5 (t. officinale) was the most effective for dissolving black pgs (9.6 mg). in comparison with the most effective positive control edta (11.5 mg), the efficacy of e5 (t. officinale) to dissolve black pgs can be considered as comparatively excellent. e5 (t. officinale) was more effective than another positive control etoh (8.6 mg). the efficacy of e1 (m. uniflorum) to dissolve black pgs (9.3 mg) was almost equivalent to that of the most effective plant e5 (t. officinale). this shows that e5 (t. officinale) and e1 (m. uniflorum) can effectively dissolve pigbijaya b.k. et al./ bibechana 20 (2023) 175-182 179 figure 2: the comparative study of the final dissolution of ccgs and black pgs in different extract and solvent preparations ment containing stones. moreover, this fact was further supported by the result obtained for cholesterol predominant ccgs which shows that e5 (t. officinale) and e1 (m. uniflorum) dissolved significantly lesser amount of ccgs than black pgs (3.0 mg in e5 and 5.6 mg in e1). m2 showed almost similar efficacy to dissolve both the ccgs (3.9 mg) and gsb (4.0 mg) whereas m1 (cystone®) and m3 (gokshuradi) showed better dissolution for ccgs than black pgs. similarly, e2 (b. asiatica) and e3 (b. ciliata) dissolved ccgs more effectively than black pgs. the efficacy of e6 (k. pinnata) was similar for both the stones (2.2 mg ccgs, 2.1 mg black pgs). a close inspection revealed that e6 (k. pinnata) was the least effective plant to dissolve both the ccgs and black pgs. among different plant extracts, e2 (b. asiatica) was the most effective (10.8 mg) to dissolve ccgs. in comparison with the most effective positive control etoh (14.3 mg), the efficacy of e2 (b. asiatica) to dissolve ccgs can also be considered significant. e2 (b. asiatica) was found to be more effective than another positive control edta (5.1 mg). moreover, e2 (b. asiatica) dissolved only 5.4 mg of bilirubinate predominant black pgs which was almost half of the dissolution obtained for ccgs. form all of these interpretations we can conclude that e2 (b. asiatica) is effective to dissolve cholesterol containing gallstones like ccgs. e4 (c. europaea), although, was found to be more efficient to dissolve black pgs than ccgs, there is no significant difference between the values obtained (8.5 mg of black pgs vs 7.3 mg of ccgs). dissolution of black pgs in e4 (c. europaea) is far close to the dissolution in e5 (t. officinale) with only 1.1 mg difference. on the other hand, a significant difference was obtained for dissolution of ccgs in e4 (c. europaea) and the most efficient plant e2 (b. asiatica) and positive control etoh (difference of 3.5 mg with e2 and 7.0 mg with etoh). therefore, it can safely be inferred that e4 (c. europaea) was also effective in dissolving pigment stones like gsb. among different medicine extract, m3 (gokshuradi) was the most effective to dissolve ccgs, it dissolved ccgs (5.8 mg) more efficiently than black gsb (3.5 mg, 16.20%). even though, m3 (gokshuradi) including other medicine extracts were far less effective than e2 (b. asiatica) (10.8 mg) and e4 (c. europaea) (7.3 mg) for dissolving ccgs. on the other hand, among different medicines, m2 (calcury) showed the highest dissolution for black pgs (4.0 mg). but, m3 was still less effective than e1 (m. uniflorum), e2 (b. asiatica), e4 (c. europaea), and e5 (t. officinale). therefore, it can be inferred that the medicine extracts were less effective than most of the plant extracts in dissolving gallstones. these ayurvedic medicines are recommended for oral dissolution therapy of kidney stones and not for gallstones. this fact could explain lesser efficacy of these commercial ayurvedic medicines over plant extracts in dissolving gallstones. chekroune and benamara [30] reported difbijaya b.k. et al./ bibechana 20 (2023) 175-182 180 ferent plant preparations to dissolve a significant amount of gs. about 209.37 mg, 97.42 mg, and 15.02 mg of the cholesterol-bilirubin containing gs were reduced in herniaria hirsuta extract, lemon juice, and their mixture respectively after 312 hours of immersion whereas the olive oil/lemon juice emulsion dissolved the stone with 291.1 mg weight completely after 168 hours of immersion [30]. in the present study, among all plant extracts and gallstone samples, the maximum weight reduction after 190 h was 10.8 mg in e2 (b. asiatica) for ccgs. due to the difference in immersion period of the stone, direct comparison of our report with the reported data seems to be inappropriate, however, it can be concluded that we also found in vitro cholelitholytic activity of different plant extracts. the in vitro dissolution study showed that ccgs was more soluble in etoh than in edta whereas black pgs is more soluble in edta than in etoh. the insoluble residue of ccgs was left even after 190 hours of incubation in edta (5.1 mg dissolution) whereas ccgs was completely dissolved in etoh after 94 hours (11.6 mg dissolution). the efficacy of edta to dissolve black pgs (11.5 mg) was almost double that for ccgs (5.1 mg); however, almost half of the black pgs was left undissolved even after 190 hours of incubation (annexure 1: table 3). this indicates that ccgs is easily dissolvable by using cholesterol solvent like ethanol whereas black pgs contains a greater proportion of insoluble components and it is difficult to dissolve completely both in cholesterol solvent like etoh and in calcium chelating solvents like edta. this finding is consistent with the result reported by lin et al. [24]. lee and co-worker [31] reported that 1.1 mg, 9.1 mg, and 30.0 mg of the cgs stone sample with 69.3 mg initial weight was dissolved in absolute ethanol after 3, 6, and 9 hours of incubation, respectively and the stone was dissolved completely after 18 h. in the present study, we have recorded 2.7 mg and 14.3 mg dissolution of the ccgs (wt. 14.48±0.21 mg) after 4 and 94 hours respectively. the stone was dissolved completely after 94 hours. based on this data, the dissolution we obtained in etoh for ccgs was lower than that reported by lee et al. the difference arises due to the lower concentration of etoh we used (95% etoh). in the case of black pgs (wt. 21.46±0.22 mg), etoh was not found effective and only 8.6 mg of the stone was dissolved after 190 h. the low solubility of the black pgs in etoh is due to the presence of a high concentration of insoluble bilirubinate as the main component which was confirmed by sem and eds in a separate study by our group [28]. 5 conclusion from the morphological and cross-sectional study, gallstones were classified as combined cholesterol gallstone (ccgs) or black pigment gallstone (black pgs). in vitro dissolution studies were also conducted using plant and medicine extracts, revealing that t. officinale was the most effective in dissolving black pgs, whereas b. asiatica was the best for ccgs. m. uniflorum and c. europaea were also found to be effective in dissolving black pgs, while k. pinnata was the least effective for both types of gallstones. b. ciliate and c. europaea were equally effective in dissolving both types of gs. medicine extracts were less effective than plant extracts. this in vitro study showed the plants can dissolve gs effectively. however, the effectiveness of the plant to dissolve gs depends on the type of the stone. the study suggests that further research is necessary to confirm the effectiveness of these plants in real biological systems through animal models. furthermore, the potential compound in plant that show cholelitholytic or anti-cholelithogenic activity and mechanism of action is still unknown and requires further research. the present research is limited to in vitro study. although normal human body temperature (37 ℃) was maintained while preforming in vitro dissolution study, other factors like normal bile ph is not considered. list of abbreviations gs = gallstone ccgs = combination cholesterol gallstone black pgs = black pigment gallstone cdca: chenodeoxycholic acid udca: ursodeoxycholic acid mtbe: methyl tert-butyl ether mmp: 2-methoxy-6-methylpyridine edta: ethylenediaminetetraacetic acid ethics approval and consent to participate not applicable. human and animals right no animals/humans were used for studies that are basis of this research. availability of data and materials all the data are provided in the supplementary material which is available on the publisher’s website along with the published article. bijaya b.k. et al./ bibechana 20 (2023) 175-182 181 conflict of interest the authors declare no conflict of interest, financial or otherwise. acknowledgements we acknowledge the national youth council, government of nepal, for providing a partial research grant to conduct this research. we also extend our thanks to dr. barun kumar shah, norvic international hospital, kathmandu, nepal, for providing gallstone samples. supplemetary materials supplementary material is available on the publisher’s website along with the published article. references [1] g. liu, d. xing, h. wang, and j. wu. vibrational spectroscopic study of human pigment gallstones and their insoluble materials. journal of molecular structure, 616(1-3):187–191, 2002. 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[31] lawrence l lee and john p mcgahan. dissolution of cholesterol gallstones: comparison of solvents. gastrointestinal radiology, 11:169– 171, 1986. introduction materials and methods collection and pre-treatment of gallstones macroscopic classification of gallstones collection of plant and ayurvedic medicines in vitro dissolution study of gallstones dissolution of gallstones with plant and medicine extracts results general observation and macroscopic classification of gallstones in vitro dissolution study of gallstones discussion conclusion bibechana vol. 20, no. 2, august 2023, 190–199 issn 2091-0762 (print), 2382-5340 (online) journal homepage: http://nepjol.info/index.php/bibechana publisher:dept. of phys., mahendra morang a. m. campus (tribhuvan university)biratnagar analysis of phytochemical, antioxidant, and α-amylase inhibition capacity of methanol extract of six plants from kaski, gulmi and rupandehi districts of nepal bimala subba1,∗, namrata subedi1, sanju parajuli2 1central department of chemistry, tribhuvan university, kirtipur, kathmandu, nepal 2botany department, central campus of technology, hattisar, dharan, nepal ∗corresponding author. email: bimalasubba@gmail.com abstract this study was designed to explore phytochemicals and biological activities of methanolic extract of six traditional medicinal plants ( clerodendrum trichotomum, mallotus philipinensis, dioscorea-bublifera, rubia cordifolia, viscum articulatum) of nepalese origin. plant extracts were prepared in methanol through cold percolation. analysis of phytochemical constituent, antioxidant capacities and brine shrimp lethality assay of the test plant samples were carried out using standard methods. the 1,1-diphenyl-2-picryl hydrazyl (dpph) method was used to study antioxidant activity of different plant extracts. furthermore, starch-iodine method was used to study the inhibition effect of extract on α-amylase. phytochemical analysis showed the presence of phytochemicals like alkaloids, flavonoids, phenolic content, glycosides, reducing sugars, etc. in six medicinal plants. brine shrimp lethality assay suggested the presence of pharmacologically active compounds. total phenolic content and total flavonoid content of c. trichotomum’s leaves extract were found to be higher with 212.742 mg gae/g and 112 mg q/g respectively with strong antioxidant activity. similarly, the α–amylase inhibition of r. cardifolia’s root extract and c. trichotomum‘s leaves extract was found to be 252.44±0.55 µg/ml and 293.33± 0.81 µg/ml comparative with ic50 value 119.063±0.73 µg/ml of standard acarbose that showed remarkable antidiabetic property among six samples. the results, obtained here suggested that six medicinal plants i.e. c. trichotomum, m. philipinensis, d. deltoidea, d.bublifera, r. cordifolia, and v. articulatum, nepal origin showed biological activity by targeting multiple drug targets which justifies their traditional uses. this is the first finding that c. trichotomum‘s leaves can be a promising source for the development of natural antioxidant and antidiabetic agents. keywords c. trichotomum, m. philipinensis, d. deltoidea, d. bublifera, r. cordifolia, v. articulatum,. article information manuscript received: march 25, 2023; accepted: june 30, 2023 doi https://doi.org/10.3126/bibechana.v20i2.56226 this work is licensed under the creative commons cc by-nc license. https://creativecommons. org/licenses/by-nc/4.0/ 190 http://nepjol.info/index.php/bibechana bimalasubba@gmail.com https://doi.org/10.3126/bibechana.v20i2.56226 https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ b. subba et al./ bibechana 20 (2023) 190-199 191 1 introduction many diseases have been cured with plants by relating in various formula such as vegetables, fruit, spices, tea, decoction, etc., since ancient time. today’s modern society is slowly returning to ancient medicinal system as the number of people who still prefer using traditional folk medicines is increasing per year. plants comprise a wide diversity of compounds, especially secondary metabolites with anticancer, antibacterial, antidiabetics, antitumor, antiviral, analgesic and many other activities to a greater or lesser extent. notable examples of these secondary metabolites include phenols, flavonoids, alkaloids, terpenoids, saponins, glycosides, tannins etc. [1, 2]. antioxidants compounds have the ability to protect the body from reactive oxygen species. reactive oxygen species (ros) become toxic and cause the harmful effects like oxidative stress in absences of proper amount of antioxidants in our body. dpph scavenging (2, 2-diphenyl-1-picrylhydrazyl) assay is a popular method for screening in-vitro antioxidant of plant extract. dpph radical in methanol shows strong absorption at 517 nm. on increasing the concentration of extracts decreases the value of maximum absorbance suggest that scavenging of free radical by electron donation [3]. the types of diabetes mellitus according to the american diabetes association in 1997 are type 1, type 2, other specific types, and gestational diabetes. it is characterized by high blood sugar (glucose) levels that consequence from defects in insulin secretion, or resistant in its action, or both. normally when there is an elevated level of glucose in the bloodstream, the pancreas releases insulin which then binds a membrane protein of a cell and results a series of reactions that induce glucose transporters to move into the membrane and facilitate the movement of glucose into the cell. type 2 diabetes [t2d] is characterized by an increase in insulin resistance and decrease beta-cell function and chronic hyperglycemia. inhibition of α amylase activity is one of the alternative pathways for the control of postprandial hyperglycemia in diabetic patients. nepal has many plants with medicinal values as it is rich in biodiversity. some of them are used in traditional medicine that usually has a pharmacological or biological activity for use in pharmaceutical drug discovery and drug design and some are still not explored scientifically for medicinal values [3, 4]. the biological activities of the test plants i.e c. trichotomum, m. philipinensis, d. deltoidea, d. bublifera, r. cordifolia, v. articulatum had been reported by many research articles. although these plants are reported for their uses such antioxidant, antidiabetic, antimicrobial, reducing high blood pressure, or improving blood circulation, the same plant grown in gulmi, rupandehi, kaski district of nepal has not been explored yet. the genetic and geographical variation plays significant role for the dissimilarity of chemical constituents of the same plant. the growing stages of the concerned plant at the time of the collection also plays a role for the variation of the chemical constituents of the same plants. usually synthetic drugs contain a single active compound targeting a specific drug target but plant extracts may contain various active ingredients aiming at multiple drug targets [5]. on the basis of these reports, the present study purposes to calculate the polyphenols and flavonoids in the methanolic extract from different parts of such six medicinal plants i.e. c. trichotomum, m. philipinensis, d. deltoidea, d. bublifera, r. cordifolia, v. articulatum to determine their antioxidant potential and to evaluate its inhibitory properties on the α-amylase activities. 2 materials and methods 2.1 collection of plant materials and preparation their methanol extract various plant parts of six medicinal plants were collected from the farmland of gulmi, rupandehi, kaski district of nepal. the plants were identified with literatures and matched with the voucher specimens deposited at national herbarium and plant laboratories, godavari, kathmandu. the collected plant parts were cleaned, sliced into small pieces, and shade dried for 10-15 days. then it was ground into a powder and stored. the leaves extract of six plants was prepared by cold percolation method in methanol solvent. 2.2 chemicals and reagents the chemicals used in this study were of the commercially available analytical grade. the methanol solvent was purchased from merck, germany. similarly, porcine pancreatic α-amylase, 2,2-diphenyl1-picrylhydrazyl (dpph), and ascorbic acid were purchased from sigma-aldrich, usa. 2.3 qualitative phytochemical analysis phytochemicals were identified by various color reactions [6]. in brief, all plant materials were completely extracted by percolation with methanol and subjected to phytochemical screening. the presence of main groups of natural constituents in different extractive solutions was analyzed by using different specific reagents. b. subba et al./ bibechana 20 (2023) 190-199 192 2.4 brine shrimp lethality assay brine shrimp lethality assay was carried according to standard protocol [7]. briefly, sample stock solutions were prepared by dissolving 200 mg of each plant extract in 2 ml acetone. the test was conducted in 15 ml test tubes and sterilized artificial sea water (final volume 10 ml). from each stock solution 500 µl (equivalent to 1000 ppm), 50 µl (equivalent to 100 ppm) and 5 µl (equivalent to 10 ppm) were transferred to total nine test tubes, three test tubes for each dose level. the control was performed by adding the solvent used to dissolve the extract (acetone) in the assay. the solvent was evaporated by standing overnight. after complete evaporation of solvent, 5 ml of sea water was added to each test tube with gentle shaking to ensure that the compounds diffused adequately in the aqueous solution. ten salina nauplii were counted macroscopically in the stem of a pasteur pipette against a lighted background and transferred into each sample tube and the solutions were made to 10 ml with artificial sea water. after 24 hours, the numbers of survivor were counted with the help of disposable pipettes. the entire experiment was performed in a temperature controlled room at 28 °c under the continuous supply of light by table lamp (60 watt). the surviving nauplii were counted with the aid of a 3x magnifying glass after 24 hours. the mean mortality at the three dose levels for each extract was determined. no death was observed in the control tubes. the lc50 (lethal concentration for 50% mortality) values was determined using the probit method, as the measure of toxicity of the extracts [8]. 2.5 determination of total phenolic content in the plant extracts total phenolic content in plant extract was calculated by folin-ciocalteu colorimetric method with some modifications [9]. an aliquot of 1 ml of each leaf extract (0.5 -1.0 mg/ml) were mixed with 5 ml folin–ciocalteu phenol reagent (10%) and 4 ml of 7% na2co3 to get a total volume of 10 ml. the blue colored mixture was shaken well and incubated for 30 minutes at 40 ºc in a water bath. the absorbance of the mixture was measured at 760 nm. the samples were prepared in triplicate for each analysis and the mean value of absorbance was obtained. standard calibration curve for gallic acid in the range of 50–100 g/ml was prepared in the same manner and results were expressed as mg gallic acid equivalent (gae) per gram of extract. 2.6 determination of total flavonoid content in the plant extracts total flavonoid content was determined using the aluminum colorimetric method using quercetin as the standard [10]. an aliquot of 1 ml leaf extract of each concentration range from 0.5-1 mg/ ml in methanol was poured to 15 ml test tube containing 4 ml of double distilled water. at the 0, 5 and 6 minutes’ time interval, 0.3 ml 5% nano2, 0.3 ml of 10% alcl3 and 2 ml of 1 m naoh was added to the test tube respectively. immediately volume was maintained 10 ml by adding 2.5 ml double distill water. absorbance of the obtained pink color mixture was determined at 510 nm versus blank containing all reagents except quercetin. the samples were prepared in triplicate for each analysis and the mean value of absorbance was obtained. standard calibration curve for quercetin in the range of 50–100 g/ml was prepared in the same manner. the concentration of flavonoid was expressed as mg quercetin equivalent (qe) per gram of extract. 2.7 antioxidant assay the antioxidant activity of extract of the test plants and standard (ascorbic acid) was carried by dpph assay method, following standard protocol [11]. different concentration of plant extracts (10100 µg/ml) and ascorbic acid (10-100 µg/ml) were prepared in methanol on the clean and clear test tubes. the sample volume was taken 2 ml. to this sample volume, 2 ml of this 0.2 mm dpph solution was added. the tube was shaken vigorously for the uniform mixing. these tubes were allowed to stand in dark for half an hour. the control was prepared as above without the plant extract or ascorbic acid. methanol was used to collect the baseline on the spectrophotometer (thermo fisher scientific, genosystem-10-50. the absorbance was taken on the spectrophotometer at 517 nm. now the radical scavenging activity was calculated by using the following formula. %radical scavenging activity = controlabs − sampleabs controlabs × 100% (1) standard graph was plotted by taking the concentration on the xaxis and percentage free radical scavenging activity on the y-axis. based on this graph, ic50 value of each sample was calculated and these values of the different species were compared. the species having the lowest ic50 is considered to have the best antioxidant property. 2.8 alpha-amylase inhibition assay α-amylase inhibition assay was performed following the standard protocol [11]. this protocol is based b. subba et al./ bibechana 20 (2023) 190-199 193 on that α-amylase converts starch into sugars and plant extracts inhibits the action of α-amylase as acarbose. substrate was prepared by dissolving 200 mg starch in 25 ml of naoh (0.4 m) by heating at 100 ºc for 5 minutes and adjusting ph was adjusted to 7.0 after cooling. 400 µl of substrate solution was pre-incubated at 37 ºc for 15 min. termination of the reaction was carried out by adding 800 µl of hcl (0.1 m). then, 1000 µl of iodine reagent (2.5 mm) was added, and absorbance was measured at 630 nm. the assay was carried out in triplicates using spectrophotometer. percentage of inhibition was calculated using the formula %inhibition = ( 1 − abs2 − abs1 abs4 − abs3 ) × 100 (2) where abs1 is the absorbance of the incubated mixture containing sample, starch, and α-amylase, abs2 is the absorbance of the incubated mixture of sample and starch, abs3 is the absorbance of the incubated mixture of the of starch and α-amylase, abs4 is the absorbance of the incubated solution containing starch. 3 results and discussion 3.1 plant collections the plants samples on this research work were selected and collected from the farmland of gulmi, rupandehi, kaski district of nepal (table 1). table1: list of plants, parts used and therapeutic use. s.n. scientific name common name used part therapeutic use 1. clerodendrum-trichotomum lapche leaves antioxidant antidiabetic antibacterial 2. mallotus-philipinensis sindoor leaves antioxidant antifilarial 3. diosocreadeltoidea vyakur rhizomes anti-inflammatory antiviral antidiarrhoic 4. dioscorea-bublifera gitto rhizomes antioxidant anti-inflammatory antidiabetic 5. rubiacordifolia majitho roots antidysentric antioxidant 6. viscumarticulatum harchur bark anticancer antioxidant table 2: phytoconstituents of test plants extract in methanol. phytochemical ct mp dd db rc va reducing sugars + + polyphenols + + + + + alkaloids + + + + + + glycosides + + + + quinones + + + + saponins + + + coumarins + + flavonoids + + + + + + where, (+) present and (-) absent, ct = c. trichotomum, mp = m. philipinensis, dd = d. deltoidea, db = d. bublifera, rc = r. cordifolia, and va = v. articulatum 3.2 phytochemical screening the results obtained from the phytochemical screening indicating the presence and absence of different types of phytoconstituents are tabulated in table 2. the different extracts of plants depict the presence of various secondary metabolites like alkaloids, glycosides, coumarins, saponins, flavonoids, terpenoides etc. these compound are supposed to be biologically active and act as anticancer, antioxidant, antidiabetic etc. [12]. due to presence of such secondary metabolites v. articulatum be the good source of such biologically active compounds and b. subba et al./ bibechana 20 (2023) 190-199 194 d. deltoidea and c. trichotomum can be inferred to be potent antioxidant, antidiabetic and antimicrobial [13–15]. flavonoids, alkaloids and phenolic compounds are a major source of compounds that act as primary antioxidants or free radical scavenger. subsequently, plants containing these phytochemicals may be used for the preparation of drug in a systematic way which may be lead to the cure of many ailments in the future [16]. so, many of selected medicinal plants can be inferred as the good source of biologically active compounds. 3.3 brine shrimp bioassay results obtained from the brine shrimp lethality assay are presented in table 3. table 3: lc50 value of plant samples in brine shrimp bioassay. name of plant extracts lc50 value µg/ml c. trichotomum 184.79 m. philipinensis 100 d. deltoidea 193.06 d. bublifera 135.93 r. cordifolia 184.79 v. articulatum 46.39 from the above calculations, sample va (bark of v. articulatum) had shown the lower lc50 value which indicates that it contains highly active compound. the result showed that lc50 of different plant extracts ranged from 46.39 to 193.06 µg/ml. v. articulatum can be inferred to have very strong cytotoxic effect. likewise, m. philipinensis, d. bublifera, c. trichotomum, r. cardifolia, and d. deltoidea were biologically active with lc50 values 100, 135.93, 184.79, 184.791, and 193.06 µg/ml respectively. so, these plants can be suggested to use as therapeutic agents as the toxicity activity on them is nominal. plant extract resulting in lc50 less than 1 mg/ml were considered toxic to the larvae. this brine shrimp lethality test was also a guide for active antitumor agent [17, 18]. 3.4 total phenolic and flavonoid contents the tpc and tfc were expressed as the gae/g, and qe/g of extract using a calibration curve of gallic acid and quercetin, respectively which is shown in fig. 1 & 3. the total phenolic content of all selected medicinal plant extracts showed varied data ranging from 54.742±0.025 to 212.742±3.34 mg gae/g in c. trichotomum and d. deltoidea respectively which is shown in figure 2. the total phenol content of rest of plant extract lied between these two extremes. the results showed that the total phenolic content in c. trichotomum (ct) was highest among six selected medicinal plants. leaf extract of c. trichotomum (ct), bark of v. articulatum (va) also showed relatively high phenolic content. figure 1: calibration curve of gallic acid. figure 2: comparison of tpc in different plant extracts. b. subba et al./ bibechana 20 (2023) 190-199 195 figure 3: calibration curve of gallic acid. figure 4: comparison of tpc in different plant extracts. from the calculation of total flavonoid content in different plant extract, it was found that sample (ct) c. trichotomum has highest tfc. it was also seen that tfc in (dd) d. deltoidea was high enough. the total flavonoid content of different medicinal plants was found and the result revealed that the tfc varied from 72.39±112 mgqe/g in m. philipinensis to 109.964±1.25 mgqe/g is d. deltoidea. all the remaining plant extract showed the tfc in two extremes as shown in fig 4. the literature revealed that presence of phenols and flavonoids in plants extract had been reported to be associated with antioxidative action in biological system, acting as scavengers of singlet oxygen and free radicals. thus, greater tpc and tfc had positive correlation with greater antioxidant activities. in this study, the high value tpc (212±2.742 mgqe/g) and tfc (112±2.42 mgqe/g) could be attributed to antioxidant property of c. trichotomum [19]. 3.5 dpph free radical scavenging activity the free radical scavenging activity of methanol extractives of c. trichotomum, m. philipinensis, d. deltoidea, d. bublifera, r. cordifolia, and v. articulatum were evaluated by dpph assay. table 4 show the value of % dpph free radical scavenging activity at different concentrations of ascorbic acid, c. trichotomum, m. philipinensis, d. deltoidea, d. bublifera, r. cordifolia, and v. articulatum. the graph of concentration against the corresponding percentage radical scavenging activity of different samples was plotted fig 5 and ic50 value was determined. ascorbic acid was used as the standard in this experiment. among six different plant extract, methanol extract of leaves of c. trichotomum had the ic50 value 40.62±0.16 g/ml and rhizome of d. deltoidea had the ic50 value 41.18±0.81 very close to that of ascorbic acid i.e. 26.64±0.14 g/ml respectively. remaining plants m. philipinensis’s leaves (60.45 ± 0.99 g/ml), v. articulatum’s bark (63.12±0.85g/ml), d. bublifera’s rhizome (69.58±0.15 µg/ml), r. cordifolia’s root (89.71±0.13 µg/ml) extract had moderate antioxidant activity table 5. literature revealed that the majority of the antioxidant activity is due to the flavones, isoflavones, flavonoids, anthocyanin, coumarins, lignans, catechins and isocatechins. the obtained ic50 of methanolic extract of test samples are supported by previously reported value of the same plants from different region with slight variation in value [20, 21]. compounds like trichotomoside (phenylpropanoid glycoside), isoacteoside, jionoside d had been isolated and proved to be responsible for the antioxidant activity of c. trichotomum [22–24]. strong antioxidant activity for acetone and ethanol extract of v. articulatum bark had been reported by kakpure, 2020 [25]. while weak antioxidant activity of methanol extract of v. articulatum bark extract had been reported for plant collected from kathmandu valley by kumal et al 2020 [26]. b. subba et al./ bibechana 20 (2023) 190-199 196 table 4: % inhibition of ascorbic acid, ct, mp, dd, db, rc, va at different concentrations concentration % inhibition (µ g/ml) ascorbic acid ct mp dd db rc va 20 64.06±0.56 52.5±3.23 35.97±0.85 64.37±0.51 45.23±2.82 1.54±4.04 23.73±2.34 40 75.45±0.05 61.76±2.74 45.27±2.65 65.52±0.08 50.59±2.8 2.97±3.63 55.49±7.74 60 86.79±0.06 69.86±1.52 55.52±0.52 64.85±1.33 51.27±2.86 20.91±2.56 57.99±0.65 80 92.5±0.06 75.40±2.17 60.44±0.99 64.22±3.31 52.44±2.85 54.89±2.01 58.49±19.25 100 94.06±1 78.09±0.64 65.23±0.66 65.55±0.20 52.77±3.73 59.02±3.56 59.62±23.22 figure 5: a plot of % free radical scavenging of methanol extracts against concentration of plant extract, and ascorbic acid. ic50 values of the plant extracts along with the standard ascorbic acid is tabulated below in table 5. table 5: comparison of ic50 values of different plant extracts with ascorbic acid sample ic50 (µg/ml) ascorbic acid 26.64±0.14 c. trichotomum 40.62±0.16 m. philipinensis 60.45±0.99 d. deltoidea 41.18±0.81 d. bublifera 69.58±0.16 r. cardifolia 89.71±0.13 v. articulatum 63.12±0.91 ic50 values are expressed as mean±sd (n = 3) 3.6 alpha-amylase inhibition test alpha-amylase inhibitory activity of plant extracts was determined from quantitative starch-iodine method. alpha-amylase inhibition % values at different concentrations of standard acarbose and six plant extracts are shown in the below table 6. the graph of concentration against the corresponding percentage radical scavenging activity of different samples was plotted fig 6 and ic50 value was determined. the percentage inhibition of different plant extracts versus concentration and antidiabetic activity in term of ic50 values of different extract are given in table 7. table 6: % inhibition at different concentrations of plant extract. concentration % inhibition (µg/ml) acarbose ct mp dd db rc va 40 53.07±1.02 30±0.29 22.89±4.89 13.81±3.06 45.01±3.99 60.96±3.05 37.37±5.33 80 60.73±0.95 41±0.82 30.46±0.39 13.61±1.23 59.01±3.5 63.09±3.09 53.56±0.25 160 67.12±0.95 49±1.13 40.22±0.20 18.77±3.42 57.31±2.4 57.21±2.01 57.12±2.8 320 81.31±0.59 65±5.47 45±2.56 32.08±2.42 62.46±3.2 75.20±1.89 54.92±0.21 640 88.20±0.58 70±0.29 47.24±4.21 38.92±1.23 64.16±4.5 70.52±1.55 56.1±0.69 where ct = c. trichotomum, mp = m. philipinensis, dd = d. deltoidea, db = d. bublifera,rb =r. cardifolia, va = v. articulatum. mean % inhibition are expressed as means±sd (n = 3). b. subba et al./ bibechana 20 (2023) 190-199 197 figure 6: inhibition of α-amylase activities by plant extracts. ic50 values of the six plant extracts along with the standard acarbose is tabulated below in table 7. table 7: comparison of ic50 values of different plant extracts with standard acarbose sample ic50(µg/ml) acarbose 119.063 ± 0.73 clerodendrum trichotomum 293.33 ± 0.81 mallotus philipinensis 554.18 ± 0.75 dioscorea deltoidea 785.15 ± 0.92 dioscorea bublifera 352.71± 0.59 rubia cordifolia 252.44 ± 0.55 viscum articulatum 344.58 ± 0.61 ic50 values are expressed as mean±sd (n = 3) here, ic50 value of standard acarbose was found to be 119.063 ± 0.73 µg/ml. c. trichotomum, m. philipinensis, d. deltoidea, d. bublifera, r. cardifolia, v. articulatum showed α-amylase inhibitory activity with ic50 value 293.33±0.81 µg/ml, 554.18±0.75 µg/ml, 785.15±0.92 µg/ml, 352.71±0.59 µg/ml, 252.44±0.55 µg/ml, 252.44± 0.55µg/ml respectively in table 7. there was a dose dependent increase in percentage inhibitory activity against α-amylase by all the six plant extracts. the result obtained here are in good correlation with previously reported results [27,28]. strong antioxidant and antimicrobial activity of leaves of c. trichotomum collected from dhankuta had been reported by subba et al, 2016 [29]. among six medicinal plants, leaves of c. trichotomum is found to be good property as antidiabatic, antioxidant, phenolic and flavonoid content with low cytotoxic effect. 4 conclusion the present study revealed that the methanolic extract of the c. trichotomum’s leaves, m. philipinesis’s leaves, d. deltoidea’s root, d. bublifera’s root, r. cardifolia’s stem, v. artculatum’s bark has notable antioxidant potential and inhibitory activity on the α-amylase enzyme. these effects would be due to its important phenolic composition, whose quantitative study has revealed the varied presence of polyphenols and flavonoids. these results could justify the use of these plants in traditional medicine for the treatment various health problem including type 2 diabetes and complications. our study is the first examination of anti-α-amylase capacities of c. trichotomum leaves extract. study of chemical constituents of these plants using sophisticated technologies like nmr, hplc, etc. can provide a way for extensive research that can be used for commercial drug production with lesser or no side effects. acknowledgement central 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[29] b. subba, c. srivastav, and r. c. kandel. scientific validation of medicinal plants used by yakkha community of chanuwa vdc, dhankuta, nepal. springerplus, 5:155, 2016. introduction materials and methods collection of plant materials and preparation their methanol extract chemicals and reagents qualitative phytochemical analysis brine shrimp lethality assay determination of total phenolic content in the plant extracts determination of total flavonoid content in the plant extracts antioxidant assay alpha-amylase inhibition assay results and discussion plant collections phytochemical screening brine shrimp bioassay total phenolic and flavonoid contents dpph free radical scavenging activity alpha-amylase inhibition test conclusion