Biophysical, Biochemical and Histopathological Studies on the transport across Duramater

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1 Biphysical, Bicheical and Histpathlgical Studies n the transprt acrss Duraater DISSERTATIN SUBMITT^ T THE ALIGARH MUSLIM UNIVERSITY, ALIGARH FR THE DEGREE F MASTER F PHILSPHY IN CHEMISTRY BY MHD. IBRAHIM DEPARTMENT F CHEMISTRY ALIGARH MUSLIM UNIVERSITY ALIGARH (INDIA) 1987

2 I 2 AUG 1981 DS1120

3 DEDICATED T * MY ALMA MATER * MEMRY F HER FUNDER, SIR SYED AHMAD KHAN * AND ALL MY RESPECTED AND BELVED TEACHERS.

4 DEPARTMENT F CHEMISTRY ALIGARH MUSLIM UNIVERSITY, ALIGARH U.P., (INDIA). C E R TIFICATE This is t certify that the dissertat entitled, "BIPHYSICAL, BICHE24ICAL AND HISTPATHLGICAL STUDIES N THE TRANSPRT ACRSS DURAMATER" ebdied the wrk carried ut by Mr, MHD. IBRAHIM under ur supervis. He has fulfilled all the requireents fr the Degree f MASTER F PHILSPHY (M.Phil,) Cheistry, regardg the nature and perid f the vestigatal wrk. The wrk cluded this dissertat has nt been subitted fr any ther degree and unless thertvise stated, all rigal. is (Dr, Hasan^'^Arif) (Supervisr) M.Sc., Ph.D., A.R.I.e.(Lndn) M.R.N.A. Sc. (Ned), F. Ind. Sc, Che. (India; Reader, Departent f Cheistry, A.M.U. Aligarh M^^JA' <H^ Ct'W (Prf. Dr, Mahdi Hasan) (C-Supervisr) MBBS, MS(Hns.), Ph.D., D.Sc, FAMS., FICS, FRI^IS. Prfessr and Chairan, Departent f Anaty, J.N. Medical Cllege, A.M.U. Aligarh lhd, Ahad) (C-Supervisr) MBBS, MD (Medice), D.M. (Cardilgy), M.N.A.M.S. (Cardilgy Prfessr Medice, J.N.Medical Cllege, A.M.U. Aligarh-2021.

5 ACKNtfLEDGEMENTS I a deeply debted t Dr. Hasan Arif, Reader, Departent f Cheistry, Aligarh Musli University, Aligarh, y Supervisr and entr, fr his benvalent supervis, sustaed terest and war encurageent thrughut the curse f this wrk. nly his untirg and pastakg effrts ade it pssible fr e t cplete and subit this dissertat tie, I d nt fd wrds t express y prfund gratitude and huble regards t Prf. (Dr.) Mhd, Ahad, Cardilgy Unit, J^]MC and Medical Supertendent, JNMC, Aligarh Musli University, Aligarh, y c-guide, fr his substantial and estiable help at every stage f this research wrk. I feel re than I can express y gratitude t Prf, (Dr,) Mahdi Hasan, Chairan, Departent f Anaty, JNMC, Aligarh Musli University, Aligarh, y c-supervisr, fr his sypathetic attitude and cnstant help, A grateful acknwledgeent is ade t Prf, M,S, Ahad, Chairan, Departent f Cheistry, fr prvidg all research facilities and cnstant encurageent. I wuld like t place n recrds y scere thanks t Drs. Nasee Khan, Arshad Mehd, Shahid, W.H, Khan, I.A, Qasi

6 and M/s. Sheb Khan, H.R. Khan, Shahabuddln, A.A, Hasti, Shabbir Aliad, Akbar All, Abul Hasan, Naseer and any clleagues and friends fr their sustaed effrts akg this wrk success. My scere heartfelt thanks t y parents, brthers and sisters wh were an dispensable asset thrughut y rdeal. I a bliged t Mr, Mhd, Athar Qureshi fr the skilful tjrpg f this dissertat. Wrds fail t ce by which I can express y appreciat t y wife Sajida Usani and well wishers Mrs. Rizwana Shabbir, Ruby Usani, Suby Usani whse lve, affect and encurageents ade y effrts succeed. ( MHD. IBRAHIM )

7 CNTENTS Page N. CHAPTER I - Review f Literature : Intrduct ---_-. Significance f the present study Duraater _- Structure f Duraater Structure f Bilgical ebrane Cpsits Transprt phenena thrugh bilgical ebranes. Theries CHAPTER. II - Materials and Methds CHAPTER III - Results and Discuss 40-A9 Bibligraphy 5-58

8 KXfisf F uxauasfis

9 Durg the last few decades, transprt prcesses ccurrg acrss the binbranes have acquired uch iprtance. Because f the applied significance f biebrane, this field has attracted the attent f vestigatrs fr varius disciples e.g. Cheists, Bicheists, Biphysicists, Physilgists, Pharaclgists, Cardilgists and Cheical Engeers, T understand the behaviur and prperties f biebranes, an extensive wrk is available and wrk this field is cntributg significantly t physical well beg f ankd. The review f a nuber f publicats f the literature (1-10) shws that this field is develpg by leaps and bunds and an addital chapter this field is expected t appear. Extensive wrk has been dne by a nuber f vestigatrs n trace eleents (11-15) bdy tissues, islat and purificat f bi-ebrane (16), transprt prcesses ebranes (6-8, 17-20) and histpathlgical prperties cludg electix)n-icrscpic studies (21-28) and biphysical prperties f del ebranes (29-35). SIGNIFICANCE F THE PRESENT STUDY: Duraater has gaed great attent durg the last half f a decade because f its useful rle the prduct f Cardiac valves (36-39). Tlhie literature has been reviewed a

10 nuber f publicats revealg se 5)rtant prperties and applicats f durarnater e.g. echanical suitability f glycerl preserved huan duraater fr cnstruct f prsthetic cardiac valves (A). Mrphlgical alterat duraater encephali used as ventricular wall grafts the cane heart (^1), the use f xengenic cervical duraater, a new anistrpic tissue fr heart valve prstheses (42), the use f dural allgraft surgical repair f large defect f the abdal wall (43), surgical treatent by renal crsetage with lyphlized huaui dura (44), the echanis f dural pereability t narctics and lcal anaesthetics (45), the use f duraater repair f large vesical wall defects and partial replaceent f uretr (46-47) and study f cerebrspal fluid pharacketics and drugs peneterat f duraater pig (48), These extra-rdary publicats f recent ties ay, t a great extent, cntribute t the valuable and helpful rles the prduct f biprsthetic cardiac valves and understandg the behaviur f bld bra barrier which is s vital fr transprt f drugs and txs the central nervus syste. It has been prved fr clical fllw-up f patients that certa biprsthetic cardiac valves (prce valve) get calcified with the passage f tie. Altered calciu etablis has been plicated the prduct f calcificat. Attepts have als been ade t exae tiie electrn icrscpic and histpathlgic alterat calcified prce cardiac valves (49-50).

11 Unfrtunately, hwever nthg has been s far achieved this cnnect. Biprsthetic valves prepared fr duraater are expected t prve superir t artificial because they will nt require lng ter anticgulant therapy. Because f lesser fancial iplicat their anufacture and atenance the tjriird v/rld cuntries like urs, such valves are f special significance, Metals are iprtant cnstituents f all bilgical tissue cludg bi-ebranes, they affect biphysical prperties f the binbranes by virtue f their electrphysilgical effects. These etals are als iprtant cpnents f several etallenzyes. They als have cnsiderable effect n the active transprt thrugh ebrane by fluencg the varius ebraneenzyes, Duraater has tw vital functs. It prvides the prvis f physical prtect t bra and cntrls active veent f fluid thrugh itself. It is thus f great value that alterat etal envirnent nt nly will fluence the electrphysical prperties f this biebrane but will als fluence active transprt thrugh it by alterg the funct f varius ebrane enzyes. N study dealg with the effect f alterat f etal envirnent and electrlytes n physical, electrphysical and bicheical prperties f duraater is s far available. The present study is significant nt nly understandg the varius bilgical functs f duraater but it will als help

12 understandg the echanis which, turn, will fluence the lng ter prgnsis f the cardiac valves prepared fr duraater. It has bvius therapeutic iplicats, DURAMAIER Duraater is a thick and dense enelastic ebrane. The prt f it which enclses the bra (cerebrai-duraater) differs several particulars fr that which surrunds the spal crd (spal duraater). The spal dura crrespnds t the ner fibrus layer f the cranial dura, with which it is cntuus at the fraen agnu. The vertebrae have their wn peristeu.. Bth ner and uter surfaces f the spal dura are cvered by a sgle layer f flat cells, and dura is separated fr peristeu by a slender epidural space which are anastsg venus channels lyg fatty, arelar tissue (28), The cerebral duraater les the terir f the skuhand serves the tw fld purpse f an ternal peristeu t the bnes, and a supprtive ebrane fr the bra. It is cpsed f tw layers, an ner r engeal and an uter r endsteal, but these are clsely united, except alng certa les where they separate t enclse the venus suses wirdch dra the bld fr the bra. The duraater adheres t the ner surfaces f the cranial bnes, and sends bld vessels and fibrus prcesses t the, the adhes beg st arked at the sutures, at

13 the base f skull, and arund the fraen agnu. The bld vessels and fibrus prcesses are trn acrss when the duraater is detached fr the bnes, and cnsequently, the uter surface f the ebrane presents a rugh and fibrillated appearance, the ner surface is sth. The endsteal layer f the duraater is cntuus thrugh the sutures and the f ra f the skull with the pericrani\a, and thrugh the superir rbital fissure with the peristeal lg f the rbital cavity. The engeal layer prvides tubular sheaths fr the cranial nerves as the latter pass thrugh the fraa at the base f the skull. utside the skull these sheaths fuse with the epeuriu f the nerves, and the sheath f the ptic nerve is cntuus with the sclera f the eyeball. The engeal layer f the cerebral duraater is reduplicated wards as fur prcesses r septa which partially divide the cranial cavity t a series f freely cunicatg spaces fr the ldgeent f the subdiviss f the bra, (1) The Falx Cerebri, s naed fr its sickle like fr, is a strng, arched prcess f duraater which descends vertically the lngitudal fissure between the cerebral heispheres. It is narrw frnt, where it is fixed t the crista galli f the ethid bne, and brad behd, where it blends the edian plane with the upper surface f the tentriim cerebelli;

14 the narrw, anterir part is th, and is frequently perfrated by nuerus apertures. The upper arg f the falx cerebri is cnvex and attached t the ner surface f the skull n each side f the edian plane, as far back as the ternal ccipital prtuberance, the superir sagittal sus runs alng this arg. Its lwer arg is free and cncave, and cntas the ferir sagittal sus. The straight sus runs alng its attachent t the tentriia cerebelli. (2) The tentriu cerebelli is a crescentic, arched laa f duraater which cvers the cerebellu, and supprts the ccipital lbes f the cerebru. Its cncave, anterir brder is free, and between it and the drsu sellae f the sphenid bne there is a large val peng, the tentrial cisure, which is ccupied by the id bra and the anterir part f the superir surface f the veris f the cerebellu. Its cnvex uter arg is attached (a) psterirly, t the lips f the transverse sulci f the ccipital bne and the pster-ferir angles f the parietal bnes, where it cntas the transverse suses; (b) laterally, t the superir brders f the petrus parts f the tepral bnes, where it enclses the superir petrsal suses. Near the apex f the petrus part f the tepral bne, the lwer layer f the tentriu is puched frwards and laterally, beneath the superir petrsal sus, t fr a recess between the endsteal and engeal layers f duraater f the iddle cranial fssa. This

15 recess is called the trigeal cave and envelps the rts f the trigeal nerve and the psterir parts f its sensrygangl; it is a little re extensive belv/ than abve the gangl. The evagated engeal layer terates by fusg with the anterir part f the gangl. At the apex f the petrus part f the tepral bne the free brder and attached periphery f the tentriu crss each ther; the anterir liits f the free brder are fixed t the anterir clid prcesses f the sphenid bne, whilst thse f the attached periphery end n the psterir clid prcesses f that bne. (3) The falx cerebelli is a sall, sickle-shaped prcess f duraater which is situated belw the tentriu cerebelli, and prjects frwards t the psterir cerebellar ntch. Its base, directed upwards, is attached t the psterir part f the ferir surface f the tentriu cerebelli, the ediu plane, its psterir arg cntas the ccipital sus, and is fixed t the ternal ccipital crest; its apex frequently divides t tw sall flds, which are lst n the sides f the fraen agnu. Hasan and Das fund the falx cerebelli duble 76 f 1 cadavers, (4) The diaphraga sellae is a sall, circular, hrizntal fld f duraater, which frs a rf fr the sella turcica and alst cpletely cvers the hypphysis, a sall peng its centre transits the fundibulu.

16 8 TPiE STRUCTURE F THE DURAMATER It is basically fibrus, wiriite cllagen fibres predatg with an adixture f elastic fibres. The cllagen fibres are densely arranged laae, which the fibres are ften arranged a parallel anner, with wide angles between these grupgs adjacent laae, prducg a latticed appearance particularly easy t see the tentriu cerebelli. The cerebral duraater is ften described as cnsistg f tw layers, an endsteal layer actg as the peristeu f the cranial bnes t which it is attached, and a engeal r cerebral layer ternal t this. This descript wes re, perhaps, t the separat where venus suses ccur and t the splittg f the cerebral duraater at the fraen agnu and ptic canals than t any arked histlgical differences (28,51), The saller branches f the engeal vessels are, f curse, largely the endsteal reg, sce they are, despite their nae, priarily peristeal distribut. Fibrblasts ccur thruiihut the duraater, but steblasts are naturally cnfed t the endsteal level. The elastic fibres separate the laae f cllagen fibres, wliich are als and re extensively separated by lacunar spaces cnsidered by se t be cntuus with the subdural space (vide fra). These spaces are aly cnfed t the ner* part f the duraater. In su, therefre, there are a nuber f features which distguish the external fr the ternal levels f the dura, but

17 there is n discntuity r any ther kd f bundary upn which a clear distct f a bilaar nature culd be based. At all fraa the craniu the endsteal eleent is cntuus thrugh the with the external peristeu. At the sutures, befre their fus, the endsteal eleent is cntuus with the sutural ebrane, and the duraater is re strngly attached at these lcats. Elsewhere it is re easily detached fr the cranial bnes. The engeal eleent is cntuus thrugh the apprpriate fraa v/ith the dural sheaths f the spal crd and the ptic nerves. At ther fra it is said t be 'pierced' by the nerve r vessel passg thrugh the, but is perhaps re accurately described as becg cntuus with pereuriu r adventitial sheaths. Thugh clse appsit t the arachnidater ternal t it, the duraater is very easily separated fr the frer, as exeplified by the ccurrance f subdural haerrhages between the. Althugh cntrary views have been recrded, there is little dubt that a layer f flattened 'esthelial' cells is a cnstant feature f the ternal aspect f the duraater and that sall aunts f fluid, thugh as a ere capillary fil, usually exist as a zne f ptential separat between arachnid and dura. The existence f epithelial cells this reg, have been cnfired by electrnicrscpy. These are regarded as neurtheliu. These are described as arranged several irregular layers, with abundant tercellular

18 10 spaces, devid f cllagen fibres. The cells display tnfilaents and desses, but these are cnsidered as the epitheliu is fragile and easily trn apart t prduce a 'subdural space' (51,52). The subdural space is a ptential space between the duraater and arachnidater. It cntas a fil f serus fluid which istens the surfaces f the ppsed ebranes. It des nt appear t cunicate with the subarachnid space, but is cntued fr a shrt distance n the cranial and spal nerves, and is free cunicat with the lyph spaces f the nerves. Arund the ptic nerve it is cntued as far as the back f the eyeball. The significance f subdural space ters f funct reas a atter f arguent rather than denstrat. Pssible cnnexs with venus channels n the ne hand and with hypthetical lyph spaces the substance f the duraater n the ther have been claied and disclaied (53). The evidence f electrn icrscpe bservats is agast the ccurrance f any specialized cells f epithelial type the duraater, apart fr its arachnid surface, all the dural fibrblasts beg f siilar appearance. The dural lacunae ay fact be artefacts, certaly the evidence available refutes the passage f significant aunts f a lyphatic fluid fr the dura t the subdural r subarachnid spaces (5.4). In the case f spal duraater, hwever, there is undubtedly a lyphatic draage regard t the extradural adipse tissue, and this ay als clude the dura itself (55).

19 11 STRUCTURE F BILGICAL MHVJBRANE In general^ebranes ay be classified as natural and artificial ebranes. Natural ebranes existg bilgical systes (biebranes) are cnsidered t have a fundaental unit ebrane structure which is a bilecular leaflet f lipid with their plar grups riented twards the tw aqueus, the extracellular and the tracellular, phases f the cell; prte is suppsed t existdse t the plar heads f the leaflet (56-58). T understand functal activities f a biebrane, knwledge f its structural rganizat is very iprtant, The first iprtant del explag ebrane rganizat and functs v/as pstulated by Danielli and Davsn (59). An iprtant f it is the prpsal that ebranes cnta a cntuus hydrcarbn phase cntributed by the lipid cpnents f the ebrane. Se years later tiiis hypthesis v/as dified and refed, particularly by Rbertsn (60), t the unit-ebrane hypthesis. The unit ebrane was prpsed t cnsist f a bilayer f ixed plar lipids, v/ith their hydrcarbn chas riented wards t fr a cntuus hydrcarbn phase and their hydrphilic heads riented utward. Each surface was thught t be cated with a nlecular layer f prte lecules, with the plypeptide chas extended fr. Later ther vestigatrs prpsed glbular r subunit del, which ebranes were viewed as cnsistg f sheets f recurrg lipprte

20 12 subunits x diaeter- 4.0 t 9.0 n, reseblg the subunit structure f se ligetric prtes r the cats f se viruses. Hwever, glbular dels have failed t accunt satisfactrily fr any prperties f ebranes, S.J. Sger and G.L, Niclsn (61), pstulated the st acceptable and satisfactry del f Bbrane structure t date appears t be the fluid saic del. This del pstulates that the phsphlipids f ebranes are arranged a bilayer t fr a fluid, liquid-crystalle atrix r cre. In this bilayer dividual lipid lecules can ve laterally, endwg the bilayer with fluidity, flexibility, and a characteristically high electrical resistance and relative ipereability t highly plar lecules. The fluid-saic del pstulates that the ebrane prte are glbular, t accunt fr their high cntent f helix. The varius ebrane prtes v/uld fr a saic- like structure the therwise fluid phsphlipid bilayer. This saic is nt fixed r static. The fluid saic del accunts satisfactrily fr any features and prperties f bilgical ebranes. It prvides fr ebranes with widely different prte cntent, dependg n the nuber f different prte lecules per unit area f ebrane, it prvides fr the varyg thickness f different types f ebranes; it can accunt fr the electrical prperties and penneability f ebranes and it als accunt fr the bservat that se prte cpnents f cell ebranes ve the plane f the ebrane at a rather high rate (6).

21 13 Ruser aiid c-wrkers (62) have suuiarised their cnsiderable analyses f any ebrane systes as under: 1) All anial cell ebranes cnta phsphlipids. The sae classes f phsphlipid are fund vertebrates and vertebrates. Se ebranes (e.g, yel) cnta glyclipids vereas thers d nt. nly certa ebranes cnta sterl. 2) Plasa ebranes, (cell surface) f the endplasic reticulu, nuclear ebranes and itchndrial ebranes fr the sae species have different cpsits. All differ quantitatively and t se extent qualitatively the classes f lipid present e.g. Plasa ebranes r elabrat f these appear t cnta st f the glyclipid f the cell, 3) The prprt f the different phsphlipids vary greatly and the ttal aunt as well as the types f bth ceraide plyhexsides and ganglisides is very different different species. Data fr whle rgans dicate that plasa ebranes fr different cell types f the sae species ay vary cpsit, 4) The fatty acid cpsit f each class f lipids fr different rganelles and rgans f ne species, as well as fr different species, is variable. This is true even v/hen the classes f lipids are the sae the different structures, Individuality is thus e3q)ressed st clearly differences fatty acid cpsit.

22 la Cpsits; Hst f the ebranes cnta abut A percent lipid and 6 percent prte, but there is cnsiderable variat. At ne extree the ner itchndrial ebrane cntas nly abut 20 t 25 percent lipid, and at the ther the yel ebrane surrundg certa nerves ay cnta upt 75 percent lipid. The lipids f ebranes are largely plar, phsphglycerides, predate, with uch saller aunts f sphglipids. In fact, nearly all the plar lipids f cells are lcalized their ebranes. Endplasic reticulu and rganelle ebranes cnta relatively little chlesterl r triacylglycerl, v/hereas the plasa ebrane f se cells f higher anials cntas uch chlesterl, bth free and esterified. Mebrane can be classified tw categries. The extrsic r peripheral prtes are nly lsely attached t the ebrane surface and can easily be reved sluble fr by ild extract prcedure. The trsic r tegral prtes, which ake up 70 percent r re f the ttal ebrane prte are very tightly bund t the lipid prt and can be reved nly drastic treatent (61). The islat and characterizat have been studied by the histcpatibility antigens f plasa ebranes (63-65). These lecules are glycprtes and are expsed n the uter surface f the ebranes. Pssibly, they are assciated v/ith ebrane particles (66) as are the glycprtes f the erythrcyte ebrane. T\f a glycprtes are fund by SDS-gel

23 15 electrphresis the ebranes f rat liver and kidney cells (67), Kela cells (68) and use liver cells (69) f ebranes. Electix)n icrscpy has revealed that ebranes have a trilaar structure with a ttal thickness between 7.0 and 9.0 niii, dependg n the type f ebrane. Transprt Phenena thrugh Bilgical Mebranes The cell is a lcus f cneical structure and ftcts which cntuity f prpertiesis ataed the idst f drastically different and ever changg envirnent. ne iprtant ecihanis by which the cell achieve the cnstancy is due t the regulat f veent f aterials t the cell and ut f it. Even with the cell,aterials are nt unifrally distributed. Here als a precise regulat f terchange f aterial is encuntered. T achieve this regulatry cntrl the cell utilizes its delicate ebrane f abut 75A thiclaiess. At ne tie it was thught that the veent f substances thi'ugh tiie ebranes was detered slely be cncentrats gradient, but veent agast cncentrat gradient have been bserved st bilgical tissues r biebranes. Fr exaple K is usually accuulated plant and anial cells t a cncentrat any ties higher than tliat f the ediu surrundg the cell such transprt requires energy by the cells and has been called active transprt r net etablically lked transprt,

24 16 while that v/liich respnds t a cncentrat gradient is passive transprt. The passive transprt is isleadg the case f cells, because while the cell ay be passive with respect t the veent, n directal veent acrss cell ebrane ccurs v/ithut the expenditure f energy by the lecules vlved. Ketic energy accunts fr the rand veent f the lecule and the cheical ptential energy f higher cncentrat f a particular substance utside the cell than side the cell. This gives effective direct t the veent, is dissipated as the lecule f that substances utside f the cell ve t the cell and reduces the cncentrat gradient f substances between the utside and the side f the cell. The ter active transprt is used different ways by varius authrs but n satisfactry defit has been given as yet (70), Advanced and cplicated liv/ledge cncerng the nature f active transprt is nw available. With re advanced knwledge, any issues have been settled but several cntrversies are yet t be slved. There sees t be general cnsensus that a carrier is vlved bth facilitated and active transprt and the carrier is prte. It sees that the carrier prte is itself nt an enzye but lked t an enzye r enzyes and thus energy fr ATP r fr se siilar higli energy phsphate bnd is ade available fr active transprt. Cnsiderable evidence has accuulated ptg t the Na-Pup as priver suggestg that

25 17 the veent f tner lecule is the case f c-transprt but situat is nt general and the prble reas pen. A nuber f theretical appraches have been given which can cllectively be suarized as (a) Irreversible therdynaic apprach (8), (b) Cheical engeerg apprach (8), (C) Activat barrier ketic apprach (8), (d) Phenenlgical equat f t apprach (8), The thrust f the theretical descript has been the terpretat r explanat f transprt prcesses and the easured effect resultg fr the pressure differences, teperature differences, activity differences, ptential differences and current thrugh ebranes. Apart fr varius theretical cncepts used the vestigat f ebrane. ne f the st iprtant apprach the ebrane study is the applicat f electrcheical prciple. Electrcheistry the ebrane study is pertent at tl:iree levels (71). (1) The develpent f teciique with applicat t experiental phenenlgy cludg current vltage tie cncentrat behaviur, (2) Matheatical dellg iplied by experients and tested agast experients, (3) Experiental varificats f dels ters f lecular prcesses and prperties and als cludes deterat f

26 18 theretical paraeters by electrical ethd and by cpleentary nn-electrcheical ethds e.g. Physical, ptical, ESR, NI4R, Raan, Flurescence, T-jup techniques etc. The rle f ketic and equilibriu paraeters can be deduced r ferred fr transient and steady state easureents f current and ebrane ptential as a funct f cheical cpsit, cheical treatent and teperature. A pssible apprach t dellg begs with the assupt f ebrane as a lear systes t which laws f netwrk thery (72) ay be applied. Anther apprach begs by slut f basic electrdiffus law f transprt with equilibriu r ketic bundary cndit, rder t deduce frs fr syste and funct which satisfy the data. It is t extensive t ent the whle literature describg the transprt phenena. The significant wrk this field is given as fllws: V/ilbrandt and Rsenberg (73) reviewed the general features f carrier transprt systes, bth equilibratg td transpi'tg uphill. They als cpared reprted bservats. It was pted ut that a nuber f ketic features are shared by "absrpt systes" which vlve bdg f substrate at fixed sites rather than n bile carriers, but that cunter transprt and cpetitive activat are criteria fr bile bdg sites. A nuber f pertent bservats reprted the literature were discussed

27 19 (74), Recent bservat ented here cncerng cunter transprt (75-78) which were nt terpreted by se authrs alng the sae les, are als pertent this cntext, Katchalsky et al, (79,80) elabrated their descript f bilgical transprt prcesses ters f irreversible therdynaics. Fr bilgists with liited atheatical sphisticat Katchalsky (81) has suarized the st iprtant aspects f his wrk, A nuber f cnsiderat based n the cncept f irreversible therdynaics have been entertaed. Rsenberg and Wilbrandt (82) crrelate facilitated diffus with active transprt by assug a cnvers f a "nnactive" carrier t an "active" ne. The transprt syste fr aalian tissues appear t be cnstitutive and fnly bxd t the ebrane. In general the transprt systaas fr st f the a acids icrrganis are cnstitutive, while fr sugars nly glucse transprt activity appears t be cnstitutive (83). The rest f the sugar transprt systes are ducible. Earlier ketic studies n anial cells prvided evidence fr ebrane ediat f slute transprt (84). The nature f the ebrane cpnents, which have been tered "carriers", is nt v/ell defed. These ketic studies have, hwever, prvided us with a descript f the nuber and kds f transprt systns present

28 20 the ebrane as well as a basis fr recgnit f the receptr sites (85). A nuber f studies n perfused hearts f rats v/ere reprted fr Park's labratry (86,87) and suarized by i-lrgan, Pst and Park (88). rate f glucse uptake several fld. It v/as fund that sul creases the The iprtant relatsliips between the alkali etal s and the physilgy f anial cells have lng been recgnized. very extensive cverage f the subject f the (Na -K ) ATPase and alkali etal transprt has been ade by Bntg (89). Addital reviews cverg this area are the fllv/g, Hez (90), Albers (91), Pst (92), V/hitta and V/heelar (93). Transprt f calciu t vesicle preparats f sarcplasic reticulu has been extensively studied by Hasselbach and Kane - Zawa et al. (94,95). These vesicles cnta a agnesiu and ATP-dependent transprt systes fr calciu wldch can pix)duce and ata large calciu gradients. Zerahn (96) and cvrrkers have designed a ethd fr direct study f Li and Na cntent f frg sk. Ussg (97) at Harvey lecture suarized his v/rk abut the water and electrlyte transprt frg sk. He cnsidered epithelial layer f frg + sk as a tw ebrane syste v/here Na enters the first livg cell layer by a sdiu, selective ebrane. Fr this layer the A

29 21 Na either flw t next cell layer via cell "bridge" r can extrude t the terspaces which is pen twards the side. The basal ebrane represents anther tight resistance t free flw f Na and is the site f the pup. Chck and Titus (98) gave an idea f echanis f transprt ti.rugh bilgical and artificial ebrane and als discussed the effect f alkali cat n enzyic activity and Na dependent transprt f plar rganic substances, Larsen and Rasussen (99) studied abut the rle f ebrane ptential fr chlride transprt acrss tad sk, Hernandez et al, (1) easured the apparent transprt nubers fr a hgenius passive ebrane separated by bary electrlyte (NaCl), Trres et al, (101) carried ut the electrphysilgical characterizat f transprt (Na and Cl~) acrss islated frg sk, Devillarde et al, (102) easured the ebrane ptential and deduced the ic transprt nubers side the prteic phases. Navebska, Kter and Kujawski (103) studied the isthennal transprt f s acrss the perflurated Naf ebrane cntact with NaCl, n the basis f irreversible thenndynaic transprt, NsJcagaki et al, (104) gt an frat n the assyetry f the ebrane structure by analyzg the assyetric ebrane ptential, Santha Kuari, Sheela and Radhakrista Murty (105) reviewed the echanis f transprt acrss cell ebrane with reference t nerve cell. Ussg (I06) detered the e,.f. f active Na transprt

30 22 sk epitheliu v/ith the help f steady state flux equat, Zeevi, Aeera et al, (107) discussed the selective transprt f Li acrss lipid bilayer ebrane. The electrical prperties e,g, capacitance, resistance and cnductance f bilecular phsphlipid ebranes v/ere easured by Laugher et al, (108), Benavente (109) easured ebrane ptential f prus ebranes and calculated apparent transprt nuber f cat fr diffus ptential equat with the frae wrk f therdynaics f irreversible prcess. Theries: A steady electrtive frce (e,.f,) arises between sluts f an electrlyte f different cncentrats at cnstant tanperature and pressure when they are separated by a unifr ebrane that cnta fixed izable grups, Txiis e,,f, usually called the ebrane ptential, has been the subject f any theretical and experiental studies ( ), The electrical ptential arisg acrss the ebrane separatg different salt slut are usually easured by cntributg a cell f type. Reference Electrde Slut Kebrane Slut C Reference Electrde uij Electrde ptential Dnnan ptential Diffus Ptential J. Dnnan ptential J Electrde ptential

31 23 The reference electrde ay be reversible electrde either Aii-AgCl chlride slut r calel electrde, cnnected t the slut via KCl-Agar Bridges. In the case f calel electrde cell ptential give directly the ebrane ptential (liquid junct ptential beg ignred). These type f the easureent are used t characterize the selectivity f the ebrane. The agnitude and sign f the ptential depends upn the nature f the ebrane and pereatg species. Varius ebrane phenena have been successfully crrelated a quantitative anner by the therdynaics f irreversible prcesses ( ). This type f treatent hv/ever, des nt prvide frat abut the actual echanis which prduces bserved ebrane ptential. The earlier thery f Terell (1 17) and Meyer and Si ever s (33) ppularly knwn as T.Ki.S. thery and its varius refeents ( ), which all had been based n a fixed charge ebrane del, were criticized by Hills (118), wh shwed tliat these theries are adequate t expla experiental results n erabranes v/hich there is an cplete ic selectivity. Kbatake (119) tegrated flw equat prvide by therdynaics f irreversible prcesses t derive an equat fr the ebrane ptential. The wrk described this dissertat is aly cncerned with the transprt studies thrugh the ebrane and ptentietric evaluat f ebrane charge density. The etjrids used are f

32 Ik (i) Kbatake et al. (31,35,120) and (ii) Nagasawa et al. (30) based n the therdynaics f irreversible prcess. Brief accunt f the varius theries vlved (i) and (ii) ethds are given as xder: Kbatake et al. (31) derived the fllwg fal express fr ebrane ptential as given by equat (1) where 0^= - - -(2) andys = 1 + (KFG/ 1^ ) (3) where a^, /3 and G are the paraeters and have been assued t be dependent f salt cncentrats, AG) is the ebrane ptential, Cp and C. are the cncentrat s that C2/ C^ = 10, R, T, and F have their usual eangs, 1^ and 1_ are the ic bilities f + ve and -ve s. Kbatake et al, (31) derived tw liitg frs f equat (1) (a) when Cp beces sufficiently sall with Y= '-'p/^i ^ixe<i» equat (I) ay be expanded t give equat (4) as where I A/; _- ^...(5)

33 25 equat (4) dicates that tercept f plt )A(Z)-r I vs Cp give the value f y3. (b) It has als been shwn by Kbatake et al. (31) that at^t fixed, the verse f an apparant transference nuber ("t ) fr the c- species the ebrane is prprtal t the verse f the cncentrats. Here t is given by equat. A/J = (1-2 t^pp ) In^ (6) substitutg fr Aj2)fr eq, (1) and expandg the resultg express fr 1/. pwers f r; gives equat (7) as, ^app ^2 1/ = -L + iljdkjll±s±llijl± { -^ ), --(7) ^app ^-'^ 2(1-X.)2ir ' ^ ^ ' equat (7) dicates that tercept f plt 1/^ vs app ^2 give the value f (J, and if this predetered value f C and /3is put eq. (4) and eq. (7)» 0 can be calculated. The tw valuesf 0 btaed this way fr ppsite liits shuld agree with ne anther, Kbatake and Ka (35) derived anther equat fr ebrane ptential (E) startg with basic flw equat prvided by therdynaics f irreversible prcesses and usg different set f assupts naely (a) a cntribut f ass veent is negligible and (b) sall s d nt behave ideally

34 26 charged ebranes (35). Their results is, E = - S^ ^"In ^ + ( 2 ^ - 1 ) In ^ ^ ^ ^ F - C^ /4C^^ +j2^^x^+ (2^-1)j/} Ac/ + GI^ X ^ + j/x - In ^ ^ ^ (8) ' /4C/ +jzf^x ^ +/x wherej21 is the characteristic factr f ebrane electrlyte pair and represents the fract f cxter s nt tightly bund t the ebrane skeletn. The GTX is therdynaically effective fixed charge density f ebrane and ther ter have their usual significance. Fr)6 = 1 eq. (8) reduces t TMS ebrane ptential. Sce it is difficult t calculate X usg eq. (8), s Kbatake and Ka prpsed siple ethd usg fllwg apprxiate equat fr diffus cntribut t ef, f cell with transprt. E =. - ^ (1-2 i^pp) - ^ (9) Where T is apparant transference nuber f c-s eaibrane app -^-^ phase cparisn f eq. (8) and (9) give

35 27 A^^^ ^ ^ + 2c^-1 A^p^ ^ 1-2^ ^ ^ / cC- 1 y4^^2_^1+1 '^app=~i 3 + :^ (10) ^^ ^ In 0 2 In ^ where ^ = C//x When the cncentrat f external salt slut is large as cpared t JZ^X, i.e. when C^/^zfX = ^^/>)>1 then eq, (10) is expended t 1 1 The equat (11) dicates that plt f rj vs-r with a app ^1 fixed ^ give a straight le and the values f C and^x the cncentrated slut fr a given cbat f ebrane electrlyte can be detered fr tercept and slpe f the le. n the ther hand (35,120) the ass fixed transference nuber f c- the negatively charged ebrane iersed an electrlyte slut f cncentrat C is defed by '^app = VC-/ (Uc^ + VC- ) (12) where C +and C- are the cncentrats f cat aiid an respectively the ebrane phase. This equat is transfred t

36 28 Usg equat given by Kbatake et al. (35,120), fr activity cefficients, bility f sall s ebrane phase, and equilibriu cndit fr electrical neutrality. The difference between T' fr eq, (1j5) and t fr eq, (7) fr varius reduced cncentrats t_ is fund t be less than 2%, Therefre '^T^r^ ^"*^ "^ar^^^ ^^^ chsidered t be practically sae. app -pp Arrangeents f eq. (13) leads t the defit f perselectivity (Ps) by express _ 1 - app '^ ^ _ ^^^^ (4?^. 1 )^2 ^_ (2^-1)(1-T'^pp) The equat (14) can be used t fd the pennselectivity (Pg) fr the ebrane ptential easureents usg eq. (7). If the transprt nuber f c-s i ^ r t ) is zer. The e- ^ ^ app app brane will be perfectly selective and hence Pg tends t be unity. While if transprt nuber f c- has value free slut the Pg tends t be zer. Therefre ne ay cnsider that Pg defed by eq, (14) takes a value betv/een zer and unity dependg n external salt cndit fr a given systsu f ebrane and an electrlyte pair.

37 29 Recently Nagasawa et al, (30) develped anther etiid fr evaluat f charge density (^X) f the ebranes. If a ebrane separates tw aqueus slut f different cncentrats f an electrlyte, Nagasawa et al, (30) gave tw extree criteria (a) at the liit f lw cncentrat, it was fund that -E = - ^ In ^2 /C^ (15) F where E is the ebrane ptential, (b) At high cncentrats f electrlyte eq, (16) is applicable as,.en./1^=-^ (-^)-i- (16) ' -^T F ^ 2 'C^ equat (16) predicts a lear relatship between E/ ^ - vs 0 1, fr which px can be evaluated. The wrk described this dissertat is aly cncerned with transprt studies thrugh bilgical ebranes with special reference t buffal- dura. The varius ic prcesses (a) ic transprt, (b) ebrane ptential and (c) spatial distribut f s and ptential with the duraater have been thrughly vestigated by takg different cncentrat ratis f uni-univalent electrlytes. The st iprtant paraeters gverng the diffus f electrlytes, the dura-

38 30 ater is the electrical charge n the ebrane, Therdynaxuically effective fixed charge density have been calculated by (a) Kbatake et al., (b) Kagasawa et al., and (c) Kbatake and Ka based n therdynaics f irreversible prcesses. Applicability f Kbatake et al, and Nagasawa et al, thery have been tested n this ebrane. ther therdynaic paraeters f siple diffus e.g. energy f activat Ea, free energy f activat AF/, Enthalpy f activat A H', and Entrpy f activat A s^ will be evaluated. In rder t establish structural cplexities and characteristics f duraater, cnductance, ductance, capicitance and ipedence easureent, will be ade.bicheical and histpathlgical studies f this ebrane will be further vestigated. Bicheical studies will clude estiat f prte cntent, a acid prfile, carbhydrate, fat cntent, nvalent, divalent, trivalent electrlytes, ATP and ATPase, and trace etal estiat. The histpathlgical studies will clude the light as well as electrn icrscpic structure f this ebrane.

39 MinXiLS AND MBXIBD8

40 31 EXPERIMENTAL The duraater was reved carefully fr experiental anial (i.e. baffal aged between abut nths). The ebrane was iediately subersed ice-cld rger slut ( ) f 7, ph fr preservat f the ebrane tissues. The rger slut (fr duraater) had the fllwg cpsit gs/lit. NaCl 8,1, KCl 0.25, CaCl^ 0,14, Glucse 0.61, MgClp 0.11, NaHC^ 1.76, Sdiu hydrgen phsphate 0.07, Urea 0.13, The ebrane preserved Rger slut was kept at lv/ teperature befre the prceedg f experiental wrk. APPARATUS AND EXPERIMENTAL METHD: The diagra f the apparatus used fr the easureent f ebrane ptential is shwn Fig. A. It cnsists f tw half cells. The vertical feale jts A and A«attach t the each half cell prvide fr purg f electrlyte sluts and calel electrde B^ and Bp. The test ebrane the fr f disc was stalled between the flangs f half cell. Bth the sluts were stirred vigrusly. The cncentrats f the different electrlyte sluts was kept at 10 fld difference (i.e. ^2^^^ ~ ^^^ ^"^ ^^ ^^^ ataed. The easureents were ade usg saw Vernier Plentieter CAT. n, 371, the whle cell was iersed water therstat bath ataed at C. The varius salts slut

41 c c v_ XI E E c E l_ 3 (U E k\\\\\^ - a f E en E 0; x: a t * «< en LL

42 32 (NaCl, KCl, NH^Cl, KN^, KF, NaF, Na^S^) were prepared fr B.D.H, r A.R, grade (India) Cheicals deized water. MEASURa^IElSfl F MflViBRANE PTENTIAL; The ebrane was washed 5-6 ties with deized water fr the reval f rger slut befre bservg the ebrane ptential. The ptential develped by settg up a cncentrat cell f type described by Sllner and Gregr (123), Michaelis (124) and Marshall and Ayers (125) was taken as a easure f ebrane ptential. Electrcheical cell f the type was.used fr easurg S,C,E, Slut C. Mebrane Slut C S,C,E, C2 = 10 C^ ebrane ptential. The sae electrlyte with different cncentrat was used n bth side f ebrane. The dilute side is taken as negative. The washed ebrane was cut t disc type ccised with uth f the cell, and tightly held by hard clips. Thus ebrane ptential was easured. The experients were repeated with fresh slut f electrlyte and axiu ptential attaed was recrded fr 30 utes fr each sets.

43 M Q * X* c* v9 i/> * C9 n n t* i *-* r* «-l ih v^ C<4 r^ ^ ^ 33 \ I/) *- «0 <«r-) H f M ^ fi H CT* «0 c* r^ M C4 r^ <7> If) G tn 0\ 1» <N t* ««H v C4»» -( t»- T^ M rh t-t i/> C <7> \ en a\ X «\ \rt C\ t v <S r- a\ r-i r- > ^ V 0\ vn «a a i ft \ rl a \ N ur> C u> <» C 3 s M ui en C la l V en V a a c «IT) 5 <M r^ C r^ tn «cs «-( ra r-t en i «H T-I *H t \ c^ r C C en -9 i <8 n 5 * T4 3 0) n IS J ^1 G

44 CN 9 0) n Q CI u <M a (4 a u c 10 w a 91 s H P «p 8 \ \ «n N. r-l \ M X \ \ S \ X <D n t f< «^ s t- i «M * > * r^ <f n v C n k M rt «r-l T-«c«rl T-( r- C n *» t-< V <N 34 <T> <N» C a\ t^ 0\ \ v r» a» \ v C fn fl» v CD v v v U1 rj v n r- fh IT) B u a (ti S c r:? 2 C7V > P «UJ C v c P fl «fl

45 TH VD P l rn v <7> 35 u N V M «r~ r««h V * f-l»-l T^ M «ft ih»^ v M CTl T-«r- ^ u B3 u 0 d \5 vn «\»-( us f C.H v <s M V t l r» f n r- t ( v M n r v r-t v t r- ri ci r- n 9 s: «*> ft at a -H 4J Id u c a p c I p c \ <N X «\ \ «-i i-t TH r- - * rh f ^ 0\ V VH n W v r-t r- Mr a\ r-t C r '«\ tn r r- r V CD V C r4 > P < c s -r-l n X ^ r r v C4 a a\ v > ui av C «1^ t) a X \ f-l C V v v ;3 EH-.- n 9) n V W

46 I a) n n e c< +J d r) U n % -P a >M c H «P a. S) a -p c P (0 a p J3 \ \ tn «\ X 5: \ t r c B 2 n s «n ^ r< * l f (N v N C «u> r-t CT> t-( C <N a\ tn r-t v 0^ r- a\ f V ^ > v 03 C r-< C C r^ r r~ r CT\ tn» V (^ V V V C V <N. V 36 tn V V n

47 I 0) a -p (0. p - tn w tj -p 10 4) Id ) «1 a 4J (^ p c c u +J r: M-l 4J P n 4J a ri) u i I 4J C \ X. r 2: \ if) X X \ I 3 w CD tn vn 0 C 1^ * a^ C C rj «CD 0»-l t CD ex CD v v t-t 10 C «n V a» Q > «C ID r C C '^V. n 0 r T»" r <«0 y < V CD r \ n t 0 0 * \ 0 r c^ 0 C7» «C u> C * C <^ r» r- C tn r~ c^»0 t- CD r f~ r 0 IC TA 0 > H P «Cn 0 c C <^ C C 37 «H -P a a u «

48 I 1) i-t itl 1^ (3 U c (2 "+4 n C +) (D M -U c I]) V c *} c a) 14-1 a (0 p c <D P s I a p c p a ;:? 2: \ <N \ tn s \ V X «-( ^ 6 frl-' C C C C V V \» C ^ V r- M V t ri lf> r) * T-«u> r-t ft «-t ID *») r-(»-( TH * «H V r\ 1/1 c^ <N t rh en N"»H ^ n * M V CN th v r- n T^ <M»-4 t^ «n «-( V <-< l * r-i r.-t 'J <N r-t -*.-4 x> n r^ U) C V U1 r«v C V V V C V 0^ V 03 C V 0\,, * - V r r~ 0) Id B ^ V a ^1 c 3-9 i" Sab X E C C4 r- 38 "J > 4-> n3 a> c H (TV t-h.-4 c~ cs. 0 p C aj V s 1> c IT) ^^ XI g E TJ 0) t U w XJ 0

49 39 <! I H EH 01 U S 15 0) 15 H Til W <H C H P nj PI P c (U 0) Q) «H «H H 'd 15 '^ ^ 0) ft u u > B C -P CU C a-p 03 t<^ v C C^ t>irv t^ * fn!> CNJ > K v <x> cr> tn N C-- T- v v v^ r- C^ <t rn (M tn c^ rn c- <M <r v <M > tn en ^ ^ V ^ v «T" CD T- C tn 0^ CTi ^ C v ^ -d^ v 0) > H P cd (50 Q) a w H -p ft u a (1) t C H H <:

50 vss AMD wassn

51 ^0 Thugh this wrk a natural ebrane has been used cntrast t a del ebrane used by Kbatake (31,35,120), the results btaed are very uch agreeent. The results als general cnfir the basic requireents f the Kbatake (31,35,120) theries f ebranes. The values f ebrane ptential ( ^)^) easured acrss duraater separatg different cncentrat f 1:1 electrlytes ^1 -^ ^2 are pltted as a funct f lg with the rati f = "^p/^l ^^^^^ ^"^ ^^' Fig^J^es 1 & 2 shw' a plt f lg - vs. ebrane ptential ( ^^) r (E) illivlts. It is C + C2 'bserved that as the value f lg creases the ebrane 2 ptential decreases after an itial crease. This is very uch expected because the cnductivity f an electrlyte is a funct f cncentrat and as the cnductivity creases the ptential f the ebrane decreases. Anther terestg bservat is that the ebrane ptential generated acrss duraater has a negative value when the ebrane is used t se>parate cncentrated slut (cncentrated side taken as psitive). This eans that duraater is a psitively charged ebrane (an selective) Beg et al. (126). Kbatake et al, (31) derived the equat (1) as described previusly fr ebrane ptential {^^) which arises between tw sluts f 1:1 electrlyte f different cncentrat C. and Cp that are separated by a ebrane. Fr the analysis f data f

52 < NaCl NaF A KCl KF A KN3 NH/,Cl uj '' c <b ^-10 C s J e A Lg{Ci*C2)/2 ^-10 LU -A Lg(Ci*C2)/2 Fig. 1 & 2. Plts f bserved ptentials agast lg C1+C2/2 fe varius electrlytes with duraater^_^

53 A1 eq. (1) under tw cndits (a) the dilute range (b) the cncentrated range, tw liitg frs f eq"(1) i.e. eq. (4) and eq. (7) were btaed respectively t (a) and (h). (a) In the extree dilute range eq. (A) dicates the values f /3 and a relat between "^ and 0 can be btaed by evaluatg the tercepts and itial slpe f a plt fr A C5^ agast C^. Figure 3 shws a variat f reduced ebrane ptential (A^r ) with variat cncentrat fr lw cncentrat f electrlyte. With the crease f cncentrat, change is nt s significant. changes but the The value f tercept is equal t In ^, fr which A ay be evaluated. The va].ues f y3btaed fr dural-ebrane fr six uni-univalent electrlyte are given Table 7, (b) It is well knwn experientally that at fixed V, the verse f an apparent transprt nuber, (i.e. t^^_) f app c- species is prprtal t the verse f cncentrat Cp, when the salt cncentrat is high. Here t^^^ is defed by app eq. (6). The transprt nuber (t ) can be calculated fr eq, (6) fr varius electrlytes with the ebrane. The values f t calculated are listed Table-8. Figures 4 & 5 shw the app variat f verse f an apparent transference nuber f with variat verse f cncentrat Cp. As the verse f the cncentrat creases the verse f the apparent transference nuber als creases. This has been bserved nly at higher cncentrat range and this phenenn when tried dilute range was fund t be absent. This ay be because f the cncentrat

54 MaCl KN KF -^ A KCI NaF " NH^Cl C-.X102 fr varius electr Fiq. 3. Plts f A)^r /2.303^2 2 C2XI lytes With duraater.

55 L Fig-5 «NaCi NH/Cl NaF Q I/C2 t Fig. 4 & 5. Plts f 1/tapp Vs 1/C2 fr varius electrlytes with duraater.

56 42 (0 0) u -p I-t v 01 > u Q. c (0 0) +) is 4-1 1^ > _ > 0) EH (0 x: +> QQ. ^ V CNJ "«t ^ ^ <* ^ r»* t»> r «1«^^ f»h \ ^ rh V ^ Tl" r- r) r T»<» V ^ "* 0) E u +> 0 J. u 10 2 h (0 ^ ih q Ui B r <M r V C

57 (1) A3 0 0) 0) H V s 1 f V C C «H r* CD ^ v v r- a» TH n C f) fi r ^ ^ l \ N ^ T-1 ^ th v ^ '"i* CD 1 v r r» ri V r v "* r VD TJ< I I v c M T H r H n " < * r ^ ^ f «- ) v r ^ cl i I \ r ^ C M" ^ C ih <7» CI V v v n C v <n n (n r>) s^ CJN n C n ^ fu (0 Z r C 1 V ^ r r-i f 5j" n 1 TH «;«< C TH ^ 0^ 'i* xj- -«* C^ C 1-1 ^ H \ r ^ v \ r- rh ^lnlnc^^^'^c<l ct»r»^thih»h '*rrrrcfrcr s \ HrH +> S3 p M th 1-1 i n c N i H C M r H \ \ \ \ \ \ \ \ \ \. C M T - l i n C M» H i n C M t H i n M \ T H " C ih r-t C4 C '*vr-ca>»-iih

58 AA which was nt enugh t give rise the required plarizat, 1 1 Eq, (7) dicates that the tercept fr a plt f vs. t C^ at fixed V allws the value f "C t be detered. app 2 The values f tercept is equal t - fri^i which -C ay be evaluated. The 1 - «v values f ^thus evaluated are listed Table 7. Fr the evaluat f charge density 0, there are tw liitg cases. In the dilute range the charge density ( 0t) ay be evaluated with the slpe f Fig, 3: and is equated with - ~ ^ 1 1 ( *C)-5j, The predetered value f 'C and/3 ay be substituted it and Ctis evaluated. Values f 6Care listed Table-10, In the cncentrated range usg eq. (7) the slpe f plt is given by ^\"*"'^" ^ (). ^^ 6. The graph ical values f 2(1 - "C; In 5 slpe detered fr Fig, 4 & 5 fr dural-ebrane is equated with abve express. The values f «^ and/3 predetered are substituted and the value f charge density cncentrated range (6 c ) evaluated and listed Table-10, The present vestigats revealed that the Gd and Sc btaed at dilute and cncentrated range respectively, agree well with each ther (Table-10) and can safely be cncluded that Kbatake express is applicable t this bilgical ebrane. These are the basic requireents f Kbatake thery f ebranes. Cparisn can be ade fr theretical and experiental data and applicability f Kbatake et al, (31). Equat t the

59 ^5 ebranes can be tested by fllwg analytical technique suggested by hi; eq, (1) can be rewritten as ^ - e " e^ - 1 = Z (17) where q and Z are defed by /AK1+ (1 + 2.cC) ^ (18) and n z = ^2/^/xe (19) -^ q If equat (1) is valid then the values f lg( " ^ ) e^ - 1 calculated fr easured A J0 with the predetered ^and A and the given value f ust fall n a straight le, which has a unit slpe and passes the crdate rig v;hen pltted agast lg Z (lg Z ay be calculated fr Q.^ and predetered value f C, Aand 0), This behaviur ust be valid irrespective f the value f V and the kd f ebrane-electrlyte syste. Figure 6 denstrates that this theretical prduct f ebrane ptential equat is brne ut quite satisfactrily by ur experiental results n dural-ebrane, syste. Ps is a easure f perselectivity f ebrane electrlyte The varius values f perselectivity were calculated fr eq. (14) by substitutg the value f Ji^wid. t fr Tables

60 -Nad AKCl NaF -» KF KN3 NH4CI > Lg Z Fig. 6. Plts f lg (Y-e*/#-l) Vs lg Z fr duraater cntact with varius 1:1 electrlyte sluts.

61 A5 7 & 8 fr duraater. The values f Ps are given Table-9. Accrdg t Kbatake's paper, if the transprt nuber f c-s (t ) is zer then the ebrane will be perfectly selective and ^ app hence Ps tends t be unity. Further, if transprt nuber f cs has value free slut then Ps will tend t be zer. The values f transprt nuber are listed the Table-8 which shws the value f Ps case f duraeter is tendg t be zer. The values f Ps ented Table-9 are very lw, thus cnfirg the applicability f Kbatake's thery t tliis ebrane syste, Kbatake and Ka (120) frulated eq, (11) fr evaluat f charge density, A curve, was pltted between -; 1 vs. 1 app *"1 (Figures 7 & 8), The different value f charge density can be btaed fr the slpe f le f the varius curves given Figures (7 & 8) fr duraater with varius electrlytes. The values f)^x fr different electrlytes btaed this way are given Table-10. Nagasawa et al, (30) derived tw liitg frs f express fr ebrane ptential at extreely lw cncentrats (eq, 15) and at high cncentrats (eq, 16) f electrlytes respectively. At extreely high cncentrats eq, (16) predicts a ^ lear relatship between -E/ ^ and -r» fr which we can als detere dx. The curve with varius electrlytes fr duraater are shwn Figures 9 and 10. Tiae different values f JZfx derived fr itial slpe f varius curves Figures (9 &. 10)

62 1 M TH TH r-4.-1 V t- fh fh * r- r- A? 8 rh 0) n n C C» <N r-{ C <7> ri U) C u 4J n S f-i i n 04 t <T\ n 10 \0 0\ fr, "V -^ ri I - r-«m ^ < > T-t (N * 1 M f-h «*> r-t > r>»-t.-4 I «) rh XI (0 i 0) E 4-1 n 1^ <N C>».H r-l ^ > H* r-.-4 -P H ei > C -H -P -H U V 0) f <-< M fl) p t C R 0) u c p c r-\ n f) K V VD 1 t V CD.H 1 C rh ty\ '0 TH V l rh (N rh P 0) > -H C r-* \ H M P 4) r rh M Q +J S C ^ (1) rh Up C \ u CI. \ C \ (N y-< \ rh \ t fv) \ rh \ rh L'> >> c5 \ t \ C^J rh \ rh t-r V Ci

63 % Fig7» KF - NaCl NH4CI A KN3 60.a ^ I/C Fig.8 NaF ^ KCl Fig. 7 & 8. Plts f l/tapp Vs 1/ci fr varius electrlytes with duraater.

64 60f rig-9 «NaCl NaF A KC( A KN3 A I/C1 10 Figl 60 KF NHi;Cl 1^ r- 5 I/C1 Ttf Fig. 9 & 10. Plts t E Vs 1/Cl fr varius electrlytes with duraater.

65 I 0) (0 p a a c CQ 0) E 0) p 01 n U +> (D rh 0) c (0 ^^ i 0) E >i +J H t C (U -d (0 > 0) CQ 0) 3 (0 > (0 0) C ( ) 0] S en ^ T > e I C I X V. c n (t) 0) 3 (1) H P (0 f I a 0) > -p (0 0) +> W > ^ ft (0 X % 0) u I C I v C X v r V r-l en I r-l X a\ C I X v ^ C I N C I X 0\ V I X C v v N <n I X v V C Ch rh r 1 rh X ^ C v 1 ri X 'J' v Tj< r» C 1 rh X rh TJ* v rh cn 1 rh X r- v v n 1 rh X C v rh r 1 rh X v v v r 1 rh X VD C rh r> r C i rh X * r C C 0\ r 1 rh X t-i \ V r f 1 r-^ X C \ ri as C r 1 rh X r- * a\ r 1 f-i ^ v C C rh V 48 0) u a) u

66 A9 fr duraater with varius electrlytes are given Table-10. It is nted fr the Table-10 that the charge density values f the ebrane electrlyte syste are lw. The values derived fr different ethds are alst sae. A little difference ay be attributed t the different prcedure adpted. It is cncluded fr the results that the ethds develped by Nagasawa et al, and Kbatake et al are satisfactry fr the evaluat f effective fixed charge density f the syste under vestigat. The ther prperties f this ebrane are beg further vestigated.

67 BiBuaiuuPHr