CENTRALE LANDBOUWCATALOGUS

0000 0030 4317 L(0frl imvhias^O

ADSORPTION OF POLYLYSINES AT SOLID-LIQUID INTERFACES

BIBLIOTHEEK

LANDBOUWHOGESCHOOL WAGENINGEN

[Sfl/^ll Joi^ Promotor:dr .J .Lyklema ,hoogleraa ri nd efysisch ee nkolloïdchemi e ^o2?o\} W

STELLINGEN

1. Bumbullis et al. en Wolfes en Schügerl noemen de bulk-viscositeit die zij berekenen m.b.v. gegevens verkregen uit transversale ca­ pillaire golf- en oppervlaktespanningsmetingen aan viscoelastische oppervlakken ten onrechte de oppervlakte-viscositeit. Bumbullis, W. , Kalischewski, K en Schügerl, K. (1981). Foam behaviour of bio­ logical media. VII. Surface viscosity and viscoelasticity. Eur. J. Appl. Micro­ biol. Biotechnol. H, 110-115. Wolfes, H. en Schügerl, K. (1983). Foam behavior of biological media. VIII. Surface properties. Eur. J. Appl. Microbiol. Biotechnol. _T7, 371-375.

2. De Voeght en Joos missen een essentieel punt in het gedrag van de capillaire golven op het vloeistofoppervlak van natriumlaurylsul- faat/1-tetradecanol oplossingen door niet op te merken dat in dit geval de modulatie van de amplitude van de golven met de afstand wezenlijk verschilt met de amplitudemodulatie die bij water t.g.v. reflecties optreedt.

Voeght, F. de en Joos, P. (1983). Waves at the air/liquid interface of a sur­ factant solution with a high surface shear viscosity. J. Colloid Interface Sei. 95, 142-147.

3. Bij een lage en constante geadsorbeerde hoeveelheid vindt t.g.v. de ladingstegenstelling tussen adsorbaat en adsorbens in poly-L- lysine, geadsorbeerd aan polystyreen (latex), geen ladingsgeïndu- ceerde helix-kluwen overgang plaats. Bij hogere adsorpties is zo'n overgang echter wel mogelijk.

Dit proefschrift, hoofdstuk 6.

4. De analyse van de Surface-Enhanced Raman Spectra (SERS) van pipe- ridine van Sanchez et al. zou vollediger zijn geweest als zij er ook de invloed van de bezettingsgraad van het adsorbaat bij hadden betrokken.

Sanchez, L.A., Birke, R.L. en Lombardi, J.R. (1984). Surface-Enhanced Raman scattering of piperidine. The effect of electrode potential on intensity. J. Phys. Chem. 88, 1762-1766.

BIBLIOTHEEK DKR LANDBOUWHOGESCHOOL WAGENINGEN 5. Bij de presentatie van polyelectrolytadsorptiegegevens, waarbij de geadsorbeerde hoeveelheid is uitgedrukt als hoeveelheid massa, dient het duidelijk te zijn of het tegenion wel of niet hierin is inbegrepen.

6. Het onderscheid dat Lok et al. maken tussen reversibel en irrever­ sibel geadsorbeerd runder plasma-albumine is niet zinvol omdat zij de aggregatietoestand van het eiwit niet hebben vastgesteld. Lok, B.K., Cheng, Y-L. en Robertson, CR. (1983). Protein adsorption on cross- linked polydimethylsiloxane using total internal reflection fluorescence. J. Colloid Interface Sei.

1. De grafieken van Y /w versus m die Kvastek en Horvat gebruiken om de Warburg coëfficiënt uit te rekenen, geven alleen de door hen verwachte rechte te zien omdat ze het (wel gemeten) hoogfrequente deel ervan weglaten. Kvastek, K. en Horvat, V. (1981). Kinetic study of the Ag/AgI electrode by com­ plex impedance dispersion analysis. J. Electroanal. Chera. 130, 67-79.

8. Bij het onderzoek naar het gebied van afschuifsnelheden waarbij men pseudo-plastische vloeibare levensmiddelen in de mond beoor­ deelt op hun dikvloeibaarheid, gaan Cutler et al. ten onrechte voorbij aan het feit dat de meeste van deze vloeistoffen ook thi- xotroop zijn. Cutler, A.N., Morris, E.R. en Taylor, L.J. (1983). Oral perception of viscosity in fluid foods and model systems. J. Text. Stud. U±, 377-395.

9. Bij het nagaan van het verband tussen de geadsorbeerde hoeveelhe­ den polyacrylzuur en de wandlading van de adsorbentia hematiet en rutiel, houden Gebhardt en Fuerstenau ten onrechte geen rekening met een verschuiving van de wandlading die kan optreden t.g.v. de adsorptie van het polyelectrolyt. Gebhardt, J.E. en Fuerstenau, D.W. (1983). Adsorption of polyacrylic acid at oxide/water interfaces. Colloids and Surfaces 7, 221-231. Dit proefschrift, hoofdstuk 4 en 5. 10.D eschattin gdi eBuscal le nCorne rmake nva nd egemiddeld evolume ­ fractiepolyacrylzuu r aanhe toppervla k vanhu npolystyreendeel - tjes, opbasi sva no.a .protontitratie sva ngeconcentreerd epoly - acrylzuuroplossingen, isinconsisten tme tee nva nd edoo rhe nge ­ maakteveronderstellingen .

Buscall,R . en Corner,T . (1982). Polyelectrolyte stabilised latices.Par t2 : Characterisation and colloidal behaviour.Colloid s and Surfaces5 ,333-351 . Ditproefschrift ,hoofdstu k6 .

11. Integenstellin g totd eresultate nbeschreve ni ndi tproefschrif t vinden Furusawa et al. geennoemenswaardig e invloedva nd ezout - concentratie op de adsorptie van poly-L-lysine aanpolystyree n (latex)bi jp H4 .Waarschijnlij k isdi the tgevol gva n demeet ­ proceduredi ez etoepassen .

Furusawa, K., Kanesaka,M . en Yamashita, S. (1984). Adsorption behavior of poly-L-lysine and its conformation at the latex-water interface. J. Colloid Interface Sei.99 ,341-348 . Ditproefschrift ,hoofdstu k4 .

12. Intheoretisch epublicatie swaari nd eresultate nva ncomputerbere ­ keningenwezenlij kzij nvoo rd etheorie ,dien td ebeschrijvin gva n debijbehorend eprogramma' sminsten sgelijkwaardi gt ezij naa ndi e welkegangbaa r isbi jd eparagraa fmateriale ne nmethode ni nexpe ­ rimenteleverhandelingen .

13.Ee n grotere integratieva nbelei dm.b.t .waterkwalitei te nwater ­ kwantiteitda nn ui nNederlan dgebruikelij k is,ka nleide nto tee n groteredoelmatighei dva nhe twaterbeheer .

14.D e consumptie van zoutjes bijhe ttelevisiekijke nka ntoeneme n doord eaanwezighei dva nondertitels .

B.C.Bonekam p Adsorptiono fpolylysine sa tsolid-ligui dinterface s Wageningen,1 2septembe r198 4 B.C.Bonekam p

ADSORPTIONO FPOLYLYSINE S ATSOLID-LIQUI D INTERFACES

Proefschrift terverkrijgin gva nd egraa dva n doctori nd elandbouwwetenschappen , opgeza gva nd erecto rmagnificus , dr.C.C .Oosterlee , inhe topenbaa rt everdedige n opwoensda g1 2septembe r198 4 desnamiddag st evie ruu ri nd eaul a vand eLandbouwhogeschoo l teWageninge n Voor mijn ouders

Voor Margo, Stijn en Bart ABSTRACT

Bonekamp,B.C . (1984)Adsorptio n ofpolylysine s atsolid-liqui d interfaces.Doctora l thesis,Agricultura l University, Wageningen. 208p , 80 figs,5 tables .Englis han d Dutchsummarie s

Adsorption properties ofth epolyelectrolyte s poly-L-lysine (PL-L)an dpoly-DL-lysin e (PL-DL)o n hydrophobic (polystyrene latex, silver iodide)an dhydrophili c (silica) negatively charged solidparticle swer e studied. Adsorbed amounts asa functio no f concentration,ioni c strength,surfac echarge , PL chain length and chain charge density weredetermined .Th eadsorptio no fP Lo n negatively charged polystyrene latex and silica was alsomonitore d conductometri- callyan dpotentiometrically . Further flocculation and coagulationmeasurement swer e performed. Information about the secondary structureo fadsorbe d PL-Lwa s obtained fromproto n titrations ofPL- Lan d PL-DLadsorbe d atpolystyren eparticles . The stereoregularity and secondary chain structureo fP Ldoe sno t influenceth e adsorbed amount.A t lowconstan tadsorbe d amount,ther e isn o coilt oheli xtransi ­ tion in adsorbed PL-L,becaus e ofth echarg econtras tan dhydrophobi c interactions between PL and the surface.Onl y when the adsorbed amount is high ateac hpH ,a transition takesplac ean d adsorbed PL-Lca nb epartl yhelical . At lowp Han d lowioni c strength theadsorbe dpolyelectrolyte s showa rathe r flat conformation and there is no pronounced effecto fth ehydrophobicit yo fth eadsor ­ bent. All negative surface groups form ionpair swit ha n -NH,grou p ofPL ,bu tth e reverse is notth ecase .Th eadsorptio no fth ebasi cpolyaminoacid s increases(i.e . loopsan d tails start todevelop )i fth ep H isincrease d or ifth eioni c strengthi s raised. With the hydrophobic adsorbents theelectrolyt e effectpersist s upt over y high concentrations. Inth ecas eo fth ehydrophili c silica no increase above0.0 1M salt was observed, because here hydrophobic interactions are absent. The ionic strengthan dp Hdependenc e ofth eadsorptio no nhydrophobi c substrates are insatis ­ factorily agreementwit h theoreticalpredictions .

Free descriptors:Adsorption ,binding ,solid-liqui d interface,polycation-polyanio n complex,polyelectrolyte ,charge dmacromolecule ,charge dpolyaminoacid ,poly-L-lysine , poly-DL-lysine, PLL,PLDL ,polystyren e latex, silica,conformation ,conformationa l transition,helix-coi l transition,secondar y structure,proto ntitration ,conductiv ­ ity,flocculation ,coagulation . CONTENTS page

1 INTRODUCTION 1 1.1 Generalbackgroun d 1 1.2 Aimso fthi swor k 3 1.3 Systemsuse dan doutlin eo fthi sstud y 3 1.4 References 5

2 ADSORPTIONO FPOLYAMINOACID SWIT HIONIZABL ESID EGROUP S 7 2.1 Introduction 7 2.2 Polyaminoacidsa tinterface s 8 2.3 Polyelectrolyte adsorption 10 2.4 References 13

3 MATERIALS 15 3.1 Polylysinean drelate dpolyaminoacid s 15 3.1.1 Synthesisan dstructur e 15 3.1.2 Solutionpropertie so fP L 16 3.2 Polystyrene latex 20 3.2.1 Preparation 20 3.2.2 Characterizationo fP Slate x 21 3.2.3 Thepolystyrene-wate r interface 28 3.3 Silicaan dBorosilicat eglas s 29 3.3.1 Preparationan dspecifi csurfac eare ao fsilic a 29 3.3.2 Borosilicateglas spurificatio nan dspecifi c 30 surfaceare a 3.3.3 Determinationo fth esurfac echarg edensit yb y 32 conductometrico rPotentiometri eproto n titrations 3.3.4 Someothe rrelevan tpropertie so fth esilica - 34 wateran dglass-wate rinterfac e 3.4 SilverIodid ean dPolyoxymethylen ecrystal s 37 3.5 Summary 37 3.6 References 38 Page ADSORPTIONO FTH EHIGHL YCHARGE DPOLYELECTROLYT EPOLYLYSIN E '4 1 ONDIFFEREN TSUBSTRATE S 4.1 Introduction 41 4.2 Experimental 41 4.2.1 Materials 41 4.2.2 Determinationo fth epolyaminoaci dconcentratio n 42 4.2.3 Adsorptionmeasurement s 42 4.2.4 Conductometrican dPotentiometri etitration s 43 4.2.5 Stabilitymeasurement s 44 4.3 Resultsan ddiscussio n 44 4.3.1 Comparisono fth especifi csurfac earea s 44 ofth eadsorbent s 4.3.2 Adsorptiontim e 47 4.3.3 Effecto fmolecula rmas s 48 4.3.4 Adsorptionisotherm s 50 4.3.5 Influenceo fth esurfac echarg edensit y 55 4.3.5.1Polystyren esurfac echarg ean dth e 56 effecto flate xpretreatmen t 4.3.5.2Silic aan dBorosilicat eglas s 59 4.3.6 Influenceo felectrolyt econcentratio n 62 4.3.6.1Polystyren elate xan dAg i 62 4.3.6.2Silic aan dBorosilicat eglas s 66 4.3.7 Stabilityo flate xan dAg isol sagains tlo w 71 molecularmas selectrolyt ei nth epresenc eo f polylysine 4.4 Summaryan dconclusion s 76 4.5 References 77

INTERACTIONSBETWEE NNEGATIVEL YCHARGE DCOLLOIDA LPARTICLE S 81 ANDPOLYCATION S 5.1 Introduction 81 5.2 Experimental 82 5.2.1 Materials 82 5.2.2 Potentiometriean dconductometri cTitration s 82 5.2.3 Adsorptionmeasurement s 83 5.2.4 Stabilitymeasurement s 83 5.2.5 Microcalorimetry 83 5.2.6 UVan dC Dspectroscop y 83 Page 5.3 Resultsan ddiscussio n 84 5.3.1 Adsorbedamoun tPL.HB rfro mdepletio n 84 measurements 5.3.2 TheP Slate x- PL.HB rsyste m 86 5.3.3 Thesilic a- P Lsyste m 94 5.3.4 Influenceo fth esurfac echarg edensit yan d 96 ionicstrengt ho nth epolylysine-charge d particleinteractio n 5.3.5 Protontitration so fsilic ai nth epresenc e 102 ofP L 5.3.6 Theoreticaldescription so fpolycation - 105 polyanionassociatio n 5.3.7 UVan dC Dspectroscop yo fth esilica/poly-L - 106 histidinesyste m 5.4 Summaryan dconclusion s 111 5.5 References 112

CONFORMATIONO FFRE EAN DADSORBE DPOLYLYSIN E 115 6.1 Introduction 115 6.2 Interactionsdeterminin gth econformatio no fcharge d 116 polyaminoacids 6.2.1 'Non-bonded'interaction s 117 6.2.2 Hydrogenbon dformatio n 117 6.2.3 Hydrophobicbondin g 119 6.2.4 Ionicinteraction s 120 6.3 Secondarystructur ean dhelix-coi l interactions in 122 chargedpolyaminoacid s 6.3.1 Theoriesfo rth ehelix-coi ltransition s 123 incharge dpolyaminoacid s 6.3.2 Conformational aspectso fpoly-DL-aminoacid s 125 6.4 Precipitationo fpoly-L-lysin ean dpoly-DL-lysin ea t 126 highp Hvalue s 6.4.1 Experimental 127 6.4.2 Resultsan dDiscussio n 128 6.5 Adsorptiono fpartiall ycharge dpoly-L-lysin e and 135 poly-DL-lysineo npolystyren e 6.5.1 Materials 135 6.5.2 Determinationo fth eP Ladsorptio no nP S 136 particlesa sa functio no fth ep Hi nsolutio n Page 6.5.3 Adsorptionisotherm so fpoly-L-lysin ean d 137 poly-DL-lysinea thig hp H 6.5.3.1Adsorbe damoun ta sa functio no fth e 138 solutionp H 6.5.3.2Phas eseparatio ni nsolutio nan d 141 neara ninterfac e 6.6 Characterizationo ffre ean dadsorbe dpolylysin eb y 142 Potentiometrieproto ntitration s 6.6.1 Introduction 142 6.6.2 Principleo fth emetho d 143 6.6.2.1Suspensio neffec ti npolyelectrolyte / 147 chargedparticl esystem s 6.6.2.2p K andth edistributio no ffixe d 149 app chargesi nadsorbe dpolyelectrolyte s 6.6.2.3Mediu meffec to np K 150 o,m 6.6.3 Experimental 152 6.6.3.1Material s 152 6.6.3.2Potentiometri etitration s 152 6.6.3.3Sampl epreparatio n 153 6.6.4 Resultsan dDiscussio n 155 6.6.4.1Dat atreatmen t 155 6.6.4.2Proto ntitration so ffre ean dadsorbe d 162 polylysine 6.6.4.3Compariso nwit hothe rexperimenta l 172 systems 6.6.5 Generaldiscussio n 176 6.7 Summaryan dconclusion s 178 6.8 References 180 POLYELECTROLYTEADSORPTIO NTHEOR YAN DTH EADSORPTIO N 185 OFCHARGE DPOLYAMINOACID S 7.1 Introduction 185 7.2 Theory 186 7.3 Choiceo fth eparameter s 188 7.3.1 Coordinationnumbe ro fth elattic e 189 7.3.2 Monolayercoverage ,are ao fa lattic esit ea 189 andth edistanc ebetwee nth elattic elayer sr 7.3.3 Thechai ncharg edensit y 189 7.3.4 Thepolymer-solven tinteractio nparamete rx 190 page 7.3.5 Thenon-electrica lenerg yo fadsorptio n 190

parameterx s 7.4 Resultsan dDiscussio n 191 7.4.1 Dependenceo nelectrolyt econcentratio n 191 7.4.2 Influenceo fth esurfac echarg e 192 7.4.3 Influenceo fth echai ncharg edensit y 192 7.5 References 194

SUMMARY 195

ACKNOWLEDGEMENTS 198

LISTO FABBREVIATION SAN DSYMBOL S 199

SAMENVATTING 203

CURRICULUMVITA E 207

NAWOORD 208 INTRODUCTION

1.1 GENERALBACKGROUN D

Interactionsbetwee npolymer so fbiologica lo rsyntheti corigi nan d interfacesar eo fimportanc efo ra wid evariet yo fbiological ,medica l andtechnologica lprocesses .Fo rinstanc eboun dprotein san dglycopro ­ teins play importantrole s innatura lmembrane s (seee.g . Cantor & Schimmel, 1980). Further,th eflocculatio no fsuspende dbacteria lcell s bybiologica l or synthetic water-solublepolyelectrolyte s iso fcon ­ siderableimportanc ei nbiotechnolog yan dwate rpurificatio n (Daniels, 1980). Alsoth eattachmen to fwhol ebacteria lcell so rviruse st osolid - surfaces isa nexampl eo fbiopolymer-surfac einteractio nan do frele ­ vance formicrobiology ,biochemistry , (bio)engineeringan ddentistr y (Daniels, 1980). Stillmor eexample swil lb e given inth efollowin g chapters.Becaus eo fth eimportanc eo fpolypeptide sincludin gth epro ­ teins atbiologica l andnon-biologica l interfaces, Ishal lno wfocu s onth eadsorptio npropertie so fwate rsolubl epolypeptide sonly . Polypeptides appear innatur e atvariou s levelso fstructura lor ­ ganization, fromrando m coilsu pt oordere d secondary, tertiary and quarternary structures*. Invie wo fth estructure-functio nrelatio n of natural polypeptides the investigation ofth ethre edimensiona l structure ofpolypeptides ,protein sincluded ,i so fgrea timportance . Theconformatio n (i.e.three-dimensiona l structure)o fa polypeptid e isth eresul to fth esu mo fal lintr amolecula rinteraction san dinter ­ actionso fth ebiopolyme rwit hth eenvironment .A sth epresenc eo fa n adsorbentimplie sa chang eo fthi senvironmen ti ti sreasonabl et oas ­ sumetha tth econformatio no fpolypeptide sca nchang eupo nadsorption . Another important aspectconcernin gth eadsorptio no fmos tnatura l polypeptides is,tha tthes epolypeptide s contain aminoacid residues withionizabl esid echains .Henc ethes epolypeptide sca nb eelectricall y charged and the electrostatic interactionwit h interfaces,whic har e alsocharge di nmos tcases ,ma yt oa greate ro rlesse rexten tinfluenc e

The primary structure of a polypeptide is defined asth esequenc e inwhic hth e amino acids are linked together. The secondary structure refers toth econfor ­ mation the polypeptide backbone adopts (e.g.a-heli xan d ß-structure).Th eter ­ tiary structure refers to folding of sections ofsecondar y structure inspace . Thequaternar y structure referst oth earrangemen t oftertiar y structures inspace . the adsorptionproperties . Itshoul dals ob erealize dtha tadsorptio n fromsolutio ni sa competitiv eprocess .Whe npolypeptid emolecule sad ­ sorb, solvent (water)molecule s andpossibl y other components (e.g. ions)mus tb edisplaced .I ngeneral ,th estructur eo fth eadsorbe dpoly ­ peptide layerwil lb eth ene tresul to fal lintra -an dintermolecula r interactions including the interactions ofth epolypeptid e withth e solventan dth esurface . When apolypeptid emolecul e insolutio nbehave sa sa rando mcoil , i.e. as a flexiblepolyme rchain ,th eadsorptio nbehaviou rshoul dre ­ semble thato f synthetic flexiblepolymers .Suc hpolymer s adsorb at almost any surface andth eadsorptio nisother mi susuall yo fth eHig h Affinity type.Eve n ifth eaffinit yo fa polyme rsegmen tfo rth esur ­ facei ssmall ,th eadsorptio ni sstil lo fa hig haffinit ytype ,becaus e the total adsorption energy isth e sumo fth ebindin generg yo fman y segments.No t all segments ofth e adsorbedmacromolecule snee dt ob e attached toth e surface.Par to f them canresid e inloop san dtail s protuding into the solution.A serie so fadjacen tsegment si ncontac t with the surface iscalle d atrain .Th eadsorbe dlaye rconsist sthe n oftrains ,loop s and tails.Th e interfacial properties arestrongl y influenced by thepresenc e ofsuc h anadsorbe d layer (seee.g . Fleer and Lyklema (1983)). When there are considerable intrasegmental attractions ina poly ­ peptide molecule, such as hydrophobicbonds ,hydroge nbonds ,ioni c interactions etc.,th e train-loop-tail model isno tsuitabl efo rth e descriptiono fth eadsorbate .Thi si sth ecas efo rmos tproteins .A sa consequence ofth e strong intramolecula r attractionsmos tprotein s havea well-define dthre edimensiona lstructure ,containin gpart swit h differentdegree s oforde rrangin gfro mrando mcoil st oa heli xan dß sheet structures.Becaus e ofth especifi c structures they adopti n solution, a general theory for the adsorption ofprotein s doesno t exista tpresent . Another complicating factor with proteins as adsorbates istha t theyar eusuall yampholyti cpolyelectrolytes .Th eimportanc eo felectro ­ statican dhydrophobi cinteraction si ndeterminin gth epropertie so fad ­ sorbedprotein s hasbee nemphasize db yfo rinstanc e Norde and Lyklema (1978). Overth epas tthre edecade ssyntheti cpolypeptide s(polyaminoacids ) haveevolve d as an interesting class ofpolymer si nthei row nright , and asmode lprotein s forconformationa l studies.Muc hwor kha sbee n done on the properties of syntheticpoly-a-aminoacid s inth esoli d State and insolutio n (Fasman, 1967). However relatively littleat ­ tentionha sbee ndirecte d tothei r interfacialpropertie sespeciall y at solid-liquid interfaces.Amon g these interfacial studies amino r part is devoted to the study ofcharge dpoly-a-aminoacid scapabl e of forming secondary structures.Thi sdespit e of the importanceo f charge interactions and structural alterations inadsorbe d proteins (seee.g . Norde, 1978). Althoughth estud yo fth eadsorptio no fcharge d polyaminoacidsgive sn odirec tinformatio nabou tth ebehaviou ro fter ­ tiarypolypeptid e structures atinterfaces ,th estud y ofthes ecom ­ pounds canprovid e insight inth ebehaviou r ofa helice s andothe r secondary structures at surfaces and therol eo fcharg einteraction s therein. Also therol e of the aminoacid side chains inth e surface interaction canb e traced. Iti stherefor e thatthi s thesis isde ­ voted to polylysine, a positively chargedpoly-a-aminoaci dcapabl e of forming secondary structure,adsorbe d atsolid-liqui dinterfaces . As atlo wp Hpolylysin e is ahighl ycharge dpolyelectrolyte ,th ead ­ sorptionpropertie swil l resemblethe nthos eo fothe rsyntheti cpoly - electrolytes and they canb ecompare dwit htheoretica lprediction so f van der Schee (1984) forth eadsorptio no fflexibl epolyelectrolytes .

1.2 AIMSO FTHI SWOR K

Theaim so fth epresen tstud yare :

(i) To gainmor e insightint oth ebehaviou ro fcharge dpolymer sa t (oppositely)charge dinterface san dt ocompar eth eresult swit h theoreticalprediction s andwit hth ehomogeneou scomple xforma ­ tionbetwee npolycation san dpolyanion si nsolution . (ii) Establishing of the role of the substrate indeterminin g the characteristicso fth eadsorbate . (iii) Establishing of the secondarystructur eo fadsorbe dwea k(cat - ionic)aci dpolyelectrolyte sa tlo wchai ncharg edensit yan dth e occurrenceo fconformationa ltransition si nth eadsorbe dstate .

1.3 SYSTEMSUSE DAN DOUTLIN EO FTHI SSTUD Y

Mostexperiment s inthi s studywer eperforme dwit hpoly-L-lysine . HBr (PL-L)an dpoly-DL-lysine.HB r (PL-DL)a sth eadsorptives .A ssai d before,polylysin ewa s chosenbecaus ei nsolutio nthi shomopolyamino - acid showsdifferen t structures depending onth esolutio npH :a tlo w pH, themolecul e isa highl ycharge dpolyelectrolyte .A thig hp Hth e molecule ishelical ,a structur ewhic h isals otypica lfo rproteins . Poly-DL-lysine doesno t form aheli x andthi spolyme rwil lbehav ea s acoi la tan ypH . Asa tlo wp HP Li shighl ypositivel ycharged ,accurat econcentratio n determinations arepossibl eb ymean s oftitratio nwit ha noppositel y chargedpolyelectrolyte . As the adsorbentspolystyren e (latex),pyrogeni c silicaan dboro - silicaglas swer eused .Fo rcompariso nreason si nsom eexperiment sals o Agiwa sused .I nthi swa yi twa spossibl et otrac eth einfluenc eo fth e natureo fth esurfac eo fth eadsorben to nth eadsorptio nproperties . Thepresen tthesi s iswritte ni nsuc ha way ,tha teac hchapte rca n berea dindependently . Inchapte r2 som e general aspects of the adsorption of ionizable polyaminoacidswil lb ediscussed . Inchapte r3 relevan tpropertie s ofth epolyaminoacid san dadsor ­ bents used obtained fromth eliteratur ean dfro mow nmeasurement sar e discussed. Chapter4 deals with the adsorption ofth ehighl y chargedpoly ­ electrolyte polylysine ondifferen t substrates.Th e adsorbed amount as a function of time,molecula r mass, ionic strength and surface charge ismeasure d and discussed.Als o the influence ofth esurfac e hydrophobicity is considered. Further the coagulation ofth elate x andsol suse di nth epresenc eo fa nadsorbe dP Llaye rar einvestigated . Inchapte r5 informatio ni scollecte dabou tth einteractio nbetwee n positivelycharge dpolylysin ean dnegativel ycharge dpolystyren e (latex) andsilic aobtaine dwit hth ehel po fconductometri c andPotentiometri e techniques.A t the sametim eth ecomple xformatio nbetwee npolylysin e and the solidparticle si scompare dwit hth ehomogeneou scomple xfor ­ mationbetwee nlinea rpolycation san dpolyanions .Th eresult sar eals o relatedt oth eflocculatio nbehaviou ro fth eparticle si nth epresenc e ofPL . Chapter6 deal swit h the conformational properties ofadsorbe dP L athig hpH .I nth efirs tpar to fthi schapte rinteraction sdeterminin g the secondary structure inP L areexamined .A s at lowchai ncharg e density phase separation occurs inP L solutions,som eprecipitatio n characteristics ofPL- L andPL-D Lar edetermine dan dth einfluenc eo f thesecondar ystructur eo fPL- Lo nth eadsorbat epropertie si sobtaine d from the comparison between the adsorptiono fPL- L andPL-D L asa functiono fth ep Hi nth ealkalin eregion . Bymean s ofPotentiometri eproto n titrations ofPL- Lan dPL-D Li n solutionan dadsorbe do npolystyrene ,informatio ni sobtaine dabou tth e PL conformation inth eadsorbe dstat ean dth eoccurrenc eo fconforma ­ tional transitions inadsorbe dPL .I nth efirs tpar to fth etitratio n section,th eapplicatio no fproto ntitration si nheterogeneou ssystem s isdiscusse d insom edetail .Fo rcompariso nreason sals osom etitratio n experiments are performed with other polyelectrolytes.Finall y the conformationo fadsorbe dP La sa functio no fth ep Han dadsorbe damoun t asi temerge sfro mthi sstud yi sroughl ysketched . In the lastchapte r some ofth e adsorptionpropertie s ofP Lar e comparedwit h thetheoretica l predictions ofth epolyelectrolyt ead ­ sorptiontheor yo fva nde rSchee .

1.4 REFERENCES

Cantor,C.R . andSchimmel ,P.R . (1980). 'Biophysicalchemistry 'par tI : Theconformatio no fbiologica lmacromolecules .W.H .Freema nan dCom ­ pany,Sa nFrancisco ,p .235 . Daniels,S.T . (1980). 'Adsorptiono fmicroorganis m tosurfaces' .Bitton , G.an dMarshall ,K.C .eds .Joh nWile y& Sons ,Chapte r2 . Fasman,G . (1967). 'Poly-a-Aminoacids' :Protei nmodel s forconforma ­ tionalstudies' .Fasman ,G .ed. ,Marce lDekke r Inc.Ne wYork . Fleer, G.J. andLyklema ,J . (1983)i n 'Adsorption from solutiona t theSolid/liqui d Interface',Parfitt ,G.D .an dRochester ,C.H .eds. . AcademicPress ,Londo np .153-219 . Schee, H.A. vande r (1984).Doctora l thesisAgricultura l University Wageningen,Th eNetherlands . Norde,W . and Lyklema,J . (1978). J.Colloi d InterfaceSei . 66, 285- 293. Norde,W . (1980).Adhesio nan dadsorptio no fpolymers ,par tB .Lee ,L.H . ed.Plenu mPubl .Corp .Ne wYork ,p .801-825 . 2 ADSORPTIONO FPOLYAMINOACID SWIT HIONIZABL ESID EGROUP S

2.1 INTRODUCTION

Thisthesi si smainl yconcerne dwit hth einteractio nbetwee ncharge d polyaminoacids (i.e.a clas so fpolyelectrolytes )an doppositel ycharge d colloidalparticle san dth econformationa lpropertie so fth eboun dpoly ­ aminoacids.Followin gth edefinition so f Bungenberg de Jong (1952) the systemsunde rconsideratio nar ecomple xcolloi dsystems . Inth e interactionbetwee noppositel ycharge dparticle si naqueou s solution,system swit hincreasin gcomplexit yca nb econsidered . Insimpl eelectrolyt esolution sinteraction ssuc ha sio npai rformatio n betweenoppositel ycharge dmicro-ion sar eo fimportance .I nhomogeneou s solutions ofpolyelectrolytes ,th einteractio nbetwee nmicro-ion san d thepolyio ndetermine st oa larg eexten tth epropertie so fth esystem . Anexampl e ofcurren tinteres ti sth einteractio no fheav ymeta lion s withpolyelectrolytes . When twooppositel y chargedpolyion sar epresen ti nth esolution ,th e complexformatio nbetwee nth eoppositel ycharge dpolyion susuall ylead s tophas e separation.Thi s type ofcomple xwa salread ystudie dexten ­ sivelyb y Bungenberg de Jong (1952) forth egelatin/gu m arabicsystem . Some 10t o3 0year slate rpolyanion-polycatio n complexeswer estudie d againwit hbette rdefine dpolyelectrolyte s (seechapte r5) . Insol so rsuspension so fcharge dsoli dparticles ,whic har ehetero ­ geneoussystems ,th einteractio nbetwee ncharge dparticle san dopposite ­ lycharge dpolyelectrolyte s is,a swil lb e shown inchapte r5 ,t oa largeexten tanalogou s totha tbetwee npolyanion san dpolycation si n homogeneoussystems . Ofcours e also inth emacromolecula r systems,th einteractio nbe ­ tweenth echarge d particles and themicro-ion s iso f importance.I n thecas eo fcharge dparticle san doppositel ycharge dpolyelectrolytes , binding of thepolyelectrolyt e to theparticl e surfacewil lusuall y occur. Finally complex formationbetwee noppositel ycharge dsoli dpartic ­ les inth e absenceo rpresenc eo fpolyelectrolyte sca nb econsidered . However this type ofcomple x formation isbeyon d the scope ofthi s study. 8

Table2. 1Charge-Charg e interactions inpolyelectrolyt e systemswit h oppositelycharge dmacro-ions .

A Homogeneoussystem s 1.polyanio n +micro-catio n 2.polycatio n +micro-anio n 3.polycatio n +polyanio n 4.micro-anio n+ micro-catio n

B Heterogeneoussystem swit hnegativel ycharge dinterface s 1.anioni cinterfac e+ micro-catio n 2.polycatio n +micro-anio n 3.anioni cinterfac e+ poly-catio n 4.micro-anio n +micro-catio n

Thepossibl eelectri cinteraction sbetwee noppositel ycharge dparticle s inpolyelectrolyt e systemsar ecollecte d intabl e2.1 . Exceptcharg e interactions alsoothe rinteraction sca npla ya rol e inth eafor ementione dsystems ,suc ha shydroge nbond san dhydrophobi c bonding. Iwil lretur nt othi s andt oth econformationa laspect so f thecomple xformatio ni nth esubsequen tchapters .

2.2 POLYAMINOACIDSA TINTERFACE S

Inthi ssectio na shor tsurve yo fliteratur econcernin gth eadsorp ­ tiono fsyntheti cpolyaminoacid si sgiven . E.' Katchalski (1953) wason eo fth efirs twh ostudie dth eadsorptio n ofbasi csyntheti cpoly-a-aminoacids .Thes estudie sdeal twit hth einter ­ actiono fbasi cpolyaminoacid sa spolylysine ,polyornithin ean dpoly - argininewit hbacteria lcel lsurface s (Katchalski et al., 1953) andre d blood cell surfaces (Nevo et al., 1955). Theseexperiment swer eper ­ formedwit hpolyaminoacid s ofa lowdegre eo fpolymerizatio n (DP36 - 70).Result so fthes estudie so fa mor egenera lsignificance ,i ncase s ofcharg econtras tare : Ata certai npolyaminoaci dconcentratio ncharg ereversa loccurs . Flocculationo fth eparticle s (i.e.cells )occur sjus tbelo wcharg e neutralization. Electrostatic interactions areo fmajo r importance,bu tals onon - electrostaticadsorptio nforce spla ya role . The study of Taketomi and Kuramoto (1978) followsth esam elin ea s theearl yinvestigation so f Katchalski and Nevo. Theseauthor sinvesti ­ gated the aggregation ofhuma nplatelet sb ypolylysin e anddextran . Broadly outlined their results are analogous tothos e of Nevo and Katchalski. The interactions of polycations withmitochondri awer e investigatedb y Tomasiak et al (1980). Theeffec to fth epolycation so n the functioning ofmitochondri a wasmainl y considered. Hartmann and Galla (1978) investigated thebindin g of spin-labeled polylysine to charged bilayer membranes (i.e.bindin g tovesicles) .A remarkable conclusionwa stha tupo nbindin go fhighl ycharge dP La conformationa l transition from a random coil to apartiall y ordered conformation (not ana helix )ma y takeplace . Itwa s furtherconclude dtha thal f ofth elysin egroup s areboun d toth emembranes .Howeve rth eresult s mayb eaffecte db yth eus eo fspinlabels . As far as Ikno w there areonl y aver y fewstudie s dealingwit h the adsorption ofwea k acido rbasi c homopolypeptides on solidsub ­ strates. Corry and Seaman (1978) and Corry (1978) studiedth einter ­ actionbetwee npoly-L-lysin ean dpolystyren eparticle s (latex)b ymean s ofelectrophoreti c mobility andaggregatio nstudies .Result so fthei r investigations of relevance for this study will be discussed in chapter4 . Inconnectio nwit hth etheor y ofchromatograph yo nhydroxyapatit e columnswit hsmal lloads , Kawasaki (1978) studiedth ebehaviou ro flo w molecularmas spoly-L-lysin e inthi styp eo fadsorptio nchromatography . Juriaanse et al. (1980a,b) investigated the adsorptiono fpoly-L - lysine,poly-L-ornithine ,poly-L-asparti caci dan dpoly-L-glutami caci d towhol e BovineDenta l Enamel.Thei r results forth ehighl ycharge d polylysine andpolyornithin edeviat e fromwha t isusuall y found for polyelectrolyte adsorption.Th ever y high adsorbed amounts ofabou t _2 20-30mg. m (in the absence of added salt)ar eexplaine d bythes e authorsb y anadsorptio nmode l inwhic honl y afe wsegment sar eat ­ tached to the surface and the repulsionbetwee nth e resulting long tails is effectively screenedb y 'bridges'o fdivalen tion scomin g fromth esurface .Th ereleas eo fdivalen tion swa sals oexperimentall y verified. Inm y opinionals osurfac eprecipitatio ndu et oth ebindin g ofdivalen t ionsca npla y arole . Itwil lb eclea rtha tth eresult s of Juriaanse et al. areno trepresentativ e forsimpl e solid-liquid interfaces. A study of the adsorption of acidicpolyaminoacid st o hydroxyapatite comparable totha to f Juriaanse et al. wasperforme d by Garcia-Ramos et al. (1981). 10

As already said inchapte r1 th estud yo fcharge-induce dconforma ­ tional transitions ina nanisotropi cenvironmen ti so fprim einteres t inth estud yo fadsorbe dpolyaminoacids .Nevertheles slittl eattentio n hasbee npai dt othi ssubjec ti nth eliterature . Although Caspers et al. (1974) studiedth econformatio no fa nion - izable polypeptide adsorbed at the air-water interface andno ta t solid-liquid interfaces, their result is worth mentioning here. Caspers et al. foundtha ta charge-induce d transitionfro mth ehelical - toth ecoile dconformatio ni spossibl ei na copolyme ro fglutami caci d andmethy lglutamat eadsorbe da tth eair-wate rinterface . Especiallywit hrespec tt oth econformationa lproperties ,th epoly ­ peptidebehaviou r atsolid-liqui d and liquid-fluid interfacesca nb e different.Fo rexample ,a tliquid-flui d interfacessterica lconstraint s mayb eles simportan ttha na tsolid-liqui dinterfaces ,a sa consequenc e ofwhic hconformationa l transitionsma yoccu rmor eeasily . Pefferkorn et al. (1982) studiedth ehelix-coi ltransitio no fpoly - glutamic acid adsorbed ata nartificia l (solid)membran esurface .Th e influence ofth epolyglutami caci dconformatio no nth emembran eprop ­ erties was investigated. Their results indicate that ahelix-coi l transition inth e adsorbed stateoccur s indeedwhe n the solutionp H ischange dove ra certai nregion . A study closely related toth eunderlyin g one istha to f van der Schee and Lyklema (1982), who studied the adsorptionbehaviou ro f oligo- an polyaminoacids on Agi and theireffect s on doublelaye r propertiesan dcolloi dstability . The dimensions and states ofuncharge d polypeptide moleculesad ­ sorbed atsolid-liqui d interfaceswer e investigated by CAao (1974). This authormeasure d the adsorptiono fa variet yo funcharge dpoly ­ aminoacids from various solvents on glass.Th e discussion ofhi s resultsi sbeyon dth escop eo fthi sthesis . Discussions about the properties of polyaminoacids adsorbed or spread atliquid-liqui d or liquid-air interfaces canb efoun di nth e reviewso f Miller (1971), Miller and Bach (1973) and Malcolm (1973).

2.3 POLYELECTROLYTEADSORPTIO N

Theadsorptio no fpolyelectrolyte so nsoli dsubstrate si smor ecom ­ plextha ntha to funcharge dpolymers ,becaus eo fth eelectrostatic so f thepolymer/adsorben tan dpolymer/polyme rinteraction . Ingeneral ,polyelectrolyte s canbecom eadsorbe do nadsorbent sop - 11 positely charged toth epolyelectrolyt e (then ionexchang ephenomen a maypla y arole ) (see Hesselink, 1977), but also onadsorbent swit h the same type of charge asth epolyelectrolyte .Th e latteroccur s onlywhe nth e non-electrostatic adsorption energy ishig h enought o overcometh eelectrostati crepulsion . Usuallyhig haffinit yadsorptio nisotherm sar efound .I nmos tcase s the amount adsorbed is lower than thatfo rnon-ioni cpolymer sdu et o the strong repulsionbetwee n segments inth eadsorbe dlayer .A thig h ionicstrength ,o ra tlo wchai ncharg edensity ,polyelectrolyt eadsorp ­ tion should resemble non-ionicpolyme radsorption .I tseem stherefor e logicalt otak eth etrain-loop-tai lmode l (asi ntheorie s foruncharge d polymeradsorption )a sa startin gpoin tfo rth edescriptio no fflexibl e polyelectrolyte adsorption.Whethe r sucha ndescriptio ni sals ovali d forpolyelectrolyte s showing secondarystructur ei squestionabl e (see chapter 7). The adsorption ofpolyelectrolyte s on solid adsorbents ismainl y dependenton : Propertieso fth eadsorbent ;e.g .surfac echarg edensit ya ,surfac e heterogeneity,hydrophobic/hydrophili c balanceo fth esurface . Propertieso fth epolyelectrolyte ;e.g .degre eo fpolymerizatio nDP , pK'so fth echarge dgroups ,spacin gbetwee nth echarges ,chai nflex ­ ibility. Interactionbetwee npolyelectrolyt e andsolvent . Interactionbetwee npolyelectrolyt e andadsorben ta sdetermine db y

theeffectiv eadsorptio nenerg y \ ff, defineda sth enon-electro - Si 6X X

staticadsorptio nenerg yx s includingth eelectrostati c interactions oftrai nsegment swit hth esurface . Properties of the solution, e.g. ionic strength,polyme rvolum e fraction,pH ,presenc e ofbivalen t ions,presenc eo fspecificall y adsorbed (i.e.throug hnon-electrostati c forces)ions . Ina theoretica l treatmento npolyelectrolyt e adsorptioni twoul d be a formidable taskt oinclud eal lthes econtributions .I nth eexis ­ tingtheorie s the independentvariable swhic h aretake nint oaccoun t are:polyme rconcentratio ni nsolution ,chai nlength ,surfac echarg e a , polymerchai ncharg edensity ,th eFlory-Huggin spolymer-solven tinter ­ actionparamete r x and thenon-ioni c adsorptionenerg yparamete rx„ - For a discussion ofexperimenta l andtheoretica l trendsth ereade r isreferre d toth e reviewb y Hesselink (1983). However,mor erecen t (i.e. after 1979)experimenta l andtheoretica l developments areno t 12 included inHesselink' s article.Som e of these ifo fimportanc efo r thisstudy ,ca nb efoun di nchapte r4 t o7 . The first theory on polyelectrolyte adsorption, capable ofde ­ scribingsom eexperimenta ltrend swa stha to f Hesselink (1977). However this theory isnevertheles sinadequat ebecaus esom eimportan tproper ­ tiesar epredicte d incorrectly.Fo rexample ,th efractio no fsegment sp attachedt oth esurfac ean dth epredicte dthic kpolyelectrolyt elayer s aregreatl yi ndisagreemen twit hgenera lexperimenta lexperience . Usually,hig hp value san dthi nadsorbe dlayer sar efound ,a tleas t atlo wioni cstrength . A model forth e adsorption ofpolyelectrolyte s analogous totha t of Hesselink, waspublishe db y Silberberg (1978). Recently, van der Schee (1984) extendedth etheorie so f Roe (1974) and Scheutjens-Fleer (1979) toth eadsorptio no fcharge dflexibl epoly ­ mers. Van der Schee derivedexpression s forth epotentia ldistributio n inan d the freeenerg y of adoubl e layer containingpolyelectrolyt e charge. Inth emode l of van der Schee cilindrical symmetry,whic hi s usually encountered intheorie sfo rbul kpolyelectrolyte ,i slost .I n contrastt oth e theoryo f Hesselink, thepolyme rsegment-densit ya sa function ofth e distance toth e surface follows from thetheor yan d isno tpreassume da s Hesselink did (seeals ochapte r7) . At lowelectrolyt e concentration the theory doespredic tth ehig h valueso fp an dthi npolyelectrolyt e layers.Als oothe rpropertie sar e predicted correctly, forexampl eth eioni cstrengt hdependenc eo fth e adsorptionan dth eeffec to fmolecula rmass .Howeve rsom eothe rproper ­ ties ofadsorbe dpolyelectrolyt e predicted by the theory of van der Schee may be atvarianc ewit h experimental observations due toth e approximationsdon ei nth etheory .Th emos tseriou so fthes eare : Theus e of alattic emode l foradsorbe d andbul kpolyelectrolyt e inwhic hth echaracteristi c lengthfo rth epolyelectrolyt esegment s isth esam ea sfo rth eelectri cinteractions . Thesmearin gou to fcharge si nplane sparalle lt oth esurface ,als o forth edescriptio no fbul kpolyelectrolyte . - Theneglec to fchai nstiffnes seffects . The neglect ofth e influence ofth epotentia l onth edielectri c constantan dth edegre eo fionization .

Theconsequenc eo fthes eapproximations ,whic hexcep tfo rth elas t mentioned,ar edifficul tt oavoi da tpresent ,i stha ti ti sver ylikel y thatproto ntitratio npropertie so fadsorbe dpolyelectrolyt ewil lno t 13 bepredicte dwell .A discrepanc ybetwee ntheor yan dexperimen ti sals o expectedwhe ndiscret e charge effects atth e surface areimportant . This may be the casewit h adsorbents thathav e localized ionizable groupsa tth esurface . Nevertheless,th epolyelectrolyt e adsorptiontheor yo f van der Schee isa fruitfu ldevelopmen ti nth epolyelectrolyt eadsorptio nfield ,war ­ rantingfurthe relaborations .

2.4 REFERENCES

Bungenbergd eJong ,H.L . (1952)i n 'ColloidScience' ,Kruyt ,H.H. ,ed . Amsterdam,vol .2 . Caspers, J., Berliner, C, Ruysschaert,J.M .an dJaffe ,J . (1974). J.Colloi d Interface Sei. 49, 433-441. Corry,W.D . and Seaman,G.V.F . (1978). J.Colloi d InterfaceSei . 63, 136-161. Corry,W.D . (1978).J .Colloi d InterfaceSei . 63, 151-160. Garcia-Ramos, J.V., Carmona, P. andHidalgo ,A . (1981). J. Colloid InterfaceSei . 83, 479-484. Hartmann, W. and Galla, H.J. (1978). Biochim. Biophys. Acta 509, 474-490. Hesselink,F.Th . (1977).J .Colloi d Interface Sei. 60, 448-466. Hesselink, F.Th. (1983)i n 'Adsorption from solution atth eSolid - liquid Interface',Parfitt ,G.D. ,Rochester ,C.H. ,eds .Acad .Pres s London. Juriaanse, A.C.,Arends ,J . andTe nBosch ,J.J . (1980). J. Colloid InterfaceSei . 76, 212-219. Juriaanse, A.C.,Arends ,J . andTe nBosch ,J.J . (1980). J. Colloid InterfaceSei . 76, 220-226. Katchalski,E. ,Bichowski-Slomnitzk ian dVolcani ,B.E . (1953).Biochem . J. 55, 671-680. Kawasaki,T . (1978).J .Chromatogr . 157, 7-42. Malcolm, B.R. (1973)i n 'Progressi nSurfac ean dMembran eScience '7 , Danielli, J.F., Rosenberg, M.D. and Cadenhead,D.A. , eds.Acad . Press,Ne wYork-London ,p .183-229 . Marra,J. , Schee,H.A .va nder ,Fleer ,G.J . andLyklema ,J . (1983)i n 'Adsorption from Soltuion', Ottewill, R.H., Rochester,C.H .an d Smith,A.L. ,eds .Acad .Pres s245-258 . Miller, I.R.(1971 )i n 'Progress inSurfac ean dMembran eScience ' 4, Danielli, J.F., Rosenberg, M.D. and Cadenhead,D.A. , eds.Acad . Press,Ne wYork-London ,p .299-350 . 14

Miller, I.R.an dBach ,D . (1973)i n 'Surfacean dColloi dScience '6 , Matijevic,E. ,ed .J .Wile y& Sons ,p .185-260 . Nevo,A. , deVries ,A . and Katchalsky,A . (1955). Biochim.Biophys . Acta 17, 536-547. Pefferkorn, E., Smith, A. and Varoqui, R. (1982). Biopolymers 21, 1451-1463. Roe,R.J . (1974).J .Chem .Phys . 60, 4192-4207. Schee,H.A . van deran dLyklema ,J . (1982)i n 'Theeffec to fpolymer s ondispersio nproperties' ,Tadros ,Th.F. ,ed. ,Acad .Press ,London , p.81-100 . Schee, H.A.va nde r (1984).Doctora l thesisAgricultura lUniversit y Wageningen,Th eNetherlands . Silberberg,A . (1978)i n 'Ionsi nMacromolecula ran dBiologica lSystems ' (ColstonPaper s 29), Everett,D.H .an dVincent ,B. ,eds. ,Scientech - nicaBristol ,p .1-10 . Scheutjens,J.M.H.M .an d Fleer,G.J . (1979).J .Phys .Chem . 83, 1619- 1635, 84 (1980),178-190 . Taketomi,Y . andKuramoto ,A . (1978). Thrombos,Haemostas .(Stuttg. ) 40, 11-23. Tomasiak, M., Tomasiak, A. and Rzeczycki,W . (1980). Bulletin de l'AcadémiePolonais e des Sciences (seriede sscience sbiologiques ) CLII,vol . 28, 1-6. 15

3 MATERIALS

3.1 POLYLYSINE (PL)AN D RELATED POLYAMINOACIDS

3.1.1 Synthesis and structure

Thepolyaminoacid suse di nthi sstud ywer ecommerciall yproduce db y theSigm achemica lcompany .Thei rviscosit yaverag emolecula rmass ,a s determined by themanufacturer ,wer e used inthi sstudy .Th egenera l primarystructur ei sshow ni n fig. 3.1.

„ .-1 1er- H --' H | H 0 i H i i r i i H 1 II -- -N- -C-C- 1 1 ' R 1 II 1 H| R l^ H ] 10 „J R -"n -».J U' © ®

©

® H Hl 121* JL 122* JÊL. JAW ^no*"^ (3 Nfe H! \ HO TH (ê) © K- 0.727 nm 1

Fig. 3.1 a.Repeatin gchemica luni to fa polyaminoaci d chain.

R= -(CH 2)3-NH*Br" ,polyornithine.HBr. ;R = -(CH2)-NH*Br" ,polylysine.HBr . o 4 H b.Plana rpeptid ebon d unit -C-Ni na polyaminoaci d chain,characteristi c for t polypeptides andproteins . H c.Dimension s and configuration ofa full yextende dpolypeptid e chain (after L.Paulin g etal . (1951).Dimension s aregive n innm .

Differenthomopolyaminoacid sdiffe rwit hrespec tt oth ecompositio n ofth esid egrou pR (fig.3.1 )o fth epolypeptid ebackbone .Th enatur e ofthi ssid egrou pi sver yimportan ti ndeterminin gth esolven tproper - 16 tiesan dsecondar ystructure . Thepolyaminoacid sar eprepare db ybas einitiate dpolymerizatio no f N-carboxyanhydrides of the corresponding a-aminoacids.Thi sresult s theoretically in aPoisson-typ e distribution for themolecula rmas s of the molecules. Thepoly-DL-lysin e samples used are atactic.Th e viscosity averagemolecula rmas so feac hbasi cpolyaminoaci dha sbee n determined by themanufacture r fromth esolutio nviscosit yo fth ema ­ terial using thepoly-L-lysin ecalibratio nmetho d describedb y Yaron and Berger (1963), Sigma (1980). Themolecula rmas so fpoly-L-glutami c acid (PGA)use d in some experiments hasbee ndetermine d alsob yth e manufacturer fromth esolutio nviscosit yo fth epolyaminoaci d following Idelson and Blout (1958). Zimmerman and Mandelkern (1975) found for aPG Asampl efro mSigm a

(Mv 102.680) aR/ M ratio of1.13 ,whic h ist ob e expected fora Poisson typedistribution .Thes eauthor sfoun dals otha tth eabsolut e value of themolecula rmas swa s alwaysappreciabl ylowe r (10-20%fo r PGA samples from Sigma)tha nth evalue sgive nb yth esupplier .I ti s possible thatthi s isals o the casefo rth eP Lsample suse dhere .I t isver ylikel ytha tth eM/ S ratioi sals oclos et o1. 0 forth epoly ­ aminoacidsample suse di nthi sstudy . Mosto fth eexperiment swer eperforme dwit hpolylysine ,her eth esid e chaingrou pR (seefig .3.1 )i s-(CH-K-NH-Br -.Som eexperiment swer e donewit hpolyornithin eR = -(CH 9)o-NH-Br~,polyhistidin eR = -CH ~ [=] - + N NH andth esodiu msal to fpolyglutami caci dR = -(CH_) 2-COON a . **^ SomeP Lsample swer echecke dfo rresidua lblockin ggroup sb yUV ,I Ran d NMR spectroscopy. Inmos tcase s therewa sn o evidence foundfo rth e presence ofsuc himpurities .Th ewate r contento fth eP Lsample sap ­ peared tob e 2-4%afte rdryin gunde rvacuu move rKOH .Th eBr "conten t ofth ePL.HB r samples asdetermine d fromth eweighte d amountwa si n good agreementwit h thevalu e found fromconductometri co rPotentio ­ metrietitratio no fa PL.HB rsolutio nwit ha calibrate dAgN0 3solution . Inmos texperiment sth epolyaminoaci d sampleswer euse da sreceived .

3.1.2 Solution properties of PL

Mosthomopolyaminoacid s areno tsolubl ei nwater .O fth euncharge d polyaminoacidsonl ypolyprolin ean dpolyhomoserin ear esolubl ei nwate r (Fasman, 1967). Poly-DL-alanine has a limited solubility inwater , whilepoly-L-alanin ei sno tsolubl ea tal li nwate r (Doty and Gratzer, 1961). Thisi sprobabl ycause db yth eimpossibilit yo fth eformatio no f 17 intramolecularhydroge nbond si nit smolecule .Th esalt so fpolyamino - acidswit ha nionizabl esid egrou par eals osolubl ei nwate ro raqueou s lowmolecula rmas s electrolytsolutions .However ,th euncharge dbasi c oracidi cpolyaminoacid sar einsolubl ei naqueou selectrolyt esolutions . Hence for thesepolyaminoacid s inwate r thewel lknow nFlor yHuggin s interactionparamete r xexceed s0.5 .Ther ear en odat aconcernin gth e measurement of thex paramete r ofbasi c (PLan dPO )o racidi c (PGA) polyaminoacidsavailabl e fromliterature .Interpretatio no fcolligativ e and hydrodynamicpropertie s ofpolyelectrolyte s interm so fa secon d virialcoefficient ,fro mwhic hx value sar ederive di nth ecas eo fnon - ionicpolymers ,i sver ymuc hcomplicate db yth epresenc eo felectri c charges.S oth eusua ltechnique s fordeterminin gx value sar eno tsuit ­ ableye tfo rpolyelectrolytes . Someinformatio nconcernin gth eFlory-Huggin sinteractio nparamete r mayb e obtained fromphase-separatio n data,becaus ephas eseparatio n occursnea r charge neutralizationo fth epolyelectrolyt .However ,th e occurrence of secondary structure can complicate theprecipitatio n behaviouro fpolyelectrolyte s andpolyaminoacid s (Zimmerman and Mandel­ kern, 1975). Measurementso nth eprecipitatio nbehaviou ro fP Lwil lb e reportedi nChapte r6 .

Asstate di nsectio n1.2 ,on eo fth eattractiv efeature so fsynthet ­ icpolyaminoacid si stha tthei rsecondar ystructur eca nb emanipulate d byvaryin g experimental conditions.Fo rpolyaminoacid swit hionizabl e side chains achang e insecondar y structure (helix-coil transition) can occurwhe nth echarg edensit yo nth epolypeptid echai ni schange d by adjusting thep Ho fth e aqueoussolutio n (Fasman, 1967). ForPL- L these changes insecondar ystructur eca nver ynicel yb efollowe dwit h CD spectroscopy, see forexampl e Beychok (1967). The CD spectrao f poly-L-lysinei nth e a, ßo rcoi lconformatio nar eeve nuse da sstandar d forth eestimatio no fth eheli xan dß shee tconten to fprotein s (Green­ field and Fasman, 1969). The CD spectra of aPL- Lsampl efro mSigm a used here shared the same characteristics atp H6 an d 11.0a sthos e reported inth e literature.C Dmeasurement swit hpoly-DL(l-l)-lysin e cannotrevea lan yindication so fhelice sa tan ypH ,sinc epoly-DL(l-l ) -lysinei si nfac ta racemi cmixtur eo fL an dD lysin eresidues . WithPotentiometri eproto ntitration showeve ri ti spossibl et omo ­ nitor conformational changesbot hi npoly-L-lysin ean dpoly-DL-lysin e asa functio no fth echarg edensit yo nth epolylysin echain . 18

Potentiometrieproto n titration data ofwea k acid orbasi cpoly - electrolytes contain information aboutth econformatio no fth emacro - molecule because themeasure d pK (=pH-lo gy~ )contain sa nelectro ­ static termwhic h isdependen t on theelectri c field onth epolyio n chain, and hence depends on the conformation ofth emolecule .Th e

'intrinsic'pK Qvalu ea ta fixe dpolyion -an dsal tconcentratio nca nb e foundb yextrapolatin gth eapparen tp Kvalue st ozer ocharg edensit yo n thechain . (Nagasawa, 1970). Moredetail sabou tth emetho dca nb efoun d insectio n 6.6. The apparentp K (=pH-lo g T^—)a sa functio no fth e degree ofdissociatio n isplotte d in fig.3. 2 forpoly-L-lysin ean d poly-DL-lysinei n0. 1M NaBr .Th ecurv efo rpoly-L-lysin eshow sa 'dip ' around pH 10,cause db yth ecoi lt oheli xtransition .A son eca nse e this 'dip'i sabsen t inth ePL-D Lcurve .S oth ePL-D Lmolecul eshow s asexpecte dn ocoi lt oheli xtransition ,an dthu sstay si na coi lcon ­ formation atp Hvalue swer ePL- Li shelical .Th ecurv efoun dfo rPL- L is in reasonable agreementwit hthos ereporte d inliteratur e (e.g. Hermans, 1966; Grourke and Gibbs, 1971). Seeals oChapte r6 .

'I i '*• T= 293.15 K PKo ,/ 0.1 M NaBr // 10.7 // i i i 1 10.6 ! ' -I2 : i 10.5 i ! - X / / / ƒ 'PL-L '' // //HELIX / * 10.3 * i ~ / / J/ 10.2 /^ PL-DL COIL / /^~ 101 - / /

10.0

. i . i . i . i . 0.6 08 0.2 0.4 a 1.0

Fig. 3.2 Protontitration so fpoly-L-lysin ean dpoly-DL-lysine . c_ =2.81 nol 4.87 mol. m PL-T T L r ' PL-DL r See alsofig .6.12. 1an d fig. 6.13 inChapte r 6. 19

Thecoi lconformatio no fPL- Lan dPL-D La tacidi cp Hvalue sar epro ­ bably notth e same.Th e characteristic ratio forth e racemicrando m poly-DL-aminoacid is about a factor oftw o lower than thato fth e corresponding poly-L- orpoly-D-aminoaci d (Flory, 1969). Thiscorres ­ pondst oa mor eflexibl echai nfo rth epoly-DL-aminoacid .A tlo wioni c strengthsth eeffec to fth echarge so nth epolyaminoacid so nth echai n stiffnessi sdominant . Van der Schee (1981) founda stron gris eo fth e opticalrotatio no fL-lysin eoligomer si nwate r (pH<7)wit hincreasin g chain lengthu p to3 2residues .Thi si sindicativ eo fa structur ede ­ veloping gradually to lengthso fmor etha n1 0residues .Othe rauthor s found alsoevidenc e forth eoccurrenc e ofopticall yactiv estructur e inpoly-L-lysin emolecule s inwate ra tlo wp Hvalue s (coil).Circula r dichroism measurements havepointe dt oa nextende dheli x {Tiffany and Krimm, 1969, 1972; Rippon and Walton, 1971), although this ideaha s beencontradicte d amongother sb y Balasubramanian (1974). Painter and Koenig (1976) contributedt othi sdisput ewit hthei rRama nan dI Rspec ­ troscopic studyo fth e solution conformation ofpoly-L-lysine .Thei r results are in favoro fth epresenc eo fsom eloca lordere dstructur e atlo wioni cstrengt hsimila rt otha tsuggeste db yTiffann yan dKrimm . CD andoptica l rotationmeasurement s areno t suitable todetec tth e presence ofsom e structurei nrando m (1:1)poly-DL-lysin ebecaus eso ­ lutions ofthes emolecule s areracemi cmixture so fL -an dD residue s as stated earlier.Hence ,wit hth e abovementione d techniques iti s notpossibl e to compareexperimentall yth eflexibilit yo fpoly-L -an d poly-DL-lysinemolecules . Information aboutth echai nstiffnes sma yb eobtaine d fromfo rex ­ ampleviscosimetric , lightscatterin gan dflo wbirefringenc emeasure ­ ments.Th e determined chain stiffness at low ionic strengthwil lb e largedu et oelectrostati ceffects ,i.e .th eelectrostati cpersistenc e length,whic h isidentica l forPL- L andPL-D Li sth elarges tpar to f thetota lpersistenc e length.Abov e ionic strengthso f 0.1M anda t acidicp H differences inchai nstiffnes sdu et odifferen tstereoregu -

laritybecom evisible ,becaus ethe nth e-NH 3 chargeso fP Lar escreened . This difference inchai n stiffness cancaus e adifferen tadsorptio n behaviouro fpoly-L -an dDL-lysin ea thighe rsal tconcentrations . Toconclud e this sectionfig .3. 3 showsmolecula rmodel so fa par t ofa poly-L-lysin echai ni nth efull yextended ,an dcompac tconformation . 20

SExxsoaxscq^

ösa=E&3KKS3Q3aas^

Fig. 3.3 Stuart models of poly-L-lysine, a) extended configuration, b) compact conformation.

3.2 POLYSTYRENE LATEX

3. 2.1 Preparation

The preparation and characterization of emulsifier-free polystyrene 21 laticeswa sessentiall yth esam ea sdescribe db y Furusawa et al. (1972) and Norde (1976), exceptfo rtw omodification si nth epurificatio nstep :

Afterpreparatio no fth elate x (latexM :cfK-S-Og )= 1.8 5mM ,c(KHC0 3) =

10mM ;Late x L:c(K oS„0o)= 0.31 mM), the4- 6 latexsample so fabou t ^ Z z ö 200c m (solid content:6-8 % (w/v))obtaine di non elate xpreparatio n run,wer emixe dtogethe rwhe nth eparticl ediamete ro feac hsampl ewa s thesam ewithi nnarro wlimits .The nthi slate xbatc hwa ssteamstrippe d under reducedpressur e at308-313K , to removeexces smonomer .Durin g thisproces s the ionic strength of thelate xwa skep ta tth esam eo r ata lowe r level thanth e initial value.The nth elate xwa streate d batchwisewit ha tenfol dexces sio nexchang eresin sfro mBiora d (Dowex 50W-X.cationi c resin,Dowe x 1-x.anioni c resin), using amixe dbe d technique.Th e tenfold excessi swit hrespec tt oinitia lbuffe rvalu e (i.e.ioni cstrength )o fth elatex .I nthi swa yth elo wmolecula rmas s saltswer eremove dan dth elate xwa sbrough tint oth eprotonate dform . Conductometric titration ofth e latex after the first and asecon d treatmentwit h the ionexchang eresin sshowe dtha ton eru no fio nex ­ change sufficed. The ion exchange resinswer e extensively purified following themetho d of Van den Hul and Vanderhoff (1968). Afterth e ionexchang e treatment anddeterminatio no fth esurfac echarg eo fth e latex,th e latexwa sbrough tint oth eN a formb ytitratio nwit hNaO H andstore di na refrigerato ra t27 6K unti luse ,t oslo wdow nan ysur ­ facegrou phydrolysi san dmicrobia lgrowth .

3.2.2 Characterization of PS latex

Propertieso fa latex ,lik eparticl ediameter ,numbe ran dnatur eo f surfacegroup setc. ,ar ei nfirs tinstanc edependen to nth econdition s prevailing during thepolymerizatio nreaction .Fo rinstance ,th esur ­ facecharg e density canb evarie db yvaryin gth einitiato rconcentra ­ tion and theconcentratio n ofbuffe r and/or supportingelectrolyte . For adetaile d descriptiono fth e influenceo fpolymerizatio ncondi ­ tionso nth e latexpropertie s Irefe r toth e reviewb y Hearn et al. (1981). The properties of theprepare d PS latex canb e alteredb y the cleaning procedure applied and the duration and conditions of latex storage (see also Hearn et al., 1981; van den Hoven, 1984). In thebeginnin g ofthi s study dialysis ofth elate xwa suse dt oremov e thelo wmolecula rmas ssalt san dunreacte dstyren emonome ri nth esam e way asdescribe d by Furusawa et al. (1972) and Norde (1976). Because ofth epossibl e contamination ofth e latexdurin gth eprolonge ddia - 22 lysis and the ineffectivityo fthi sproces s (Hearn et al., 1981)', the dialysis stepwa s skipped and replacedb y steamstripping and anio n exchangeprocedur et oremov eexces selectrolyt ean dt obrin gth elate x inth eprotonate d form as already describedunde r3.2.1 .Fo rstudie s onprotei n adsorption onP S latex, Norde (1976) usedlate xwhic hwa s only dialysed,becaus e useo fio nexchang eresin swoul dpossibl ycon ­ taminate the latexb ypolyelectrolyte s leaching fromth eresin ,eve n afterextensiv epurificatio no fth eresins .Thi sha sindee dbee nfoun d by McCarvill and Fitch (1978) whencation -an danio nexchang eresin s areuse dseparately .O nth eothe rhand ,whe nmixe dbe dresin sar euse d McCarvill and Fitch (1978) and VanderHoff (1970) foundn oindicatio no f surfacecontamination .Th ereason sfo rthi sapparen tcontradictio nar e notclea r (Hearn et al., 1981). Theus eo fio nexchange d latexfo rad ­ sorption studies has the advantageove rdialyse dlate xo fa mor eac ­ curatelyknow nsurfac echarge . Knowledge of the surface charge ofth elate xrequire sbeside sth e titration chargeo f ionexchange dlatex ,a tleas tadditiona linforma ­ tion aboutth e sulphur contento fth elate xbefor ean dafte rio nex ­ change (Norde, 1976). Adetaile ddiscussio no fth eadvantage san ddis ­ advantages of thevariou s latex cleaningprocedure s canb e foundi n the review by Hearn et al. (1981). Despitepossibl e differences in latexpropertie s by applyingdifferen tcleanin gprocedure sther ewer e no indications found inthi s study thatth e adsorptionbehaviou ro f polylysineo nlate xi sver ydependen to nth ecleanin gprocedur eadopted . Adsorptionisotherm so fP Lo nonl ydialyse dlate xo rsteamstrippe dan d ionexchange d latexwer ewithi n experimental accuracy the same.Th e samewa sfoun dfo rproto ntitration so fadsorbe dpolylysine . Themea nparticl esiz eo fP Sparticle swa sdetermine db ytransmissio n electronmicroscopy .Operatin gcondition swer eselecte dsuc ht ominim ­ izeth e effects ofth eelectro nbea mo nth eparticles .Th einstrumen t was calibrated atth emagnification suse dagains ta carbo nreplic ao f a diffractiongrating .Al lsample suse dshowe dmonodispers espherica l particles. The meanparticl e size ofth eP Sparticle swa sobtaine d from EMpicture sb ymeasurin g theparticl ediamete ro f25-9 0partic ­ les,i nth esam ewa ya sdescribe db y Norde (1976, 1978). Theuniformit ycoefficien tU ,define da sU = d / d, wher ed andd areth eweigh tan dnumbe raverag eparticl ediamete rrespectively ,i sa measure ofth ehomogeneit yo fth elatex ,d / d canb ecalculate dfro m theindividua lparticl ediameter sd .using : 23

d {(Z.n.df)/(I.n.d?)}1/3/ {(^n^)/!^} (3.1) V n 'lil' ill' where n. isth enumbe r ofparticles .Fo r all negatively chargedP S laticesuse dth esiz edistribution s arever ynarrow ,wit h1.000

Table3. 1Relevan tpropertie so fP Slatice suse di nthi sstud y

PS d(nm ) surfacecone . (EM) Aor^(MB ) a Latex -OSO~(Mmol.g ) m .g m .g mC.m

613 ± 19 1.005 6.99 9.3 Ml -74 M? 551 ± 14 1.003 5.98 10.4 -60 M, 657 ± 30 1.008 5.0 9.2 4.0 -52 M4 602 ± 21 1.005 4.2 10.0 -40 MS 622 ± 16 1.003 3.6 9.2 -39 L 488 ± 18 1.006 1.3 11.7 1.7 -11 P 405 ± 35 1.03 - 14.8 +

Another observationmad eb y van den Hoven (1984) istha tth elate x particlediamete ri slowere db y^3% , afterth eio nexchang etreatment . A possible qualitative explanation for thisphenomeno nma yb etha ta complete layer ofolig oan dpolystyren emolecule si sstrippe do ffro m thelate xsurface .Thi si si naccordanc ewit hth elos si nbul ksulphu r content of the latexparticle s up to30 %observe d by Norde (1976). Anotherpossibilit y istha tfractionatin go fth eparticle soccurre ddu e toth eio nexchang etreatment .I ti sals opossibl etha tth eP Sparticle s haveanothe rbehaviou ri nth eelectro nbea mafte rio nexchange .However , theeffec to nth especifi csurfac eare ao fth elate xi swithi nexperi ­ mental error inou r case.Becaus e ofth emonodispersit yo fth elate x samples,th e specific surface areaA (surface areape r grampoly - sp styrene)ma yb esimpl ycalculate dusing :

sp p.d (3.2) inwhic hp i sth edensit yo fpolystyrene ,wher ei tdoe sno tmatte rwhethe r 24

dw ord n ischose n ford . Norde (1976) foundb ymean so fpyknometry , densities of theP S latexparticle s between 1,048 and 1,052 kgm~ 3 The specific surface area foundwit h (3.2)i sth e geometricsurfac e area, so the actual (physical), surface area,whic h depends onth e methoduse d for itsdetermination ,ma yb e higher due tosurfac eir ­ regularities ('hairy'surface )an dfo rporosit yo fth elate xsurface . There are indications thatth eN ,B.E.T .specifi csurfac eare adeter ­ mined with freeze dried PS latex samples ishighe r (upt o30% )tha n theE M surface (Norde, personalcommunication) . Barclay (1970) repor­ teda simila rresult . Kamel (1981) however foundalmos tn odifference s betweenth etw osurfac eareas .Becaus eo fthei rshap eth esurfac e 'seen' bymacromolecule sma yb eclose rt oth egeometrica lon etha nt oth eB.E.T . (N,)specifi csurfac earea .

The adsorption of lowmolecula rmas s compounds fromsolutio ni sa commonan dexperimentall yrelativel yeas ytechniqu e forestimatin gth e interfacial areao fadsorbent sa tth esolid-liqui dinterface .

T=295K

5 12 A ^£L_ __A. E >T*—AA ï-

—O*— J) ûs°

100 200 300

cMB/mmol. m"

Fig. 3.4 Adsorption isotherms of MB on PS latex. T = 295 K o-o PS latex M„ a 120 C.kg ; pH(ads) - 5.5; A (geom.) = -•? ° 9.2 m2.g 1 sp A-A PS latex L a = 500 C.kg"*; -pH(ads1 ) - 5.5; A (geom.) = 11.0 m2.g_1.

In fig. 3.4 adsorption isotherms of methylene blue (MB) from aqueous solution on PS latices with different surface charge are shown. The adsorbed amount of MB was determined by depletion from solution, re- 25 moving theP Sparticle si nessentiall yth esam ewa ya sfo rP Ladsorp ­ tion (section4.2.3) .Th eM Bconcentratio nwa sdetermine dspectrophoto - metricallya ta Beekma nmode l360 0spectrophotomete r at66 3nm .Th eM B usedwa sanala rgrad efro mBaker . Theadsorptio nisother mo fM Bi sa hig haffinit yone .Thi swa sals o foundfo rth eadsorptio no fM Bo nAg i (Koopal, 1978). Theplatea uvalu e isver y sensitive to the surface charge of the latexa son eclearl y can see from fig.3.4 . When inbot h casesmonolaye r adsorptiona t theplatea u adsorption isassume dan dfo rth ep Hunde rconsideration , acros ssectio na permolecul eM Bo f0.5 5 nm2 (monolayero fM Bdimers ) istake n (Koopal, 1978) thenth eM Bsurface so flate xM _an dL ar ere - 2-1 2-1 spectively4. 0m g and1. 7m g ,wherea sth especifi csurfac earea s obtained from EM are 9.2 and 11.0m g respectively2- 1 . Even ifM B monomer adsorption should occur thevalue s found forA arelowe r than the geometrical ones. Itseem s thatonl y thecharge dgroup so n thelate xsurfac ean dthei rdirec tenvironmen tar epossibl eadsorptio n sites (patchwise adsorption). Coagulation ofth e latex aftercharg e neutralizationb yM Boccurred .I nprincipl ethi sca nlea dt oa surfac e area reduction and the estimated MBplatea uadsorptio nvalue sma yb e somewhatto olow . Fromth eabov eth econclusio nma yb edrawn ,tha tM Badsorptio ni sno t suitable forspecifi csurfac eare ameasurement so flatices ,bu trathe r acrud emeasur efo rth esurfac echarg eo fa latex .A simila rconclusio n was reached forth eadsorptio no fM Bo nsilic aan dglas s (seesectio n 3.3.1 and3.3.2) . Kasper (1971) usedth ecomplexatio no fM Bwit hth esurfac esulphonat e groupsfo rth edeterminatio no fth esurfac echarg eo fth elate xh euse d for flocculationstudies .A sth eadsorptio no fM Bi ssuperequivalent , thevalue sh e obtained areto ohigh .I tma yb enote dtha tther ei sa reasonableagreemen tbetwee nth eMB ,N 2 B.E.T.an dgeometrica lsurfac e area ofAg i suspensions (Koopal, 1978; de Keizer, 1981). Themobilit y ofth esurfac echarge sand/o radsorbat emolecule so nAg ima yb erespon ­ siblefo rthis . Inconnectio nwit hth eabove ,th eadsorptio no ftetr aalkylammoniu m ions (TAA )o nsolid-liqui dinterfaces ,migh tals ob econsidered . These ions,hav e aspherica l shape,s otha tn oproblem saris ei nde ­ termining the orientation atth e interface.Th ecros s sectiono fa TAA iona tth einterfac ei sthu swel ldefined . De Keizer (1981) found forth etetrabutylammoniu m areao fAg isuspension sa valu etha ti s10 - 20%highe r thanth eM B surface area.Thi sdifferenc ei swithi nexpe - 26 rimental error.Howeve r theplatea uvalue s oftetrapropy lan dtetra - butylammoniu mion sar eth esame ,indicatin gtha tTA A ionsd ono tfor m a densemonolaye r onAgi ,an dtherefor ethe ygiv ea to olo wvalu efo r A„ .A simila rsituatio nexist sfo rP Slatex , sp + Van den Hoven (1984) measuredth eadsorptio no ftetrapropy l (TprA) , tetrabutyl (TBuA )an dtetra-amylammoniu m (TAmA )ion so nP Slate x( a = 82.9m Cm ~ ).Th eaffinit yo fth eTAA + ionsfo rth elate xincrease s inth egive norder .Howeve rth eplatea uvalu ei sth esam efo ral lthre e + . -2 TAA ions: 0.8 pmolm .Thi svalu ecorrespond swel lwit hth esurfac e charge of the latex, indicating thatth eTA A ions adsorb onlya t charged (-OSO~)group s on the surface.Th e ionic radiuso fTA A in­ creases inth e orderTpr A ,TBu A ,Tam A ,s oth ecalculate dapparen t specificsurfac eare aincrease sals oi nth egive norder .Th eTA A area isi nal lcase ssignificantl y lowertha nth egeometrica lsurfac earea . Inthi srespec tTA A ionsbehav esimila rt oM Bion sa tp H5.5 .Th eob ­ servations above areo fimportanc efo rth einterpretatio no fth epla ­ teauadsorptio nvalu eo fpolylysine . The surface charge of theP S laticeswa sdetermine dafte rio nex ­ change by conductometric and/orPotentiometri e titrationwit h0. 1M NaOHunde rN _ atmosphere.Fo rdetail so fth etitratio nmetho dse esec ­ tion4.2.4 .Ther ewer en osignifican tdifference sbetwee nth eequiva ­ lencepoint s ofth e titrations after a firstan d asecon dtreatmen t with ion exchange resin,indicatin g thatafte r the firsttreatmen t protonationwa scomplete .I nfig .3. 5 atypica lexampl eo fa conducto ­ metrictitratio ni sshown .

5 6 7 limolNaO H

Fig.3. 5 Conductometric titration of ion exchanged (mixed bed) PS latex M„ (H+ form)wit h0. 1 MNaOH . 27

From the titration curves itwa s conductedtha tth elatice suse ddi d notcontai n significant amounts ofwea k acidgroups .Th epresenc eo f surfaceO Hgroup s (resulting from theKolthof freactio ndurin gpoly ­ merization,o rhydrolysi so fR-OSO lgroup sdurin gstorage )i so fcours e notexcluded .Howeve rth eKolthof freactio ni ssuppresse ddu et oth eus e

ofKHC0 3 inth ereactio nmediu m (Hearn et al., 1981). The.slop eo fK vs [0H~] (see fig.3.5 )befor e the equivalencepoin t is appreciable lower (4-5 fold)tha nth eslop efo rth ecorrespondin gstron gaci dti ­ tration,a si sgenerall yobserve dfo rP Slate xtitrations .I npar tthi s isdu et o alowe rcontributio nt oth eoveral lconductivit yo fH ions inth eelectrica ldoubl elayer .Wit hlinea rpolyelectrolyte s asimila r behaviouri sobserved . Anotherquestio ni swhethe ro rno tth esurfac echarg eo fP Slate xi s the samebefor e andafte rio nexchange . Norde (1976) assumedtha tth e reduction intota lbul k sulphur thath efoun dafte rio nexchang ewa s completely attributable toth elos so foligo -an dpolystyren esulpho - natemolecule s during theexchang eprocess ,an dtha tth eburrie dbul k sulphategroup sar eno texpose da tth einterfac eafte rio nexchange .H e assumedthe ntha tth esurfac echarg e (Cg~ )befor eio nexchang ei sth e measuredon eafte rexchang eplu sth edifferenc ei nsulphu rconten tbe ­ forean dafte rth eio nexchang etreatmen tpe rgra mlatex .Fo rP Slate x M-.(i.e .a late xwit h the sameformulatio na suse dhere) ,h efoun da -2 ao valu_e_ (afte r ionexchange )3 o f -45mC m anda (beforoe i.e. )= -84mCm " .Ther e are several factstha tplea dagains ta reductio ni n a duringth eio nexhang eprocess : (i) repeatedio nexchang etreatment sgiv eth esam evalu eo fa . (ii)Th e adsorptionplatea u valueo fP Lo nP Slate xM i sth esam ebe ­ forean d afterexchange ,whil e theplatea uvalu ei ssensitiv et oth e surfacecharg ea tleas ti nth eregio n-(10-60 )mC m (seesectio -2 n4.3.5) . Analternativ eexplanatio ni stha tdu et oth eio nexchang etreatmen ta thin layerpolystyren e isstrippe doff ,thu sformin ga ne wP Ssurfac e withne w -OSOlgroups ,wh owher eburrie da sio npair si nth eP Spar ­ ticlebefor eth etreatment .Th econsequenc ethe ni stha tth ea values givenb y Norde (1976, 1978) forlate xbefor eio nexchang ema yb eabou t 50%to ohigh . Latex thatonc eha sbee nexpose dt o0. 1M electrolyt esolutio nca n havea surfac echarg etha ti sappreciabl ehighe r (sometimesb ya smuc h as a factor two)tha n latex thatha sno tbee na thig h (>0.1M )sal t concentrations.Thi swa s concluded by van den Hoven (1984) fromcon - ductometric titrationso f latexsample swh ower eio nexchange dbefor e 28 andafte rexposur et o0. 1M KNO, .Whe na secon dexposur et o0. 1M KNO , wasapplie dn oincreas ei n a wasobserved .N oclea rinterpretatio no f thissal teffec tca nb eoffere da tth emoment .Th elatice suse di nthi s studywer e nottreate dwit h0. 1M electrolyt esolution ,befor eus ei n adsorption experiments,excep t for latexM ,. Th econsequence so fth e uncertaintyi n a athig hioni cstrengt hwil lb ediscusse di nsection s 4.3.5,4.3. 6 and6.6.4 . The question whether the surface ofth eP Sparticle s is smootho r 'hairy'wil lb edeal twit hi nth enex tsection .

3. 2. 3 The polystyrene-water interface

Insection s 3.2.1 and3.2. 2 somepropertie so fth eP Slatice swer e discussed, related toth especifi csurfac earea ,natur ean damoun to f surface groups. Inthi ssectio nsom eothe rpropertie so fth ePS-wate r interface willb e discussed thatma yb eo frelevanc efo rth eadsorp ­ tiono fpolyaminoacids . As stated inth epreviou ssection ,o nth esurfac ether ear emainl y -OSO.,groups .Fo r alate xwit h asurfac e chargeof ,say ,-5 0mC m , -2 the total surface area ofthes egroup si s0.1 5m perm geometrica2 l2 surface area.Th eres to fth e surfacei shydrophobic .Thus ,th esur ­ face 'seen'b ypositivel y charged adsorbates likeMB ,TA A ionsan d polylysinei sver ylikel yt ob eheterogeneou si nnature . Another consideration iswhethe rth epolystyren esurfac ei ssmoot h or 'hairy'. B y 'hairy'i ti smeant ,tha tth esurfac ei scovere dwit h boundoligo -an dpolystyren emolecule sdanglin gi nsolution . Goossens and Zembrod (1981) founda nincreas ei nparticl ediamete r of acarboxylate d latexwit hphoto ncorrelatio nspectroscop yupo nin ­ creasingth ep Hfro m3 t o8 .Thi si sexpecte di nth ecas eo fa 'hairy ' surfaceo nwhic hth e 'hairs'consis to fpolyme rchain swit hcarboxylat e groups..Th epolyme rchain sar esolubl ei nwate rbecaus eo fth echarge d end groups.Uncharge dpolystyren emolecule s arenearl yunsolubl e in water (x> 0.5 )an dwil ltherefor ela ymor efla to nth esurface ,thu s forminga mor eo rles s 'smooth'surface . From electrokinetic measurements onP S latexplug s van den Hoven (1984) foundevidenc etha tals olatice swit h-OSO lgroup sa suse dher e showa 'hairy'character .Th esurfac echarg eo fthes elatice showever , ismuc h lower than thato fth e carboxylated latices ofGoossen san d Zembrod.Th ehair y layeri stherefor eprobabl yles sdens etha ni nth e caseo fa highl ycharge dlatex . 29

Thehydrophobicit yo fth epolystyren e -waterinterfac ei sa nimpor ­ tant factor determining the adsorptionpropertie s ofadsorbate swit h hydrophobic groups such asth e -(CH?).- groupi nth esid echai no fa PL residue, because of thepossibilit y ofhydrophobi c interactions betweenthes egroup san dth eadsorbent . Sinceth epolystyren eitsel fi shydrophobi cbu tth e-OSO lgroup san d also-OH ,-COO Han dspecificall y adsorbed ions,i fany ,ar ehydrophili c in nature, the polystyrene-water interface consists ofhydrophili c patches in anotherwis ehydrophobi cenvironment .Th edensit yo fthes e polar groups on the surface determines the overall hydrophobicity, butth emeanin go ftha tfo radsorptio npropertie si sobscure . Because ofth epatchwis e distribution ofhydrophobi cregions ,th e stateo fhydratio n variesals oalon gth esurface .Th estat eo fhydra ­ tion along apolypeptid e chain isver y important indeterminin gth e secondary and tertiary structure of thesemolecules .Becaus eo fthi s iti sver y likely thatth e state of interfacial waterplay sa role , indeterminin gth estructur eo fadsorbe dpolypeptides . From this section andth epreviou son ei tca nb econclude dtha tP S latices areno t as ideal amode lsubstrat efo radsorptio nstudie sa s wasgenerall ybelieve dsom e1 0year sago .Nevertheles sthe yconstitut e stillinterestin gsubstrate sfo rth einvestigatio no f (bio)polymerad ­ sorption,mainl ybecaus eamon gth eorgani ccolloid savailable ,P Slat ­ icesar eth emos tstudie done san drelativel ybes tcharacterize dones .

3.3 SILICAAN DBOROSILICAT EGLAS S

3.3.1 Preparation and specific surface area of silica

Pyrogenic (fumed)silica sar eprepare d fromSiCl .b yoxidatio ni na hydrogen airmixtur e athig htemperature s (1270K) . This resultsi n amorphous spherical SiO,particles .Befor e contactwit hwate rhydro ­ phobic =Si-0-Si = (siloxane)bond spredominat e-a tth esurfac ean donl y a few=Si-O Hgroup s arepresent .Th especifi csurfac earea sa sfoun d from N2 orA r adsorption data aregenerall y ingoo d agreementwit h the surface found fromE M (Koberstein and Voll, 1970). Gasadsorptio n measurements withA r arepreferabl e abovethos ewit hN 2 asth ead - sorbate, because the latter interacts specifically withs-Si-O H groupso nth e surface.Th eresult s of Koberstein and Voll (1970) in­ dicatetha tfume dsilica sar eessentiall ynonporous . AEROSIL0X5 0 (Degussa)use di nthi sstud yha sa specifi csurfac eare a 30 of5 0± 1 5m g as2state — 1 db yth emanufacturer .Thi svalu eaqree swel l withtha treporte db y Sonntag (1980) forthi ssilica .Th esurfac eare a obtained frommethylen eblu e adsorption (a =0.5 5ran ) i s3. 6m g 2 . 2-1 (pH^6, no added electrolyte)indicatin g adsorption ofM B at= Si- 0 sites only, a similar resulta swa s found forM B adsorptiono nP S latex (section3.2.2) .

3. 3.2 Borosilicate glass purification and specific surface area

Theglas spowde ruse di nthi sstud ywa sa borosilicat eglas s (732-01) obtained fromSovire l (particles< 6 0pm) . Thecompositio no fth eglas s

as statedb y themanufacture ris :80 %Si0 2, 13.00%B 203,2.25 %A1 203,

0.05%Fe 203, 3.50%Na 20an d1.15 %K 20.Purificatio no fth eglas spowde r was done inessentiall y the samewa y asdescribe d by Nyilas (1966): Firstth epowde rwa swashe dwit hwate r twice,the nwit h 6M HC la t room temperature until noiro ncoul db edetecte di nth ewashin gwate r by SCN~.The nth epowde rwa sexhaustivel ywashe dwit hdestille dwate r untillth ep Ho fth esupernatan twa sabou t6 .Th epowde rwa sthe ndrie d at37 8K .Th e specific surface area of thepowde r wasdetermine db y B.E.T.N _ adsorption,M Badsorptio nan delectronmicroscopy . For the glasspowder sampleuse dher eth eB.E.T .metho di sno tver y 2- 1 accuratebecaus eo fth esmal l (<1 m g )valu eo fA (seefo ra dis ­ cussion Beurton and Bussiere, 1970). Inaddition ,th ecompute dvalu e ofth esurfac eare ai sprobabl yals oto olow ,becaus eN _ha sa prefer ­ ence for freeO Hgroup so nth esurfac e {Doremus, 1973). TheB.E.T .N _ 2-1 surface area (0.5 ± 0.2 mg )an d theM B adsorption area (A = 2-12 p 0.21m g ,a = 0.55ran , pH~6 ,n o added salt)diffe rb y abouta factor oftwo .Th eplatea u adsorption ofM Bo nth eglas sfro ma 50 % methanol-water mixture was just half of that in pure water (see fig.3.6) .Thi spoints ,a swa s alsopr eassumed ,i nth edirectio no f dimeradsorptio no fM Bmolecule sfro mwater . Anotherargumen tfo rdime radsorptio no fM Bfro mpur ewate ro nglass , stemsfro ma compariso nwit hth eadsorptio no fToluidin eblu ewhic hi s very similar toMB .Toluidin eblu eshow sa colou rchang efro mblu et o pink-violet upon adsorption onth eglas s (metachromacy).Thi si spos ­ siblewhe n aggregates onth esurfac eexist .A simila reffec tha sbee n observed forth eadsorptio no fcyanin edye so nsilve rhalide s (Padday, 1970). Also forth ebindin go ftoluidin eblu eo nanioni clinea rpoly - electrolytes the metachromasy is well established (Horn, 1978). 31

100 200 300 400 3 cMB-/ mmol.m"

Fig. 3.6 Adsorption of MB on to borosilicate glass (A (B.E.T.) 0.5 ir^.g"1). sp pH (afterads. )= 6.8 ;n oadde d electrolyte;A/ V= 125m .1 •1 o-o Adsorption fromwater/methano l (1:1); A-AAdsorptio n fromwater .

Nxjilas (1976) found fromwate rvapou radsorptio nmeasurement so na soda lime type glass (75%Si0 2# 10%Ti0 2/BaO,5 %CaO ,3 %B 203 and2 % Na-O)whic hha dbee n subjected toth esam epretreatmen ta sth eglas ­ ses used by us that the glass surfacewa s essentiallynon-porous . This isprobabl y alsoth ecas ewit hou rglas spowders .Th econclusio n from the above istha tals o onth eglas ssurfac eM Bmolecule sadsor b mainly onth echarge d sites,thu sexplainin gth ediscrepanc ybetwee n theN 2 andM Bsurfac earea . Thevalu e found forth egeometrica lsurfac eare afollowin gth eme - 2- 1 thoddescribe d forirregula rparticle sb y Heywood (1970) is0.2 4m g , usinga calculate dshap efacto ro f11.0 .Th eorde ro fmagnitud ei sth e same asth eM Bsurfac earea .Als oth egeometrica lsurfac ei sto olow , becausea kin do fparticl eenvelopp ei smeasured ,thu sneglectin gsmal l irregularities.Althoug h theM Badsorptio nisother mca nb edetermine d relatively easily and accurateth ecalculate dM Bsurfac eare ai sver y limitedbecaus e ofman ycomplicatin gfactor ssuc ha sth ep Han dioni c strengthdependenc eo fth eadsorption ,a conclusio nwhic happlie sals o toth esilic aan dlate xadsorbents ,an dwhic hwa sals oreache db ysev ­ eralothe rauthors . The reasonable agreementbetwee nM B adsorption andN _ adsorption valueswhic h exist forth e specific surfaceare ao fAg iparticle si n 32

Agi suspensions as already stated in thepreviou s section (3.3.-2), seemst ob ea nexceptio nt oth eabove .

Despiteth erelativ einaccurac yo fth eB.E.T .N 2 specificsurfac eare a ofth eglas spowder ,i tappear st ob eth emos trea lvalu eavailabl ean d thereforethi svalu ewil lb euse dhenceforth .

3. 3.3 Determination of the surface charge density by conductometric or Potentiometrie proton titrations

Thecharg eo nth esilica-wate rinterfac earise sfro mth eionizatio n ofsurfac esilano lgroup saccordin gt oth ereaction s=Si-O H« -= Si- 0+

H or=Si-OH+ H *nsi-OH 2,th elatte rreactio ni showeve rno tobserve d atpyrogeni csilic ainterface s {Abendroth, 1970). Inth ecas eo fglas sth eionizatio no fsB-O Han dsAl-O Hgroup spre ­ sent at the interface plays also arole .Th epoin to fzer ocharg e (p.z.c.)o fglas s isdependen to n theamoun tan dnatur eo fbasi cox ­ ides present at the surface.Becaus e of thep H and ionic strength dependence of the ionization equilibria,th e surface charge ofth e oxides iso fcours e alsop H dependent.A surfac echarge-p Hcurv eca n be obtained from aPotentiometri eproto n titrationo f asol ,i na n indifferentelectrolyt esolution .A descriptio no fth emetho di sgive n forexampl e inth erevie wo nth echaracterisatio no faqueou scolloid s by James and Parks (1982) p.144 ,145 . Inth epresen t studycharg edensitie sar ebase do nth eB.E.T .(N, ) surfaces andcalculate d froma =F(r „-r_ „) wher e T„, andr_ „ are o ri+ Uii— rl+~ un— the surface excesses ofhydroge nan dhydroxy lion spe rm .A poin to f zero charge cani nprincipl eb e established by theintersectio no f er-p Hcurve sdetermine da tvaryin gconcentration sindifferen telectro ­ lytes.Fo rou rsyste mthi swa sfoun dno tpossibl ei nth ecas eo fsilic a andglas sbecaus e inth eregio no fth ep.z.c .th ecurve sa tvariou s ionicstrength sar ealmos thorizonta lan dsuperimpos eove ra relativel y broad pH traject.Fortunatel y thea -p Hcurve s are therefore rather unsensitive for the precize value chosen forth ep.z.c . (seeals o Abendroth (1970) and Sonntag (1980).). Thep.z.c .wa schose na tp H3. 0 following the reasoning of Abendroth (1970). Thesam evalu ewa suse d forAerosi l OX50b y Sonntag (1980). Forth epyre xglas sals oa p.z.c . atp H3. 0wa staken . Inth e case ofAEROSI LOX5 0wit honl yH asth ecounterions ,with ­ outadde d salt,accurat ePotentiometri emeasurement sar edifficul tt o 33 perform.A n estimate ofth e surface chargeca nthe nb eobtaine dfro m a conductometric titration of the solwit hNaOH .Th e firstbrea ki n the graphobtaine d is ameasur e ofth esurfac echarg epresen ta tth e pHo fth efirs tbrea kpoint . All conductometric andPotentiometri etitration swer eperforme di n awellclose ddoubl ewalle dvesse lthermostate da t293.1 5± 0. 1K unde r aCO _ free,wate rvapour-saturate dN ? atmosphere.Fo rexperimenta lde ­ tails concerning the Potentiometrie measurements and the apparatus used, see section 6.6.3.2. Experimental details ofth e conductivity measurements will be giveni nsectio n5.2.2 .Proto ntitration so fth e suspensionswer eperforme d with0. 1M NaO Ho r0. 1M HC lusin ga Mett - ler DV10/DV201 automatic burette.Befor e the starto f atitration ,

C02-freeN_ ,saturate dwit hwate rvapou ro f293.1 5K wa sbubble dthroug h the suspensions for atleas t2 0min .Th etitratio nvolum ewa salway s 3 20c m .Fo r theproton-titration s2.5 % (w/v)silic asol san dsuspen - 3 sionso f1 0g glas spowde ri n20. 0c m electrolytesolutio nwer eused . The suspensions wereprepare dwit h CO_-freeconductivit ywater .Wit h silica60 s after each NaOH addition (1-2nmol )a constan tp Hvalu e was reached. Inth e case ofglas spowde rp Hreading swer etake nwhe n thep Hchang eo fth esuspensio nwa ssmalle rtha n0.0 1p Hunit/min .

T= 29315K 0.01M NaBr

Fig.3. 7 Surface charge pH curves for AER0SIL 0X50 silica and Borosilicate glass (Sovirel). 34

Betweenp H4 an d7 thi swa sth ecas ebetwee n5-1 0mi nafte rth ead ­ ditiono f1- 2nmo lNaOH .I nfig .3. 7th esurfac echarg e a isplotte d asa functio no fp Hi n0.0 1M NaB rfo rth eborosilicat eglas spowde r and forth eAEROSI L0X5 0used .Th eresult swit hsilic aar ei nreason ­ able agreementwit h Sonntags (1980) results.H euse dKC linstea do f NaBra sth esupportin gelectrolyte .Th ebehaviou ro fth eborosilicat e glass adsorbenti ssimila rt otha to fth eprecipitate dsilic astudie d by Tadros and Lyklema (1968) and Yates and Healy (1976). Alsoher ea n unusually high surface chargedevelop swit hincreasin gpH ,indicatin g theexistenc eo fa gellayer ,onl yporou sfo rmicro-ions .Becaus ethi s layeri sno tporou sfo rpolyelectrolytes ,thes ewil lexperienc ea muc h lowercharg eupo nadsorption . In fig.3. 8 aconductometri c titration ofa nAEROSI L 0X50so l without added electrolyte isshown .Th esurfac e charge calculated _2 fromth efirs t breaki s2 mC m whichi sfairl ylow .S oonl ya ver y smallpar to fth esurfac e =Si-OHgroup si sdissociate da tpH~ 6unde r theseconditions .

T E E \ y* * 1

\ ^^

0.5 \-^

T=233.15 K

1 2 3 4 5 6 7 8 nmol NaOH

Fig. 3.8 Conductometric titration of AEROSIL 0X50 silica in water, and no added electrolyte.

3. 3.4 Some other relevant properties of the silica-water and glass- water interface

When a fresh pyrogenic silica surface is brought into contact with water, siloxane groups on the surface can react with water molecules to form hydrophilic silanol groups: 35

=Si-0-Si= +H 20 t 2=Si-0H

Athig htemperature s (1270K )th erevers ereactio ntake splac ean dth e surfacebecome smor ehydrophobi cagain .Wate rmolecule sca nals oadsor b onsilano lgroup sthroug hhydroge nbonds .O na full yhydroxylate dsilic a surface, twokind s of silanol groups exist: 'free'silanol swhic hd o not interactwit h otherO Hgroup san d 'perturbed'o r 'bound'silanol s whichar eclos eenoug ht oothe rO Hgroup st ofor ma hydroge nbound .I n the infrared spectrum, thesetw okind sappea ra squit edifferen tspe ­ cies (Kiselev, 1975). Iti spossibl e thatthes etw okind so fsilano l groupshav eals oa differen tacidit y {James and Parks, 1982 p. 140). Thesurfac econcentratio no fsilano lgroup si sver ysensitiv et oth e pretreatment ofth e silica aswil l be obvious from theabove .Fo ra hydroxylatedsilic asurfac ei nvacuu ma t47 3K (nophysicall yadsorbe d water), Kiselev (1975, p. 80) foundvalue sbetwee n3. 7 and5. 0sSi-O H 2 groupspe rm .Th esurfac esilano lconcentratio n fora full yhydroxyl ­ atedsilic ai nwate rma yb esomewha thigher .Fo ra discussio no fa fe w ofth emor eusua lmethod so fidentifyin gan dcountin gsurfac efunctiona l groups, againth e review oncharacterizatio n of aqueous colloidsb y James and Parks (1982) canb e recommended. Itwil lb eclea rtha tth e silica-water interface isheterogeneou swit hrespec tt oadsorption ,a feature thati tha s incommo nwit hP S (latex).Th edegre eo fhetero ­ geneitydepend so nth epretreatmen tconditons .O na full yhydroxylate d andlargel yhydropili csurface ,lik eAEROSI L0X5 0use dhere ,th einter ­ actionswit h acharge dadsorbat elik epolylysin ewil lb emainl yo fa n electrostaticnature .Surfac ehydroge nbondin gca nals ob epar to fth e adsorptionmechanism . Thesurfac echemistr yo fglas sha srecentl ybee nreviewe db y Filbert and Hair (1975). A sectiono fth e review 'Wateran d interfaces'b y Texter et al. (1978) dealswit h theinteractio no fwate rwit hsilic a and silicate systems.Olde rreview sare : Doremus (1973) and Deribere- Desgardes and Bre (1967). Monographso nth esubjec thav ebee nwritte n for instanceb y Holland (1964) and Korànyi (1963). Howeverthes ere ­ views dealmainl ywit h theglass-ga s interface andpertinen tinfor ­ mation concerning the borosilicate-glass/waterinterfac e isscarce . Thepropertie s described abovefo rth esilic aadsorben tappl yals ot o alarg eexten tt oth eborosilicat eglass .O fth elatte rsubstanc ehow ­ ever the surface chemistry ismuc hmor e complicated because ofth e presenceo facidi cB 203,th eamphoteri cA1 20_ andbasi coxide ssuc ha s

Na20an dK 20.Howeve rth elas tthre ear emino rcomponents .Th esurfac e 36 composition of a fresh fractureborosilicat e surfacei sprobabl yno t muchdifferen tfro mth ebul kcomposition .Howeve rdu et oth epretreat - ment (contactwit hwate ran dstron gacid )th esurfac econten to fvari ­ ous oxides differs from thebul k composition.Washing s with 6M HC l asdon eher ewit hth eborosilicat e glass,result si na surfac ewhic h is composed mainly of SiO,an dB-O- , inth e formo fth e ionizable silanol andborano l groups,becaus e ofth ereplacemen to falkal ian d alkaline earth ionsb yH .Th e originalglas sbackbon estructure ,i s unaffected (Filbert and Hair, 1975). Becauseo fth eleachin go falkal i andbasi c oxide compounds,a mor eporou s andhydrate d surfacelaye r (gellayer)develops ,whic h canhav e gross effectso nadsorptio npro ­ perties.Thi sporosit y isno tmeasure dwit hN _ adsorption,probabl y because thepore s arever ysmal lan dshrinkag eo fth egellaye roccur s due to the dryingo fth eglasspowde rnecessar yfo rth ega sadsorptio n measurements.Th epresenc e of amor epronounce dgellaye rtha ni nth e case ofAEROSI L isevidence db yth echarge-p Hcurve so fth eadsorben t as already described undersectio n3.3.3 .Als oth erelativel ylon gp H driftwit h theborosilicat eglas suse da scompare dwit hpyrogeni csi ­ lica,point sint othi sdirection .Becaus eth egellaye ro nth eborosili - cate-waterinterfac ei sno tpermeabl et olarg emolecule ssuc ha spoly - lysine,onl yth enegativ echarge dgroup so nth e 'surface'o fthi sgel ­ layerca ndirectl yinterac twit hth eR-NH^ . groupso fpolylysine . Jednacak and Pravdic (1974) measured the electrokineticpotentia l ofglasse s inaqueou selectrolyt esolution sb ystreamin gcurren tmea ­ surements. In the experiments thebehaviou r ofquart z andvitreou s silica is almostth esame .Pyre xglas sshow shighe rnegativel ypoten ­ tialstha nsilica ,bu tno ta shig ha son ewoul dexpec tfro mth ediffer ­ ence'i nsurfac e chargebetwee n Pyrex andSilic a (seesectio n3.3.3) . This isbecaus emos to fth echarg ei nth egellaye ri scompensate dfo r insidethi slayer .Th eiso-electri cpoin t (i.e.p.)i nNaC lsolution si s situated atabou tp H2. 5 for 'pyrex'an da tabou tp H3. 0 forvitreou s silica and quartz.Becaus eAEROSI LOX5 0i sals oa pur esilic ait sbe ­ haviourwil lno tdepar tmuc hfro mtha to fsilic aan dquartz .Th elowe r i.e.p. forpyre x isexpecte d because ofth epresenc eo fth econstan t chargeo fth e=B-0 ~groups . Thepropertie so fglasse sa sio nexchanger sar eo fparticula rrele ­ vance forth e adsorptionpropertie s ofcharge dadsorbate slik epoly ­ lysine.Th e ionexchang epropertie so fglas sar ereviewe dfo rexampl e by Doremus (1973). 37

Asjudge d fromcontac tangle so fwate ro nvariou sglasse smeasure db y Moser (8= 10-12 °fo rpyre xborosilicat ean d8 = 7-10 ° forpur esilic a (see Holland (1964), p.359) ,th epyre xsurfac eseem st ob eslightl y more hydrophobic than thesilic asurface .Howeve rth epretreatmen to f theglas san dth ecleanin gprocedur eca nhav ea grea tinfluenc eo nth e measured contact angle,s otha ti ti squestionabl e ifthi ssmal ldif ­ ferenceapplie sals ot oth esyste mstudie db yus .

3.4 SILVER IODIDEAN DPOLYOXYMETHYLEN ECRYSTAL S

For comparison reasons someexperiment so nth eadsorptio no fpoly - lysinewer eperforme d withAg isol san dpolyoxymethylen ecrysta lsus ­ pensions.Th eAg i sol usedwa s kindyprovide db y H.A. van der Schee. The solwa sprepare db yth eadditio ni nth edar ko fa nAgNO -solutio n toa well-stirre dK Isolutio na sdescribe db y De Wit (1975). Theinter - facialpropertie so fth eessentiall yhydrophobi cAg isurfac ehav ebee n reviewedb y Bijsterbosch and Lyklema (1978). Atheor yo fpatchwis ein ­ terfacialhydratio no fAg iha sbee npu tforwar db yd e Keizer (1981). Polyoxymethylenecrystal s (POM)ar euse da sa nadsorben tfo rprotein s by Roe (1981). Theadvantag eo fthi sadsorben ti stha tadsorptio nca n takeplac eo na wel ldefine dregula r flatcrysta lsurface ,whic hbear s no charge onth e crystal-water interface,a tleas twhe nn ospecifi c adsorption of ions takesplace . Inthi swa yth eelectrostati cinter ­ actions between adsorbed charged macromolecules canb e studied,i n principle in a more directway .Th epolyoxymethylen e crystalsuse d were kindlyprovide db y M. A. Cohen Stuart. Thecrystal swer eprepare d fromPO Mpellet s bya recrystallizatio nproces sfro mcyclohexano lan d subsequent changing the solventfro mcyclohexano lvi aaceto nt owate r inalmos tth esam ewa ya sdescribe db y Roe (1981).

3.5 SUMMARY

Inthi s chapter adescriptio n isgive no fadsorben tan dadsorbat e propertiesrelevan tfo rth eadsorptio no fcharge dadsorbates .Solutio n properties of polyaminoacids of importance for the adsorptionar e described. The determination isdescribe d and discussed ofth especifi csur ­ faceare aan dsurfac echarg eo fth eadsorbent smos tfrequentl yuse di n thisstud yviz. :P Slatex ,silic aan dpyre xglass .Als osom eothe rre ­ levantpropertie so fth esolid-wate rinterfac eo fthes eadsorbent sar e discussed. 38

3.6 REFERENCES

Abendroth,R.P . (1970). J.Colloi d Interface Sei. 34, 591-596. Balasubramanian,D . (1974).Biopolymer s 13, 407-410. Barclay, L. (1970)i n 'Surfaceare a determination', Proc. Int.Symp . IUPAC,Everett ,D.H . andOttewill ,R.H .eds. ,Butterworth ,London . Beurton, G. and Bussiere, P. (1970)i n 'Surfaceare adetermination' , Proc. Int. Symp. IUPAC, Everett, D.H. and Ottewill,R.H .eds. , Butterworth,London . Beychok, S. (1967)i n 'Poly-a-aminoacids', Fasman,G.D .ed. ,M .Dek ­ ker Inc.Ne wYork . Bijsterbosch,B.H .an dLyklema ,J . (1978).Adv .Colloi d Interface Sei. 9,147-251 . Deribere-Desgardes, M.L. and Bre, M.M. (1967). Symp.o f theUnio n Scientific Continentale du Verre. 'Thesurfac e ofglas s and its moderntreatments' ,Luxemburg . Doty,P . and Gratzer,B . (1961)i n 'Polyaminoacids,polypeptide s and proteins',Stahmann ,M.A .ed .Univ .Winconsi nPress ,Madison . Doremus, R.H. (1973). 'Glass Science'.Wiley-Interscienc eNe wYork - London. Fasman,G.D . (1967)i n 'poly-a-aminoacids',Fasman ,G.D .ed. ,M .Dek ­ ker, NewYork . Filbert,A.M . andHair ,M.L . (1975).Adv .i nCorrosio n Sei.:Technol . 5, 1-54. Fontana,M.G . and Stachle,R.W . eds.,Plenu m PressNe w York-London. Flory,P.J . (1969). 'Statisticalmechanic s ofchai nmolecules' .Inter ­ sciencePublishers ,Wile y& Sons ,Ne wYork . Furusawa, K.; Norde ,W . and Lyklema, J. (1972). Kolloid-Z.Z.Polym . 250, 908-909. Goossens, J.W.S. and Zembrod, A. (1979). Colloid Polym. Sei. 257, 437-438. Greenfield,N .an dFasman ,G.D . (1969).Biochemistr y 8, 4108-4115. Grourke,M.J .an dGibbs ,J.H . (1971).Biopolymer s 10, 795-808. Hermans,J . (Jr)(1966) .J .Phys .Chem . 70, 510-515. Heywood, H. (1970)i n 'SurfaceAre a Determination',Proc .Int .Symp . IUPAC,Everett ,D.H . andOttewill ,R.H. ,eds. ,Butterwort hLondon . Hearn, J.; Wilkinson, M.C.an dGoodall ,A.R . (1981).Adv .Colloi dIn ­ terfaceSei . 14, 173-236. Hoven, T. van den (1984). Doctoral thesis Agricultural University Wageningen. Inpress . 39

Hul, H.J. van den andVanderhoff , J.W. (1968). J. Colloid Interface Sei. 28, 336-337. Holland, L. (1964). 'The properties of glass surfaces', Chapman and Hall London. Horn, D. (1978). Progr. Colloid Polym. Sei. 65, 251-264. Idelson,M . and Blout, E.R. (1958). J. Amer. Chem. Soc. 80, 4631-4634. James, R.O. and Parks, G.A. (1982) in 'Surface and Colloid Sei. 12, 119-216.Matijevic , E.ed . Jednacak, J.; Pravdic, V. and Haller,W . (1974). J. Colloid Interface Sei. 49, 16-23. Kamel, A.A.M. (1981). Ph.D. thesis. LeHigh Uiversity Bethlehem, Pennsylvania. Kasper, D.A. (1971). Ph.D. thesis.Californi a Institute of Technology Pasadena, California. Keizer, A. de (1981). Doctoral thesis Agricultural University Wage­ ningen. Kiselev, A.V.; Lygin, V.l. (1975). 'Infrared Spectra of Surface Com­ pounds', HalstedPress ,Wile y and Sons,Ne w York,Toronto . Koberstein, E. and Voll, M. (1970), Z. Phys. Chem., Neue Folge 71, 275. Koopal, L.K. (1978). Doctoral thesis Agricultural University Wageningen. Korânyi, D. (1963). 'Surface properties of silicate glasses', Akar démiai Kiadó,Budapest . McGarvill, W.T. and Fitch,R.M . (1978). J. Colloid Interface Sei. 67, 204-212. Nagasawa,M . (1970). Pure Appl. Chem. 26, 519-536. Norde, W. (1976). Doctoral thesis Agricultural University Wageningen. Norde, W. and Lyklema, J. (1978). J. Colloid Interface Sei. 66, 257-266. Nyilas, E.; Chiu, T-H. and Lederman, D.M. (1976)i n 'RecentAdv . Col­ loid Interface Sei.' 3. Kerker,M. , ed. Acad. Press New York. Padday, J.F. (1970) in 'Surface Area Determination', Proc Int.Symp . IUPAK., Everett,D.H . and Ottewill,R.H. , eds.Butterwort h London. Pauling, L.; Corey,R.B . and Branson,H.R . (1951). Proc.Nat .Acad . Sei. U.S. 37, 205-211. Painter, C.P. andKoenig , J.L. (1976). Biopolymers 15, 229-240. Rippon,W.B . andWalton , A.G. (1971). Biopolymers 10, 1207-1212. Roe, R.J.; Shastri,R . andWille ,W . (1981). J. Colloid Interface Sei. 84, 346-354. Sigma Chemical Co. (1980). Fine chemicals catalog. Schee, H.A. van der (1981) in 'The effect of polymers on dispersion properties', Tadros,Th.F . ed.Academi c Press London. 40

Sonntag,H .an dKolesnikova ,R.S .(1980) .Z .Phys .Chem . 261, 226-232. Tadros,Th.F .an dLyklema ,J . (1968). J.Electroanal .Chem . 17, 267-275. Tiffany,M.L .an dKrimm ,S . (1969).Biopolymer s 8, 347-359. Tiffany,M.L .an dKrimm ,S . (1972).Biopolymer s 11, 2309-2316. Texter, J.; Klier, K. and Zettlemoyer,A.C . (1978). Progr.Surfac e MembraneSei . 12, 327-391. Wit, J.N.d e (1975).Doctora lthesi sAgricultura lUniversit yWageningen . Yaron,A .an dBerger ,A . (1963)Biochim .Biophys .Act a 69, 397-399. Yates,D.E .an dHealy ,T.W . (1976).J .Colloi d InterfaceSei . 55, 9-19. Vanderhoff,J.W. ;Hul ,H.J .va nden ;Tausk ,R.J.M .an dOverbeek ,J.Th.G . (1970)i n 'CleanSurfaces' .Goldfinger ,ed .M .Dekke rNe wYork . Zimmerman,S.S .an dMandelkern ,L . (1975).Biopolymer s 14, 567-581. 41

4 ADSORPTIONO FTH EHIGHL YCHARGE DPOLYELECTROLYT EPOLYLYSIN EO N DIFFERENTSUBSTRATE S

4.1 INTRODUCTION

Themai nsubjec to fthi schapte ri sth eadsorptio nbehaviou ro fpoly - L-lysinea tmaxima lcharg edensit yi nsolutio n (pH<8)an dth einfluenc e ofth enatur eo fth esubstrat eo nthi sadsorption . A special feature ofpoly-L-lysin ea sa polyelectrolyti cadsorbat e isth echai ncharg edensit ydependen tconformatio no fthes emacromole - cules in aqueous electrolyte solutions.A t low chain charge density (pH>10)th epolyaminoaci d isi nth ea-heli xconformation ,thu sresem ­ blinga structur ewhic hals ooccur si nmos tprotei nmolecules .Howeve r athig hcharg edensit y (pH<8)th emolecule sare ,dependin go nth eelec ­ trolyte concentration, more or less flexible polyelectrolytes.The n they are suitable model substances to investigate the influenceo f molecular mass, ionic strength,surfac echarge ,an dnatur eo fth ead ­ sorbento npolyelectrolyt eadsorption . On theothe r hand, theunravellin g ofth eadsorptio nbehaviou ro f thepolylysin e helix andth erol eo fth ehelix-coi l transitionma yb e offundamenta l importancei nth eunderstandin go fth ebehaviou ro fbio - macromoleculeswit h secondary and tertiary structure atsolid-liqui d interfaces.Thi swil lb eth esubjec tmatte ro fchapte r6 . Theadsorptio nbehaviou ro fbot hflexibl ean drigi dbiologica lmacro - molecules,a swel la sflexibl esyntheti cpolyelectrolyte s iso fimpor ­ tance in anumbe r of (bio)medical,biologica l and(bio)technologica l processes.Som eexample sare :Th eus eo fsyntheti cpolyelectrolyte sa s polymeric flocculants forparticle s oppositely charged toth epoly ­ electrolytei nwate rpurificatio nan dth eadsorptio no fsalivar ypoly ­ peptides onhar d toothtissu ei nrelatio nt oth edevelopmen to ftoot h caries.Polyelectrolyt e adsorptiondoe sals opla ya rol ei nth eblood - clottingproces s atinterfaces .Mor e examples aregive nb y Hesselink (1983).

4.2 EXPERIMENTAL

4.2.1 Materials

poly-L-lysine.HBr,poly-DL-lysine.HBr ,poly-L-ornithine.HB ran dpoly - L-histidine were obtained fromth eSigm achemica lC oan duse dwithou t 42 furtherpurification .Viscosit y averagemolecula rmasse so fth epoly - aminoacidsa s stated byth emanufacture rwer euse di nthi sstud y(se e also3.1) .Al lothe rchemical suse dwer eo fanalytica lgrade .Th ewate r usedwa s distilled onceo rconductivit y waterobtaine dfro ma milli - pore super Q water purification apparatus. Thepreparatio n ofth e polystyrene latices was essentiallyth esam ea sdescribe db y Furusawa et al. (1972). Purificationo fth elatice swa sdon eb ysteamstrippin g and ionexchang e of the laticeswit h extensively purifiedDowe xio n exchangeresin s (BIORAD).Fo rdetail sse e (section3.2) . Theglasspowde ruse dwa sa borosilicat eglas s (732-01)obtaine dfro m Sovirelan dpurifie di nth esam ewa ya sdescribe db y Nyilas (1976). The B.E.T.N surfaceare aamount st o0. 5i 0. 2i g 2- 1 Thesilic ause dwa sAEROSI LOX5 0 (Degussa),wit ha specifi csurfac e area of 50± 15m g as state2- 1 db y themanufacturer .Th esilic awa s used without furtherpurification .Dispersion s ofth e silicapowde r in conductivity water were prepared by suspending silica thatwa s dried for twohour s at41 3K inwate r andultrasonicatio n (25kHz ) ofth esuspensio nunde rcoolin g(~28 7K )fo ron ehour . TheAg i sols andpolyoxymethylen ecrysta lsuspension suse di nsom e of the experimentswer e gifts ofH.A .v.d .Sche ean dM .Cohe nStuar t respectively from our laboratory. Some characteristics ofthes esus ­ pensionsar egive ni nsectio n3.4 .

4.2.2 Determination of the polyaminoacid concentration

Theconcentratio no fth epositivel ycharge dpolyaminoacid si naqueou s solutionwa sdetermine db ya comple xtitratio nwit hth epotassiu msal t ofpolyvinylsulphate ,usin gtoluidin eblu ea sth eindicato r (Terayama, 1952; Horn, 1978). In each series of polyaminoacid determinations standardsample so fknow nconcentratio nwer einclude d forth econstruc ­ tiono f acalibratio n curve.Adding s ofpolyvinylsulphat e weredon e witha Mettle rDV10/D V201/( 1cm 3)automati cburette .

4. 2. 3 Adsorption measurements

Adsorptionisother mmeasurement swer ecarrie dou tb yaddin galiquot s 3 ofth eso lt o4. 0c m polyaminoacidsolutio n (inth ecas eo fglasspowde r 3 3 5.0c m PA solutionwa s added to2. 0g glasspowder )i n1 0c m poly­ carbonatecentrifug etube swit hpolyethylen ecaps .Fo rp Hvalue shighe r than8 polyethylen e tubeswer eused .Prio rt omixing ,bot hsolution s 43 werebrough tt oth esam ep Han dioni cstrength ,withou tusin gbuffers . The tubeswer e rotated end over endfo r1 6hours ,i na thermostatte d room (295K) , toensur eequilibrium .Whe nP Slate xwithou tadde dsal t (pH~6)wa s added toa polyaminoaci d solutionwit hsuc ha p Han dioni c strength (obtained by addingknow namount so f0. 1M NaO Ho rHCl ,NaB r solution andwate r to aP L stocksolution )tha tafte rmixin gth ede ­ sired ionic strength andp Hvalu e wasreached ,n odifferen tadsorbe d amounto fP Lwa s foundwit hrespec tt oth e formermetho d ofsampl e preparation.Thi sshow stha tth eadsorbe damoun tfoun dwa sindependen t ofth ewa yi nwhic hth efina lstat ewa sreached . Theamoun to fPA-aci dadsorbe dwa sdetermine d fromth edepletio no f the solution.T o thisend ,th esample swer ecentrifuge d for20-3 0mi n at18.00 0r.p.m .(37.00 0g a tr = 10 2mm )i na Beekma nJA-2 1centrifug e with JA-21 rotor.Th e PA concentration inth e clear supernatantwa s measured asdescribe d under4.2.2 .Th emeasurement swer eperforme da t room-temperature (295± 2 K). pHmeasurement swer edon ewit ha nelectro - fact3620 0p Hmete r ora nAnke rSmit hA16 1digita lp Hmeter .Combine d glass- Ag/AgC l electrodes fromElectrofac t (7GR131)o r Schott(N58 ) wereused .

4.2.4 Conductometric and Potentiometrie titrations

All conductometric andPotentiometri etitration sfo rparticl esur ­ face charge determinations were performed in awel l closed double walledvessel ,thermostatte da t293.1 5± 0.0 5K unde ra C0 2-freewate r vapour saturated N_ atmosphere.A nAnkersmit h A161 digital pHmete r and SchottN5 8o rN5 9 combined glass-Ag/AgClo r glass-calomelelec ­ trodeswer e used for themeasurements .Th ep Hmete rwa sstandardize d withtitriso l (Merck)buffer sp H7.00 ;4.0 0 and9.0 0befor eeac hti ­ tration. Conductometric titrations ofP S latex (H form)o rsilic a were performed with aRetc h conductivity meter operating at4 kHz . Conductivity cells with cell constants of 10.40 m or 71.94m (platinum black)wer e used.Adding so fcalibrate d0. 1M NaO Ho r0. 1M HCl (Titrisol)wer e donewit h aMettle rDV10/DV20 1o rMethroh m65 5 Dosimatautomati cmicr oburette . Surfacecharge-p Hcurve sar eobtaine d fromth ePotentiometri eproton - titration curves of silicaan dborosilicat eglas spowde rdispersions , andth ecorrespondin gblan ktitration si nth eusua lway ,definin g oQ = r _ F(rH+- rQjj-)wher e (rH+ - 0H ) isth eadsorptio nexces so fhydroge n overhydroxy lion si nequivalent spe rm (N,surface) .Se efo rexampl e 44

Abendroth (1970), James and Parks (1982), Ardizzone et al. (1982). Some more experimental details concerning the silica andglasspowde rdis ­ persiontitration shav ebee ngive ni nsectio n3.3.3 .Th esurface/volum e ratioi s125 0m 2.l_1 forth eAEROSI LOX5 0titration san d25 0m 2l-1 for theglasspowde rtitrations .

4.2.5 Stability measurements

Stability measurements ofbar e andpoly-L-lysin ecovere dpolysty ­ renean dAg iso lparticle sagains tlo wmolecula rmas selectrolyt ewer e 3 performed by astati cmethod . Five cm sol (witho rwithou tPL )wa s mixedrapidl ywit h5 c m electrolyt3 esolutio nan dth emixtur ewa skep t at298.1 5K in athermostatte dwate rbath .Thi swa srepeate d fordif ­ ferent electrolyte concentrations.Afte r atota l equilibrationtim e of 18hours , the OD,-™ " of the top 2c m ofth e dispersionswa s measured using aBeekma nmode l 3600 spectrophotometer. Optical data are expressed as the residual turbidity x, whic h isth eoptica l density ofth e suspensionx ,obtaine di nth epresenc eo felectrolyte , relative totha tmeasure d for anidentica l system inth eabsenc eo f electrolytean dpolyelectrolyt e x .:x =t/ x. Thusx equals1. 0 forstabl esuspension san dcontro lsystems .Th eob ­ tained relative turbidity valueswer eplotte dagains tth eelectrolyt e concentrationt ojudg eth estabilit yo fth esuspensions . Stock solswit h addedpoly-L-lysin ewer eprepare da sfollows :T oa diluted latex (^0.01%w/v )o rAg i sol (pi= 4)(~0.01 % (w/v))a PL- L solution ofth e desired concentration inwate rwa s added rapidlyb y means of apipetma n dispensingpipet .Th e totalpipette dvolum ewa s such,'tha trapi d mixing due toth e liquid jetwa sachieved .Th eob ­ tained stable solwa srotate d end overen dfo ra tleas ttw ohour sa t 295K to ensure adsorption equilibrium. Blank sols for stability measurements wereprepare d inth e samewa yb ypipettin gconductivit y water toth e dilutedso linstea do fP Lsolution .Th eparticl econcen ­ tration ofth e obtained stable testsol ,afte relectrolyt eaddition ,

wasalway ssuc htha tth eOD 1Q *™wa sbetwee n .3an d.8 .

4.3 RESULTSAN DDISCUSSIO N

4. 3.1 Comparison of the specific surface areas of the adsorbents

Whenon ewant st ocompar eth eadsorptio nsaturatio nvalue so fa nad - sorbateo ndifferen tadsorbents ,th especifi csurfac eare ao fthes ead - 45 sorbentsmus tb eknown .Als oothe rcharacteristic so fth esurface ssuc h asth esurfac echarg edensit yan dth edistributio no fadsorptio nsite s in the case ofheterogeneou s surfaces,shoul d be known,t o allowa meaningfullcomparison .Fro ma practica lpoin to fvie wth elas tdeman d isalmos tno trealizable . There isalway s some intrinsic uncertainty onth evalu et ob eas ­ signedt oth especifi csurfac earea .Th esurfac eare aa s 'seen'b yth e polylysine molecules isno tnecessarel y the same astha tdetermine d with, forexampl e N„-o rdy eadsorption .Henc esom ereserv ei salway s neededi nmakin gcomparison so fabsolut eadsorptio ndata .Thi spartic ­ ulardifficult yi sno tencountere di fshape so fadsorptio nisotherm sar e compared,wher eth eadsorbe damoun ti sexpresse d asfractiona lcoverage . Consider forexampl e thenegativel ycharge dpolystyren esurfac ea s an adsorbent for the positively charged polylysine.Th e negatively charged sulphate groupswil l act as strong adsorption sites forth e positively charged aminogroups.Th espac ebetwee nth esulphat egroup s consisting ofbenzen e and-CH ~group so fth epolystyren esurface ,ha s probablya muc hlowe raffinit y forth epolylysin echain .I nth ecas eo f the adsorption of smallmolecule sadsorbin go nth enegativel ycharge d sulphategroup sonly ,on eca ndefin eth especifi csurfac eare afo rthi s compound asth e sum ofth e surfaces ofal l sulphategroup spe rgra m adsorbent.Th eobtaine dplatea uadsorptio ni sthe n 'monolayer'adsorp ­ tionpe rdefinition .I nth ecas eo fa positivel ycharge dmacromolecul e thisdefinitio nha slittl emeanin gbecaus eo fth epossibilit yo fbridge s betweennegativel y charged surfacesites .Suc hbridge sca nb epolyme r loopsbu tals otrains .I nothe rwords ,th epositiv eunit suse dt ocoun t thenumbe r ofnegativ e surface sites aren o longer independent.Fo r this reason, Ichos e the geometrical orN ,ga sadsorptio n (whichar e notmuc hdifferen t forth eadsorben tuse dhere )fo rconversio no fth e adsorbed amount into mg.m .Th eN ,ga s adsorption area approaches mostprobabl y thephysica l surface area inou rcases ,i.e .i ti sth e sumo fth eare ao fal lstron gan dwea kP Ladsorptio nsite spe rgra mad ­ sorbent. Whenth edistributio no fstron gadsorptio nsite si shomogeneou sove r the surface,th e obtainedplatea uadsorptio nvalue sar ea measur eof f thefractio no fth e 'total'surfac eare acovere db ypolyelectrolyte . Intabe l4. 1 somerelevan tpropertie so fth eadsorbent suse di nthi s study aregiven .A discussion ofth evalue s foundha sbee ngive ni n section3. 2 and 3.3. Forth e adsorptionmeasurement s on silicath e value of 50m g wa2- s1 use d arbitrarely forth especifi csurfac eare a inmos tcases . u co co CQ m O O ia m H O - _ _ = 2 z § § O O E E o> V V H !-t UI UI M O O m u u xi ai .^ ... o co V in m = = = = il il 0) CS s ft sa X l-H I ft ft (-* o ft ft ft (0 CM o 4-1 CN o ai ai CM U m co rH + CM CM <* 10 i i i i 1 <>

>1 X) 3 +J UI U) 10 UI o. o H CM 10 •H a X! O O co P < s —

•O 0) Ol — UI P M O E 0) UI ^ Oi •p f) < o. M co ft p X! u Oi e a U < o > u-i •H 01 4) O -o ö rH co III 01 •H 01 O) e m PC •o M M ft i co u o 'S ö ö u O) s O I o (d oi 0> •p o m •• UI ft S p 01 u O PQ •O l 0) o T) 01 I m < l-H "O Z

01 •a 01 o p ft id l-H 01 u (0 UI ü 1/1 H O • 01 UI o u co rH m M rH P U* H M n * 1/) H os X 01 o i-a>i rd PuSEEESJP* rH H CN CO < UI E OM« 3 o ft 47

4. 3.2 Adsorption time

The amount ofPL- L (DP= 240 )adsorbe d from0. 1M NaB r (pH= 6 )o n

PSlate xparticle sM 3wa smeasure dafte r0.5 ;1.3 ;1. 6 and3 0hour so f contact time.Th e latexconcentratio nwa sth esam ea suse d fordeter ­ mining adsorption isotherms.A t each mixing time several starting concentrations ofP Lwer e used, all resulting inth esam eadsorptio n valuea tal lcontac ttimes .Th eequilibriu mconcentration svarie dfro m 20-200gm " .Th eresult sar eplotte di nfig .4.1 .

1 • 1 •

5 IN 1 o e o .4 D 'o - o IL. 3 -

.2 -

.1

1 1 1 50 100 , 150 10s

Fig.4. 1 Adsorbed mass ofPL.HBr- L (DP240 )a sa functiono fcontac ttim e in0. 1M

NaBr;p H= 6 ;T = 29 5K ;P S latex Yiy

Itca nb esee ntha twithi nexperimenta lerro rth eadsorbe damoun tr isconstan t inth etim e range studied.A simila rresul twa sobtaine d forth e adsorption ofPL- L (DP1923 )fro m0.0 1M NaB ro nborosilicat e glass (pH~ 6) . Also van der Schee (1982) foundth esam eresul tfo rth e adsorptiono fPL- Lo nAgi . Investigationso fcontac ttime sshorte rtha n 30mi n isno tpossibl ewit hou rtechnique ,becaus eth ecentrifugatio n timei sthe no fth esam eorde ro fmagnitud ea sth econtac ttime . Another technique for separating adsorbate and adsorbenti sthe nre ­ quired. Thetim escal eobserve dher efo rpolyelectrolyt e adsorptionproces s (faster than 30min. )i softe nobserve d forth e adsorption ofpoly - electrolytes,oppositel y charged to the surface (Rawls et al. , 1982; Williams et al. , 1982, Horn and Melzer, 1976; Eggert, 1976; Lindguist, 1975). Thestron gattractiv eelectrostati cinteractio nbetwee npolyme r 48 andcolloida lparticle swa shol dresponsibl efo rthi sb y Eggert (1976). Howeverthi sargumen ti sonl yapplicabl eu pt oth epoin to fcharg eneu ­ tralization. The increase of the adsorbed amountwhic h takesplac e beyondthi spoin t (dependento nth eelectrolyt econcentration) ,shoul d be aslowe rprocess . Corry (1978) foundfo rth eadsorptio no fPL- Lo n latex,equilibriu mtime so f5 mi na sjudge db ychange si nelectrophore - ticmobilit ywit htim eafte radditio no fPL . The polyelectrolyte adsorptionproces s is faster thangenerall yob ­ served foruncharge d polymeradsorption .Fo rsuc hsystem si ti softe n found thatth e adsorbed amount still increases during the firstte n hourso fcontac ttim eo reve nlonge r (Cohen Stuart, 1980; Koopal, 1978). The causeo fthi s relatively long adsorption time isthough t tob e reconformation processes ofth epolyme r atth e interface and inth e case ofheterodisper s polymers,th e exchange of smallmolecule sad ­ sorbed atth e interfaces againstbigge r ones.Thi sgive s riset oa n increasei nth eadsorbe damoun t (Cohen Stuart et al., 1980). Ofcours ereconformatio nan dpolyme rheterogeneit y alsopla ya rol e inth epolyelectrolyt e adsorptionprocess .Th elon gequilibriu mtime s for the adsorption ofpoly( 2 sulphoethylmethacrylate)( M/ M = 1.2) onpolyethylen e powder at0. 1M NaC l foundbi j Greene (1971) canb e attributedt othi seffect .Fo rPL- Luse dher eth eM / M ratioi sprob ­ ably closer to 1.Moreove r themolecula r mass dependence ofth ead ­ sorptioni sals over ysmal labov eM 30.000,s otha texchang eo fshor t chains against longer onescanno tgiv e ameasurabl e increase inth e adsorbedamoun tan dhenc ethi seffec tha sn oeffec to nth eequilibriu m time. This absence of any detectable polydispersity effectca nb e responsiblefo rth erelativel yshor tequilibriu mtime stoo . Althoughth eadsorbe damoun to fP Lremain sconstan tafte r3 0mi no f contacttime ,th ecompositio no fth eadsorbe dlaye rma ystil lno tye t have itsequilibriu m composition. For this reason and forpractica l convenience anadsorptio n timeo f1 6hour swa schose ni nmos to fth e experiments.

4. 3. 3 Effect of molecular mass

Infig .4. 2 themolecula rmas sdependenc eo fPL- Ladsorptio no nlate x M„i n0. 1M NaB ra tp H6 i sshown .Th estartin gconcentratio no fPL.HB r wassuc hi nthes eexperiment stha tplatea uadsorptio nwa sassured .Th e adsorptiono fP L increaseswit hM u pt o %50.00 0 (DP240 )afte rwhic h themolecula rmas sdependenc ei sonl yslight .Suc ha nbehaviou ri stheo - 49

Fig.4. 2 Plateau adsorption of poly-L-lysine on PS particles (latex M. a = -2 -60mC. m )i n0. 1 MNaB ra tp H6 ,a sa functio no fth eP Lmolecula rmass . reticallyexpecte dfo runcharge dpolymers ,adsorbin gfro ma goo dsolvent . Becauseo fth elon grang eelectrostati cinteraction sbetwee nP Lsegment s themeanin go fth ex paramete ri sno tobviou si nthi scase .I nth eab - senceo fadde d electrolyte (K10 )ther -4e isn omeasurabl emolecula r mass difference atal lbetwee nD P1 9an d240 ,indicatin g flatadsorp ­ tion.Wit h respectt oth emolecula rmas sdependence ,th ebehaviou ro f PL.HBr, adsorbed onglas s andAgi , isqualitativel y the samea sde ­ scribed here for theP Ssurface .I nth ecas eo fth eglas spowde rthi s showstha tth e glass surface isno tsignificantl y porous forP Lbe ­ causeotherwis e PL,wit h alo wdegre eo fpolymerization ,shoul dgiv e higheramount so fadsorptio na thighe rdegree so fpolymerization .Suc h aneffec to fpor e sizewa sobserve dindee db y Horn and Melzer (1978) forth eadsorptio no fpolyethyleneimine so ncellulos efibers . Themolecula rmas s dependencei nth epolylysine-polystyren e system athighe rsal tconcentratio ni smainl ycause db yth edecrease delectro ­ static repulsionbetwee n thechai n segments,s otha tmor e loopsan d tailsca ndevelop .A tver yhig hioni cstrengt hth elowe rsolven tquali ­ tyo fth eelectrolyt esolutio nca nals ocontribut et oa highe rr .Thi s effecti sstronge rfo rP Lo fhig hDP .Thes eresult sar ei nqualitativ e agreementwit hth eprediction so fth epolyelectrolyt e adsorptiontheor y ofva nde r Schee (Bonekamp et al., 1983). Experimentalresult so f van der Schee (1983) for the PL-AgI and Eggert (1976) for the poly 50

(l,2-dimethyl-5-vinyl(pyridinium)bromide-polystyrene latexsyste msho w thesam etrend sa sobserve dher efo rth ePL-PS.late xsystem . Becauseo fth esligh tmolecula rmas sdependenc eo fth eP Ladsorptio n onpolystyren e and theappearanc eo fver yshar padsorptio nisotherms , aninfluenc eo fth eadsorben tsurfac et ovolum erati oo nth eisotherms , duet oth epolyme rheterodispersit y (Cohen Stuart et al., 1980), isno t expected atlo w saltconcentration san dlo wpH .Ther ewer en odiffer ­ ences indeed between isotherms ofPL- L adsorbedo nP San dglas smea - suredwit hA/ Vratio sbetwee n8 0an d40 0m .1 forth e2glasspowde -1 rad - sorbent and 48-278m 1 fo2- r1 PS latex. The saltconcentratio nwa s 0.01M NaBr .Accurat emeasurement sa tlo wP Lequilibriu mconcentration s showedhoweve rtha ta thighe rioni cstrengt h (0.1M ;p H6 )th eadsorp ­ tionisotherm so fP Lo nP Sbecom esomewha tmor erounded ,indicatin ga n effecto fth eP Lheterodispersity , whichwa seve nmor epronounce da t 0.5 and1. 0M NaBr .Howeve ra tthes ehighe rsal tconcentration sth eP L concentrationdeterminatio ni sles saccurate ,whic hca nals ocontribut e toth eobserve droundednes so fth eisotherms .

4. 3. 4 Adsorption isotherms

In fig.4. 3 (a,b,c)characteristi c adsorption isotherms ofhighl y charged polylysine ondifferen t adsorbents areplotted .Th eadsorbe d -2 amount is expressed inmg. m PL.HBr inal l casesusin g theN ? or geometric specific surface areasgive ni ntabl e4.1 .I nal lcase sth e isotherms have aver yhig haffinit ycharacte ran da well-define dpla ­ teauvalue .O f course thisdoe sno tmea ntha tth eaffinit yo fP Lfo r thevariou s surfaces isno tver ydifferent ,bu tonl ytha tan ydiffer ­ ence isno tmeasurable . Inth ecas eo fP Ladsorptio no nP Ssom eiso - _3 thermswer emeasure dwit hequilibriu mconcentration sP Lu pt o90 0g. m No raise inadsorptio n after reaching theplatea uvalu ecoul db eob - _3 served.A ssaid ,a tlo wsal tconcentration s (<10 M)th eplatea uvalue s areindependen t ofmola rmass , indicating adsorption ina flatcon ­ formation,i.e .wit h trains only.Ther e isn omeasurabl e difference in plateau valuebetwee nPL- L andPL-D Lbetwee n all adsorbentsin ­ vestigated. This isexpecte dbecaus ebot hpolymer s areknow nt ob e ina coi lconformatio na tp Hvalue slowe rtha n9 (Applequist and Doty, 1962). Thenon-electrostati cpersistenc elengt ho fPL-D L (1 ~0. 8nm ) isabou ttwic e as low as thevalu efo rPL- L (1 *2 nm )(e.g . Flory, 1969; Brant and Flory, 1965). 51

i • i —r i -l 1 1 r © © . -6 E 01 ,. E .5 - E -5 ^-A—BT-A-S 3 .4 - O O •> (t—o—Ö ö—o o— i

3 X xn5* *° x 0 o x x o * x x —x_ —x x_ 1 .2 -

.1. 1-

• i i 1 100 200 300 400 100 200 300 400 c /g.rrT c /gm -3 PL PL

Fig. 4.3 a. Adsorption of polylysines on silica and borosilicate glass. T = 293 K. x-x PL-L (DP 240)/glass; 0.01 MNaBr , pH 5.9 o-o PL-DL (DP 240)/glass; 0.01 MNaBr , pH 5.9 A-A PL-L (DP 190)/AER0SIL; 0.01 MNaBr , pH 4.7 D-Ü PL-L (DP 190)/AER0SIL; 0.01 MNaSCN , pH 5.0 b. Adsorption of poly-L-lysine (DP 1683) on PS (latex M ). T = 293 K. x-x 10"3M NaBr, pH 6; o-o 10"2M NaBr. pH 6; A-A 10~XM NaBr pH = 6.

In principle this could lead to different adsorption values for PL-L and PL-DL at salt concentrations above 0.1 M. Above this concentration the total persistence length L_ is mainly determined by the non-electro­ static concentration 1 . At low salt concentrations PL-L and PL-DL are about equally flexible, because the calculated electrostatic persis- -3 -1 tence length (lg ^ 36 nm, 10 MNaBr ; 1 ~ 0.4 nm, 10 MNaBr) , which is dominant at low salt concentration, is identical for PL-L and PL-DL. No differences in r due to differences in L„ are expected then. Monolayer coverages for the fully extended PL-L and the most compact conformation (pH<9), obtained from Stuart models, amount 0.67 and 1.0 mg -2 -2 m PL.HBr (0.41 and 0.61 mg.m PL (without Br )) respectively (van der Schee and Lyklema, 1981). Because of entropical reasons an inter- 52

Fig. 4.3c Adsorption ofpolylysine s onsilve riodide . x-x PL-L (DP300) ;o- o PL-L (DP2000) ;+- + PL-DL (DP250) . -10raC.m" , electrolyt e lu"2M HN0 „ T= 29 3K . o - '3' (afterva nde rSche e (1984),va nde rSche e andLyklem a (1982). mediatesituatio nseem sth emos tprobable .Compariso nwit hth eexperi - _3 mentalvalue si n1 0 Melectrolyt esolutio nsuggest stha tth emolecul e adsorbs ina rathe rextende d fashioni.e .i tha sa larg econtac tare a withth eadsorben tused .Implicitl yi ti sassume dthe ntha tth esurfac e charge isdistribute d homogeneousove rth esurface .Th eassumptio no f ahomogeneou ssurfac echarg edistributio ni sbette rfo rAgi ,wher eth e surfacecharg eha sa more ,smeare dou tcharacter ,tha nfo rsilica ,glas s andcertainl ypolystyren ewer ediscret echarge dgroup sexist . The adsorbed amount as such is indicativeo fth ethicknes so fth e adsorbatelaye ronly ,whe non ei ssur etha tth edistributio no fadsorp ­ tion sites (i.e.patche s withhig h adsorption free energy)i shomo ­ geneous and the densityo fpatche si sknow n (seeals o4.3.1) . Inthi s caseth econclusio no ffla tadsorptio no fP La tlo wsal tconcentration , obtained from the lowvalu e ofth eadsorbe d amount remainsvalid .A homogeneous distribution of adsorption sites isver y likely forAg i and the SiO,adsorbent .I nth ecas eo fP Slate xi ti sno tsur ewethe r the -OScC groups arehomogeneousl y distributed over the surface.A homogeneous distribution seemsprobabl e inthi s caseconsiderin gth e emulsion polymerization mechanism of styrene. However van der Put (1981) suggested the presence of apatchwis e distribution of-OSC C 53 groupso nth eP Ssurfac efro melectrokineti cmeasurements . Differencesi nth eabsolut evalue so fth eadsorbe damount so fP Lbe ­ tweendifferen t substrateshav eonl ya meanin gwhe ngreate rthan ,sa y 40-50%,becaus eo fth elarg euncertaintie s inth especifi csurfac earea s ofth eadsorbents .Fo rth eglas spowde radsorben tth euncertaintie sar e probablyeve ngreate rbecaus eo fth erelativel ygrea terro ri nA . The effecto f thehydrophobicit y ofth eadsorben to nth eadsorbe d amount,ca nonl yb edirectl y studied bycomparin gadsorptio ndat ao n adsorbents,whic hdiffe ri nhydrophobicity ,a tconstan tsurfac echarge , which isexperimentall y very difficult to achieve.Whe nw e taketh e _2 adsorption ofPL- L onP S latex from 10 MNaB rp H6 a sa referenc e value (0.37mg. m )-,the2 n adsorptionvalue sunde rth esam econdition s _2 for theothe r adsorbents higher than 0.56 or lower than0.1 9mg. m mayb econsidere dt odeviat esignificantl y (seefig . 4.3). Becauseo fth eabove ,nothin gdefinit eca nb econclude d fromfig .4. 3 aboutth einfluenc eo fdifference si nnon-electrostati ccontributions , toth eadsorbe damoun tbetwee nth eadsorbent sused .I nn ocas eP Lform s a completemonolayer .Th edominan trol eo felectrostati c interactions (repulsivean dattractive )i nthes esystem smus tb eth emai ncaus efo r this. Such a conclusion was also reached by several other authors (e.g. Cafe and Robb, 1982; Williams et al., 1982). For example Williams et al. (1982) concluded thatth e adsorption ofnegativel y charged carboxymethyl cellulose onnegativel ycharge dBaSO .i smainl y limitedb y the strong repulsionbetwee n thesegment so fth eadsorbe d phase. The adsorbed amountP Lo nP S andAg i expressed inmC. m ismuc h -2 higher thanth e surface charge.A s theP Lmolecule s arever ylikel y tob e adsorbed flat,th eadsorptio nmus tb esuperequivalen ttoo .Tha t thiswa sth ecas eindee dwa sshow nb y van der Schee and Lyklema (1982) forth ePL-Ag Isyste mb ymean so felectrophoreti cmobilit ymeasurements . Thesam ewa sshow nb yu sfo rth ePL-P Slate xsyste mals ofro m electro­ phoreticmobilit ymeasurement san dfro melectrokineti cmeasurement so n PSplug swit h adsorbed PL.Whe nth e surface charge isthough tt ob e smeared out, superequivalent adsorption is onlypossibl ewhe nals o other attractive interactions contributet oth eadsorptio nenerg ype r segmentx •

Inth ecas eo fP Shydrophobi cbondin gbetwee nth e-(CH,) 4~groupso f thelysin esid echai nan dth epheny lgroup so fth eP Sparticle sca nb e responsible for this.Wit hAg i the same argument applies,bu tals o complexformatio nbetwee nA gan d-NH _ isa possibility .Th eoccurrenc e 54 ofsuperequivalen tadsorptio ni sno tcertai ni nth ecas eo fsilic aan d glass.Th e adsorption ofP L onthes esubstrate si sprobabl yno tcom ­ pletelyo fa nelectrostati cnature ,becaus en ocomplet edesorptio noc ­ curs athig h ionic strength as Iwil l show lateron .Hydroge nbond s between thepeptid e groups ofP L and the surface silanolgroup sma y contributet ox „i nthes ecases . When there is anon-electrostati c contribution toth e adsorption energysom eadsorptio na tth euncharge dsurface si sexpected ,a tleas t athighe r saltconcentrations .Wit h thePL-Ag Isyste mthi si sindee d thecas e (van der Schee, personalcommunication) .I nth ecas eo fsilic a noP L adsorption at thep.z.c . couldb edetected .N odirec tmeasure ­ mentsar eavailabl efo rth eadsorptio no fP Lo nuncharge dP Slate xpar ­ ticles (seesectio n4.3.5) . No adsorption ofP Lcoul db edetecte da t theuncharge dpolyoxymethylen e surface,a tleas tbelo wo ra t0. 1M NaBr . To obtain information aboutth e electrostatic and non-electrostatic contributions,plot s of theadsorbe damoun tversu sioni cstrengt han d surface charge forth evariou s adsorbents,ar emor eimformativ etha n theadsorptio nisotherm si nfig .4. 3 (a,b,c).Thi swil lb eth esubjec t ofsection s4.3. 5 and4.3.6 . Therewa sn odetectabl edesorptio no fP Lfro mP Supo ndilution .Th e same featurewa s foundfo rth eAg isyste mb y van der Schee (1984) and forexampl eb y Williams et al. (1982) forth ecarboxymethylcellulose - bariumsulphate system. The non-desorbability of the macromolecular adsorbate isno tdu et oa rea lirreversibilit y (i.e.th eadsorbat ei s not a frozennon-equilibriu m state)bu tt oth eextrem edilution stha t are required to remove higher M compound from the surface (Cohen Stuart et al., 1980). Someindication swer eobtained ,tha tP Ladsorbe d onglas sca nb edisplace d fromth esurfac eb yMB . For comparison purposes isotherms of poly-L-histidine (DP115) , poly-L-ornithine (DP103 ) andPL- L (DP62 )wer e simultaneouslymea ­ sured at29 3K and 0.1M NaB rwit hP S (latexM, )a sth esubstrat ea t pH= 3 (allpolyaminoacid sar ecompletel ycharged) .Th eplatea uvalue s -2 -2 -2 foundwer erespectivel y2. 1pmo l .m ,1. 8umo l .m and1. 8 pmol. m andno tsignificantl y apart.Thi ssupport sou rconclusio no fth edomi ­ nating importance ofelectrostati cinteraction sove rpossibl eeffect s of the different sidechai nstructur e onth e adsorbed amount(i.e . differences in x_)- The poly-L-histidine isotherm wasmor e rounded thanth e other two.Thi s is indicative for abroade rmolecula rmas s distribution forthi ssample . 55 ""'-'

4.3.5 Influence of the surface charge density

From thepoin t ofvie w of thepolyelectrolyt e adsorption theory of van der Schee (1982, 1984) it is important to know themeasure d plateau adsorption, not only as a function of the pH or pAg in the case of silica and Agi respectively, but also as a function of the surface charge. This isbecaus e the numerical calculations with the v. d. Schee (1984) theory are for the moment expressed as a function ofa only. For PS this isn o problem since the surface charge is constant,bu t on

Si02 and Agi, a changes when PL adsorbs. However the experimental trends are roughly the samewhethe r theplatea u adsorption is consid­ ered as a function of a or as a function of thep H orpAg . As said the polystyrene-water interface,negativel y charged due to the presence of covalently bound -OSOl groups in the interface, is in principle a so-called constant-charge surface. This means that the surface charge cr is not altered due to changes in salt concentration, electrical double layer overlap and specific adsorption. This isclear ­ ly not the case withAg i and silica,wher e atconstan t pAg and pHre ­ spectively, the surface charge increases with increasing saltconcen ­ tration. The charge-pAg curves of Agi in the presence of polylysine are shifted to higher a values and a lower p.z.c. due to the ad­ sorptiono fP L (van der Schee and Luklema, 1982). Theshif tt ohighe r a values is much more pronounced in the case of silica with adsorbed PL (plateau adsorption at each pH value). OnAg i the adsorbedpositi ­ vely charged PL,promote s the adsorption of I~ions and a becomes more negative. By the same token, on the silica surface, the dissociation of the silanol groups is strongly promoted in thepresenc e ofPL , also causing a to become morenegativ e (fig.4.4) .Th e -NH_ charges of PL are not titrated belowp H 7,becaus e proton titrationso fP L adsorbed on PS latex between pH 3 and 7 did not show any change in"o in this region.Henc e o isver y likely tob e solely due to the dissociation of silanol groups, also in thepresenc e of adsorbed PL. It is remarkable that thea (pH)curv e of silica with adsorbed PL resembles that ofbo - rosilicate glass without PL. Both charge-pH curves are linear over a largep H range incontradistinctio n to the curve ofbar e silica (fig. 4.4). The charge development on the glass-water interface,whic h has a thick gel layer, is apparantly similar to thato f the silica-water interface which has a much less porous double layer but which is covered with apolyelectrolyt e layer (see chapter5) . From the above itwil l be clear that only inth e case ofP S the ad- 56

Fig. 4.4 Charge-pH curves of borosilicate glass B and AEROSIL 0X50 in 0.01 M NaBr. T = 293.15 K. 1. Borosilicate glass; 2. AEROSIL; 3. AEROSIL in the presence of excess PL-L (DP 192). sorbed amount can be measured simple as a function of the surface charge. In the case of Agi and silica only the plateau value adsorp­ tion as a function of the pAg and pH respectively is measurable. The surface charge-pH (or pAg) curves in the presence of a surplus PL must also be known, for plotting the maximally adsorbed amount of PL as a function of the surface charge.

4.3.5.1 Polystyrene surface charge density and the effect of latex pretreatment

In fig. 4.5 adsorption isotherms of PL-L (DP 1682) on latex L (a 2 -11m Cm~ ) andM (ao = -60m Cm ') i n0.0 1M NaB rar eshown .Ther e isa significan tincreas ei nth eP Ladsorptio nwit hincreasin gsurfac e charge.O fcours e thisi sexpecte dbecaus eo fth eattractiv eelectro - 57

-1—• 1—• r

E"* x x en x £

•2 9—b—b" -o o o-

.p

I . I I . I 100 200 300 400 3 cpL/g.m-

Fig. 4.5 Adsorption of PL-L (DP 1683) on PS (latices M2 and L) in 0.01 M NaBr atp H= 6 .T = 29 3K . x-x PS latexM .( a -2 -2 2 o -60DiC. m );o- o PS latexL (a =-1 2mC. m ).

static interactionbetwee n the -OSO_ groups onth e PS and the -NH, groupso fth elysin esid echain so fPL . In connection to the above itwoul d be interesting to knowwhethe r orno tth e surface charge of alate x isaltere d due to atreatmen t withio nexchangers . Mostexperiment s with latexdescribe d inthi sthesi sar eperforme d with (mixedbed )io nexchange d (i.e.)latex .O nth eothe rhan d Norde (1976, 1978) used latextha twa sonl ydialysed .Th esurfac echarg eo f Nordeslatice swa scalculate d fromth elos si nsulphu rdu et oth ei.e . treatmentan dth etitratio ncharg eo fth ei.e .latex .Fo rlate xM (same formulationa suse dhere )Nord efoun dfo ra nexchange dan dunexchange d _2 polystyrene latex, a valueso f-4 5an d-8 4mC m respectively.Nord e foundals otha to ni.e .latices ,th eadsorptio nplatau so fHuma nPlasm a Albumine (HPA)ar e lowerb y about25% .Th edifference ,i nplatea uad ­ sorption (pH4.0 )betwee nNorde sunexchange dhig hcharg edensit ylate x _2 (a =-15 5mC. m )an dNorde slo wcharg edensit ylate x( a -23m Cm~ 2) waso fth esam emagnitude :30% . Incontras twit hNorde sresult sw efoun dtha tth eplatea uvalue so f PL adsorption onpolystyrene ,befor ean dafte rio nexchange ,wer eth e 58 same,despit eo fth eclea reffec to fth esurfac echarg eo nth eabsorp ­ tiono fPL ,a swa sshow ni nfig .4.5 .Thi sindicate stha tth edifferenc e insurfac e chargedu e to i.e. treatmenti ssmall .Thi sfac ttogethe r withth eobservatio ntha trepeate di.e .treatment sgiv eth esam evalu e of a (see3.2.2) ,sugges tstrongly ,tha tNorde sinterpretatio no fth e i.e. effect isno tcorrect .A nalternativ eexplanatio ni stha tdu et o thei.e .treatmen ta laye ro folig oan dpolystyren esulphat emolecule s are stripped off from aP Sparticl e asconclude d byNorde ,bu ttha t also inthi swa y ane w surfacewit hne w-0S0 ~groups ,burrie di nth e PSparticl e as ionpair s before thei.e .an dhenc eno tdetectabl eb y titration, is formed.Th e consequence wouldb etha tth evalue so f a used by Norde for the study of HPA and RNAse adsorption onP Sparticle sar eabou t50 %to ohigh . Although there isn oeffec to f i.e.o n the surfacecharg eo fPS , there exist aneffec to fth eioni cstrengt hhistor yafte rpolymeriza ­ tiono fa late xo nth ea value found. Van den Hoven (1984) foundtha t o thenegativ esurfac echarg eo fP Sparticle sincrease db ya facto r 1.5-2 when alate x hasbee nonc e exposedt oa nioni cstrengt ho f0. 1M .H e concluded this from conductometric titration of latex samples,tha t werebrough t inth e deionized H form,befor e and afterexposur et o 0.1M KN03- A secondexposur et o0. 1M electrolyt eha dn ofurthe ref ­ fecto na .Th emechanis m for this irreversible effect isno tclea r o yet. Also from electrokinetic measurements onP Splug s van der Put (1980) and van den Hoven (1984) obtainedindication sfo rthi selectro ­ lyteeffec to na .A mor edetaile ddiscussio ni sgive nb y van den Hoven (1984). Because of theincreas ei nth eplatea uadsorptio nvalu eo fP L with increasing surface charge onth ePS ,th e adsorptionplatea uo n electrolyte pre-treated laticeswil lb ehighe rtha ntha to nuntreate d PSsurfaces . Unfortunatelyn osystemati cmeasurement so fP Ladsorptio no nP Sar e available toquantif y this.Mos to fth e latices used inthi sstud y (exceptM .) wer e nottreate dwit h0. 1M electrolyt ebefor eus ei nad ­ sorption studies.Th e consequences ofthi s forth e electrolytede ­ pendence of the PL adsorptiono nPS ,wil l be discussed insectio n 4.3.6.1. Inconclusio n itca nb esai dtha ta P Ssurfac ewit ha bette r defined a canb e obtainedwhe nbefor e i.e. firsta sal ttreatmen t isgiven . 59

4.3.5.2 Silica and Borosilicate glass

Bothsilica -an dpyre xglas sar ehydrophili cwit ha weakl yaci dchar ­ acter,becaus eo fth epresenc eo fsilano lgroup si nth einterfacia lre ­ gion.I nth ecas eo fpyre xdissociatio no fth emor estrongl y acidbora - nol groups can alsocontribut e to thesurfac echarge .Becaus eo fth e presence ofth ewea k acid groups thesurfac echarg ei spH -an dioni c strengthdependent ,(e.g . James and Parks, 1982). Theparamete r most relevant forth e amounto fP Ladsorbed ,i sth e electric potential atth eplac e of the adsorbing PL segments.Thi s potential depends onman y factors such asth epH ,ioni cstrengt han d theadsorbe damoun titself .It svalu ei sno tknown .I na nexperimenta l situation the adsorbed amountca nb eplotte da sa functio no fth ep H orth erelate dsurfac echarge . Inth e case ofoxides ,whic hbehav e non-Nernstian, the relationbe ­ tweenth e solutionp H and the surfacepotentia l mustb ea mor ecom ­ plicatedon etha nNernst .Henc eth esurfac epotentia lcanno tb eeasil y calculated fromth eequilibriu mp Hi nthes ecases . Furthermore iti sno tth esurfac epotentia lwhic hi so frelevanc efo r the adsorption ofP L segments onth e surface,bu tth epotentia l at some distance from thesurface .Thi si sbecaus eth echarg ea ta poly - lysine segmentcanno t reach the surface due to the finitevolum eo f the ionized groups. Inth epolyelectrolyt e adsorption theory ofva n der Schee (seeChapte r7 )th eeffectiv eadsorptio nenerg ype rsegmen t x is defined as: s,eff Xsef f =X s - (l-aJet^/kTi nwhic h xs isth e nonionic adsorption energy, a isth e dissociation grade ofth e-NH 3 groupsan dip .i sth epotentia la tth eplac eo fth etrai nsegments . Exceptchemica lcontribution s (i.e.H-bond s andhydrophobi cbonding ) thenon-ioni cadsorptio nenerg yx cancontai ncontribution so fdipola r interactions.I nth ecas eo fcharge dpolypeptide sno tonl yth edisplace ­ ment ofwate r dipolesb y charged groupsca npla ya rol ebu tals oth e strongdipol eo fth epeptid egroup s (M =3. 4D (Wada, 1961)) cancompet e withwate rdipole sa tth esurface .Th emagnitud eo fthes econtribution s isdetermine db yth esurfac echarge ,becaus eth eadsorptio nenerg yo fa dipole isjj*E *an d the fieldstrengt hE a tth elocu so fadsorptio ni s according toGaus s lawproportiona l toth e surface chargeo .Henc e theadsorptio no fpolylysin ei si npar tdetermine db yth epotentia la t theplac ewer ea segmen tadsorb san di npar tb yth esurfac echarge . Experimentallyth eamoun to fP Ladsorbe do nsilic ao rglas sca nonl y bemeasure da sa functio no fth esuspensio npH .Th eadditiona linforma - 60 tiono fth ecorrespondin g charge-pH curves (i.e. titrationcurve so f silicai nth epresenc eo fPL )i sneede dt oconstruc tth esurfac echarg e dependenceo fth eadsorbe d amount (seesectio n4.3.5) .I nman ypoly - electrolyte adsorption and/or flocculationstudie sth eauthor sd ono t perceive thisi ndiscussin g theinfluenc eo fth esurfac echarge .Th e thick gellayer (i.e.porou s double layer)tha texist si nth ecas eo f glass isa complicatin g factor.Th emeasure d titration charge(se e fig.4.4) ,whic hi sactuall ya spac echarg edensity ,i sno tth echarg e densitywhic hth eP Lmolecule s 'see'.Th echarg ei nth egellaye rca n onlyb ecompensate d forb ymicro-ion san dno tb ylarg emacromolecule s sucha sPL .I nth ecas eo fsilica ,whic hha sonl ya thi ngellayer ,si - lanolgroup sca nprobabl yinterac tdirectl ywit h-NH _ groupso fth ePL .

i 1 l l l \. /o

7 .8 /o E en E - .6 /, •

M : // : .2 _x- *?5 • *-x—* ^/A i^O^AA 1 1 1 1 3 U 5 6 7 8 pH

Fig. 4.6 Adsorption of poly-L-lysine as a function of the equilibrium pH. Electro­ lyte: 0.01 MNaBr ; T = 293 K.

x-x PL-L (DP l683)/Borosilicate glass B2; o-o PL-L (DP 192)/AEROSIL 0X50 A-A PL-L (DP 1683)/AEROSIL~OX50.

In fig. 4.6 the plateau adsorption values of PL in 0.01 MNaB r (T = 293 K) on silica and glass powder are plotted against the solution equilibrium pH. The suspension effect on the pH measurements was pro­ bably small because the pH difference measured between the suspension and the equilibrium supernatant was always smaller than 0.02 pH units. From the figure one can see that at the p.z.c. of silica (pH ~ 3) the adsorbed amount is also negligibly small. This is an indication that 61 electrostatic interactions between adsorbent and adsorbate are dom­ inating in this case. The hydrogen bonding between R-C=0 orR-N H of a peptide group and a surface silanol group is apparently not strong enough to give a measurable adsorption. However in the case of glass the adsorbed amount has a finite value atp H 3. The difference inpla ­ teau value at pH 3betwee n silica and glass isprobabl y caused by the difference in p.z.c. between the two substrates, and not to adiffer ­ ence inhydrophobicity as onemigh t think at firstsight . Jednacak et al. (1974) measured the i.e.p.'s of various glasses from streaming potential and/or streaming current measurements on glass plugs as a function of the pH and ionic strength. The value of the i.e.p. they found for (vitreous) silica isp H 3,wherea s the value for pyrex glass is about pH 2.5. This difference has no consequences for thepositio n of theproto n titration curve of glass as explained before. Because of the flatcharge-p H curves at lowpH , a shift in thep.z.c . due to the interaction of the silica or glass with PL has almost no effect on the actual value of the surface charge density atp H 3. This is demonstrated in fig. 4.4 for the surface charge-pH curves of AEROSIL with and without added PL. Because of the large uncertainties in the specific surface area of the glass powder nothing more quantitative can be said about the difference in slope found between the T-pHcurve s of glass and silica. Inth e case of silica the slope of the r-pH curve

' 1 ' i • i ' i '

•8 - s ° en o/ e - Ù -6 : y 1 ' -/

I-I. 40 80 120 160 o„/m Cm "

Fig.4. 7 Adsorption of poly-L-lysine (DP 190)o n AEROSIL0X5 0a sa functio no fa in0.0 1 MNaBr . T= 29 3K . 62

(110mC/p Hunit )i s almost twicetha to f theproto n titrationcurv e (61mC/p Hunit) . Iti stherefor ever ylikel ytha tth eadsorptio no fP L onsilic ai ssuperequivalen tove rth ewhol ep Hrang estudied . Infig .4. 7 theadsorbe damoun tP L (DP192 )i n0.0 1M NaB ri splot ­ ted against a insteado fth epH .A tmoderat e a valuesth eamoun to f PLadsorbe dincrease salmos tlinearl ywit hth esurfac echarge .Thi swa s alsofoun db yva nde rSche efo rth eadsorptio no fP Lo nAgi .Her ehow ­ ever there is adefinit evalu eo fth eadsorbe damoun tP La tzer osur ­ facecharge ,probabl y because ofth ehydrophobi c surfacepresen ti n thiscase .Th e 'linear'behaviou ri spredicte db yth epolyelectrolyt e adsorptiontheor yo f van der Schee (1984).

4.3.6 Influence of electrolyte concentration

Withpolyelectrolyt eadsorptio nth eelectri crepulsio nbetwee nsimi ­ larlycharge dgroup swil loppos eth eformatio no fthic kadsorbat elay ­ ers.O nth eothe rhand ,i fthi srepulsio ni ssuppresse db yelectrolyte s rca nbecom ehigh . When the non-ionic adsorption energy parameter x iszer oo rsmall , there is asensitiv ebalanc ebetwee nth eelectrostati c attractionbe ­ tweensurfac e andP L charges and thelatera lelectrostati crepulsio n betweenadsorbe dpolyelectrolyt echarge si nloop san dtails .I tdepend s onthi sbalanc ewhethe rth eadsorptio nincrease so rdecrease supo nin ­ creasingioni cstrength .

4. 3. 6.1 Polystyrene latex and Agi

In fig.4. 8 theplatea uadsorptio nvalue so fPL.HB ro npolystyren e andAg i respectively areplotte da sa functio no f-lo gI .A sexpecte d theadsorptio nincrease si nbot hcase swit hincreasin gioni cstrengt hI . Athighe r Ith eadsorptio nbecome sals omor esensitiv et oth emolecula r mass as already discussed in (4.3.4). Forth equantitativ einterpre ­ tationo fth eeffec to fth e ionicstrengt ho nth eadsorbe damoun ti t isnecessar yt orealiz etha ti nthe .cas eo fP Sth esurfac echarg eabov e _3 0.1M NaB rca nb ehighe rtha na t1 0 MNaB ra sdiscusse dbefor e(sec ­ tion4.3.5.1) . This will lead to asomewha thighe r adsorptiontha n would otherwiseb e athighe r saltconcentration .Th eincreas ei nad - _2 sorbedamoun ti n0. 1M NaB rwhe na isincrease d from-4 0t o-8 0m Cm o isonl y a fewpercen ta spredicte db yth epolyelectrolyt eadsorptio n theory ofva nd e Schee,unde r similarcondition sa sth eexperimenta l 63

Fig.4. 8 Adsorption ofP L (expressed inm gPL.HBr )a sa functio no fth eelectrolyt e concentration (i.e.-log(ioni c strengthNaBr) )o nP San dAgi .T = 29 3K .

1. PS (latex M4): o-o PL-L DP 19:D- D PL-DL DP240 ;A- A PL-L DP 1683 electrolyte:NaBr ;p H6 . 2. Agi: o-o e-Acp-lys ,-NHMe;D- D PL-L DP300 ;A- A PL-L DP200 0 elec- trolyte:HNO. ;surfac echarg eAg ia ^ 10mC. m

ones.Th e effecto f the lateral interactionbetwee nadsorbe dP Lseg ­ mentsi sprobabl ydominant . Considering the ionic strength dependence ofth eP Ladsorptio no n Agi (van der Schee, 1984; Bonekamp et al., 1983) alsoi nthi scas eth e experimentsar eno tperforme d atconstan tsurfac echarge ,bu ta tabou t constantpAg ,s oa increaseswit hincreasin g I.Th emagnitud eo fthi s effect is not exactly knownbecaus e thecharge-potentia lcurve so f Agiwit h adsorbed PL atdifferen t electrolyteconcentration sar eno t available (onlyth ecurv eo f0. 1M KNO .) . Itwil lb eo fth esam eorde r ofmagnitud e aswit hbar eAgi , andprobabl yless ,whe nth esituatio n mayb ecompare d withth esilica-P Linteraction .I nth epresenc eo fP L thepA gi sals ono texactl yconstan twhe nth eioni cstrengt hi svaried , unlessspecia lprecaution sar etaken ,whic hwa sno tth ecas ehere . 64

So the surface potential isno tentirel y constanteithe rwhe nth e ionicstrengt hi sincreased . When atconstan t surface coverage ofP Lth eioni cstrengt hi sin ­ creased,th esurfac e charge also increases.Howeve ri nth esituatio n offig .4.8. 2th eadsorbe damoun to fP Lincrease sals oa sa direc tcon ­ sequenceo fincreasin gth eioni cstrength .Thi slead st oa decreas ei n thepA gan dconsequentl yt oa decreas ei nth esurfac echarge .I nothe r words,ther ear ea numbe ro ftrend stha tpartl ycompensat eeac hother . The approximation of constant surface charge istherefor eprobabl y reasonablei nth esituatio no ffig .4.8.2 .Th ecompariso no fth eelec ­ trolyte dependence inth ePL/P S (fig.4.8.1 )an dPL/Ag I (fig.4.8.2 ) system,bot ho na basi so fconstan tsurfac echarge ,seem stherefor ea n acceptable firstapproximation ,especiall ys osinc eth elatera lelectro ­ static interaction betweenth epolyme rsegment si sprobabl yth edomi ­ nantfactor . The adsorptiono fP Lcontinuou st oris eprogressivel ybeyon d0. 1M electrolyte both forAg ian dlate xa sth esubstrate .Th ediffus epar t ofth eelectrica l doublelaye ri sthe nhoweve ralmos tcompletel ycom ­ pressed (K <1run) .Fo rpolyelectrolyt e systemsi ti squit ecommo nhow ­ evertha tth einfluenc eo fth esal tpersist sabov e0. 1M .A qualitativ e explanationi sa sfollows :Th edistanc ebetwee nth echarge so na poly ­ electrolyte chaini susuall yver ysmall ,abou t0. 4ru nfo rP Lan deve n smaller forfull y charged polyacrylic acid (0.25run) .Henc ea t0. 1M saltwer e K ^ 1ru ntw oadjacen tcharge so na polyelectrolyt echai n are still notcompletel y screened.A nexampl eo fthi s effecti na polyelectrolyte solution isth etitratio nbehaviou r ofwea k acido r weakbasi cpolyelectrolytes .A plo to fp K versusdegre eo fdissoci ­ ation a isstil lno thorizonta lu pt oconcentration so f1 M electrolyte . Thesam ei sfoun db yu sfo rplot so fth especifi cviscosit yo fP Lsolu ­ tions againstP Lconcentratio nwit hNaB r concentrations above 0.1M NaBr.I nth edomai no fpolyelectrolyt e adsorption, Greene (1971) found astron gincreas ei nth eplatea uvalu eabov e0. 1M NaC lfo rth eadsorp ­ tiono fpoly(2-sulphoethylmethacrylate )o npolyethylen epowder . Marra et al. (1982) reported adsorptionso fpolystyren esulphonat eo n silicawhic h kept increasing atleas tu pt o3 M MgCl ,o rNaCl .Th e polyelectrolyte adsorption theoryo f van der Schee (1984) iscapabl e of explaining this rise of adsorptionwit h increasing I(se eals o chapter 7). Itseem s thereforeno tnecessaril y toassum ea nincreas e ofx wit hrisin gsal tconcentration st oexplai nth eelectrolyt edepen ­ denceo nth eadsorptio na thig helectrolyt econcentrations .I ti sthere - 65 foreconclude d thatth e strong increase ofth eadsorbe damoun tabov e

0.1M electrolyt ei sstil ldu et oth escreenin go fth e-NH 3charges . Iti sinterestin g tonot etha tpolylysine-DN Acomplexe sdissociat e in1. 0M electrolyte solutiondespit eth epresenc eo fH-bond sbetwee n thelysin eresidue san dphosphat egroup so fth eDN A (Helene and Maurizot, 1981). Asimila rsituatio nexist swit hth ePL-silic asyste ma swil lb e shownlate r (section4.3.6.2) .Obviousl y inthes ecase sth ebindin gi s predominantlyo fa nelectrostati ccharacte ran dhenc ei ti sreduce db y theadditio no findifferen telectrolytes . Onth eothe rhan dwit hP L adsorbingo nP San dAgi ,non-coulombi cat ­ tractive interactions,suc ha shydrophobi cbonding ,contribut esigni ­ ficantlyt oth eadsorptio nenerg ype rsegment ,a salread ywa ssuggeste d whenth eadsorptio nisotherm swer ediscusse d (section4.3.4) . As one can seefro mfig .4.3.1 ,n osignifican tdifference sbetwee n thebehaviou r ofPL- L andPL-DL ,wit hrespec tt oth eelectrolyt ein ­ fluence could be detected asexpecte d (see also section4.3.4) , at leastfo relectrolyt econcentration sbelo w0. 1M . The adsorption ofP L onpolystyren e isreversibl ewit hrespec tt o changesi nioni cstrength .Thi sreversibilit ywa steste da sfollows : After adsorption ofPL-D L (DP240 )o npolystyren e latexM in0.0 1M NaBr, the supernatant obtainedafte rmil dcentrifugatio nwa sreplace d by aknow nvolum e ofNaB r solutioni nsuc ha wa ytha tth efina lNaB r concentration was~1 0 M-.4Th elate xwa sthe nresuspende d androtate d end over end for1 6hours .Th edesorbe damoun twa sdetermine dan dap ­ pearedt ob eequa lt oth edifferenc ei nplatea uvalu eadsorptio nbetwee n 10~2 and10" 4M NaBr . SCN-ions are known tobin d specifically ontoth eR-NH 3sid echai n groups of polylysine (Conio et al., 1974). Thiscause s areductio n ofth e effective charge density on apolylysin echai n (Conio et al. , 1974) anda neffec to nth eamoun to fP Ladsorbe do nP Si nth epresenc e ofNaSC Ni stherefor eexpected .Thi swa sindee d found:Th eplatea uad ­ sorptionvalu e ofPL- L (DP1683 )o nlate xM _i n0.0 1 and0. 1M NaSC N isrespectivel y0.4 8 and0.7 1mg. m .Th ecorrespondin -2 gvalue si n0.0 1 _2 and 0.1M NaB r (fig.4.3 ) arerespectivel y 0.36 and0.4 6m gm .Th e plateau adsorptionvalue s ofP L adsorption onP Swit hNaC lo rNaNO _ as the electrolyte areno tdifferen t from the isotherms foundwit h NaBr, asexpected .Fro m the effect foundwit h SCN~ it isconclude d thatNaSC N acts as ascreene r ofth einteractio nbetwee nP Lsegment s moreeffectiv etha nNaBr .Thi sstronge rscreenin gmor etha ncompensate s theexpecte dweakene d -NH-surfac e-OSO lattraction .I nth ecas eo fsi - 66 lica as adsorbent thisi sno tso ,becaus eher eth eamoun to fPL- Lad ­ sorbedi sabou tth esam ewhethe rNaB ro rNaSC Ni suse d (seefig .4.3a) .

4.3.6.2 Silica and Borosilicate glass

Silica andborosilicat e glassar ehydrophili cadsorbents .Th enon - ionic adsorption energy x_ ofP Ldoe sno tcontai n acontributio no f hydrophobicbondin gi nthes ecases .Furthermor eth estron gcompetitio n ofP L segmentswit hwate rmolecule s forsurfac esite swil llowe rth e valueo fx_ -Therefor ex swil lb elowe rtha ni nth ecas eo fAg ian dPS . Consequently thebalanc ebetwee nth eelectrostati c attractionbetwee n surfacecharge san dP Lcharge san dth elatera lelectrostati crepulsio n between adsorbed polyelectrolyte charges in loops andtails ,wil lb e moresensitiv e forelectrolyt etha ni nth ecas eo fP San dAgi . Whenknow namount so fa NaB ran da P Lsolutio ni ndem iwate r (pH6 ) are added inth e givenorde r to aweighte d amounto fpurifie dboro ­ silicateglas spowder ,n osignifican tdifference si nth eplatea uvalue s arefoun do nvaryin gth eioni cstrengt ho fth esuspensio n (seefig . 4.9). Clearly several opposing effectspla y arol ei nthes esystem .First , thep H of theglas s suspensions isno tconstant ,bu tdecrease swit h increasingioni cstrengt ha son eca nse ei nfig .4.9 .Thi sdecreas e

• 1 • 1 • 1 ' 1 I pH

f.3 o 0 ° °^ .2-

• ••••• 1 2 3 4 -logI

Fig.4. 9 Adsorption ofPL- L (DP1683 )o nBorosilicat e glassB .a sa functio no fth e ionic strength.T = 29 3K . o-o Tv s -loglfo r5. 2

Fig.4.1 0 Effect of the pH on the adsorption ofPL-D L (DP307 )a tdifferen tioni c strengtho nsilic a (AEROSIL0X50) ,T = 29 3K . o-o 10"3M NaBr ;A- A 10"2NaBr ;D- G 10_1 MNaBr ;x- x 0.3 MNaBr . 68

Infig .4.1 0 theplatea uvalue so fth eadsorptio no fPL-D L(D P307 ) onsilic aar eplotte da sa functio no fth ep Ha tsevera lvalue so fth e ionic strength.Th e curves atdiffren t ionic strengthwer emeasure d simultaneously inon eserie so fexperiments ,wit hth eobjectiv et ous e the same stock solutions andpipette s foreac hmeasurement ,s otha t smaller differences in adsorbed amountbetwee n twocurve scoul db e estimated. Series of curveswit hPL- L (DP240 )an dPL- L (DP192 )wer e obtained in the same way. They showed similar behaviour asPL-D L (DP= 308 )i nfig .4.10 .Thi sindicate stha tth eioni cstrengt hdepen ­ dence ofth er v sp Hcurve si sreproducibl ewithi non ese to fcurves , andtha ti ti sindependen to fth emolecula rmas san dstereoregula rcon ­ figurationo fth ePL . Ateac hioni cstrengt hvalue ,th eamoun to fP Ladsorbe dincrease swit h increasingp Hdu et oth eincreasin gsurfac echarg ea salread ydiscusse d under (4.3.5.2). In fig.4.1 1 theplatea uvalue so fth eadsorptio no f PL-DL as a functiono f -logI ar eplotte d forthre ep Hvalue s(i.e . verticalcros ssection sthroug hth ese to fcurve si nfig .4.10) .A tp H valueslowe rtha n4 ,th eadsorbe damoun tseem st odecreas emonotonical - lywit h increasing ionic strength.Th eexperimenta lerro ri showeve r toolarg ea tthes elo wadsorbe damount st oallo wconclusion st ob edraw n

Fig. 4.11 Influence of the ionic strength Io nth eadsorptio no fPL-D L (DP307 )o n AER0SIL0X5 0a tdifferen tp Hvalues , o-op H4.0 ;D- Dp H6.0 ;A- Ap H7.5 . 69

from theobserve d trend.Th elowe radsorbe damoun ta t0. 3M NaB rcom - paredt otha ta t1 0 MNaB ri sprobabl y real.A thighe rp Hvalue sth e rv s-lo gI curve ssho wa maximum ,indicatin gtha tth efacto rdominatin g theadsorbe damoun ti sdifferen ta thig han dlo wioni cstrength . With the help of the a -pHcurve s of silica in thepresenc e of -3 -2 -1 excess PL for 10 ,1 0 and 10 M NaBr (see fig. 4.12), the adsorbed

Fig.4.1 2Charge-p Hcurve so fAER0SI L0X5 0silic ai nth epresenc eo fexces sP L( f ateac hpH) .T = 293.1 5K .c 2.50%(w/v) .Electrolyt econcentration s sil areindicated . amounta sa functio no fp Hca nb ereplotte da sa functio no f a forth e ionicstrengt hvalue sindicated .Fro mthi sse to fcurve sth eamoun to f PL adsorbed as afunctio no f-lo gI a tconstan t a valuesa sshow ni n fig.4.1 3 areobserved . Itca nb esee nfro mthes ecurve stha ta tcon ­ stant surface charge theplatea uadsorptio no fP Lonl yincrease swit h increasing ionic strength andn omaximu m ispresen tanymore .I ncon ­ trastwit hth e comparablecurve sfo rAg ian dP Sa sth esubstrate sfo r

PL, ther v s-lo gI (a Q= constant )curve sfo rsilic aar econcav ean d theincreas eabov e0.0 1M sal ti sonl ysmall .Thi sshow stha tth ebal ­ ancebetwee n thetw oopposin gsal teffect smentione d inth ebeginnin g ofthi ssection ,i sturne di nfavou ro fth eweakenin go fth e-NH*/=Si-o " attractiona thighe rsal tconcentration .Wit hth eP L- glas ssyste ma t lowp H asimila reffec twa sfound .Th edisplacemen to f-NH ~ fromsur - face sitesb yN a ,a thighe r ionic strength,ca npla ya rol ei nth e 70

Fig.4.1 3 Influence of the ionic strength on the adsorption of PL-DL (DP307 )o n silica atconstan t surfacecharge . -2 2 2 o-o a =-2 0m Cm -50mC.nf ;A- A a -100mC.m" . o D-D a o observed trend. Iti sstresse d again thatsuc ha sal teffect ,a sob ­ served in fig.4.13 ,ca nonl yb e foundwhe nth enon-ioni cadsorptio n energyx s isa mino rfacto rcontributin gt oth ebinding . Theproble m remainsho w toexplai n themaximu m inth er v s-log l curves atconstan tp H (fig.4.11) . The reasonmus tb e soughti nth e factthat ,i ncontradistinctio n toth ebehaviou ro fbar esilica ,th e surface charge atconstan tp Ho fsilic awit hexces sP Lpresent ,i sa decreasing functiono fth eioni cstrength .Thi si sanothe rindicatio n forth ecompetitio nbetwee nN a and-NH -charge so fPL ,fo rdissociate d surface silanol groups.A mor e detailed discussion ofth echarge-p H curveso f silicawil lb egive ni nchapte r5 .I ti sprobabl ythi sdro p insurfac e charge,whic h causes adecreas ei nth eadsorbe damoun ta t higher ionic strength atconstan tpH .I twoul db einterestin gt ocal ­ culate the electrolyte dependence of the adsorption ofpositivel y chargedpolyelectrolyte s onnegativel ycharge dsurface sfo rx values abouto rles stha nx 0„, .(se echapte r 7). Inthi srespec ti ti sinter - o/ C l esting that Lindquist (1975) founda nincreas ei nth eamoun tadsorbe d atconstan tp Hfo rpolyethyleneimin eo nsilic a (LudoxAM )particle su p to0. 1M NaCl .Thi si sprobabl ydu et oa highe rx _valu ei nthi scase . 71

4.3.7 The stability of latex and Agi sols against low molecular mass electrolyte in the presence of polylysine

When anegativel y chargedsoli ddisperse d inwate ri sbrough tint o contactwit h an increasingconcentratio no fa cationi cpolymer ,thre e zones of actionma yb erecognized ,viz.(i) :a stabl ezon ea tver ylo w total polymer concentration (i.e.ver y lowpolyelectrolyt e coverage ofth eparticles) , (ii)a flocculation *zon ean d (iii)a thighe rpoly ­ electrolytecontent s (highsurfac ecoverage )a zon eo frestabilization . Theunstabl e region isboun d by thecritica l flocculationconcentra ­ tion (CFC)an dth erestabilizatio nconcentratio n (RSC).Th econcentra ­ tiono fpolyelectrolyt erequire d formaximu mdestabilizatio ni sdefine d asth eoptimu m flocculationconcentratio n (OFC). Themai nreaso nfo rflocculatio no fcharge dparticle sb yoppositel y chargedpolyelectrolyte s ischarg eneutralizatio n (Gregory, 1973; Ho and Howard, 1982; Lindquist, 1975; Bleier and Goddard, 1980). Besides theexten to f adsorption, also the configuration of thead ­ sorbedpolyme rdetermine swhethe r flocculationo rstabilizatio noccurs . Thecoagulatio nconcentratio ni sals odependen to nth estructur eo fth e adsorbed layer. Corry and Seaman (1978) pointed outtha ti nth ePL / latex system,polylysin e canac teithe ra sa flocculatin go rstabili ­ zing agent, depending on the configuration and disposition ofth e adsorbedpolymer . In this study, the stability of polystyrene- andAgl-particle s coveredwit h PL above theRS C againstlo wmolecula rmas selectrolyt e wasinvestigated .Th einfluenc eo fth eP Lmolecula rmas swa sals ocon ­ sidered.I nthi swa ysom eadditiona linformatio nca nb eobtaine dabou t theconfiguratio n ofth eadsorbe dpolylysine .Th estabilit ymeasure ­ mentswer edon eb ya stati cmetho ddescribe di nsectio n4.2.5 . The stability of polystyrene andAg i sols,containin gparticle s coveredwit hPL-L ,agains tincreasin gKN0 3 concentrationwa smeasured . Inth eabsenc eo fP Lth ecoagulatio nconcentratio no fP Slate xwa sno t measurablydifferen twit hKNO .o rNaB ra selectrolyte .Als oi nth epre ­ sence ofPL ,th e same trendswer efoun dwit hKNO _ orKB ra selectro ­ lyte.

* Theter m flocculation isuse dher ewhe ndestabilizatio noccur sb yth eadditio no f polyelectrolyte. The term coagulation is used inth e caseo fdestabilizatio nb y lowmolecula rmas selectrolyte . 72

Experimentswit hsol si nth epresenc eo fadsorbe dP Lwer eperforme d byu sunde r conditions of large excess (i.e.wit hrespec tt oth ead - _3 sorbedamount )P Li nth ebul k (cÜT >3 0g. m )o rsmal linitia lexces s -3 PLi nth ebul k (cpL <0. 7g. m ).A sth eadsorbe damoun tincrease swit h increasing ionic strength,th econcentratio n PL inth ebul ksolutio n decreases with increasing ionic strength tover y lowvalue s inth e latter case.Abov e 0.1M electrolyte,platea u adsorptioni sno tcom ­ pletely reached inth ecas e of small initial excess PL.Whe n large excess ispresent ,th eP L concentration inth ebul kstay sabou tcon ­ stant on increasing the electrolyte concentration. Inthi ssituatio n theplatea uvalue sar elargel yreache da teac hsal tconcentration . In fig.4.1 4 and4.1 5 therelativ eturbidit yx , i.e .th emeasure d extinction ofth e supernatantdevide db yth eturbidit yo fth ecorres ­ pondingstabl eblan cso lt , i splotte da sa functio no fth eKNO -con ­ centration forAgi - andP S sols,bot hbar e and coveredwit hPL- Lo f differentmolecula rmasses . Inbot hcase s thetota lconcentratio nP L ensures large excess of PL-L.Fro m fig.4.1 5 one can seetha tth e stability of theAg i sol increases withincreasin gmolecula rmas so f the adsorbed PL-L. Inth e case ofPL- L (DP19 )ther e is apparently noextr agai ni nstabilit yo fth esol ,despit eth eincreasin gadsorbe d

Fig.4.1 4 Stability curves ofP S (latexM„ )wit h andwithou tadde dPL- La tp H6 an d 3 T =298.1 5K . CpL =3 0g.m" .x- xP S latexM 2; o-o PS latexM 2/PL-L (DP19) ;

G-DP S latexM 2/PL-L (DP240) ;A- AP Slate xM 2/PL-L (DP 1683). 73

Fig.4.1 5 Stability against 1-1 electrolyte of Agi sol with and without PL-L. 3 T =298.1 5K , CpL =5 0g.m" ,p H6 . x-x Bare Agi; o-o Agl/PL-L (DP 19);D- D Agl/PL-L (DP 192);A- AAgl/PL- L (DP 1683).

amountwhe nth esal tconcentratio ni sraised .However ,i nth epresenc e oflarg eexces sPL- L (DP192 )o rPL- L (DP1683) ,th eAg isol sar emor e stable thanbar eAg i sols.Apparentl y loops and tails areforme da t higher ionic strength, largeenoug ht ogiv esom esteri cstabilizatio n inthes ecases . Inth ecas e ofsmal linitia lexces sP L (seefig .4.16 )n oevidenc e for steric stability was found.Unde r these conditions the solsar e purelyelectrostaticall y stabilized.Th eeffec to fth eP Lconcentratio n inth ebul ko nth eso lstabilit yi sshow ni nfig .4.16 . There isa n increasing stability when ateac hKNO -concentratio nth e volume fractionP L in thebul k is such,tha tth eplatea uadsorptio n is largely reached. Inthos e caseswher e thevolum e fractioni nth e bulk Ä,i sver y low,thu s resulting inamount s of adsorption just below theplatea u or even less,o nincreasin gth eKNO ,concentratio n T —3

(CpL = 1.0g. m ),ther ei sals on oappreciabl emolecula rmas sdepen ­ dence ofth e stability (seefig .4.17) .Henc eth econformatio no fth e adsorbedP Llaye ri smor efla tunde rthes econdition stha ni nth ecas e 74

c (M) KN03

Fig.4.1 6 Influenceo fth etota lP L(D P62 )concentratio n onth eelectrolyt estabil ­ ityo fAg isol ,p i= 4 ,T 298.15K . T x-xbar eAgi ;o- oAgl/PL- LC * =1 g.m." 3;D- DAgl/PL- LC =5 g.m "; CPL pL 3 A-AAgl/PL- L CpL= 5 0g.m" . oflarg eexces sPL .S oi nth ecas eo fsmal linitia lexces sP Lth epos ­ sibilityo fsignifican tsteri cstabilit yn olonge rexists .Th eresult s obtainedb yu sunde rcondition so fsmal linitia lexces spolylysin ear e consistentwit hthos eo f van der Schee (1984), whomeasure dth eelectro ­ lyte stability ofPL-covere d Agi sols,unde rsimila rcondition sb ya kineticmethod . The stability curveso fPL-covere dP Sparticle sa tlarg eexces sP L asa functio no fth eP Lmolecula rmass ,ar equalitativel yth esam ea s those with Agi (fig.4.14) , howeverth eeffec to fth eP Lmolecula r mass isher emuc hmor epronounced .Th eP Ssol swit hparticle scovere d withPL- L(D P168 3) ar ecompletel y stableu pt oelectrolyt econcen ­ trationso fa tleas t1. 0M .A complicatin gfacto rher ei sth eP Ssur ­ faceitself .Th ePS-wate rinterfac ei st osom eexten t 'hairy'(se esec ­ tion3.2.3) . Thiscause s akin do fintrinsi csteri cstabilit yo fth e latex.Coagulatio nconcentration so flate xi nth epresenc eo fP L(D P19 ) andP L(D P240 )ar eeve nlowe rtha nthos eo fbar elatex .Thi sindicate s thatth einterfacia lP Llaye ri smor efla ttha nth ebar ePS-wate rinter - 75

Fig.4.1 7 Stability of Agi sol as a function ofth eKN O concentration.N oexces s T -3 T =298.1 5K ,p i= 4 . PL-Lpresent ,c = 1.0g. m a-xAgl/PL- L (DP 62); o-oAgl/PL- L (DP 192);D- Qbar eAgi .

face.O nth eothe rhan dwit hP Scovere db yPL- L (DP1683 )thi si sclear ­ lyno tth ecase .Quantitativ edifference si nth estabilit ybehaviou ro f Agi-an dP Ssol scovere dwit hP Lar epossibl ycause db ydifference si n Hamaker constantbetwee n thesesubstances .Als oothe rfactor ssuc ha s differences insedimentatio nvelocit y ofth e aggregate andadsorbe d amount,betwee nth etw osubstrate sca ncontribut et oit .Th estabilit y ofP S latex inth epresenc e ofP L isconsisten twit helectrokineti c measurements performed byu so nPL-coate d PSplugs .Th e resultsob ­ tainedher ear equalitativel yi nagreemen twit hthos eobtaine db y Wil­ liams et al., (1982) fornegativel y chargedBaSO .particles ,covere d withpolyacryli c acid. Corry (1978) measuredth erat econstan to fth e flocculationo fP Ssol sa ta constan tlo wmolecula rmas ssal tleve lo f 0.1M NaC l as afunctio no fP Lcoverag e (theelectrophoreti cmobilit y ofP Sparticle swa stake na sa measur eo fsurfac ecoverage) .Th erat e constant athig h surface coverage was appreciablylowe rtha ntha ta t moderatesurfac ecoverage . Corry concludedtha ta thig hsurfac ecover ­ age the low aggregationrat econstan tmus tb eaccounte d forb ysteri c stabilization, which is inlin ewit h our experience.N o satisfying 76 explanation wasoffere d forth eextr astabilit ygai na tver ylo wsur ­ face coverage. Inm yvie w theunexpectedl y high rate constantfoun d by Corry mustb e due to theharynes so fth eP Sparticle sthemselves , afeatur esimila rt otha tfoun db yus .

4.4 SUMMARYAN DCONCLUSION S

Inthi s chapter adsorptionpropertie so fhighl ycharge dpolylysin e (pH< 6 )o nhydrophobi c (polystyrene andAgi )an dhydrophili c(boro - silicateglas san dsilica )adsorbent swer ediscussed . Inal lcase shig haffinit yisotherm sar efoun di nwhic hth eplatea u adsorption is less than or aboutmonolaye r coverage.A t lowioni c strength there isn oeffec to f themolecula r mass ofP L on thead ­ sorption, indicating flat adsorbate layers.N o differencesbetwee n the adsorption ofPL- L and PL-DL arepresent . Inmos tcase sth ead ­ sorptiono fP Li ssuperequivalent . _2 At low ionic strength (i.e.I < 1 0 M)th eformatio no fthic kad ­ sorbate layers isprevente db yth estron gelectrostati crepulsio nbe ­ tweenadsorbe dP Lsegments ,a si susuall y foundfo rhighl ycharge dpoly - electrolytes.A sthi selectrostati crepulsio ndominate sth eadsorptio n atlo wioni cstrength ,difference si nadsorbe damoun tbetwee nth esub ­ stratesuse d (atth esam esurfac echarge )ar eno tver ypronounced .Als o thecompositio no fth esid echai no fth ecationi cpolyaminoacid sha sn o influenceunde rthes econditions . As expected the adsorptiono fpolylysin eincrease swit hincreasin g negative surface charge.Th e adsorbed amount increasesals owhe nth e ionicstrengt hi sincreased ,becaus ethe nth erepulsio nbetwee npolyme r segments is screened.Th esal tdependenc ewa sfoun dt ob ereversible . On increasing the ionicstrength ,difference si nadsorptio nbehaviou r betweenhydrophobi can dhydrophili cadsorbent sa sth esubstrat efo rP L become visible. With silica the increase ofth e adsorbed amountP L levels off above 0.01M NaBr, while on PS andAg i the adsorption progressively increases at leastu p to 1.0M electrolyte.Thi ssug ­ gests that x is less inth ecas eo fsilica .Th ereaso nfo rthi sca n beth eabsenc eo fhydrophobi cinteraction sbetwee nP Lsegment san dth e surface,whil e inth ecas eo fAg ian dP Shydrophobi cinteraction sbe ­

tweenth e-(CH 2)4-NH_ sidechai nan dth esurfac ear epossible . In other words, the balancebetwee n the segment-surface attraction andth eelectrostati csegment-segmen trepulsio ni sturne di nfavou ro f weakening of the segment-surface interaction athig h ionic strength 77 in the case of silica. This shows that the electrostatic contribution to xs prr is relatively more important in the case of adsorption of PL on silica than in the case ofPL-adsorptio n onAg i andPS . The electrolyte stability of Agi and PS sols in the presence of adsorbed PL was also investigated. When the adsorbed amount at the coagulation concentration is justbelo w theplatea u value the sols are only charge stabilized, independent of the degree ofpolymerizatio n of the PL. Inth e case of large excess PL,wher e the plateau value at the coagulation concentration is largely reached, the sols with PL (DP 192) and PL (DP 1683) are more stable than solswit h bare particles. This together with the fact that at high ionic strength (I > 0.1 M) the adsorbed amount increases with increasing DP shows,tha t under these conditions the adsorbate layer is less flat and thatmor e pronounced tails and loops mustb e present than at low ionic strength or adsorp­ tion values justbelo w theplatea uvalue .

4.5 REFERENCES

Abendroth, R.P. (1970). J. Colloid Interface Sei. 34, 591-596. Applequist, J. and Doty, P. (1962), in 'Polyaminoacids, Polypeptides, and Proteins', Stahmann, M.A. ed.,Univ . Wisconsin Press,Madison , p. 161-177. Ardizzone, S., Formaro, L. and Lyklema, J. (1982). J. Electroanal. Chem. 133, 147-156. Bleier, M. and Goddard, E.D. (1980). Colloids and Surfaces, 1, 407- 423. Bonekamp, B.C., Schee,H.A . van der and Lyklema, J. (1983). Croat. Chem. Acta 56, 695-704. Brant, D.A. and Flory, P.J. (1965). J. Amer. Chem. Soc. 87, 2788-2791. Cafe, M.C. and Robb, I.D. (1982). J. Colloid Interface Sei., 86, 411- 421. Conio, G., Patrone, E.,Rialdi ,G . and Ciferri,A . (1974). Macromole- cules 7,654-659 . Cohen Stuart,M.A . (1980). Doctoral thesisAgricultura l UniversityWage ­ ningen, TheNetherlands . Cohen Stuart, M.A., Scheutjens, J.M.H.M. and Fleer, G.J. (1980). J. Polym. Sei., Polym. Phys. Ed. 18, 559. Corry, W.D. and Seaman, G.V.F. (1978). J. Colloid Interface Sei. 63, 136-150. Corry, W.D. (1978). J. Colloid Interface Sei. 63, 151-160. 78

Eggeft; A.R: (1976): Ph.D. thesis Lawrence University, Appletort Wisconsin. •"• '•' -.•:•:•..••.- Furusawa,K. ,Norde ,W . and Lyklema,J .(1972) . KolloidZ;Z .Polynt . 250, 908-909. Flory;P.J . (1969). 'Statisticalmechanic só fchai nmolecules 'inter ­ sciencePublishers ,Wile yan dSons ,Ne wYork . Greene,B.W . (1971).J .Colloi d InterfaceSei . 37, 144-153. Gregory,J . (1973).J .Colloi d InterfaceSei . 42, 448-456. Hesselink, F.Th. (1983) in 'Adsorptionfro m solution atth esolid - liquid Interface', Parfitt, G.D., Rochester, C.H., eds., Acad. PressLondon . Helene,C .an dMaurizot ,J.C . (1981).CR CCrit .Rev .Bioch. ,213-258 . Ho, C.H. andHoward ,G.J . (1982)i n 'Theeffec to fpolymer son.dis ­ persionproperties' ,Tadros ,Th.F .ed. ,Acad .Press ,London-Ne wYork . Horn,D . (1978),Progr .Colloi dPolym .Sei . 65, 251-264. Horn,D . andMelzer ,J . (1978). Fibre-Water Interaction Pap.Makin g Trans.Symp .ser .i ,p .135-150 . Hoven,Th.J.J .va nde n (1984).Doctora lthesi sAgricultura lUniversit y Wageningen,Th eNetherlands .I npress . James,R.D .an dParks ,G.A . (1982)i n 'Surface and ColloidScience ' 12, 119-216.Matijevic,E .ed. ,Plenu mPres sNe wYork-London . Jednacak,J. , Pravdic,V .an dHal1er ,W . (1974).J .Colloi dInterfac e Sei. 49, 16-23. Koopal,L.K . (1978).Doctora lthesi sAgricultura lUniversit yWageningen , Commun.Agric .Univ .Wageningen ,78-12 . Kasper,R.D . (1971).Ph.D .thesi sCaliforni a Instituteo fTechnology , Pasadena,California . Lindquist, M.G. (1975). Ph.D. thesis Lawrence University Appleton Wisconsin. Lindquist,M.G . and Stratton,R.A . (1976). J.Colloi d InterfaceSei . 55,45-59 . Marra, J., Schee,H.A .va nder ,Fleer ,G.J .an dLyklema ,J . (1983)i n 'Adsorption from solution'. Ottewill, R.H., Rochester,C.H .an d Smith,A.L. ,Eds. ,Acad .Press ,245-258 . Nyilas,E. ,Chiu ,T-H . and Lederman,D.M . (1976)i n 'Recentadvance s inColloi dan d InterfaceScience' .Kerker ,M. ,Acad .Pres sNe wYork . Norde,W . (1976).Doctora lthesi sWageningen ,Commun .Agric .Univ .Wage ­ ningen,76-6 . Norde,W .an dLyklema ,J . (1978).J .Colloi d InterfaceSei . 66, 257-265. 79

Put, A.G.va nde r (1980).Doctora lthesi sAgricultura lUniversit yWage ­ ningen,Th eNetherlands . Rawls,H.R. , Bartels,T . andArends ,J . (1982).J .Colloi dInterfac e Sei., 87, 339-345. Schee, H.A. vande r (1984). Doctoral thesisAgricultura l University Wageningen,Th eNetherlands . Schee,H.A .va nder ,an dLyklema ,J . (1982)i n 'Theeffec to fpolymer s ondispersio nproperties' ,Tadros ,Th.F .ed. ,Acad .Pres sLondon - NewYork . Terayama,H . (1952).J .Polym .Sei . 8, 243-253. Wada, A. (1961) in 'Polyaminoacids, Polypeptides and Proteins', Stahmann,M.A. ,ed. ,Univ .Wisconsi nPress ,Madison .131-146 . Williams, P.A., Harrop,R. ,Philips ,CO. , Pass,G . and Robb, I.D. (1982),J .Chem .Soc , FaradayTrans .1 , 78, 1733-1740. 81

5 INTERACTIONSBETWEE NNEGATIVEL YCHARGE DCOLLOIDA LPARTICLE SAN D POLYCATIONS

5.1 INTRODUCTION

Interactionsbetwee noppositel ycharge d (biological)macromolecule s play definite roles inbiologica l and technologicalsystems ,fo rin ­ stancei nth econtac tbetwee nconnectiv etissue s (Gelman et al., 1973). Also inman ybiochemical ,biomedica l and technologicalapplications , complexes between oppositely charged macromolecules are ofgrea tim ­ portance.A biochemica l application is,adsorptio n chromatographyo f biopolymers. The interaction ofcharge d salivary proteins withhar d tooth tissue (Juriaanse, 1980) andth ebindin go fhiston eprotein st o DNA (Record et al., 1978) areothe rexamples .Flocculatio no factiv e sludge with oppositely charged polyelectrolyte iso f importance in waterpurificatio n (Daniels, 1980). Also theus eo fpolyelectrolyte - complexmateria lfo rultrafiltratio nan ddialysi smembrane sfo rmedica l purposesca nb ementione dher e (Philipp, 1982). Negativelycharge dsilic aan dpolystyren eparticle sca nb eregarde d as rigid sphericalpolyelectrolytes .Thi si sbecaus eo fth eexistenc e of fixed discrete charged groups on theparticl e surfaces whichar e relativelyclos etogethe r (seefo rexampl e Rice and Nagasawa (1961) or Tanford (1965)). Inthi schapte r Iwil lconside rth eadsorptio no fth eflexibl epoly ­ electrolyte poly-L-lysine.HBro npolystyren e (PS)o rsilic aparticle s asa specia l caseo fth eformatio no fa polyelectrolyt ecomple x(i.e . polycation-polyanion complex), rather than asa specia l case ofun ­ charged polymer adsorption, asi susuall ydon ei nth epolyelectrolyt e adsorptionfield . Thepolyelectrolyt e complex approacho fpolyelectrolyt e adsorption wasals osuggeste db y Horn (1978) and Philipp (1982). The results obtained herewil lb ecompare dwit hth epropertie so fth e more 'regular'polycation-polyanio n complexes like,fo r instanceth e polylysine-polyglutamic complex (Domard and Rinaudo, 1981). Itmus tb e kepti nmind ,however ,tha tth echarge dmacromolecul echarge dparticl e systems considered here are in factheterogeneou s systems,whil eth e polycation-polyanion complexes, are homogeneous one phase systems, atleas ta tlo wconcentrations ,i.e .belo wth esolubilit ylimi to fth e complex. 82

5.2 EXPERIMENTAL

5.2 . 1 Materials

Thematerial s used inthi s study: polyvinylsulphate-potassiumsalt (ICN inc.); polylysine.HBr (Sigma), poly-L-histidine (M = 15.000; Sigma);P SLate xan dsilic a (AEROSILOX5 0Degussa )hav ebee ndescribe d in sections 3.1-3.3 and4.2.1 .Th e concentration of aqueousPL.HB r solutions calculated on aweigh tbasi swa si ngoo dagreemen twit hth e values found from aconductometri c titrationwit hcalibrate dAgNC Uo r hydrogenpolystyren esulphat e (HPSS)solutions . Thepreparatio n ofHPS S fromNaPS S (Waters ass.)wa s doneb ydi ­ alysisagains texces sHC lsolution san ddem iwate ri nth egive norder . ThePSS-residua lconcentratio nwa sthe ndetermine db ya conductometri c protontitration . Conductivity water and analyticalgrad echemical swer euse di nal l experiments.

5.2.2 Potentiometrie and conductometric titrations

Allconductometri c andPotentiometri emeasurement swer eperforme di n awell-close d doublewalle dvessel ,thermostatte d at293.1 5± 0.0 5K , under aCO,-fre ewate rvapou r saturated N,atmosphere .Proto ntitra ­ tions for the surface charge determination ofP S (latexM )hav ebee n describedi nsectio n3.2.2 . Protontitration s of silica inth epresenc eo rabsenc eo fPL.HB ran d the construction ofcharge-p Hcurve sfro mth eobtaine ddat ahav ebee n described insectio n3.3. 3 andsectio n4.2.4 . Conductometric titrations of 20.0c m PS latex (1%(w/v) )o rAEROSI L 0X50 (1.25% (w/v))wit h poly-L-lysine.HBr (0.02rM )wer eperforme d witha Retc hconductivit ymete roperatin ga t4 KHz . Conductivity cellswit h cell constants of 10.4m " or 71.9m ~ were used.Addition s ofPL.HB rwer e donewit h anAgl amicrosyringe .Con ­ ductometric titrations at highp Hvalue swer eperforme d with latex andPL.HB r solutions thatbefor e the titrationswer ebrough t toth e samep H under aN „ atmosphere with1. 0M NaOH .Th eAgl amicrosyring e was then filled withPL.HB r (highpH )withou tallowin gcontac to fth e PL.HBrsolutio nt oth eair . 83

5.2. 3 Adsorption measurements

Adsorptionisotherm so fPL.HB ro nth esubstrate sP S (latex)o rsili ­ cawer edetermine db ydepletio na sha sbee ndescribe d insectio n4.2.3 . The concentrations ofpositivel ycharge dpolylysine.HB r afteradsorp ­ tion were determined by titrationwit h thepotassiu m salto fpoly - vinylsulphate using toluidine blue as the indicator (Terayama, 1952; Horn, 1978). Seeals osectio n4.2. 2 and4.2.3 .

5.2.4 Stability measurements

The stability of silicasol sa sa functio no fth edegre eo fPL.HB r 3 coverageo fthe'particle swa sdetermine d asfollows :I n1 0c m sovirel 3 tubeswit hscre wca p (Teflonseal )4. 0 cm silicaso l (1.25%(w/v) )wa s 3 added.The n4. 0c m PL.HBrwit hth edesire dconcentratio nwa sadde db y means of apipetma ndispensin gpipet ,s otha trapi dmixin gdu et oth e PL.HBrsolutio nje twa sachieved .I nthi swa ya serie swit hincreasin g totalPL.HB rconcentratio nan dconstan tdesire dmicr oelectrolyt econ ­ centrationwa sprepared .Th etube swer erotate den dove ren dfo r5 hour s (at295. 6± 0. 3 K)t oobtai nadsorptio nequilibrium .The nth etube swer e incubated atthi s temperaturewithou tagitatio nfo ranothe r1 8hours . After thistim e theO D (550nm )o fth eto ptw oc m ofth esupernatan 3 t was measured. The results were plotted asth eODccn., ™ againstth e totalpolylysin e concentration.Als o the amountP L adsorbed ofeac h samplean dth ep Hwa smeasure dafte rth eO Dmeasurements .

5.2. 5 Microcalorimetry

Some preliminary measurements of the heat ofadsorptio n inth e PL.HBr-PS latex system wereperforme d ina LK Btwi nmicrocalorimeter . Theprocedur e followed wasth esam ea stha tdescribe db yNord e(1976 , 1978).Th ePL.HBr- Lan dlate xwa sdialyze dagains tth esam eelectrolyt e solution before themeasurements .Du e toth e twinprincipl e ofth e calorimeterth ehea to fdilutio no fth ePL.HB rsolutio nwa sautomatic ­ allyaccounted .Th ehea to fdilutio no fth elate xwa snegligibl ysmall .

5.2.6 UV and CD spectroscopy

Some attemptswer eundertake nt omeasur e theU Van dC Dspectr ao f poly-L-histidine, adsorbed on silica. Instead ofPL ,PHi swa schose n 84 because thispolyaminoaci d showsa conformationa l transitioni n.solu ­ tion between pH4 and 6. (Beychok, 1967). UV (difference)spectr a were recorded on aBeekma nmode l 3600U Vspectrophotometer .Circula r Dichroism spectrawer e recorded ata Joua nRousse ldichrograp hunde r

N2 flush. Matched quartz cuvettes with anoptica lpathwa yo f10. 0m mwer euse d inmos to fth eexperiments .

5.3 RESULTSAN DDISCUSSIO N

5.3.1 Adsorbed amount PL.HBr from depletion measurements

Oneo fth eexperimenta ladvantage so fth epolylysine/charge dparticl e systemsis ,tha tth ecomple x (i.e.adsorbe dPL.HBr )i nequilibriu mwit h PL.HBri nsolution ,ca neasil yb eseparate db ycentrifugation .Th ecom ­ positiono fth ecomple xa sa functio no fth eequilibriu m concentration PL.HBr (i.e.th e adsorption isotherm)ca nthe nb eobtaine db ydeter ­ mining the equilibrium concentration PL.HBr inth eclea rsupernatan t andcalculatio no fth edeplete damoun to fPL.HBr . For theheterogeneou s systems studied here iti smos tconvenien tt o express the composition ofth e complex inpmo l lysine -NH_ charges adsorbed per m particl2 e geometrical surface area,instea d ofth e ratio -NH,an d -OSOZo r Si-0~charge s inth e complex as isusuall y donefo rpolycation-polyanio ncomplexes .

1.2 PS pH= 5 I* 8-

6

4-

2j*-a silica pH=4.1 A .6 .8 1.0 1.2 U

c_ /molrm~-

Fig.5. 1 Adsorption of PL.HBr (DP 192)o nP S (latexM )an d silica (AER0SIL0X50 ) from conductivity water (noadde d electrolyte) 293K

o-o PL.HBr/PS a = -0.54Mmo l0S0 o/m 0 3- 2 A-A PL.HBr/AEROSIL a = 0.04 (JmolSi- 0/ m * inth eabsenc eo fPL.HBr . 85

Infig .5. 1 adsorption isotherms ofPL.HBr- LD P19 2 onP S (latex M_)an dAEROSI L0X5 0pyrogeni csilic aar eplotted .Th eadsorptio nha s ahig haffinit ycharacter ,du et oth erelativel yhig hD Po fth ePL.HBr . Sou p toth eplatea uvalu evirtuall yal ladde dpolylysin ei sboun dt o theP So rsilic aparticles .Increasin gth eP Lconcentratio nafte rreach ­ ing theplatea u valuedoe sno tchang eth ecompositio no fth ecomplex . At thebeginnin g of theplatea u thePL.HBr/P S complex ispositivel y chargedbecaus eth erati op = [NH,]/[OSOl]i sthe n2.1 ,wher e[NH, ] max Ó j , -j and [OSOl]stan d for thetota l concentration NH,an d 0S0~group si n thesyste mrespectively .Belo wo ra tr " .[NH, ]an d [OSOl]correspon d , max o j also to thenumbe ro fNH -an d0S0 ~group spe runi tare arespectively . For latexM .a rati o p of2. 8wa sfound .Th eadsorptio no fPL.HB r 4 'max * onP S is alsosuperequivalen t( p >1)i nth eplatea uan dth esam ei s thecas efo rth ePL.HBr-silic asystem . A similarbehaviou r wasfoun db yPhilip p forth ecomple xformatio n between a strongly branched polyethyleneimine with anioniccarboxy - methylcellulose and ligninsulphate (Philipp, 1982 p.5). Thephysica l reasonfo rth eformatio no fno nstoichrometri ccomplexe si smainl ytha t iti sstericall yno tpossibl etha tever ypolycatio ncharg eneutralize s locallya polyanio ncharg eo rvis aversa .Whe nth epolycatio nan dpoly - anionar ebot hlinea rflexibl epolyelectrolyt emolecule san dothe rat ­ tractive forces are ofmino r importance,n osterica lconstraint sfo r 1:1 complex formation arepresen t and 1:1 stoichiometry isobserve d indeed.I ngenera lth eelectrokineti ccharg eo fa polyelectrolyt ecom ­ plexwil l be lowertha nth echarg ecalculate d fromth ecompositio no f thecomple xdu et oco-adsorptio no fmicro-ions . Asha sbee nshow ni nth epreviou schapte rther ei sn omolecula rmas s dependence ofth eplatea u adsorption atlo wioni cstrengt hvalues .A largepar to fth ePL.HB rresidue si sthe ni nth eclos evicinit yo fth e particlesurface .O fcours eth enumbe ro fP Lmolecule sboun dpe rP So r silicaparticl e is stronglydependen to nth edegre eo fpolymerizatio n andth edimension so fth ecolloida lparticles . Theinfluenc eo fparticl ecurvatur e (i.e.size )o nth eamoun to fpoly ­ cation adsorbed peruni t area isonl ymino r aswa s shownb y Eggert (1976). Thiswa s concluded formadsorptio nmeasurement so fpol y(1,2 - dimethyl-5-vinylpyridinium)bromid e onnegativel ycharge dpolystyren e particleso fvariou ssize s (100-1100nm) . Animportan tdifferenc ebetwee nth esilic aan dpolystyren esubstra ­ tesdiscusse d inchapte r4 , is the ionic strength dependence ofth e 86 amount ofPL.HB r adsorbed. Inth e PL.HBr/PS system the amount adsorbed is increasing up to at least 1.0 M NaBr, showing the importance of hydrophobic interactions in this case. However at constant pH the amount adsorbed on silica is decreasing with cVI„ above 0.01 MNaBr . NaBr The non-coulombic attraction energy per PL segment (probably H-bonds) is only weak in thiscase . 5.3.2 The PS latex - PL.HBr system

Conductometric studies

Fig. 5.2 shows the variation of specific conductivity when PL.HBr-L is progressively added to PS latex M. (4.2 umol (OSO~...H+).kg" )i n the presence of H counterions only. Also the addition ofPL.HB r to -3 positively charged latex (10kg. m )i s shown.Th e curves for the

pH -5.0

,„- 4. 8

4.6

4.4

4.2

20 3 Cp./motr m P[RNH3/ROSO3 ]

Fig. 5.2 Variation ofth econductivit y andp Hwhe n a solution ofPL.HBr- L (pH=6; a=0) is added to diluted latex M, (4.2mmo l -OSO'.H /kg) or latex P(+) (M0 kg.m"3) T= 293.15 K

1. PL.HBr-L (DP1683)/late x P(+) ;K v sc pL; 2.PL.HBr- L (DP192)/late x M4>

K vs cpL; 3.PL.HBr- L (DP1683)/late x M^, Kv sc ;4 .PL.HBr- L (DP192) /

latex M4, pHv sc pL; 5.PL.HBr- L (DPl683)/late x M4> pHv sc pL- 87 negatively charged latex showa brea ki nslope ,whil e forth epositi ­ velycharge dlate xa completel ystraigh tlin ei sobserved .Th epositio n of thebrea k is,a s expected forfla tadsorption ,independen to fth e molecular mass ofth e PL.HBr used.Becaus e thebreakpoin t liesa ta total PL.HBr concentration where the plateau adsorption (r_ „= ^j niciA 1.15 |jmol.m~) i sno tye treached , allPL.HB radde du pt othi spoin t isboun d toth eP Sparticles .A tth ebreakpoin ti nth econductometri c titration curve the ratio p, between the totalnumbe r ofR-lffl Uan d R-OSOlgroup spresen tgive sth ecompositio no fth ecomplex .It svalu e is2.0 ,thu s also thebreakpoin t inth econductometri c titrationre ­ flects theno n stoichrometric superequivalentbehaviou r of thecom ­ plex formation. The straight line observed withpositivel y charged latex shows,a sexpected , the absence ofa comple xi nthi scase ,no r couldan yadsorptio nb edetecte danalyticall yunde rthes econditions . As opposed toth e complex formation described above,PL.HB ran ddis ­ solvedpolystyren e sulphate (PSS)for ma stoichrometri c 1:1 complexa s is shown in fig.5.3 .Her e a1: 1 complex canb eforme dbecaus ebot h polyelectrolytes involved are flexible and linear as alreadysai d before. Another difference between thePL.HBr/H.PS S andPL.HBr/P S (latex) systemsi stha tth edistanc ebetwee nth e-OSO ~group so nth ePSS~chai n + andbetee nth e-NH „charge so nPL.HB r (a=o)ar eno tver yapart ,0.2 5ru n

i ' i ' i ' r 4.2 E PH i/i E ,60°°" s 4.1 X 7 " \ y 4.0

, < x x x x *»*y «xxxx* " - " " - 3.9

.4 .8 1.2 1.6 P[RNH 3/ROSO3 ]

Fig. 5.3 Variation ofth econductivit y andp Hwhe n a solution ofPL.HBr- L (DP192) ; pH~6 is added toa HPSS solution (0.1mo l .m"3; M(PSSH) = 88000) Kv sp ;x x pHv sp ; T= 293.15K . 88 and 0.36n m respectively,wherea sth emea ndistanc ebetwee nth e TOSO- groups on theP Sparticle si s2 nm .Whe nth epolyanio nan dpolycatio n arebot hlinea ran dentirel y flexiblei ti sno tnecessar y forth efor ­ mation of a 1:1 complextha tth edistanc ebetwee nth echarge so neac h chain is aboutequal .Th e structure ofth ecomple x formedi showeve r dependent onthes e distances.Whe n the distances onbot h chainsar e about equal aladder-lik e structure canb e formed (Tsuchida et al., 1974; Philipp, 1982), while ifthes e distances areno tequa la mor e random structure is likely (Philipp, 1982). Becauseth e-OSO ~group s onth e PS (latex)surfac e are ata n average distanceo f2 nm ,i ti s sterically impossible to accommodate one -NH„ ontoeac h-OSO lgroup , alsowhe nth e-OSO ~group sar eslightl ymovabl ebecaus ethe yar eboun d to chains protruding into the solution, socalle d hairs (seesec ­ tion3.2.3) . Inth eterminolog yuse db y Polderman (1975) thePL.HBr/PS S system is asymmetri c system while thePL.HBr/P S (latex)syste mi s asymmetric.

The slope of the KV Sc_ r curves,i.e . themola r conductivityA after the breakpointmus tb e identical totha to fdissolve dPL.HB r (when double layer effectsma yb e neglected). This is shownb yth e equality of the slope of curve 1afte r p, and the slopeo fbul k PL.HBr (curve3 )i n fig.5.2 .Befor eth ebreakpoin tth eslop ei smuc h higher.Thi si scause db yth ereleas eo fmicroions ,du et oth ebindin g of-NH 3 groupso flysin et oth esulphat egroup so nth eP Ssurface :

R-OSO~ ...H++ R-NH g ...Br" *•R-OSO ~ ...NH*-R+ H ++ Br " (1)

The sameproces s occursals oi nth ePL.HBr/PSS~ H system.Tha tH and Br isrelease d duet oth ecomple xformatio ni sals oshow nb yth ede ­ crease inp Hoccurring .Whe n all -OSOlgroup s areboun d to an-NH - groupn oH isrelease d anymore and thep H reachesa minimu mvalue .

Thisp Hminimu mcoincide swit hth ebreakpoin ti nth e KV SC_ T curves. A™ (293.15K )foun d from the KV SC curves,29. 5mS. m .eq . ,i s 2-1 about20-25 %lowe r than A^ (293.15K ;0. 1rnol.m" 3).Th elowe rmola r conductivity ofHB r canb e due toth e lower activityo fth eion si n the electrical double layer ofth eP Sparticle san dpolylysin e (Man­ ning, 1972). Whenhoweve r the amounto frelease dproton spe r kgP S particles atth e conductivity breakpoint iscalculate d fromth emea ­ suredp H difference neglecting activity coefficients andth esuspen ­ sioneffect ,a valu e of4.0 9 ±0. 3 pmolH +.kg~ isfound .Thi svalu e isclos et oth evalu eo fth esurfac econcentratio n-OSO Tfoun dfo rthi s 89 latex from conductometric protontitrations .S oth ebindin go fB r to thepeptid e group ofP L (Ciferri et al. , 1968) canals ocontribut et o thelowe rA„ B found. Another point is that no break isobserve d inth e conductivity curvesa tp=l .Thi sshow stha tth echarg estoichiometr yo fth ecomple x formation doesno tchang eu p to p, .A t p=lwhe nth eparticle s are overallneutra lth elate xi sflocculated ,becaus en ooveral lrepulsiv e interactionbetwee n theparticle s ispresen t anymore (Ho and Howard, 1982; Gregory, 1973; Kasper, 1971). As pointed out by Kasper (1971) and alsob y Gregory (1973) positively chargedan dnegativel ycharge d patches exist on theP Sparticle s surfacea tp=l .Th econductometri c titrationcurve sfoun dher esuppor tthi smodel .Visua l flocculationo f the latex-PL.HBr system during the titration experimentswa s always observedabov ep= lbu twel lbelo wth ebreakpoint . In homogeneous polycation-polyanion systems theentrop y increase due toth e release ofmicroion s isusuall yth edrivin gforc efo rth e complex formation (Philipp, 1982; Manning, 1978; Record, 1978). This isprobabl y also animportan t factor inth ePL.HBr/late xsyste muse d here. Preliminary microcalorimetric measurements performed by uso n the PL.HBr latex system atp H6 showe d aclearl y exotherme heato f adsorption.Thu sals oenthalpi cinteraction sfavou rth eadsorption . Ross and Shapiro (1974) found thebindin go fpolylysin et oDN At ob e almost athermal,s o inthi s case theio nreleas ei sth esol edrivin g force. Infig .5. 2on eca nse etha tth eamoun tPL.HB radsorbe di nth ebreak - _2 point ofth e conductometric titration curve r, is 0.84 jjmol.m is not identical with themaxima l adsorption (r „= 1.2 |jmol.m") bu t c max _ lower.Thi s shows thatafte rth ecomplet eneutralizatio no fth e-OSO g groups at T, ,th e adsorption still increases.Thi s canonl yb eth e casei fothe rtha nelectrostati cinteraction scontribut et oth einter ­ actionfre eenerg ybetwee na P Lsegmen tan dth eP Ssurface .A swa sals o concluded fromth esal tdependenc eo fth eadsorbe damount ,hydrophobi c interactions between the hydrophobic PS and the-(CH,)~ 4group ofa lysineresidu esid echai nar eprobabl yresponsibl e forthis . Direct estimation of the fraction PL charges not bound to an -OSO~ group

An interestingexperimen twa s thedirec tmeasuremen to fth efre e R-NH- lysine groups inadsorbe d poly-L-lysine.T othi sen ddilut eP S 90 latices (11.25kg.m -3)wer efirs ttitrate dwit hPL.HBr- L (DP1683),on e sample till justbelo w r. (c^ =0.080T 6mo l .m )an d on-e3til labov e _ DrT PL _ r r, but below r (cr=0.098 3mo l .m ).The n the latexwit had - DIT Hl3. X rL IT sorbed PL.HBrwa s titratedwit ha PVS- Ksolution .Blan ctitration so f water and aPL.HB rsolutio nwit hPVS. Kwer eperforme dt oserv ea sre ­ ferencetitrations . Infig .5. 4conductivit ycurve sfo rthes evariou stitration sar ecol ­ lected.Competitiv epolyelectrolyt e adsorptionplay sa rol ei nth eti ­ trations of adsorbed PLwit h PVS.K.Thi s explains the ratherbende d shape of the curves. The titrationcurve s of adsorbed PL.HBrwit h PVS.Ksho wtw obreaks .Th efirs treflect sth edisappearanc eo ffre e

3^2 1.6.

in E 1.4 *>& 3.0 y 3 1.2

Ü" 1.0

2.8, 0.8 x' y 2.7 |0.6 2.6v ^ I0.4 2.5 0.2

.04 08 .16 .20 .24

Fig.5. 4 Variationo fth e conductivitywhe na solutio no fpotassiu m polyvinylsulphate -3 (10.5mo l .m )i sadde dt o r 3 1.20. 0c m water initialp H= 5.8 ; 2.20.1 8cm 3 PL.HBr-L (DP 1683) c£ =0.080 6mo l .m"3 initialp H 5.02 3.20.1 8cm 3 latexM , (11.25kg. m )wit hadsorbe d PL-HBr-L (DP1683 ) c =0.080 6mo l .m initialp H4.24 . 3 r -3 4.20.2 2c m latexM , (11.25kg. m )wit h adsorbedPL.HBr- L 3 (DP 1683)cJ L= 0.098 3mol.m" initialp H4.47 . in3 an d4 PL.HBr isadsorbe dquantitatively . T= 293.1 5K Notice thedifferen t Kscales . 91

-NH~groups .Th eoccurrenc ean dpositio no fth esecon dbrea kshow stha t PL.HBri sdisplace d fromth e-OSO lgroup so fth epolystyren e athighe r PVS.K concentrations.Whe n also inth e adsorbedstat estoichiometri c charge complexesbetwee n thefre eR-NH -group so fPL.HB ran dsulphat e groupso fPVS. Kar efound ,th erati obetwee nth efirs tbrea kan dsecon d break inth e KV Sc p„s„ curvei sth efractio no ffre e-NH _charges . This results invalue s of 0.3 and 0.4 forthi srati ofo radsorptio n —2 valueso frespectivel y 0.72 and0.8 4|jmol. m .Th eincreas eo fth efrac ­ tionfre echarge swit hincreasin gamoun tP Ladsorbe d suggeststha tth e fraction of the totaladsorbe damoun tP Li nloop san dtail sincrease s with increasing surface coverage.Thi s trend is alsopredicte d from polyelectrolyte adsorptiontheor y (van der Schee, 1984). Iti sals opossibl et ocalculat eth efractio nfre e-NH _ chargesfro m theamoun tPL.HB radsorbe dan dth esurfac econcentratio n-OSO _groups . For the adsorption valuesmentione d above:0.7 2 and 0.84 pmol.m , -2 values forth e fraction free -NH- charges of 0.4 and0. 5 arefound . Thedifferenc ebetwee nthes evalue san dthos efoun dfro mth etitration s withPVS. Kar eprobabl yth econsequenc eo fth euncertaint y inth evalu e ofth e surface charge ofth eP Sparticles .Anothe r possibility isa deviation from the 1:1 complex formationbetwee nadsorbe dPL.HB ran d PVS.K, because of the rather flatconformatio n of the adsorbedP L molecules. A similar type of experiment asdescribe d abovewa s suggestedb y Eisenlower (1982) and performed by Horn (1978) with thepolyethyl - eneimine/polystyrene latex system.Howeve rHor nuse d ametachromati c cationic dye forth e optical detection ofth efre epolyanio nconcen ­ tration.A tth e adsorptionplatea u Horn founda valu eo f0. 4 forth e fractionfre echarge so fth eadsorbe dpolyethyleneimin e (PEI). Inthi s casen o displacement ofth epolycatio nb yPVS. Kcoul db edetected .I n bothadsorbe dPL.HB ran dPE Ith erathe rlo wfractio nfre echarge sfoun d is the consequence ofth e flatconformatio n of adsorbed polycation molecules. Infig .5. 5th ep Hvariation smeasure dupo nadditio no fPVS. Kt oP L insolutio n or adsorbed areplotte d forth esam etitration sa sgive n in fig.5.4 .A p Hincreas eu pt oth econductometri cbreakpoin ti sob ­ served whenPVS. K is added to aPL.HB r solution,whil e additiono f PVS.K to adsorbed PL.HBrresult si na p Hdecreas eove rth ewhol econ ­ centration range measured. The effect isto o large tob e accounted foronl yfro mchange si nth eproto nactivit ydurin gth etitratio nwit h PVS.K. 92

-i ' r- 48 6 6

pH pH 4.7 6.4

4.6 *-x^ 62 / " 4,5 6.0 In -o-o-o-^ 1 — \ 5.8 \ -i-o-0-0'0"0"0" 43 v 5.6 4.2 t\ /vA 5.4 52

0 0 40 5.0

.04 .08 12 .16 20 24 3 cpvSK/molr m'

Fig. 5.5 Variation of thep Hwhe na solutio n ofpotassiu mvinylsulphat e (10.5mo l -3 r m )i sadde d towate r (1),a PL.HB r solution (2)o rPL.HB radsorbe d onP S latex (3 and 4).Se e the legend of fig.5. 4 for further details. T= 293.1 5K . Notice thedifferen tp Hscales . Conductometric titrations at high pH

The complex formationbetwee nPL.HB ran dP Sparticle swa sals oin ­ vestigateda tp H10.2 .Th edegre eo fdissociatio no fth eP L-NH 3group s isthe n about0.6-0.7 . Infig .5. 6 theresult so fth ehig hp Htitra ­ tionsar eshown .Als oher ebreak si nth eK V Sc pT curvesar eobserved .

Theslop eo fth eK v sc pLcurve safte rth ebrea ki sagai nidentica lt o thato faqueou sPL.HB ra ta p Ho f10.2 .Th eamoun tPL.HB radsorbe di n thebreakpoin t is again independent ofth emolecula rmas so fth ePL , buthighe rtha ntha ta tlo wp Hvalue ,viz .1.3 3 (jmol.m and1.2 4(jmol . m-2 forPL.HB r (DP19 ;a=0.64 )an dPL.HB r (DP1683 ;a=0.70 )respectively . Whenthes eadsorptio nvalue sar emultiplie db yth edegre eo fdissocia ­ tiona ,th eadsorptio nvalue sr . atp H4. 2 areobtaine d inbot hcases . Thisshow stha tals oa thig hpH ,i.e .a tlowe rP Lchai ncharg edensity , 93

10.8 pH 10.7

10.6

10.5

10.4

10.3

10.2

10.1

Fig.5. 6 Variation ofth econductivit yan dp Hwhe na solutio no fPL.HBr- L (initial 3 1 PH10.2 )i sadde dt odilute dP Slate xM 4 (5.65kg.m" ;4. 2mmo l(-0S0~)-kg" ; initialp H10.2 )o rwate r (initialp H10. 1± 0.1 )T = 293.1 5K .

Additiono fPL.HB rt olate xM ,: 1 .an d5. :K v sc pT;2 .an d6. :p Hv sc p_ Additiono fPL.HB rt owater :3 .an d7. :K v sc _ 4.an d pHv sc "PL'^ '" r" '" PL Opensymbols :PL.HBr- L (DP19) ;Fille d symbols:PL.HBr- L (DP1683) . theno ncharg e stoichiometryo fth ecomplexatio ni sth esam ea si ti s ata= 0an dtha tal l-OSO Ö groupso nth eP Sparticle sar eboun dt oa n

-NH3 groupo fth epolycation .Becaus e atp H10. 2r . xshighe rtha n atp H4. 2th efractio no fP Lsegment si nloop san dtail si shighe r atp H10.2 .Th e interaction ofth epoly-L-lysin e withpolystyren e is analogous toth ecomple x formationbetwee npolymethacryli c acid and polyammonium polymers (charges inth echai nbackbone )investi ­ gatedb y Tsuchida et al. (1974). Theplatea u adsorptionvalu e found forth eadsorptio no fPL.HBr- L _2 (DP192 )fro mwate rp H10. 2o nP Si s4. 8pmol. m .S or ismuc h higher than r, athig hpH .No nelectrostati c contributions toth e adsorption energype rsegmen tar eno wrelativel ymor eimportan ttha n at lowpH .I nothe r words thelatera l repulsionbetwee nth e-NH _ groupsi nloop san dtail so fadsorbe dpolylysin emolecule si sstrongl y 94 reduceddu et oth elowe rchai ncharg edensit yan dconsequentl yof -les s importance (seeals osectio n6.5.3) .

5.3.3 The silica-polylysine system

Thesilica-polylysine.HB rsyste mi smor ecomplicate dtha nth elatex - PL.HB r system because the surface silanol groups on the silicapar ­ ticles have a weakly acidic character. The dissociation of these groups is,a swil l be shown later,strongl y influenced by thecom - plexationwit hpolylysine .Adsorptio n isotherms ofP Lo nsilic asho w the same general features asthos e onpolystyren e (seesectio n5.1) . However the adsorbed amount isno w alsostrongl ydependen to nth ep H inth eacidi cregion . In fig.5. 7 thevariatio n ofth e conductivity andp Hwhe na solu ­ tiono fpoly-L-lysine.HB r (DP192 )i sadde dt oa silic aso li splotte d against thetota lP Lconcentratio ni nth esystem . Curves 1an d 3 show the results for aninitia lp Ho f5.50 .Her eth e counterions ofth eAEROSI L areonl yH .Th e curves arever ysimila r to those found forP S latex as asubstrat e forPL .Th e slopeafte r thebrea k is again the same astha tfo rPL.HB ri nwater .Befor eth e breakth ehighe rslop ei scause db yth ereleas eo fH andBr~ion swhe n the complex is formedan dabou tidentica lt otha tfoun dfo rth ePL/P S latex system.Th e release of acid isals oshow nb yth edecreasin gp H with increasing c_.u p toth e conductometricbreakpoint ,afte rwhic h thep H remains constant.Durin g the additiono fPL.HB rth edegre eo f dissociationo fth esilano lgroup sincreases ,becaus eP Lac ta sspecifi ­ callyadsorbin gcounterions . At the breakpoint the adsorbed amount is suchtha tth emaxima l amounto f Si-0~group s areboun d to-NH 3 groupso flysine .Afte rth e break the adsorption still increases as iseviden t from depletion measurements,bu t thedegre e ofdissociatio n of silanol groupsdoe s not change anymore, because otherwise thep H after thebreakpoint s in fig.5. 7 would stillb echanging .Th eincreas ei nadsorptio nafte r theconductometri cbreakpoin ti spossibl ebecaus eth elikel yformatio n of hydrogen bonds between undissociated silanol groups andpeptid e unitso fth ePL .Als odipola rinteraction sma ypla ya role . Theformatio no fH-bond s andothe rinteraction sar eno timportan ti n theotherwis e comparable homogeneoussyste mpolyglutami cacid/poly-L - lysine investigated by Domard and Rinaudo (1980, 1981). Herealway s stoichiometriccomplexe sar efoun dsuc htha tal l-NH -group sar eboun d 95

• i • i i '

3

'E 3.0 -|7.5 in [p H E ^ -\s * 2.5 7.0

2.0 • Y / • • -6.5 1.5 6.0 • Â/ N / / 1.0 \//V. • 5.5 0.5 x ty* - -x-x-x.xJ<-xJ<-xi 5.0

1 . 1 . 1 . 1 . 1 . .04 .08 .12 .16 .20 -3 Cp./mol r. m

Fig.5. 7 Variation of the conductivity andp Hwhe na solutio n ofPL.HBr- L (DP 192) is added toa nAEROSI L0X5 0so l (12.65kg. m ).T = 293.1 5K . 1.K v sc PL initial pH 5.50, 2. Kv s c initial pH = 7.66;3 .p Hv sc pT initial

pH= 5.50 ;4 .p Hv sc pL initialp H= 7.66 . toa n -COO groups ofpolyglutami c acid.N o interaction takesplac e witha nundissociate dcarboxyli cgroup . At astartin gp Ho f7.6 6 (curves2 an d4 o ffig .5.7 )N a ionsar e thedominan tcounterion so fsilic abu tth esilic ai sstil lincompletel y charged.Th e conductivity titrationcurv eshow sno wtw obreaks .Afte r the second one the slope is the same astha tafte r thebrea k for curve1 .A tth esecon dbrea kth ebreakpoin tadsorptio ni sthu smaxima l againan d the compensationo fsurfac e=Si-0 ~group smaximal .Th emax ­ imalbreakpoin tadsorptio ni sagai nlowe rtha nr 3 max The slope of theconductivit ycurv e2 befor eth efirs tbrea ki slowe r thantha tafte rth ebreak ,becaus einitiall yN a andBr "rathe rtha nH and Br" areth e released microions.Th ep Hi sdecreasin gbecaus edu e toth ePL.HB rbindin gth edissociatio no fth esilano lgroup sincreases , i.e. the apparentp K of the silanol groups decreasesbecaus eo fth e strongpossitiv efiel do fth eP Lmolecules .Whil eth ep Hi sdecreasin g 96 allN a is displaced and furtherH isbecomin g tocompet ewit hN a anda nincreas ei nslop eo fth eK V SC_ T curvei sobserved .Thu safte r + - thefirs tbrea ki ncurv e2 mainl yH andB r ionsar erelease d]us ta s wasth ecas ea ta ninitia lp Ho f5.5 . Apoin to fdiscussio ni swhethe rth erelease dproton sar eoriginatin g fromth esilano lgroup so rth eNH _ groupso fPL .Th elatte ri sunlikel y because inth ePL.HBr/late x system no groupsar etitrate dbelo wp H7 also notwhe nP Li sadsorbe d (seechapte r 6). Thisi sbecaus eth ein ­ trinsic pK ofth eNH -group si sabou t10.8 .Th especifi cconductivit y atth e secondbrea k incurv e 2 (fig.5.7 ) and thebrea k incurv e1 (fig.3.9 ) are about equal.S o themaxima lamoun to frelease dmicro - ionsi sno tdependen to nth einitia lp Ho fth esilic asol .T ofin dou t whether thePL-silic a complex atth ebreakpoin tar echarg estoichio ­ metric ornot ,th e surface charge densityo fth esilic ai nth epres ­ ence of PL must be known. A conductometric proton titrationwit h NaOH (0.1M )fro mp H4. 9 to 8afte ra titratio no fa silic aso lwit h PL.HBryielde d a -Si-0~surfac econcentratio no f9.8 8 pmol.g- ,whil e atth e samep H the surface concentration inth e absence ofPL ,ob ­ tained by the samemethod ,i sonl y2 pmoLg " .Th erati op , (pH4.9 ) isthe n 0.6, i.e.smalle rtha n1 .Th ecomple xi sno ncharg estoichio ­ metricbu tno tye tsuperequivalen twit hrespec tt oth eadsorbe damoun t PL atr , ,essentiall y because the charge isno t the origino fth e adsorptionbu tth e cause.A tth emaxima ladsorptio na tp H4.9 ,deter ­ minedanalytically ,th evalu eo fp i s2.88 .The nclearl ya superequiv ­ alentcomple xi spresen t (seeals osectio n5.3.4) .Becaus eo fth ehig h silanolgrou pdensit yth epossibilit yt ofor ma 1: 1comple xi sgreate r thani nth ecas eo flatex .

5.3.4 Influence of the surface charge density and ionic strength on the polylysine-charged particle interaction

Asalread ydiscusse di nsectio n4.3.6 ,th eamoun to fP Ladsorbe do n polystyreneincrease swit hincreasin gsal tconcentration . Aconductometri c investigationo fthi ssal teffec ti slimite dt ofair ­ lylo w saltconcentration s because otherwise thevariation s incon ­ ductance due to the binding ofP L are outweighted by the swamping electrolyteadded . In fig.5. 8 the conductometric titration of latexM .wit hPL.HB r (pH= 6;a =o )i nth epresenc eo f1 0 MNaB ri sshown -3 .I ti sfoun d thatth ebreakpoin t ismuc hmor erounde dtha ni nth eabsenc eo fsalt . 97

Cp5uH.25kg.m-» E 137 Na*/H* counter ions t/i E pH 13.5

131 "-p-»"-,

12.9 /

y , i _L . I . l_ 3 04 .08 .12 .16 .20 cPL/mol,.m-

234 p[RNH*/ROSO' J

Fig.5. 8 Variationo fth econductivit y andpH ;whe n asolutio n ofPL.HBr- L (pH= 6 ; a =o )i sadde d todilute d latexM , (4.2|jmo l(-OSO'. H) kg " ,i nth epre ­ senceo f lO"M NaBr .T = 293.1 5K o o Kv s c ; x x pHv s c .

Extrapolation of the linear parts ofth e curveyielde d aslightl y higher (0.89umol. m )'breakpoint ' adsorptiontha ni nth eabsenc eo f lowmolecula rmas s electrolyte:r . = 0.84 pmol.m .Th e difference found isprobabl ywithi n experimental error. Michaels et al. (1965) found deviations from the 1:1 complexationbetwee npolyvinylbenzyl - trimethylammoniumchlorid ean dpolystyren e sulphonateonl yabov e0. 1M added NaCl. Inm yview ,abov e 0.1M electrolyte ,charg einteraction s arescreene d to such anexten d thathydrophobi cinteraction sca nbe ­ come dominant andgover nth e complex formation.Thi sresult sthe ni n acomple xwit ha charg estoichiometry ,whic hdiffer ssignificantl yfro m thata tlo wsal tconcentration .Whe nsuc hinteraction sar eabsen ta si s thecas ei nth epolylysin e-DN Acomplex ,dissociatio no fth ecomple xa t highersal tconcentratio noccur s (Manning, 1978). Athig hsal tconcentrtaion si.e .> 0. 1M als osterica l factorscon ­ tributet oth estoichiometr yo fth ecomple x formation (Michaels et al. , 1965). Duet oth estronge rcoilin go fth epolyion sa thig hsal tconcen ­ trationno t all ionicsite sar eaccessibl et osite so fth eoppositel y charged polyion.Th e stoichiometry ofth e complex formationdeviate s therefore from the lowo rn oadde delectrolyt esituatio n (Michaels et al., 1965). Thestronge rcoilin go fth epolyion sa thig hioni cstrengt h isespeciall y importantwhe nno n equilibrium states are involved in 98 thecomplexatio nreaction .Howeve r theoccurrenc e ofno nequilibriu m states isdifficul t totrac efo rhomogeneou sa swel la sheterogeneou s polycation-polyanionsystems . A clear difference between thePL/late x systemi nth epresenc eo f -3 . ... 10 MNaB ro ri nth eabsenc eo fi ti sth ep Hvariatio nwhe nPL.HB ri s _3 added.A t 10 MNaB rn o decrease inp H isobserve d on additiono f PL.HBr.Du e toth epresenc e ofNaB r the diluted latexi si nth eN a form.Bindin g ofPL.HB rcause sno wth ereleas eo fN a andBr "instea d ofH andBr" ,a proces swithou tan yp Heffec toccurring . In fig.5. 9 the conductometric breakpoint adsorption found fromth e variation in conductance, when poly-L-lysine.HBr (DP=192;pH=6 )i s added to silica solsa tvariou ssal tconcentrations ,i sshown .Th ep H atth ebreakpoint s isals oindicated .Jus ta si sfoun dfro mdepletio n measurement (see section4.3.6) , the amount adsorbed r. increases with increasing ionic strengthi nth estudie drange .I ncontras twit h thebehaviou r ofth ePL/late x system inth epresenc eo fNaBr ,th ep H isdecreasin g whenPL.HB ri sadde dt oth esilic asol si nth epresenc e ofNaBr .Thi s canb e explained by a further increaseo fth esilano l groupdissociatio nwhe nPL.HB rbind st oth esilic asurface .

E

Fig.5. 9 Adsorbed PL.HBr-L (DP 192)i nth e conductometric breakpoint asa functio n of the ionic strength (curve 1).Th ep Hvalue s inth econductometri cbreak ­ pointsar eals o indicated (curve 2). Substrate:silica . 99

log c Ig.m' a PL a

Fig. 5.10 Sol stability (upper curves) and adsorbed amount PL (lower curves) for the silica-PL.HBr system as a function of the total concentration PL.HBr (DP 192) on a log scale, o-o No added NaBr; A-A l.io"3 M NaBr; 5.10"3 M NaBr.

It is interesting to compare the conductometric results described above with the sol stability and simultaneously obtained adsorption curves for the silica/PL system (fig. 5.10). Note that in fig. 5.10 rm=-»- is plotted against the logarithm of the total concentration PL.HBr present. The stability of the silica sols as a function of the total concentration PL.HBr i.e. as a function of surface coverage, show the same picture as generally observed for the adsorption of polycations on negatively charged particles. See for example Ho and Howard (1982) Bleier and Goddard (1980) and Lindquist (1975, 1976). After floe- 100 culationo fth esilic ab ylo wadsorbe damount sPL ,restabilizatio noc ­ curs athighe r adsorptionvalue s due tocharg e reversal andperhap s also the occurrence of steric repulsion.Th ePL/silic a system shows abroadenin g ofth einstabilit ydomai nrang ewhe nth esal tconcentra ­ tioni sincreased .Th eprincipa lmechanis m forflocculatio ni sthough t tob e chargeneutralizatio ndu et oth eP Ladsorption .Tota lneutrali ­ zation ishoweve r notrequire d formaxima lsedimentation .Thi si son e ofth eobservation s leadingt oth epictur eo fth eexistenc eo felectro ­ staticpatche so nth eparticl esurfac e (Bleier and Goddard, 1980). Be­ causeo fth elon gincubatio ntime so fth eflocculatio nexperiments ,th e optimal flocculation concentrationwa sonl yobservabl ei nth eabsenc e of addedNaBr .Whe n iti s assumedtha tth eisoelectri cconcentratio n T corresponds to thec pr valuewher e the steep rise ofth e stability curves starts, the corresponding adsorption values (V• ) coincide ratherwel lwit hr . ,th ebreakpoin tadsorptio n (seeTabl e5.1) .

Table5. 1Adsorptio nvalue s ofpoly-L-lysine.HB r (DP=190)a tAEROSI L 0X50a tp H4.6 0fo rvariou selectrolyt econcentrations .

c .Mmol. .umol umol. .umol, NaBr br l K CT 2 ' r • max 2 ' o < 2 > Pbr M m ISO m2 m m -4 < 10 0.11 0.15 ±0.03 0.2 ± 0.05 0.20* 0.6 1.10"3 0.27 0.25 ±0.04 0.6 ± 0.1 0.29 0.9 -3 + 5.10 0.40 0.45 ± 0.06 0.6 ± 0.1 0.22 1.8 * obtained from aconductometri c proton titration ofsilic aafte ra titrationwit hPL.HBr . + Interpolation ofth ea valueso fsilic ai nth epresenc eo fexces s -'i -7 PLa tp H4.6 0betwee n1 0 and1 0 MNaB rrespectivel y

Thevalue s ofI~ . mayb e considerably inerro rbecaus e of theun ­ certainty inth e iso-electricP L concentration,howeve r thecorres ­ pondencebetwee nr , andr . suggests that inth e silica/PLsyste m the conductometric breakpoint occursa tabou tisoelectri cadsorption . Whenn o specific adsorption ofmicroion s takesplac e avalu eo f1. 0 would have been found forp . atth e ionic strengths investigated. However this seemscontradictor y with the increasefoun di np , with increasing ionic strength,whe n p, iscalculate d fromr , anda . More experiments areneede d tointerpre tthes etrend sfurther .Espe ­ cially micro electrophoresis could give additional informationabou t 101

thesig nan dmagnitud eo fth echarg eo fP Lcoate dparticle sa tr ..

Infig .5.1 1 themaximu m adsorptionobtaine d fromdepletio nmeasure ­ ments inth e absence ofNaB r and thebreakpoin t adsorptionr . are plotted against the equilibriumpH .Th eincreas eo fr iscause db y theincreas ei n a wito hpH .Her eals oth eeffec to fPL.HB ro nth esur - facecharg ebecome svisible ,becaus ei nth eabsenc eo fP Lth eincreas e of the surface chargewit h increasing pHi shardl ymeasurabl ei nth e absenceo fsalt . Betweenp H4. 2 and4. 8 r ishighe rtha nr , .A sstate dbefor ehy - max br drogenbondin gbetwee nP Lpeptid eunit san dsilano lgroup si sprobabl y responsible for this. Iti sremarkabl e thatr , remainsconstan tbe ­ tweenp H4. 2 andp H4.8 ,wherea s a increaseswit hincreasin gpH .Thi s showstha tth echarg estoichiometr ya tr , changeswit hincreasin gpH , towards lower p, values.Thi san dth eobservatio ntha tp . atp H4. 6 issmalle rtha n1. 0 suggeststha tth eamoun tfo rNH ,group sunaccessibl e Si-0~group s increases withincreasin gpH .Th egellaye ro nth esilic a surface,thoug h small (see Yates and Healy, 1976) mustb eresponsibl e forthis .

-I ' 1 ' 1 ' T"

4.4 4.8 5.2 56 pH

Fig.5.1 1 Comparisono fth eamoun tPL.HBr- L adsorbed inth ebreakpoin t (curve 1)an d the analytical adsorption (curve2 )a s afunctio no fth eequilibriu mpH . Noadde dNaB rpresent . 102

5. 3.5 Proton titrations of silica covered with PL

Infig .5.1 2 twoset so fcharge-p Hcurve so fAEROSI L0X5 0ar eshown . Seton ecomprise sth etitratio ncurve so fbar esilic aa tvariou selec ­ trolyte concentrations. Set2 represent s those inth epresenc eo fs o muchexces sPL.HBr ,a st oensur eplatea uadsorptio na teac hpH . The p.z.c. ofbar e silica istake n atp H3.0 , following Sonntag (1976, 1980) and Abendroth (1970). Because of the flat charge-pH curves found inth e region around thep.z.c , theexac tpositio no f thispoin tdoe s notgreatl y influence thepositio n of thecharge-p H curves. Inth epresenc e ofPL.HB r also avalu e of3. 0wa stake nfo r thep.z.c , although thep.z.c .i sprobabl yshifte dt olowe rpH ,i.e . ina mor e positive direction. Such ashif ti sexpecte dtheoreticall y and observed experimentally inth epolylysine-Ag lsyste minvestigate d by Van der Schee (1982). The flattitratio n curves ofsilic amak ea detection of ap.z.c .shif ti nth esilica-P Lsyste mimpossible .I ti s alsobecaus e of thisflatnes stha tth eresult sar einsensitiv et oth e actualchoice . These to fcurve sfoun dfo rbar esilic aagre ewel lwit hthos efoun d by Sonntag (1976, 1980) forAEROSI L0X5 0i nKC lsolutions .Quantitativ e differencesma yb e attributed toth e different silicabatche sused . Alsoth ekin do fmicr o1: 1electroly tuse dgive sris et osmal ldiffer ­ ences (Abendroth, 1970). Thebehaviou rfoun di stypica lfo roxides .Th e dissociationo fth esilano lgroup sincrease swit hincreasin gp Hresul ­ ting inhighe r surfacecharg edensitie sa thighe rpH .Increas eo fth e electrolyte concentration atconstan tp H results alsoi na nincreas e ofth e surface charge.Thi s iscause db y the screeningo fth eSi- 0 charges,whic h againresult si na nincreas eo fth esilano lgrou pdis ­ sociation. Inothe rwords ,th e apparentp Ko fth esilano lgroup sde ­ creaseswhe nth esal tconcentratio ni sincreased . Theproto n titration curves ofAEROSI LOX5 0chang edrasticall yi n thepresenc e ofPL.HBr .Thi swa s alreadyexpecte dfro mth econducto - metricresults .A sexplaine dbefore ,u pt oa p Ho f ^8, the-NH _group s ofPL.HB rar eno ttitrate dbecaus eo fth ehig hp K valueo fthes egroups : 10.8. Inothe rword sunde rth econdition schosen ,P Lact sa sa strong , multivalent cation.Th e titrationcurve so fsilic ai nth epresenc eo f PL.HBr show twomai neffect sbot hcause db yth epresenc eo fth epoly - cation. Inth e firstplac e the surface charge,i.e . dissociationo f silanol groups, increases drastically when PL.HBr is added toth e silica sol.Qualitativel y thisca nb eexplaine d as follows.Du et o 103

Fig.5.1 2 Charge-pH curves forAEROSI L0X5 0silica . 1.N oadde d PL.HBr-L;2 .Exces sPL.HBr- L (DP190) . A (silica)= 6 0n^.g" 1;Electrolyte :NaBr ;T = 293.1 5K . sp the adsorption ofPL.HB rth esilic aparticle sobtai na positiv eenve ­ lope facilitating the dissociationo fsilano lgroups .Th econsequenc e is thehighe r surface charge density observed. Inth e secondplace , thesal tconcentratio neffec ti sreverse da scompare dwit hbar esilica . This reversal was also found from thep H changes observed,whe na silicaso lwit hadsorbe dPL.HB rwa stitrate dwit helectrolyt esolutio n atvariou s initial pHvalues .Th eeffec to fth ereversa li scause db y the competitionbetwee nN a ions and -NH_ lysine groups forsurfac e sites,whic hgive sris et oa decreas ei nadsorptio no fPL.HB r (constant pH)a telectrolyt e concentrations above 0.01M asdiscusse d insec ­ tion4.3.6 . The decrease inscreenin gpowe ro fP Lresult sagai ni na n 104

increase ofth e apparentp Kvalu e of the silanol groups,an dthu sa decreased surface charge densitywit h increasing saltconcentration . The reversed salteffec t isno tdu e toth e suspensioneffect ,a si n thepresenc e ofpositivel y charged particles themeasure d pHvalue s are overestimated at low saltconcentration s due to the suspension effect.A t0. 1M NaB rth esuspensio neffec ti svanishin glo w (seeals o section6.6.2) . Theobserve dtrend sar eals oclearl yvisualize db yplottin gth eap ­ parentp K of thesilano lgroup sagains tth edegre eo fdissociatio no f these groups (fig.5.13) . Amaxima l silanol groupsurfac edensit yo f 4.4 nm is taken (Sonntag, 1980). Following James & Parks (1982), c instead ofa , (10a+ V s)i splotte di norde rt oenabl eextrapolatio n ofth eplot s tozer oa an dzer oioni cstrength .I ndoin gs oi ti sim ­ plicitly assumed thatal l silanol groups have the same intrinsicp K value, otherwise pK _ has no clearmeaning .Whe nth eplot so fbar e silica (fig.5.13a )an d those of silica inth epresenc e ofPL.HB r (fig.5.13b )ar e compared the following canb eobserved :Th epoly - electrolytecharacte ro fth esilic aparticle si sreduce db yPL.HBr ,a s c judgedb yth edecreas eo fth eslope so fth ep Kv s10 a+ V s plots,an d because these slopes are less dependento n the ionic strengthwhe n PL.HBr is adsorbed. Both effects aredu e toth epositiv e electric field experienced by the silanol groupswhe nP L ispresent .Th ein ­ trinsic pKvalu e ofth e silanol groups inth epresenc eo fPL.HB ri s lower: 5.4 ± 0.4, than forbar e silica (6.7± 0.4) .Thi smean stha t whenP L adsorbs ata = 0 th esilano lgroup swoul dbehav emor eacidic . The values found arerelativel y uncertainbecaus e the extrapolation isno tlinea r (seefig .5.13b) . James and Parks (1982) foundfo rth eintrinsi cp Ko fpyrogeni csilic a (Cab.O-SilM7 )a valu eo f7. 2 ±0. 3b yreplottin gth edat ao f Abendroth (1970). The agreementbetwee nthi svalu ean dtha tfoun dher efo rbar e silicai swithi nexperimenta lerror . Theextrapolatio nt oa = 0 ,mad ei nth epresenc eo fPL ,ha sonl ya prac ­ ticalmeanin gwhe n the adsorbed amount is independent from a,whic h isno t the case.Depletio nmeasurement s show thatth e amountP Lad ­ sorbedbelo wp H3. 5i sinmeasurebl ylow . Theproto n titrationbehaviou r ofhomogeneou s (weaklyacid )poly - anion-(weakly basic)polycatio ncomplexe s isquit e analogoust otha t of the silica-polylysine system. Polderman (1975) conducted fromPo ­ tentiometrieproto ntitration si nth epolyethyleneimine/polyacrylamide - acrylic acidcopolyme r system thatbot h thepolyanio nan dpolycatio n 105

I 8

1.2 1.6 20 ïoo.vr., 10cV F

Fig. 5.13a .Doubl e extrapolation plot forth eestimatio n ofth eintrinsi c pKo fsur ­ face silanol groups ofAEROSI L 0X50, showing thevariatio n ofth eapparen t pK value with fractional surface charge andNaB r electrolyte concentration, b. Asfig .5.13 abu tno wi nth epresenc e ofexces s PL.HBr-L (DP190) . in the complex behave like stronger polyelectrolytes and thatth e screening by the charges ofth e otherpolyio n is analogous toth e screening by small ions but much more effective. Tsuchida (1974) reached the same conclusionfo rth epolymethacryli cacid-polyammoniu m polymersystem .Tsuchid aobserve da decreas ei nth eapparen tp Ko fth e carboxyl groups and adiminishe d polyelectrolyte charactero fPM Ai n thepresenc eo fth epolycation . Reinert (1981) founda stron gincreas e ofth eapparen tp K (i.e.stronge rbasi ccharacter )o fspermin e (apoly - amine)whe ncomplexe d topolyphosphate .Reiner tpointe dou ttha tth e combined protonation/deprotonation and associationbehaviou r ofth e spermin-polyphosphate system,ma yserv ea sa mode lfo rsimila rproces ­ ses inbiologica l systems.Th e influence oflo wmolecula rmas selec ­ trolyte upon theproto n titrations ofth ehomogeneou s complexeswa s notinvestigate db yth eauthor smentione dabove .

5. 3. 6 Theoretical descriptions of polycation-poly anion association

Record (1978) describedth einteractio nbetwee npolyelectrolyte san d oppositely charged oligo(poly)electrolytes inhomogeneou s systemsi n terms of multiequilibrium theory for interacting sites (Schellman, 1975) accounting for ioncondensatio naccordin gt o Manning (1972) and theusua lscreenin geffects .Thi stheor yi sno tusefu l fora quantita - 106 tivedesriptio nwhe nth ecomplexatio nconstan ti simmeasurabl ehig ha s isusuall y the casewhe nbot hpolyelectrolyte shav ea hig hdegre eo f polymerization. Besides thever y high (i.e.unmeasurabl ehigh )affi ­ nity found for the adsorption ofpolylysin e on the surfaceo fnega ­ tively chargedparticles ,th e theory is alsono t applicable inthi s case because the appropriate statistical factors (i.e.entrop ycon ­ tributions) for the formation of trains,loop s and tails,i.e .th e structureo fth eadsorbe dP Lmolecules ,ar eno tincorporated . Instea d of followingthi sapproac hi ti tmor eexpedien tt oexten d asuitabl etheor yfo rth eadsorptio no funcharge dpolymer swit helectro ­ static interactions,a sha sbee ndon eb y Van der Schee (1984), with the polymer adsorption theories of Roe (1974) and Scheutjens-Fleer (1979, 1980). The theory ofVa n der Schee is the onlyviabl epoly - electrolyte adsorption theory availablea tth emoment ,a swil lb eex ­ plained in chapter7 . Provisional reports ofVa n der Schee'spoly - electrolyt adsorption theory have also been given by Marra et al. (1982) and Bonekamp et al. (1983). Athermodynami c treatmento fth ecomple x formationbetwee npolyanion s andpolycation si nhomogeneou ssolution s (i.e.befor ephas eseparatio n occurs)ha sbee npresente db y Polderman (1975), toexplai nhi sproto n titration data.Thi stheor yi sonl yvali dfo rhomogeneou ssystem san d inswampin gelectrolyte ,therefor ePolderman' sanalysi scanno tb euse d forth eproto ntitration so fth ePL-silic asystem ,presente dhere . Inchapte r7 I wil ldiscus ssom easpect so fth epolyelectrolyt ead ­ sorption theory ofva nde rSche ean dth eapplicabilit yo fthi stheor y forth edescriptio no fth eadsorptio no fcharge dpolyaminoacids .Specia l attentionwil lb epai dt oth eeffec to fth esal tconcentratio nan dsur ­ facecharg eo nth eadsorptio npropertie so fpolylysin eo nAg ian dpoly ­ styreneparticles .

5.3.7 UV and CDspectroscopy of the silica/poly-L-histidine-system

Measurements ofth eU V and CD spectrao fpoly-L-histidin e (PHis) DP11 0 adsorbed atth esilic awate rinterfac ewer eperforme dwit hth e followingpurposes . i. Investigationo fth epossibilitie so fperformin goptica lspectro ­ scopyo fpolyaminoacid sagains ta larg ebackgroun dscattering . ii. Detection of conformational differences between PHis inaqueou s solutionan di nth eadsorbe dstate . Silicawa schose n asth e substrate forPHi sbecaus eth eoptica lpro - 107 pertieso fsilic asol sar emos tpromisin gfo rperformin goptica lspec ­ troscopy. Poly-L-histidine was chosen because this polycation has similaradsorptio npropertie sa sPL.HB r (seesectio n4.3.4) ,but ,a si s moreimportant ,PHi sshow salso ,jus tlik eP La conformatio ntransitio n from acoil-lik e state to anordere dconformation ,probabl ya right - handedheli xo nincreasin gth ep Hfro m4 t o6 .Thi swa sconclude dfro m CDmeasurement s by Beychok (1967). Because ofth eoccurrenc eo fthi s transitioni nth eaci dregion ,silic ai sa suitabl esubstrat e forPHis , to studyb y spectroscopic means the conformationo fadsorbe dPHi sa t differentp Hvalues .A tp H1 0wher e thehelix-coi l transition inP L occurs, the silica isto osolubl et ous ei ta sa substrat e forP Lad ­ sorption. A major difficulty inth e envisaged optical spectroscopic study, whichi sno tusuall yencountere di nhomogeneou s (bio)polymercomplexes , isth ehig hturbidit yo fth eparticl esuspensio no rsol .Thi si sespe ­ ciallys oi nth eU Vregion .AEROSI LOX5 0sol sar erelativel yfavourabl e in thisrespec tbecaus ethe yar erelativel ytransparen twhe ncompare d withlate xan dAg isol so fcomparabl emas sconcentration . Infig .5.1 4 theOD 10 (200/240nm )i splotte dagains tth esilic aconcentration .

.4 .6 8 3 csll(kgm- )

Fig.5.1 4Optica l densityo fAEROSI L 0X50 solsa sa functio no fth eso lconcentratio n attw owavelengths ,o- o20 0nm ,x- x24 0nm . 108

One canse e thata tparticl e concentrations of 0.5 kg.m theO Di s -3 still lower than 1,whic h iscertainl y not thecas ewit h theusua l latexan dAg isols .Thi si smainl ydu et oth efac ttha tth esilic apar ­ ticles are small comparedwit hthos ei nlatice so rAgi-sols .Further ­ more,th eLambert-Bee rla wi svali dfo rth esilic asol sove rth eentir e concentrationrang estudied . Themeasuremen to fa nU Vspectru mo fa n (adsorbed)chromophor epresen t ina so li shindere dby : (i) Thepresenc eo fa larg ebackgroun dscattering . Bytakin gdifferenc espectr abetwee na so li nth epresenc ean dabsenc e (blanc sol)o f thechromophore ,correction s forbackgroun d scattering canb eaccounte d for,provide dtha tn oparticl eaggregatio noccurs , (ii)Th eDuysen s effect {Duysens, 1956), i.e. theapparen tlowe rex ­ tinction coefficient ofth e adsorbed chromophore incomparenc ewit h the same chromophore ina homogeneou ssolution .Thi sadsorptio nflat ­ teningi sessentiall ydu et oth efac ttha tpar to fth echromophore si s notexcitate d by the incidentbeam ,becaus ethe yar esituate di nth e 'shadow' of the particlespresent .Correction s for this effectar e more difficult. Fortunately this effect is rather small when the particles are small compared with the wavelength of the incident light beam (Dut/sens, 1956; Urry and Yi, 1968) as isth e casewit h thesilic aparticle suse dhere . (iii)Multipl escattering .Thi seffec tca nb eignore di nth eregio nwer e Beer'sla wapplies . Inon eserie so fexperiment si twa schecke dwhethe rth eU Vdifferenc e _3 spectrum ofKHphtalat e dissolvedi nAEROSI LOX5 0sol s (30.8mmol. m ) ofdifferen tparticl e concentrationswa sdifferen tfro mtha to f30. 8 •_ 3 _3 mmol,m phtalate inwater .A tsilic aconcentration su pt o0. 7kg. m the difference spectra ofth eno n adsorbingphtalat ewer e identical within1 %wit hth ecorrespondin g spectrumi nwater .A thighe rparticl e concentrationsth ephtalat espectru mbecome s flattened (seefig .5.15) . Also the relative contribution of stray lightfro mth emonochromato r becomes thenunacceptabl y high.Thes e experiments showtha ti ti si n principle possible tomeasur ea reliabl eU Vspectru mo fa chromophor e inth epresenc e of silica,whe nth eparticl e concentration doesno t _3 exceed0. 7kg. m _3 Infig .5.1 6 theU Vspectr ao fPHi ssolution s (9.09mmo l .m )an d UV difference spectra of the same solutions in the presence of AEROSIL 0X50 (0.625kg. m )a tp H4. 0 and5. 8 areshown .I nth epre ­ sence of silica the adsorbed amountexceede d85 %o fth etota lamoun t 109

20Ö 220 240 260 280 300 X(nm)

Fig.5.1 5 Absorption (difference)spectr a ofKHphtalat e (3.08*10 M)i nth epresenc e ofAEROSI L0X5 0 (2.49 kg.nf3)an d inth eabsenc eo fit . 1. KHphtalate (AEROSIL0X5 0so l (REF.:AEROSI L0X5 0sol) . 2. KHphtalate inwate r (REF.:water) . 3. Baseline forspectru m 1:bot h cuvettesAEROSI L0X5 0sol . 4. Baseline forspectru m 2:Bot h cuvettesdem iwater .

PHispresent ,a sindicate db yth eU Vspectru mo fth eclea rsupernatan t of the sol after centrifugation.Neithe r the shape ofth espectru m atp H4. 0 nor thata tp H5. 8 inth epresenc eo fsilic awa smeasurabl y different from the correspondingsolutio nspectra .Unfortunatel yals o no significant differences werefoun dbetwee nth esolutio nspectr ao f PHisa tp H4. 0 andp H5.8 .Thi si sprobabl ybecaus eth eimidazol erin g ofhistidin ei sno tpar to fth epolyaminoaci dbackbone .Anothe rdiffi ­ culty isth e occurrenceo fa baselin eshif to fth esilic ai nth epre ­ senceo fPHis .Probabl yincipien tflocculatio ni nth ePHis-silic asyste m isresponsibl efo rthis .Therefor en oconclusion sca nb edraw nabou tex ­ tinction coefficient differences between adsorbed PHis andPHi si n solution. CDmeasurement swer eperforme dwit ha singl ebea mapparatus .Direc t corrections forth e silicabackgroun d scatteringwer e thereforeno t possible. At the maximum silica concentration which could beuse d 110

.14

• f\X .12 \ \ .10 • , \\

.08 VV \ ^V •

.06 • \ \ • .04 j \V " .02 : PK\^ 240 260 nm

Fig.5.1 6U V absorption (difference) spectra of PHis (DP 110)i naqueou s solutions of pH4. 0 andp H5. 8 inth epresenc e ofAEROSI L0X5 0 (0.625kg. m )o ri n theabsenc eo fit . 1. PHis (cT= 9.0 9 mmol.m"3)/AEROSIL0X5 0so lp H4.0 ;REF. :AEROSI Lp H4.0 . 2. PHis (c =9.0 9 mmol.m" )i nwate rp H4.0 ;REF. :wate rp H4.0 . 3. Baseline forcurv e 1:Bot h cuvettesAEROSI L0X5 0so lp H4.0 . 4. As 1,bu tp H5.8 ;5 .A s2 ,bu tp H5.8 . 6. Baseline forcurv e4 :Bot h cuvettesAEROSI L0X5 0so lp H5.8 .

without running outo f scale (0.625k gm " ),th emaximall y possible concentration of adsorbed PHiswa s too lowb yabou ta facto ro ffiv e to detectth e CD spectrum of adsorbed PHis.Anothe r difficultywit h the CDmeasurement s on silica solswa stha tth ebaselin eo fth ebar e silica solwa sno t astraigh thorizonta llin ebu tincrease dprogres ­ sivelywit hdecreasin gwavelengt htoward snegativ eellipticit yvalues . Summarizingth efollowin gma yb econcluded : i.Th emeasuremen t ofU V spectrao f adsorbedpolyaminoacid so rothe r (bio)polymerswit hintrinsi cchromophore si spossibl ei nprinciple .Th e Ill measurementsca nb eimprove dwhe nparticl eaggregatio nca nb eavoided , ii.C Dmeasurement so nadsorbe dpolyaminoacid s shouldals ob epossibl e withmoder n CD spectrophotometers when sampling techniquesar eused . Besides corrections for adsorption flattening also corrections for scattering distortions should bemad ewhe n aquantitativ e interpre­ tationi swante d (Vrry and Yi, 1968; Vrry et al., 1970). iii. Inadditio nalternativ espectroscopi ctechnique sma yb econsidere d toobtai ninformatio nabou tth econformatio nan dinteractio no fP Lwit h silica,suc ha sIR ,RAMA Nan dNM Rspectroscopy .

5.4 SUMMARYAN DCONCLUSION S

Inthi schapte rth eadsorptio no fpolycation so nth esurface so fneg ­ ativelycharge dpolystyren ean dsilic aparticle swa sinvestigate da sa specialcas eo fth eformatio no fpolycation-polyanio ncomple xformation . Thiswa sdon eanalyticall y (seeals ochapte r4 )an db ytitratio no fth e colloidal particles inth eH formwit hPL.HB ri nwhic hth econducti ­ vityan dp Hwer esimultaneousl y followed.Als oproto ntitration so fth e silica-PLsyste mwer eperformed . Justa si sth ecas ewit hth ecomple x formationbetwee nlinea rpoly - electrolytes,th econductometri ctitratio ncurve s Kv se- - showa break . Thebrea ki sals oth epoin tafte rwhic hth ep Hremain sconstant .I twa s concludedtha ta tth ebreakpoin tal l-OSO lgroup so fth elate xan dth e - + maximal amount ofSi- 0 groups of the silicawer eboun d to an-NH _ charge ofPL .Th e composition ofth ecomplexe si nth econductometri c breakpointi sno tcharge-stoichiometri c andi nth ecas eo flate xclearl y superequivalent with respectt oth eP Lcharges .I nth ecas eo fsilic a thecharg e composition inth ebreakpoin t isdependen to nth eequili ­ briump Han dioni cstrength .A tp H10. 2th echarg e (non)stoichiometr y is the same astha ta tlo wp H inth ePL-late x system, howeverth e amount adsorbed ishighe r due toth ereduce dchai ncharg edensit yo n theP Lmolecule sa tthi spH . Themai n cause for the formation ofsuperequivalen t complexes is therigidit yo fth ecolloida lparticle san dth erelativ elarg eaverag e distancesbetwee nth echarge dsurfac egroups .Thi si si ncontras twit h whati sgenerall yobserve d forth ecomple x formationbetwee ndissolve d linearpolycation san dpolyanions .I nth elatte rcas e1: 1complexe sca n beforme dbecaus ebot hpolyelectrolyte s areflexible .Howeve r fordis ­ solved systems also deviationsfro m1: 1 stoichiometry areobserve da t highioni cstrength . 112

Aclea rdifferenc ebetwee nth eP Lcharge dparticl esystem san dmos t homogeneouslinea rflexibl epolyelectrolyt ecomplexe si stha tth ecom ­ positiono fth ecomple xstil lchange s (i.e.increasin gadsorbe damoun t PL)afte r theconductometri cbreakpoint .Thi s shows that alsoothe r than coulombic interactions,suc ha sion-dipole ,H-bondin g andhydro ­ phobic interactions become effectivei ntha tcase .Thi swa sals ocon ­ cluded inchapte r4 from the saltdependenc eo fth eamoun to fP Lad ­

sorbed. The results of the conductometric titrations (K vsc_ L)ar e not.conflictingwit hth emosai kcharg emode lo f Kasper (1971). Compar­ ison of flocculation resultsan dth econductometri ctitratio nresult s of the PL-silica system suggesttha t 1:1 complexesma yb eformed ,i n contradiction with other observations.Mor e information isneede dt o elucidatethis . The fraction of -NH„,remainin gfre eafte radsorptio no fP Lo nPS - particleswa s estimated by conductometric titrationswit hpolyvinyl - sulphate.Th evalue sfoun daroun dth ebreakpoin tadsorption : (0.3;0.4 ) show againtha tth eP Lmolecule s adsorbi na rathe rfla tbu tno ten ­ tirelyfla tconfiguration . Protontitration so fsilic aar estrongl yinfluence db yth epresenc e ofPL .Th esilano lgroup sbecom emor eacidi c (i.e.the yassum ea lowe r apparentpK )i n thepresenc e ofPL .Th eP Lmolecule sadsorbe dac ta s aneffectiv e screener of the Si-0~ charges,thu spromotin g thedis ­ sociationo fth esurfac egroups .Thi seffec ti sals oobserve di nhomog ­ eneouspolyelectrolyt e complexes.Th einfluenc eo fth eioni cstrengt h onth echarge-p Hcurve so fsilic ai sreverse di nth epresenc eo fPL.HBr , becauseo fth ecompetitio nfo rsurfac esite sbetwee nN a andNH _ lysine charges. Insectio n5.3. 7 the difficulties metwit h themeasuremen t ofU V and CD spectra of on silica adsorbedpolyhistidin e aredescribed . Spectrao fadsorbe dPHi sar ereported .

5.5 REFERENCES

Abendroth,R.P . (1970).J .Colloi d InterfaceSei . 34, 591-596. Beychok,S . (1967),i n 'Poly-a-aminoacids', Fasman ,G.D .ed. ,M .Dekke r NewYork . Bleier,A .an dGoddard ,E.D . (1980).Colloid san dSurface s 1, 407-423. Bonekamp, B.C.; Schee,H.A .va n der and Lyklema,J . (1983). Croat, chem.Act a 56, 695-704. Ciferri,A. ;Puett ,D. ;Rajagh ,L .an dHermans ,J . (Jr.)(1968) .Bio - polymers6 ,1019-1036 . 113

Domard, A. and Rinaudo,M . (1980). Macromolecules 13, 898-904. Domard, A. and Rinaudo,M . (1981). Macromolecules 14, 620-625. Duysens, L.N.M. (1956). Biochim. Biophys.Act a 19, 1-12. Daniels, S.T. (1980) in 'Adsorption of microorganism to surfaces'. Britton, G. and Marshall, K.C. eds. J. Wiley and Sons.Chapte r 2. Eggert, A.R. (1976). Ph.D. thesis Lawrence University, Appleton Wis­ consin. Eisenlower (1982). Discussion remarks,William s et al. (1982) in 'The effect of polymers on dispersion properties'. Tadros,Th.F . Acad. Press London,p .373 . Gelman, R.A.; Glaser, D.N. and Blackwell, J. (1973). Biopolymers 12, 1223-1232. Gregory, J. (1973). J. Colloid Interface Sei. 42, 443-456. Horn, D. (1978). Progr. Colloid Polym. Sei. 65, 251-264. Ho, C.H. and Howard, G.J. (1982) in 'The effect ofpolymer s on dis­ persion properties',Tadros ,Th.F . ed., Acad. Press London,p .343 - 359. Juriaanse, A.C.; Arends, J. and Ten bosch, J.J. (1980). J. Colloid Interface Sei. 76, 212-219. James, R.O. and Parks, G.A. (1982) in 'Surface and Colloid Science' 12, Matijevic, ed., Plenum Press New York-London. Kasper, R.D. (1971). Ph.D. thesis California Institure of Technology, Pasadena, California. Lindquist, M.G. (1975). Ph.D. thesis Lawrence University Appleton Wisconsin. Lindquist, M.G. and Stratton, R.A. (1976). J. Colloid Interface Sei. 55, 45-59. Manning, G.S. (1972). Ann.Rev . Phys.Chem . 23, 117-140. Manning, G.S. (1978). Q. Rev.Biophys . 11, 179-246. Marra, J.; Schee, H.A. van der; Fleer, G.J. and Lyklema, J. (1983), in 'Adsorption from Solution', Ottewill,R.H. ; Rochester, C.H. and Smith, A.L.,eds. ,Acad . Press,p .245 . Michaels, A.S.;Mir , L. and Schneider,N.S . (1965). J. Phys.Chem . 69, 1447-1455. Norde, W. (1976). Doctoral thesis Agricultural University Wageningen, TheNetherlands . Norde, W. and Lyklema, J. (1978). J. Colloid Interface Sei. 66, 295- 301. Philipp, B<; Dawydoff,W . and Linow, K.J. (1982). Z. Chemie 22, 1-13. Polderman,A . (1975). Biopolymers 14, 2181-2195. 114

Record, M.T.; Anderson, C.F. and Lohman, T.M. (1978). Q. Rev. Biophys. 11, 103-178. Rice, S.A. and Nagasawa, M. (1961). 'Polyelectrolyte solutions', Acad. Press London-New York. Reinert, K.E.W. (1981). Bioel. Bioeng. (J. Elect. Anal. Chem.), 8, 301-308. Roe, R-J. (1974). J. Phys. Chem. 60, 4192-4207. Ross, P.D. and Shapiro, J.T. (1974). Biopolymers 13, 415-416. Schee, H.A. van der (1984). Doctoral thesis Agricultural University Wa­ geningen, The Netherlands. Sonntag, H. and Pilgrimm, H. (1976). Progr. Colloid and Polym. Sei. 61, 87-92. Sonntag, H. and Kolesnikova, R.S. (1980). Z. Phys. Chem. 261, 226-232. Schellman,J.A . (1975). Biopolymers 14, 999-1018. Scheutjens, J.M.H.M. and Fleer, G.J. (1979). J. Phys.Chem . 83, 1619- 1635. Scheutjens, J.M.H.M. and Fleer, G.J. (1980). J. Phys.Chem . 84, 178-190. Tanford, C. (1965). 'Physical Chemistry of Macromolecules', J. Wiley and Sons,Ne w York-London-Sydney. Terayama, H. (1952). J. Polym. Sei. 8, 243-253. Tsuchida, E.; Osada, Y. and Abe, K. (1974). Makromol. Chem. 175, 583-592. Tsuchida, E.; Osada, Y. and Ohno,H . (1980). J. Macromol. Sei.-Phys., B 17, 683-714. Urry, D.W. and Yi,T.H . (1968). Arch. Bioch. Biophys. 128, 802-807. Urry, D.W.; Hinners, T.A. and Masotti, L. (1970). Arch. Bioch. Biophys. 137, 214-221. Yates', D.E. and Healy, T.W. (1976). J. Colloid Interface Sei. 55, 9-19. 115

6 CONFORMATIONO FFRE EAN DADSORBE DPOLYLYSIN E

6.1 INTRODUCTION

Inth epreviou s chapters the adsorption ofP La sa highl ycharge d polypeptide was considered.No w Iwil l focuso nth eadsorptio nprop ­ ertieso fPL- L andPL-D L asa functiono f thechai ncharg edensity , i.e. as a functiono f thep H abovep H7 . Inthi sp Hregio nno tonl y theadsorptio npropertie schange ,bu tals oth epropertie si nsolution . Above pH> 8 ther e appears asecondar y structurei nPL-L ,bu tno ti n PL-DLa tsufficientl y lowcharg edensit yan da tstil lhighe rp H(abov e pH11 )precipitatio no fbot hstereoregula r formso fP Lset sin . Exceptth eadsorptio npropertie sa thig hpH ,als osom eprecipitatio n characteristicso fbot hPL- Lan dPL-D Lwer estudie dbecaus ea tver ylo w chain charge density multilayer adsorption, i.e.precipitatio no na n adsorbedP Llaye rca ntak eplace . Themai nquestion st ob eanswere di nthi schapte rare : i.Doe sth eadsorptio nand/o rprecipitatio nbehaviou ro fPL- Lan dPL-D L differ? ii. Isther e acoi lt oheli xtransitio ni nadsorbe dPL- Lan dwha tar e the factors that govern conformational transitions inth eadsorbe d state,i fany . Theprecipitatio nbehaviou rwa sstudie db ymean so fturbidit ymeasure ­ mentsa sa functio no ftemperature ,p Han dtime .I nadditio nadsorptio n isothermso fPL- Lan dPL-D La thig hp Han dadsorbe damount sa sa func ­ tiono fp Hwer e determined tocompar e thebehaviou r ofbot hstereo - regular PL forms.Th e occurrence of.conformational transitionswa s followedb ymean so fproto ntitration so fadsorbe dPL- Lan dPL-DL .Thi s methodi son eo fth efe wcapabl et ogiv estructura linformatio no fad ­ sorbedP Li nth ever yturbi dconcentrate dlatices . Thebehaviou r ofpolyaminoacid s exhibiting secondary structurei n solution in ananisotropi c environment, isno tonl yinterestin gfro m aphysical-chemica l point ofview ,bu tals oi nbiology .Th esecondar y structureo fbiopolymers ,adsorbe da ta ninterfac ema yb eo fimportanc e in immunological and cell recognitionprocesses .Fo rtechnique ssuc h as immunoadsorptionan d theus eo fimmobilize denzyme spersistenc eo f thenativ econformatio ni nth eboun dstat ei sessential . 116

6.2 INTERACTIONSDERTERMININ GTH ECONFORMATIO NO FCHARGE DPOLYAMINO - ACIDS

Syntheticpolypeptide shav ebee nfoun dt oexis ti nman yordere dcon ­ formations characteristic of those found inprotein s (Fasman, 1967). Inthi s connection,poly-L-lysin e isa nespeciall yinterestin ghomo - polyaminoacid, in that itca n form three conformations.A t lowpH , i.e.whe nth ee-aminogroup s areprotonated ,th emolecul ei s'randomly ' coiledan dtypica lo fsolubl esyntheti cpolyelectrolytes .Howeve rsom e local ordered conformation isprobabl y present atlo wioni cstrengt h underthes ecircumstance s (Painter and Koenig, 1976). Seeals osectio n 3.1.2.A thig hp Han droo mtemperatur eth ee-ammoniu mgroup sdissociat e andth enearl yuncharge dmolecul eform sa na-helix .Whe nthes esolution s ofhig hp H areheated , almost immediatelya precipitat e forms,whic h hasth eß conformatio n {Fasman, 1967; Doty and Gratzer, 1961). Manyfactor sdetermin eth econformatio no fa polypeptid ei nsolution . Generally stated, the conformation of apolyelectrolyt e ina dilut e solution depends on intra-an dintermolecula rsolute-solute ,solvent - solute and solvent-solvent interactions. The presence of asecon d solute, forinstanc e lowmolecula r mass electrolyte,ca n alsoexer t its influence. Inpolyelectrolyt esolution sth ecoulombi cinteractio n between charged segments is amajo r factor.Thi s interaction isin ­ fluencedb yth epresenc eo fsalt . The possibility of hydrogen bond formation between thepeptid e carboxyl oxygeno fon eresidu e and theamid ehydroge no fanothe rca n be another important factor indeterminin g theconformatio no fpoly - aminoacids. Still other factors that play apar t are:v.d .Waals , dipole-dipole, hydrophobic and steric interactions. Especially the two lastmentione d areo fimportanc ebecaus ethe yar estrongl ydepen ­ dento n thenatur e ofth e amino acid side chain.I nth ecas eo fad ­ sorbed polyelectrolytes, thenatur e ofth e interface and theinter ­ action with iti s importanttoo .Th e ultimate conformation willb e the coumpounded result. Their relative influence canb e changedb y varying the solventpropertie s and temperature. Inth enex tsection s the most important interaction forces arediscusse d insom edetai l with particular reference toth econformationa l properties ofpoly - aminoacids. 117

6. 2. 1 'Non bonded' interactions

Thenumbe r ofpossibl econfiguration so fa polypeptid echai nback ­ bonear estrongl ylimite db yconstraint si nth eallowe dangle s$ an di| / between theN-C a andC a-C'bond s respectively.Shor trang erepulsiv e interactions and dipole-dipole interactions between adjacent amide groups are important inthi s respect.Th e sterically allowedconfi ­ gurations of apolypeptid e chain are alsodependen to nth enatur eo f theaminoaci dsid echai ngrou psubstitute d atth eC atom. Calculated steric diagrams (Ramachandranplots ) forresidue swit h large side chainsbu tno tbranche d atth e ßcarbo n areessentiall y the same as thato f alanine (CH-sid echain )(se efo rexampl e Cantor and Schimmel, 1980). Ofcours e also excluded volume effects,whic h are dependent on the solute-solvent interactions limitth enumbe ro f possibleconfiguration so fa polypeptid echain ,excep ta t Sconditions .

6. 2. 2 Hydrogen bond formation

Ahydroge nbon di sgenerall ysai dt oexis tbetwee na dono rmolecul e D-H and an acceptor A,whe nther ei sevidenc etha tth etw omolecule s associatei na fashio nspecificall yinvolvin gth ehydroge nato mo fth e donor.Th ehydroge nbond so fmos timportanc efo rprotei nan dpolypeptid e structure (i.e.thos e ina a helix , ßsheet )ar e thosebetwee nth e backbone amide nitrogens andcarboxy l oxygens.Sinc e intramolecular hydrogen bonding competeswit hhydroge nbondin g towate rmolecules , theoveral lcontributio no fhydroge nbondin gt oth estructur eo fpoly ­ peptidesi sdifficul tt oassess . Klotz and Franzen (1962) studied the influence of the solvento n H-bond formation choosing the solvent-dependent aggregation ofN - methylacetamide asa peptid e analog. Inwate r the formation ofa H - bond between two N-methylacetamide molecules appears tob ethermo - dynamically unfavourable (AG°= + 1 3kJ/mol )an dth eenthalp yo ffor ­ mation isnearl y zero.Howeve ri nCCI .bot hAG °an dAH °ar enegative : -1 -1 -3.86kJ.mo l and-17. 6kJ.mo l respectively.Th eunitar ycontributio n (i.e.withou tth ecrati ccontributio nt oth eentropy )t oth efre eenerg y ofH-bon d formation (AG°)i nN-methylacetamid e dimersi nwate ramount s to+2.9 3 kJ.mol" (see Olander and Holtzer, 1968). Thisvalu eca nb e comparedwit hth evalu eo fth esam equantit yobtaine dearlie rb y Kauz- mann (1959) from thermodynamic data of Schellmann (1955) onth enon - 118 ideality behaviour ofure a solutions inwater .Th evalu e ofAG°'ob - tained is-1. 7 ± 0.4kJ.mo l hydroge-1 nbonds .Obviousl y theresult s of Klotz and Franzen andthos eo f Schellman-Kauzmann areconflicting . The data ofKlot zan dFranze nar ei nm yopinio nmor ereliabl ebecaus e they are based on a direct spectroscopic estimation of theamoun t of amideproton s inH-bonds ,whil eth einterpretatio no fKauzman nan d Schellman isdeduce d fromno nidealit yeffect si nure asolutions .Be ­ sides this,ureu m is apoore r peptideanalo gtha nN-methylacetamide . Olander and Holtzer (1968) discuss the datao fKlot zan dFranze nan d Kauzmann-Schellmann inrelatio n to theheli x stability ofPGA .Whe n thenon-electrostati c stabilization freeenerg y of the aheli xi nP L orPGA , asobtaine d fromPotentiometri edat a (usuallyo fth eorde ro f -(0.2-0.7kJ.mol " ))i scompare dwit hAG °fo rth eformatio no fH-bond s in N-methylacetamide it isclea r thatwhe n thepeptid e units ina n a helix areno t shielded fromwater ,H-bond s areno t astabilizin g interaction for the a helix formation,bu tt ospea kwit h Olander and Holtzer (1968): "what,i nfact ,th edat ao fKlot zan dFranze nsugges t is that interpeptide hydrogenbond s are somuc hweake r thanwater - peptide hydrogenbond s thatthe y represent anoverwhelmin g destabi­ lizinginfluence. " Klotzan dFranze nsuggest ,tha twhe npeptid egroup sar eeffectivel y shielded from water due to aspecifi c highconcentratio n ofhydro ­ carbon-like residues,H-bond s canhoweve r contribute toth estabili ­ zationo f secondary structures.Th e samewa squote db y Fasman (1967) inrelatio n toth eheli x stability ofpolyaminoacid sbu tquestione d by Olander and Holtzer (1968), who arguetha tth eeffec tcanno tpla y alarg erol ei nth ePG Ahelix . Another point is that theuncertaint y in thevalue s ofAG °fo r H-bonds obtained frommode lcompound si so fth esam emagnitud ea sth e entire stabilization freeenerg y ofth ehelix ,s opredictio no feve n the signo fth eheli x stabilization freeenerg y from thesu mo fth e individual (relative large) contributions, some stabilizing, some destabilizing,i sver yuncertai n (Olander and Holtzer, 1968). In poly-L-lysine and other water-soluble homopolyaminoacids H- bonding in aqueous solutions seems tob eno tunimportan t indeter - ming the stability ofordere d compactconformation s inwater .Thi s isbecaus e thehelica lP Lconformatio nbehave sindee dlik ea H-bonde d structure intha t itappear s tob emor e stable at lowtemperature s thana thig htemperature s (Davidson and Fasman, 1967; Hermans, 1966). Also thegreate r stability ofth e< xheli x in50 %methano l supports 119 this. Thepeptid e unitsi na P Lheli x should thereforeb eshielde d from watera tleas tpartially .Thi sca nb erealize d becauseo fth e presenceo fth emor e hydrophobic partso fth esid e groups(-(CH 2)4) inPL ,causin ga loca llo wdielectri cconstan tenvironment . Onth econtrar yth epolylysin e ßstructur e ismor e stablea thighe r temperatures (322K )suggestin gtha thydrophobi c interactionsar emor e importanti nthi scas e (Davidson and Fasman, 1967). Inth epresenc eo f aninteractin g interface intramolecula r hydrogenbondin gca nb een ­ hanced becauseo fth epossibl elo wloca l dielectric constanta tth e interface.Fo rsurface scapabl eo fformin gH-bond s themselvesthi sca n beo fimportanc e fordeterminin gth estructur eo fadsorbe dpolyamino - acids.

6.2. 3 Hydrophobic bonding

The term hydrophobic bonding isuse dt odescrib eth etendenc yo f nonpola r groupst oaggregat ei na naqueou senvironment .Thi saggre ­ gation originates fromth efac ttha twater-wate rcontact sar ethermo - dynamically muchmor e favourable thancontact sbetwee ntw onon-pola r groupso rbetwee na non-pola r groupan dwater .So ,du et oth eaggre ­ gationo fth eno npola rgroup sth eexten to finteractio nwit hth esur ­ roundingwate ri sdiminished .Th eva nde rWaal sattractio no fnon-pola r groupsfo reac hothe rplay sonl ya mino rpar ti nth ehydrophobi ceffect . Thehydrophobi ceffec tarise sprimaril y fromth einfluenc eo fth enon - polar groupso nth estructur eo fth eadjacen twate r (Tanford, 1973). Consequently,th eeffec ti scorrelate d withth estructur eo fwater . Thereforea quantitativ edescriptio no fth eeffec trequire sth echoic e ofa wate rmode l (Franks, 1973). Whenhydrophobi cbondin goccurs ,hy ­ drogenbond sbetwee nwate rmolecule sar edisrupted .Th eeffec ti schar ­ acterized,a tleas ta t29 8K ,b ya relativel ysmall ,usuall ypositiv e enthalpy changean da larg epositiv e entropy change inth erang eo f 6-50J.K ~ perbon d (Némethy and Scheraga, 1962). Thefre eenerg yo f the formationo fhydrophobi c bondsa tmoderat e temperaturesbecome s morenegativ ewhe nth etemperatur ei sraised . Theimportanc eo fhydrophobi cbondin gfo rth estructur eo fprotein s andpolypeptide swa sfirs tpointe dou tb y Kauzman (1959). Hydrophobic bondingbetwee nth eapola rpar to flysin esid echai nresidue s(-(CH 2)4~) mayals ob ea facto ri ndeterminin gth estabilit yo fth eordere dconfor ­ mationso fPL- L (Hesselink, 1973; Hermans, 1966). However Olander and Holtzer (1968) state that: 'advocates ofhydrophobi c stabilization 120

(throughinteractio no fside-chai nmethylenes) ,i fthe ywis ht ob econ ­ vincing,wil l have torecko nwit hth eannoyin g facttha ta relativel y small fractiono fth ehydrophobi csurfac ei seffectivel yremove dfro m contact with the solventwhe n thecoil-to-heli x transitionoccurs' . When this istru e the stability of theheli x cannotb eexplaine di n termso fhydrophobi cbonding .Als o Hermans (1966) deduced fromth enea r equalityo fAG °an dAH ° (theno nelectrica lpar to fth efre eenerg yan d freeenthalp yo fheli xformation )fo rPG Aan dP Lan dth enorma lvalue s ofp K forth ecarboxy lan damin ogroup stha tneithe rhydrophobi cbond ­ ing nor hydrogenbondin gb y the side chains plays animportan trol e inth estabilit yo fth ea-helix .Herman sthe nstate dtha tthi sargumen t does notrul e outa contributio no fth eß an da CH „groups ,whic har e present inth e side chains ofbot h glutamic acid andlysine ,t oth e freeenerg y ofth e formation of the a helix.Th e estimation ofth e contribution ofhydrophobi c bondst oth estabilizatio n freeenerg yo f thepolylysin e a helixi sfurthe rcomplicate db yth efac ttha thydro ­ phobicinteraction sar epossibl ei nbot hheli xan dcoi lform s (Némethy and Scheraga, 1962; Hesselink, 1973). Inth e antiparallel ßshee to fpolylysin e the lysine sidechain s aremuc hmor e closelypacked .Hydrophobi c bonding ismor e important inthi s structure (Némethy and Scheraga, 1962) aswa sals oconfirme d experimentally forpolylysin eb y Pederson et al. (1971). Inth e caseo f adsorption ofpolylysin e on ahydrophobi csurfac e asfo rexampl epolystyrene ,hydrophobi cinteraction sbetwee npolylysin e andth esurfac ear eo fimportanc ei ndeterminin gth eadsorptio nproper ­ tieso fP La swa spointe dou talread yi nChapte r4 an d5 .Als oth econ ­ formational properties ofP Lwil lb einfluence db yth epresenc eo fa n interactingsurfac ea swil lb eshow nlater .Hydrophobi cbondin gbetwee n PLsid echain san dth ehydrophobi cP Ssurfac eca npla ya rol ei nthis .

6.2. 4 Ionic interactions

Iti swel l known thatth eordere d structure,i.e .a-heli x inun ­ chargedpolyaminoacid swit hionizabl esid egroup si sdisrupte d(helix - coil transition)whe nth eionizatio no fthes esid echain sbecome sto o high.Th ecaus eo fthi stransitio ni stha ta ta certai ndegre eo fion ­ izationth elin echarg edensit yi nth eheli xconformatio nexceed stha t inth ecoi lconformation .Th ep Han dioni cstrengt hdependen ttransitio n showsclearl yth eimportanc eo fioni cintraction si nthes epolypeptides . Charge-chargeinteraction sar eo fa lon grang enatur eeve nwhe nthe y 121 are shielded by thepresenc eo flo wmolecula rmas selectrolyte ,a si s thecas ei naqueou selectrolyt esolutions . A theory forth echarg einduce dhelix-coi ltransitio nrequire sth e computation of theelectrostati c free energy ofth eheli x andcoi l stateswhic hi sa difficul tmatter . Because ofth emathematica ldifficultie sinvolved ,a detaile dtheory , inwhic h the electrostatic energy of allcombination so fcharge dan d uncharged helix and coil stateswoul d havet ob ecompute di sno tde ­ veloped yet.Progres s inthi sfiel dha sbee nmad eb yconsiderin gbot h the helix and coil state as auniforml y charged cilinder,wit hit s counterions.Sinc eth eloca lcharg edensit yi shigh ,th eDebije-Hiicke l approximationma y notb eapplie dan dnumerica ltechnique sfo rth eso ­ lutiono f thePoisson-Boltzman nequatio nar erequired .Thes ecomputa ­ tionsca nprovid eth epH-value sand/o rsal tconcentration sa twhic hth e freeenerg y of theperfec theli xi sequa lt otha to fth ecoi l (Poland and Scheraga, 1970). Thisi sth epoin twher eth ehelix-coi ltransitio n willtak eplace .Loca lcharg eeffect s (i.e.sit ebinding )an dspecifi c ionadsorption ,whic h canb e of importancei npractica l systemshav e hithertobee nneglecte d inth eabov eapproximation . Earlyliteratur econcernin gth ePoisson-Boltzman ndescriptio no fth e ionicinteraction si na charge dmacromolecul eca nb efoun di nth eboo k 1Polyelectrolytes'b y Rice and Nagasawa (1961). Latercalculation so f thepotentia l ofa polyio n insolutio nhav ebee nmad efo rexampl eb y Katchalsky (1971), Fixman (1979) and Stigter (1975). Another complicated problem is theeffec t ofth e polyelectrolyte charge onth e conformation of themacromolecule ,i.e . theeffec to n thechai n flexibility and chaindimensions .A review of thismatte r hasbee ngive nb y Nagasawa and Takahashi (1972). Besidesth ePoisson-Boltzman ndescriptio no fa polyelectrolyte ,th e concepto fcounterio n condensation forth e descriptiono fth einter ­ action of small ionswit h linearpolyelectrolyte s asintroduce db y Oosawa (see Oosawa, 1957) can beused .Thi s conceptwa s developed furthertheoreticall yb y Manning (1969, 1972, 1978). Counterion condensation is supposed to be governed solely by the dimensionles sparamete r£ ,define das : e2 4 - 47tekTb (6-1) wheree i sth eelementar ycharge ,s i sth esolven tdielectri cconstant , k isth eBoltzman nconstant ,T i sth eabsolut etemperatur ean db i sth e averagespacin gbetwee nth echarge so na linea rpolyelectrolyt emolecule . 122 2 e/4n£k Tx sth eBjerru mlengt han di tha sth evalu eo f0.71 4n mm nbul k watera t29 8K . Manningshowe dtha twhe n4 < 1 n ocounterion scondens e onth epolymer ,bu twhe n4 > 1 counterio ncondensatio noccurs . The fraction of the line charge that is compensated by condensed counterions is 8 = 1-Ç". Afte r condensation the polyelectrolytes with the condensed envelope of counterions arestil lhighl ycharged . Aneffectiv echarg eo f4 ~ remains,i.e .on echarg epe rBjerru mlength . These charges are now screened by the counterion atmosphere.Thi s screening canb e treated then ina Debye-Hiicke lapproximatio nusin g theeffectiv echarg edensit yafte rcondensation . Fixman (1979) pointed out that from a correct application ofth e Poissón-Boltzmannequatio nt opolyelectrolyte s acertai nversio no fth e condensation model mayb ederived .Th eeffectiv echai ncharg edensit y determines the adsorbed amounto fpolylysin e aswil lb eshow nlater . Inth e calculated curveso f adsorbed amounta sa functio no fth eti ­ tration charge aspresente d inchapte r7 counterio ncondensatio nwil l beincluded . Besides purely coulombic interactions also specific ionicinter ­ actions canpla y arol e inpolyelectrolyt e systems (see forexampl e Rice and Nagasawa, Chapter8 (1961); Morawetz, Chapter7 (1965). For example,bromid ebindin g onth epeptid e group ofpolylysin eca n reduce theeffectiv e chaincharg e density (Ciferri et al. , 1968) and henceinfluenc eth eoccurrenc eo fth ehelix-coi ltransition . During and after adsorption ofcharge d polyaminoacids atcharge d interfaces,th epolyaminoaci dmolecule sar ei nth eoverlappin gelectri c field of the interface.Thi s can influenceth eadsorptio npropertie s aswel la sth econformationa lpropertie so fth eadsorbin gan dadsorbe d polyaminoacid.

6.3 SECONDARYSTRUCTUR EAN DHELIX-COI LTRANSITION S INCHARGE DPOLY ­ AMINOACIDS

Changesi nth esecondar ystructur eo fa polypeptid ei nsolutio nca n beinduce db yeithe ra chang ei ntemperatur eo ra chang eo fth esolvent . Inth e case ofchargeabl ewea k acido rbasi cpolyaminoacid sa chang e inp Hca nals ocaus ea conformationa l change.I nman ycases ,onl ysmal l alterations ina nexterna l variable arerequire dt obrin gabou tver y dramaticsecondar ystructura lrearrangements .Th ep Hdependen tconfor ­ mationaltransition sar eals ostrongl ycooperative .Als oth eintroduc ­ tion of an interface can disturb thedelicat ebalanc ebetwee nth e 123 variousinteractio nforce stha tar eresponsibl efo ra nordere dconfor ­ mation in solution, andhenc e adsorption can cause aconformationa l transition. A good introduction toth etheoretica l andexperimenta laspect so f helix-coil transitions inuncharge d polyaminoacidsha s beengive nb y Cantor and Schimmel (1980). Muchmor e elaboratetreatment so nhelix - coiltransition si nbiopolymer sca nb efoun di nth ebook so f Birshtein and Ptitsyn. (1966) and Poland and Scheraga (1970).

6.3.1 Theories for the helix-coil transition in charged polyaminoacids

Despiteth eimportanc eo fcharge-induce dconformationa l transitions inbiologica l systems,onl yver y fewtheoretica l studiesar edevote d to the effect ofcharg e onth e ordered conformations ofbiologica l macromolecules, and as fara sI kno wn otreatmen ta tal li savailabl e for adsorbed polypeptides.Thi s ismainl y because ofth e greatcom ­ putational difficulties encountered because of the longrang enatur e ofth echarge-charg e interactions.Therefor etemperature-induce dhelix - coil transitions,whic h arebiologicall y clearly less importantar e treated theoretically extensively inliteratur e (Poland and- Scheraga, 1970). Zimm and Bragg (1959) were the firstwh o developed astatistica l thermodynamical theory for thecooperativ e helix-coil transitioni n singleuncharge d polypeptide chains in solution.Thi stheor ydefine s twoparameters ,s an da .Her es i sth eequilibriu mconstan tfo raddin g anamid eresidu et oa helica lsegment :

s= exp(-AG conf/kT ) (6.2) acharacterize sth einitiatio no fa helica lsegment ,whic hi ntur nde ­ terminesth esharpnes so fth etransition :

\ a= exp(-AG init/kT) (6.3)

Inth epresenc e ofa ninteractin ginterfac eth etransitio nca nb ede ­ scribed by effective values of the aboveparameter s sf f and a ff (Zhulina et al. 1980). These effectivevalue sar ethe ndetermine db y the interactions with the interface. Inmos t theories ofhelix-coi l transitions, the interaction between dissolved polypeptides isne ­ glected.Thi s iswors e for adsorbedpolypeptide sbecaus eo fth ehig h 124 volumefractio ni nth eadsorbe dlayer . Zimm and Rice (1960) extendedth eZimm-Brag gtheor yfo relectrostati c interactions using the Debije-Hiickel theory for thecalculatio no f the electrostatic free energy. While the assumption,mad eb yZim m and Rice,tha tth e total electrostatic freeenerg yca nb emad eu po f Debije-Hiickel pair interactions isprobabl y quitepoor ,thi stheor y illustrates some features anddifficultie so ftreatin gth ehelix-coi l transition in charged macromolecules (see also Rice and Nagasawa, 1961). Inthei r treatmentZim man dRic econsidere d interactionsamon g four units inth echain ,correlatin g the i; (i+1); (i+2)an d (i+3) units,usin g thenearest-neighbou r Isingmode l toassig nth estatis ­ tical weights ofnon-ioni c origin and theweigh tobtaine d fromth e Debije-Hiickel pair interactions for the ionic contributions.Late r several authors simplified theZimm-Ric etheory ,mainl y forpractica l reasons. The s value for theuncharge d coil toheli x transition canb ede ­ termined from the Potentiometrieproto ntitratio ncurv e ofa poly - aminoacid: AG , kconi.T -"'•»_l n s _2 3- j* (—P KJ ^~ap-p Kp )d a ^c' (6.4) wherep K isth emeasure dapparen tp Kvalu ean dp K theextrapolate d apparentp Kfo rth epur ecoil . Arecen ttheor yfo rth edescriptio no fth echarge-induce dhelix-coi l transitioni sgive nb y Nakagaki and Ebert (1982). Thistheor yi sbase d onth ehelix-coi ltransitio ntheor yo f Applequist (1963), whichi ses ­ sentiallya generalizatio no fth eZimm-Brag gtheor ymentione dearlier . Theelectrostati ccontributio nt os i saccounte dfo rb yusin gth eGouy - Chapmanequatio nfo rplan egeometry ,relatin gth esurfac echarg ea to thesurfac epotentia l>| i onth eheli xsurface . Thetheorie smentione dabov ear eonl yo fver ylimite dvalu efo rth e description ofconformationa l transitions inth e adsorbedstate ,es ­ pecially forcharge-induce d transitions.Onl y foruncharge dpolypep ­ tides there are sometheoretica ltreatment so fhelix-coi ltransition s inth epresenc eo fa ninteractin ginterfac e (Dimarzio and Bishop, 1974; Birshtein et al., 1979 and Zhulina et al., 1980). Theseauthor sgiv ea treatment of the adsorption of infinitely long singlepolypeptid e chains,thu sneglectin gth einteractio nbetwee nadsorbe dchains .There ­ fore the results of these theories areprobabl yo flimite dvalu efo r practical situationswher e these interactions play certainly arol e asmentione dbefore . 125

Inprincipl e itshoul db epossibl et oexten dth eexistin gtheorie s forth eadsorptio no funcharge dpolymer so fi?o e (1974) and Scheutjens- Fleer, whichtak eint oaccoun tinterpolyme rinteractions ,fo rpolymer s exhibitingsecondar ystructure .Bu tthe nfirs tth edescriptio no fchai n stiffnessan dadsorptio no fcopolymer so fvariabl ecompositio nmus tb e incorporated inthes e theories.Th epolyelectrolyt e adsorptiontheor y of van der Schee (1984) isa nextensio no fth eRo ean dScheutjens-Flee r theory.Th eextensio nt oth eadsorptio no fcharge dchain swit hsecondar y structurean dth edescriptio no fcharge-induce d conformationaltransi ­ tions seems therefore attainable whenth e theoryfo runcharge dpoly ­ peptidesi scompleted .

6. 3. 2 Conformational aspects of poly-DL-aminoacids

Inthi sstud yexperiment swer eals operforme dwit hatacti cpoly-DL - lysine inorde r tocompar e the resultswit hisotacti cpoly-L-lysine . Some aspects ofth econformatio no fhighl ycharge dPL- Lan dPL-D Lar e already discussed in section3.1 .I nthi ssectio nI wil l focuso nth e conformationalpropertie so f (nearly)uncharge dPL-DL .Racemi catacti c PL-DL does notsho w ahelix-coi l transition inth e regionwher eth e transitioni nPL- Li sobserved .Th ereaso nfo rthi sma yb eunfavourabl e side chain interactions inth ePL-D Lmolecule .However , atver ylo w charge density PL-DL mayb epartiall y helical. Olander and Holtzer (1968) concluded from ultravioletextinctio ncoefficien tmeasurement s thatnearl yuncharge dpoly-DL-glutami caci di spartiall yhelical .Als o Gratzer (1967) came to thisconclusio n onth ebasi so fth ehypochro - micityobserve d inpoly-DL-glutami c acidb y Rosenheck and Doty (1961) andth edielectri cmeasurement so f Wada (1962). Howeverth eliteratur e on the subject is conflicting. For example Heitz and Spach (1971) argue thatbot h alternatingan drando mc opoly-DL-benzylglutamat eca n existi nth ehelica l form,whil e Hardy et al. (1971) asserttha talter ­ natingpoly-DL-benzylglutamat e formsa new ,a sye tuncertai nstructure . Ina late rpublicatio n Heitz et al. (1975) concludetha tstrictl yalter ­ natingpoly-DL-benzylglutamat e i.e.withou tan yracemizatio noccurring , canals osho wa ne wtyp eo fheli x (n_.) beside sth ea helix . Conformationalenerg ycalculation so f Hesselink and Scheraga (1972) indicate thatth eenergie s of left-an drigh thande d a helicalform s of alternating poly-DL-alanine arealmos tidentica lan dcomparabl et o thato fth e right-handed a-helical form ofpoly-L-alanine .Th esam e was found to be true for poly-DL-a-aminoheptanoicacid)a s amode l 126 foruncharge d poly-DL-lysine and some otherpoly-DL-aminoacids .Hes ­ selink and Scheraga concluded from this thatn o steric hindrancet o a helixformatio ni spresen tfo rthes eD Lcopolymers . Stulz et al. (1983) studied the tacticityan dsecondar ystructur e 13 of atactic poly-DL-leucines with FT-IR and CNM R cp/MAS (cross- polarization/magic angle spinning). They concluded from theirdat a thatth e lengtho fisotacti cblock si nthes epolyaminoacid swa s 1.5-5 monomericunits .Th esample swit ha D P> 5 0appeare dt ohav ea na-heli x contento fabou t50-60 %independen to fth etacticity . Itseem sver ylikel yfro mth eabov etha tals oatacti cpolyaminoaci d can show some secondary structure.Thi s canb e of relevance forth e precipitationbehaviou ro fthes epolyaminoacid s (seesectio n6.4.2) .

6.4 PRECIPITATIONO FPOLY-L-LYSIN EAN DPOLY-DL-LYSIN EA THIG H pHVALUE S

Iti swel l knowntha tmos tcharge dpolyaminoacid sprecipitat efro m aqueous solution at low degree of ionization.Th e insolubility of uncharged polylysine shows thatth ewel l known Flory-Hugginsinter ­ actionparamete r xmus t exceed thevalu e 0.5. Charged polylysinei s solublebecaus eo fth estron grepulsio nbetwee nth echains . The description ofth eprecipitatio no fpolyaminoacid sca nb ecom ­ plicatedb yth epossibl e formationo fcrystallin eo rpseud ocrystallin e precipitates,th eoccurrenc eo fwhic hma yb econnecte dwit hth eexten t of secondary chain structure.I nthi srespec ta ninterestin gquestio n iswhethe ro rno tther ear edifference si nprecipitatio nbehaviou rbe ­ tweenpoly-L -an d (atactic)poly-DL-lysine . Theprecipitatio nbehaviou ro fpoly-L-lysin ean dals opoly-L-glutami c acid hasbee n studiedbefor e bij Puett and Ciferri (1968) asa func ­ tiono fsal tconcentration ,p Han dtemperature .The yuse dthei rPoten ­ tiometriean doptica ltitratio nresult s (Ciferri et al. 1968) tojudg e the position of the precipitation points. Zimmerman and Mandelkern (1975, a, b) studied theprecipitatio n time,i.e . thetim erequire d to observeprecipitatio n from aqueous solution after the adjustment ofcertai nconditions ,o fpoly-L-glutami c acida sa functio no fth epH . They showed that there are two distinctly differentprecipitatio n regions,whic h depend on temperature,concentratio n andpH .Als oth e degree ofhomogeneit y ofth epolyme rsample splay sa nimportan trole . Inon e of these regions,th ea region ,a variet yo fphysica lproper ­ ties demonstrate thatprecipitatio noccur swithou tan yconformationa l 127 change,whil ei nth es ocalle dß regio na majo rconformationa ltransi ­ tionoccurs . Poly-L-lysine displays manypropertie s thatar e similar tothos e of PGA-L. Both areknow n to form gels under appropriate conditions andbot h form ßprecipitates ,thoug hi ti sno tknow nye twhethe rals o inpoly-L-lysin etw odistinc tprecipitatio nregion sexist. -

6.4.1 Experimental

Theprecipitatio nexperiment swer eperforme dwit hth eunfractionate d poly-Lan datacti cpoly-DL-lysin esample sobtaine d fromth eSigm achem . corp.an ddescribe dbefor e (section3.1) .Al lothe rchemical suse dwer e ofpr oanalys equality .Th ewate ruse dwa shig hqualit ydeionize dwate r obtainedfro ma Millipor esupe rQ wate rpurificatio napparatus . Theprecipitatio nmeasurement swer eperforme d similar toth epro ­ cedureo f Zimmerman and Mandelkern (1975a) andru na sfollows .A stoc k solutiono fPL-D Lo rPL- Lo f1 kg. m in0. 1M NaB -3 rp H6 ,containe di n aclose dtitratio nvesse lunde rN ~atmosphere ,wa skep ta tth edesire d temperaturei na wate rbath .Th esolutio nwa skep thomogeneou sb ymean s ofN - bubbling throughit .Th eN ~wa sCO.-fre ean dwate rvapou rsatu ­ rated.The n about2 M NaO Hwa s slowlyadde dt oth eP Lsolutio nunti l thedesire d pHwa s reached.A t thispoint ,a portio no fth esolutio n usually 5.0c m wa3s removed andplace d ina well-close d markedtes t tube kept atth e same temperature.Additio n ofNaO H toth estoc kP L solution was continued until ahighe rp Hwa s reached and aportio n ofth e solutionwa s again removed. Thisproces swa s continuedunti l a sample ateac hdesire dp H from the lowestt oth ehighes twa sob ­ tained.Th etim ea twhic hth ep Ho feac hsampl ewa sadjuste dwa snote d andconsidere d astim ezer ofo reac hsample .A tregula rtim einterval s rr A nvvi O theOD. A1 0 mm from an aliquot•* (0. 5c \ m )o feac ih samplewa sthe cn mea - suredwit h aBeckman n360 0spectrophotometer . Inthi swa yth eOD» 0 couldb eplotte d asa functio no ftim efo reac hp Hvalue .Calibratio n ofth ep H electrodes (Schottcombine d glass-Ag/AgCl)occurre da tth e measuring temperature with titrisol buffers (Merck)p H7.0 0 ±0.02 ; pH10.0 0± 0.0 5 andp H11.0 0± 0.05 .Th ep Hmeasurement swer eperforme d witha nElectrofac t3606 0pH/m Vmeter . 128

6.4.2 Results and Discussion

When turbid PL solutions,i.e . afterphas eseparation ,ar elooke d at,th eturbi dsolutions ,especiall ya thig hP Lconcentration ,appeare d tohav e amor e or lessgell ycharacter ,s on oflak yprecipitate sar e observed. The results of theprecipitatio n measurements performedb yu sar e showni nfig .6.1 ,6. 2 and6.3 . Infig .6. 1 themeasure dO Di splotte d againsttim ea t303.1 5K an d313.1 5K fo rdifferen tP Lsample san ddif ­ ferentp Hvalues .I nfig .6. 2 theO Dafte ra chose nincubatio ntim ei s plottedagains tth ep Hfo rPL- Lan dPL-D Lsample sa t303.1 5an d313.1 5K , andi nfig .6. 3 thetim eafte rwhic ha narbitrarel ychose nturbidit ywa s reached isplotte d againstth epH ,fo rth esam eP Lsample sa si nfig . 6.1 and6.2 .Th eprecipitatio ntim eplotte di nfig .6. 3 mayb ecompare d withth eprecipitatio ntime sobserve dvisuall yb yZimmerma nan dMandel ­ kerni nthei rexperiment so nPGA . Determinations of the amountprecipitate d at29 3K i na nanalogou s waya sth eamoun tP Ladsorbed ,wil lb ereporte di nsectio n6.5.3.2 . Above pH11. 0bot h PL-L (DP192 )an dPL- L (DP1683 )showe da nap ­ preciable precipitation after 16h .Thi si si nlin ewit hth efinding s of among others Ciferri and Puett (1968) who foundprecipitatio no f PL-L at29 8K abovep H11.3 .Howeve r inon eserie so fmeasuremen tw e did not find anyvisua l turbidity ofPL- L (DP1683 )abov ep H11.0 . Thereaso nfo rthi si sno tclea rt ome . Precipitation measurements with aPL- L (DP240 )sampl ewhic hap ­ peared to have about 1residua lblockin g group (as judged fromth e UV sprectrum)pe r PL chain showed aprecipitatio nbehaviou rmarkedl y deviating from thepur e samples:Th ep H atwhic h theprecipitatio n started was about 1p H unit lower than theprecipitatio np H ofa correspondingP Lsampl ewithou tblockin ggroups . Zimmerman and Mandelkern (1975a) showedtha tther eexis tquantitativ e differences inth eprecipitatio nbehaviou ro funfractione dPGA( M/ M=1.1 ) andfractionate dsample s (M/ M =1.0), althoughals oth eunfractionate d PGA samples show thetw oprecipitatio nregion smentione dbefore .Thu s despite the facttha tou rexperiment swit hP Lhav ebee ndon ewit hun ­ fractionatedsamples ,valuabl einformatio nconcernin gth eprecipitatio n characteristicsca nb eobtaine d fromthem . Incontras twit h thegradua l increase inturbidit ywit h timefo r thePL- L samples,th ecurve s forPL-D Lsho winitiall ya rathe rstee p increase in turbidity afterwhic h further increase ismor e orles s 129

- T= 313 1 K ® : T=313. 1K © 0.1 M NaBr. .- '2 09 .12f- 0.1M NaB r "30 OD ^^~ „~ 11.59 11.36 ^^^ 11.12

o- 1090

. A- 10.71

04 .08 .12 16 .20 .24 { ~ 10 si 6 ^^»- 'P 39 , t(10s ) ^-.0—::4 08 .12 .16 .20 24 t(106s)

OD

Fig. 6.1 Precipitation of polylysines as a function of time at various pH values and two temperatures. Precipitation was monitored by measurement of the absorbance at 550 nm; optical pathway: 10 mm. -3 e-, = 1.00 kg.m ;p H values are indicated. a. PL-L (DP 192);b . PL-DL (DP 308);c . PL-L (DP192) ; d. PL-DL (DP 308);e . PL-L (DP 1683); f. PL-L (DP 1683). 130 linear (compare fig.6.1 a and b). However,a t313.1 5K theinitia l slopes of the curves is almost thesam efo rbot hPL- Lan dPL-D L(se e fig.6.1 c and d). At longer incubation times the differencebetwee n the curves ofPL- Lan dPL-D Lobserve da t303.1 5K i salmos tabsen ta t 313.15K . The observed increase inturbidit ywit htim eca nb ecause dbot hb y growtho faggregate san dth eagglomeratio no faggregates .Therefor ea n increasei nturbidit ydoe sno tnecessaril ymea na nincreas eo fth epre ­ cipitatedamoun twit htime . Measuremento fth eP Lconcentratio nafte rremova lo fth eprecipitat e by centrifugation showed thata nincreas ei nturbidit ycorrespond st o anincreasin g amount ofprecipitate d PL-L (DP192 )o rPL-D L (DP308 ) at least at31 3K .A t this temperaturean dp H10. 7th efractio npre ­ cipitated was about30 %an d 50%fo rPL-D L andPL- Lrespectively .A t high pH (above 11.5)th eprecipitate d fractionwa s 80-85% forbot h PL-L and PL-DL.Th equantitativ erelatio nbetwee nO Dan dprecipitate d amounti sver ylikel yt ovar yfo reac hsample . Theprecipitatio ncurve so fPL- L (DP1683 )sho wmuc hhighe rturbid ­ ities than those ofPL- L (DP192) . At313.1 5K th emaximu mturbidit y isreache d much fastertha ni nth ecas eo fPL- L (DP192) .Th eprecip ­ itated amountPL- L (DP1683 )a t308. 9K appeare dt ob econstan tabov e pH11. 3an damounte d95% .Th eobserve ddifference sbetwee nPL- L (DP192 ) andPL- L (DP1683 )ar e in linewit h the expectations forincreasin g phaseseparatio nwit hincreasin gmolecula rmass . The differences observed by us between the OD-time curves of PL-L and PL-DLpoin t inth e direction of differences inth egrowt h kinetics ofth e aggregates.Thes edifference sca nonl yoriginat efro m thedifferenc ei nstereoregularit ybetwee nPL- Lan dPL-DL . From fig.6. 2 one can see thatth epH ,wher e theprecipitatio n of PL starts at 303.15K is 10.9 ± 0.1 forPL- L (DP192 )an dfo r PL-DL (DP308) , and somewhatlowe r (pH= 10.6 )fo rth ehig hmolecula r mass PL-L (DP1683 ) as expected for aphas e separationphenomena . At 313.15K the initiation of the precipitation starts atclearl y lowerp Hvalues :p H10. 5 ± 0.1 forPL- L (DP192 )an dPL-D L (DP308 ) and pH 10.1 ± 0.1 forPL- L (DP1683) . This decrease in initiation pHwa sals ofoun db y Puett and Ciferri (1968). As these authors showed,th edecreas ei ninitiatio np Hi sessentiall y anelectrostati c effectcause db yth edecreas ei np K withincreasin g temperature. Inth e temperaturerang einvestigated ,th eprecipitatio n takesplac ea tth esam echarg edensit yan dtherefor ea tlowe rp Hvalue s 131

T= 303. 1 K 0.1 M NaBr. /3 p OD / .3 / -

/

.2 1 D / / .1

/ / A

1 (TTÄ—TWln- °:S£A- 10 pH

Fig.6.2. 1 Precipitation ofpoly-L-lysin ean dpoly-DL-lysin ea sa functio no fp Ha t T =303.1 5K . 3 cpL =1. 0 kg.m" ;0. 1M NaBr . 1.PL- L (DP192 ) t= 9 0h ;2 .PL-D L (DP308 ) t= 9 0h ; 3.PL- L (DP1683 )t = 75h .

with increasing temperature.Unfortunatel y inou r caseth edegre eo f dissociationcanno tb ecalculate daccuratel yenoug hfro mth eavailabl e data,becaus eth ecorrespondin g titrationdat aar elacking . Iti sremarkabl e thatwithi nexperimenta lerro rth einitiatio no fth e precipitation occurs atth e samep Hvalu efo rbot hPL- Lan dPL-D La t 303.15K a swel la s313 .1 5K .A t313.1 5K th eshap eo fbot hcurve si s also the same over thewhol ep H region.A t theprecipitatio np Hth e degreeo fdissociatio no fPL- Lan dPL-D Lca nb edifferent .Thi si sbe ­ cause of apossibl edifferenc ei np K ofbot hpolyelectrolyte s (see section6.6.4.2) . The turbidity ofPL- L (DP1683 )exceed sgreatl yth e values found for the low molecularmas s samples as statedbefore . Probablybigge raggregate sar eformed . 132

T=313- 1 K 0.1M NcBr

Fig.6.2. 2 Precipitation ofpoly-L-lysin ean dpoly-DL-lysin ea sa functio no fp Ha t T =313.1 5K . 3 cpL = 1.0 kg.in" ;0. 1 MNaBr . 1.PL- L (DP 192) t= 7.5 h;2 .PL-D L (DP308 ) t= 8. 0 h; 3.PL- L (DP1683 ) t= 1.0h .

Anotherquestio ni swhethe rther ear ealread ysolubl eP Laggregate s presentbefor eth eobserve dprecipitatio npoint .Thi sca nb eo fimpor ­ tance for the adsorptionpropertie s ofPL .Som e indications forth e existenceo fsuc hsolubl eaggregate sa tp H> p K (T= 28 8K )wer efoun d by Propokova and Ciferri (1972) from theelectrokineti cbehaviou ro f PL-L solutions athig hpH .Th e resultsma yhoweve rb ea nartifac to f themetho d (Propokova and Ciferri, 1972). Athig hp Hvalue san dT > 29 8K th eß-for m isth ethermodynamicall y most stable conformation ofPL-L .A t27 8K the a helix isth emos t stable comformation at highp Hvalue s (Davidson and Fasman, 1967); Pederson et al., 1971). Ifpoly-L-lysin e inth ecoi lconformatio ni s brought onto highp Hwit hbas e at32 2K ,th epolyaminoaci d assumes directlyth eß conformatio nwit hn odetectabl ea helica lintermediate . 133

Between 298 K and 322 K addition ofbas e to 'coil' PL-L gives first an a helical PL-L polymer followed by slow conversion to the ß structure (via a coil-like intermediate). Then the formation of ß structure is followed by precipitation with conservation of the ß structure (David­ son and Fasman, 1967). Incontras twit h the result of Zimmerman and Mandelkern (1975a) onPGA , theprecipitatio n time -p H curves ofpoly-L - lysine and also poly-DL-lysine (fig. 6.3.1 and 6.3.2)d o not show any evidence for the existence of two distinct precipitation regions.Poly - L-lysine shows only what Zimmerman and Mandelkern denote as ßprecipi ­ tation and no a precipitation, the latter being nucleation controlled. The above is concluded from the fact that there is no abrupt change in thep H dependence of the precipitation in the investigated pH region and also from the gradual decrease inprecipitatio n time when the tem­ perature is increased from 303 K to 313 K. Apparently in the case of PL-L the rate at which the random coil intermediate isgenerate d is greater than the rate of nucleation over the whole pH region, resulting in aprecipitat e inwhic h the PL-L molecules are in the ß structure.

i • i ' i ' i I ' 1 ' 1 T=303. 1 K .6 • V 0.1 M NaBr. V) \ • 9 5 . 4 '" 3-\V V x - .3 • V v\ i • ' .2 - \V- .1

1 11.0 11.4 118 12.2 12.6 13.013. 4 pH

Fig.6.3. 1Tim edependenc eo fth eprecipitatio no fpoly-L-lysin ean dpoly-DL-lysine . 3 cpL= 1. 0kg.m" ;T = 303.1 5K ;0. 1M NaBr . 1.PL- L (DP192 ) t(0D^°n m =0.015 )v spH . 10m m 2.PL-d L(D P308 )t ( 0.08) vspH . 3.PL- L(D P1683 )t ( 0.16) vspH . 134

1 i • i • i • 1 • 1 ' 1

.24 1 T = 313.1 K -

V) • o -20 - - ""*' • .16 \2 - X

.12 • \ .08 - - \\ .0«

- v\ L X. _ 1 1 10.• A. 10.i 8 . 11.i 2 .11. i-6 —12. 0 12.412. 8 pH

Fig.6.3. 2 As fig.6.3. 1 buta tT = 313.1 5K . 1.PL- L (DP192 ) t(OD^°n m =0.065 )v sp H 10m m 2.PL-D L (DP308 ) t(0D 0.08) vspH .

Bothth ea- ß conversionan dprecipitatio no fPL- Lhav equalitativel y thesam ep Han dtemperatur edependenc e (above29 3 K), i.e.th erate so f bothprocesse s are increasedwhe nth ep Ho rtemperatur ei sincrease d (see Davidson and Fasman (1967) forth ep Han dT dependenc eo fth ea- ß conversion).Thi si sno tsurprisin gbecaus ei nbot hprocesse sth esam e interactions play arole .A salread yconclude d fromth eprecipitatio n time-pH curves at30 3K and 313K , thea- ß conversion ratemus tb e fastertha nth eprecipitatio n rate.A quantitativecompariso no fth e precipitation times foundher ean dth ea- ß conversionrate sdescribe d inth eliteratur ei sno tpossibl ea tth emoment ,becaus ebot hprocesse s depent strongly on suchexperimenta l conditions asP L concentration molecularmass ,an delectrolyt econcentration . Theprecipitatio n times ofPL- Lan dPL-D Ld ono tdiffe rmuch ,als o thedecreas e inprecipitatio n timewit h increasingtemperatur eo rp H lies inth esam erang efo rPL- Lan dPL-DL .Althoug hPL-D Lma yb epar ­ tially helical atver ylo wcharg edensit y (see6.3.2) ,a- ß conversion asi ttake splac ei nPL- Li sno tver ylikel yt ooccu ri nPL-DL .There ­ foreth e similarity inth eprecipitatio nbehaviou ro fPL- Lan dPL-D L supportsth eearlie rconclusio ntha ta- ßconversio nvi aa ncoi linter ­ mediate is faster thanth eprecipitatio n stepitsel fi nPL-L .Whe na 135 coil conformation already existsbefor e precipitation asi sprobabl y the casewit h PL-DL, the initial precipitationrat eca nb ehighe ra s canb e seen from fig.6.1.6 .Howeve r at313.1 5K thi s differencei n initial precipitation ratebetwee nPL- Lan dPL-D Li spracticall ydis ­ appeared as canb esee nfro mfig .6.1. C and6.1.d .Probabl ythi si sa consequence of the facttha t thePL- L a helix isunstabl e atthi s temperature. Now thequestio n ariseswhethe rth estructure so fth eprecipitate s ofPL- Lan dPL-D Lar eth esam eo rdifferent .Poly-DL-lysin ecanno tfor m crystalline precipitates because iti sa natacti cpolymer .Th epreci ­ pitatewil lb eamorphou sprobabl ya concentrate d liquidphas ei nequi ­ libriumwit ha ver ydilut eone .O nth econtrary ,poly-L-lysin ei siso - tactic and can form a crystalline precipitate (solid-liquid phase separation)i nwhic hth epolylysin emolecule sar ei nth eß conformation . The fact that theprecipitatio npH san dtemperatur edependenc eo fth e precipitation areno tver ymuc h apart forbot hpolylysines ,suggest s thatth eprecipitat eo fpoly-L-lysin e isals olargel yamorphous . To unravel theprecipitatio n behaviour ofPL- Lan dPL-D Lfurther , more direct information about thePL- L andPL-D Lconformatio ni nth e precipitate and the degree of order in theprecipitat e isneeded . This information canb e obtained for examplewit htechnique ssuc ha s Laser Raman spectroscopy andX-ra y diffraction.Als omuc hmor ein ­ formation onth eprecipitatio n process itself isneeded . Especially thevolum efractio ndependenc ea sa functio no fpH ,sal tconcentratio n and temperature should be determined inorde rt omak ei tpossibl et o constructphas e diagrams andt oallo wfo ra mor ecomplet einterpreta ­ tion. The precipitation data discussed above arenevertheles sver y valuable forth e interpretation of adsorption experiments atlo wP L chaincharg edensity .

6.5 ADSORPTIONO FPARTIALL YCHARGE DPOLY-L-LYSIN EAN DPOLY-DL-LYSIN E ONPOLYSTYREN E

6.5.1 Materials

Thepoly-L-lysin e and atacticpoly-DL-lysin esample swer eth esam e asdescribe dbefore .Th eP Slatice suse dar edescribe d insectio n3.2 . All other chemicals usedwer eo fanalytica lgrade .Th ewate ruse dwa s distilledonc eo rconductivit ywate robtaine dfro ma Millipor eSupe rQ waterpurificatio nset-up . 136

6.5 . 2 Determination of the PL adsorption on PS particles as a function of the pH in solution

Adsorption experiments at high pH performed without the useo f buffers which could influence the adsorption, arecomplicate db yth e disturbing influence of atmospheric CO.o n the solutionp Hvalue . Therefore contact of thehig hp HPL/late xsolution swit hth eai rwa s avoideda smuc ha spossible . Theamoun to fbas eneede dt oreac ha desire dp Hwa sfirs tcalculate d ona basi so fth etota lamoun tP Lpresen tan dproto ntitratio ndat ao f PL.Th ep Hwa smeasure d (only)afte requilibratio n (16h )an dcentrifu - gationo fth esample .Immediatel yafte ropenin go fth ecentrifug etub e a sample for theP Lconcentratio ndeterminatio nwa stake nan dinstan ­ taneouslythereafte rth ep Hwa smeasured . Measurements with PL-L and PL-DL wereperforme d simultaneously. The samewa s done for adsorption seriesa tdifferen tioni cstrength . Inthi swa yth eeffec to fsystematica lerror sca nb esuppressed . The preparation of samples for adsorption series athig hp Hwa s 3 done as follows:T o a8 c m polyethylene tubewit h screw capknow n amounts of water, PL stock solution (pH6 )an dNaB r solutionp H6 were added.The n aknow n amounto f0. 1M NaO Hwa sadded ,immediatel y 3 followedb yth eadditio no f1. 0c m PSlate x (pH 6). Thetub ewa sthe n closed immediately and rotated end over end for 16hour s toattai n adsorption equilibrium. The total volume of the samplewa s always 3 5.0c m .Th e PL concentration determination was performed asde - 3 scribedbefor e (chapter4 )excep tfo ron edifference :5 0m m 0.1M HC l was added tobrin g the sample ata p Hbelo w6 ,i.e .t oobtai nfull y chargedpolylysine . Adsorption samples with different starting concentrations ofP L wereprepare d ina few separate seriest ojudg eth evariatio no fth e adsorbed amount with the equilibrium PL concentration.Th e average degreeo fdissociatio no fP Li na nadsorptio nsampl eca nb ecalculate d fromth eadde damoun to fbas ean dth emeasure dequilibriu mpH . Besidesth eadsorptio nmeasurement s asa functio no fth eequilibriu m pH,adsorptio nisotherm swer eals odetermine da sfollows .Befor emixin g separatestoc ksolution so fPL ,NaB ran dlate xwer ebrough tt oth ede ­ sired highp Hwit hNaOH .The n known amounts ofthes esolution swer e pipettedint opolyethylen etube sa sdescribe d above,t oobtai na serie s ofincreasin gP Lconcentration .Th esubsequen tsampl ehandlin gwa sth e samea sdescribe dabove . 137

6.5.3 Adsorption isotherms of poly-L-lysine and poly-DL-lysine at high pH

In section4.3. 4 the adsorption characteristics of fully charged poly-L- and poly-DL-lysine were discussed.No w the adsorption iso­ thermso fthes epolypeptide s athig hp Ho nnegativel ycharge dP Slate x particles willb edeal twith . In fig.6. 4 characteristic adsorption isotherms ofPL.HBr- L andPL.HBr-D L adsorbed onP S atp H10. 7± 0. 1 areplotted . Forcompariso n anisother m ofPL.HBr- L atp H6 i sals o given. All isotherms are of thehig h affinity type,althoug h they are somewhatmor e rounded athig hpH . Iti sno tcertai nwhethe rth e plateau values found here are reallyplateaus .A thighe r PLvolum e fractionsphas eseparatio ni spossibl eresultin gi nmultilaye radsorp ­ tion. The observed plateau adsorption ofbot hPL- L andPL-D L isabou t 4 times higher than at a = o andno tmeasurabl e different forPL- L andPL-DL .Th e somewhathighe r adsorptionvalue s observed forPL-D L athighe requilibriu mconcentration s iscause db yth eaccidenta lhighe r pH (within0. 1p Hunit )o fth ecorrespondin gadsorptio nsamples . Theplatea uadsorptio nvalue sobserve dar eo fth eexpecte dmagnitud e foruncharge dpolymers .Th elarg eincreas ei nadsorptio nobserve dwhe n thechai ncharg edensit yi sdecrease d iscause dby : (i)Decreas e of theelectri crepulsio nbetwee npolylysin esegment si n the adsorbed layer.Th e effect is similar to theeffec to fadditio n ofelectrolyt ea sdiscusse d insectio n4.3.6 . (ii)Decreas eo fth esolven tquality . The amounto f adsorption atp H10. 7 isabou t1. 5time sth emonolaye r coverage while at pH6 the adsorption ismuc h less thanmonolaye r coverage.Henc e the fraction of segments in (short)loop s andtail s ismuc hhighe ra thig hpH . Theincrease droundednes so fth eisotherm sa thig hp Hca nb edu et o amor epronounce dpolydispersit yeffec twhe nth esolven tqualit yi sde ­ creased,bu tals ot ovariation so f awit hsurfac ecoverag ea stheoret ­ ical calculations of Evers (1984) forwea k acidpolyelectrolyt e ad­ sorption suggest.Th e facttha t also athig hp HPL- Lan dPL-D Lhav e verysimila radsorptio ncharacteristic sshow stha ti si sno tjustifie d todra wconclusion s aboutth e secondary structureo fPL- Li nth ead ­ sorbedlaye ro nth ebasi so fplatea uadsorptio nvalue so fPL- Lonly . The facttha tPL- Lan dPL-D Lhav eth esam eadsorptio ncharacteris ­ tics athig hp Hshow stha ti nthi scas eth esecondar ychai nstructur e 138

T=296 K 0.01 M NaBr. 2.4 4 pH 10.710.1 E Ol £ 2.0 -

PL-DL 1.6

- PL-L 1.2 f

firA_A—A—A—A -A PL-L pH6

100 200 300 400 /g.m"

Fig. 6.4 Adsorption isotherms of poly-L-lysine.HBr (DP 240) and poly-DL-lysine.HBr (DP 240) at high pH on PS latex M (0 = -60 mCm" ) electrolyte 0.01 M NaBr. A corresponding adsorption isotherm at pH 6 is also drawn.

has no influence. Apparently the solvent interaction parameter and adsorption energy per segment are about the same for the two PL forms and these parameters are dominating the adsorption. That x is about the same for PL-L and PL-DL follows from the similarity in precipitation behaviour, v s, erccf of PL-L and PL-DL will be the same when there is a preferential adsorption of coil segments. As coil segments are on the average positively charged and the surface is negative, this is likely to be the case. It will be clear that more information is needed to clarify the structure of adsorbed PL. To this end proton titrations- of adsorbed PL-L and PL-DL have been performed. They will be discussed in section 6.6.

6. 5. 3. 1 Adsorbed amount as a function of the solution pH

In figs. 6.5, 6.6 and 6.7 the amounts of PL.HBr-L and PL.HBr-DL adsorbed as a function of the solution pH are plotted for various poly- lysine samples and salt concentrations. The adsorptions in these figures 139

are aboc- plateau adsorption, i.e. they are maximum adsorptions or are between the middle of the rounded part in the isotherm and the maximum adsorption. The increase in adsorbed amount with increasing pH is clear­ ly demonstrated. The shape of the curves is at least qualitatively as theoretically expected, as will be shown in chapter 7. The adsorption as a function of the pH is reversible, that is: after adsorption at pH 11.00 we found desorption to occur when the pH was lowered to 6 down to the adsorption value at pH 6. As one can see from fig. 6.5 the curves for PL-L and PL-DL are not measurably different, also not in the helix-coil transition region. Again it is seen that at any a the secondary structure of PL is not important in determining the adsorbed amount.

Fig. 6.5 Adsorption ofpoly-L-lysine.HB r Fig.6. 6 Adsorption ofpoly-DL-lysine.HB r 2 (DP192 )an dpoly-DL-lysine.HB r (DP240 ) (DP240 )o nP S (latexM 4; OQ =-4 2mC.m" ) asa functio n ofp Ho nP S (latexM, ; asa functio no fpH . 1.3.10 " MNaBr ; -2 a = -42mC. m ). 2. Î.IO"2M NaBr ;3 . l.io"1M NaBr . o 1.PL-L ,0.0 1 MNaBr ;2 .PL-DL , 0.01 MNaBr ;3 .PL-DL ,0. 1 MNaBr . 140

1 r- i i I 1 T=294K 1.8 7 •J DPL.HBr-P 1683 L0 /° / , E P ' ói 1.6 " E / / " 1.« / /

1.2 / VA 10 o / PLHBr- L ' < 8 - ƒ j DP 192

6 " // ' ' M 11 o ° /1 2 / i

10 11 12 pH

Fig.6. 7 Molecularmas sdependenc e ofth eadsorptio n (open symbols)o fPL.HBr- La s -2 a function ofth epH .0.0 1 MNaBr ;late xM , (a =-4 2mC. m ). Filled symbols:precipitatio n ofPL.HBr- La sa functio n ofth ep H inth e absence ofadsorbent .

Theeffec to flo wmolecula rmas selectrolyt eo nth eadsorbe damoun t isclearl ypresen t atlo wp Hbu talmos tabsen ta thig hpH .Thi sshow s that coulombic interactions inadsorbe dP L athig hp H areo fmino r importance asexpected .A t stillhighe r saltconcentration showever , specific dehydration effectsma ybecom e important indeterminin gth e adsorption andmultilaye r adsorption (i.e.precipitation )properties , ofPL .Althoug h theeffec to fth eioni cstrengt hi ssmal la tp H11.0 , theamoun tadsorbe da tconstan tp Hi ssomewha tlowe ra t0. 1M NaB rtha n atlowe rioni cstrengt h (seefig .6. 5 and6.6) .Thi sreverse dsal tef ­ fecti sdu et oth efac ttha ta tconstan tp Hth edegre eo fdissociatio n decreaseswit hincreasin gioni cstrength . In fig.6. 7 the r-pH curves and thecorrespondin g precipitation curves areplotte d forPL- L (DP192 )an dPL- L (DP1683) . Whereasa t lowp H (a=0 ) therei sn omolecula rmas sdependenc eo fth eadsorptio n 141

above DP192 , this dependence is clearly observed athig hpH .Th e amount adsorbed of PL-L (DP1683 )a tp H1 0i s significantly higher -? -2 than thevalu e forPL- L (DP192) ; 1.5mg. m and 1.2mg. m respec­ tively.Thi si sa consequenc eo fth elowe rsolven tqualit ya tthi spH . Abovep H10. 3 forPL- L (DP1683 )an dp H10. 9fo rPL- L (DP192 )multi ­ layer adsorption (i.e.phas e separation)occur s atabou tth esam ep H valuesa sphas eseparatio nstart si nsolutio n (seeals osectio n6.5.3.2) . Foruncharge dpolymer ssuc ha behaviou raroun dth eprecipitatio npoin t is expected (Silberberg, 1972). The shape of the adsorption curves showni nfig .6. 7 isexpecte dtheoreticall y forth eadsorptio no fwea k polyelectrolytes froma ba dsolven t (x> 0.55 ) (Evers, 1984). At 293K and 0.01M NaBr thep Hvalue swher eth eprecipitatio no f PL-LD P168 3an dD P19 2start sar ealmos tth esam ea sth evalue sfoun d by us at30 3K and 0.1M NaBr .Thi si sa tleas tqualitativel ydu et o two compensating effects:A decreas eo fth eprecipitatio np Hwit hin ­ creasing temperature asdiscusse di nsectio n6.4. 2 anda nincreas eo f theprecipitatio np Hwit hincreasin gelectrolyt econcentratio n (Puett and Ciferri, 1968). 6. 5.3.2 Phase separation in solution and near an interface

As shown in fig.6. 7 phase separation insolutio nan di nth epre ­ sence of an interacting interface sets ina tabou tth esam epH .How ­ ever, themea n degree of dissociation â ofP L inth epresenc eo fP S latex can differ from that inth e solution at theprecipitatio npH , because the averagep K ofadsorbe dP Li shighe rtha ntha to fP Li n solution atth e sameoveral lconcentration .Th ecalculatio no fä fro m the amountNaO H added,p H and totalamoun to fP Lpresen ti spossibl e butno taccurat e enought oallo wfo rconclusion sabou tdifference si n â atth eprecipitatio np Hi nth etw ocases . Becausea o fP Li nth eoute radsorptio nlayer si sno tver ydifferen t froma i nth ebul k (aswil lb eshow ni nsectio n6.6.4.2) ,n oappreciabl e precipitation onth e surfacewil loccu rwhe nth econdition sfo rphas e separation inth ebul kar eno tfullfilled . Silberberg (1972) analyzed theoreticallyth emultilaye radsorptio no funcharge dpolymers .H econ ­ cludedtha tonl ya tclos et o8 condition s (x/x3= 0.99 )o ra P Lvolum e fractioni nth ebulk , Äclos et oth ecritica lon ea tx = X« ,abou ton e additionalpolyme rlaye rcoul db eformed .Furthe rgrowt hoccur sonl ya t worse thana conditions .Howeve rthi si sprobabl yonl yvali dfo rver y longchains . Inth ecas e of shorterchain s (DP< 100 )thicke rlayer s 142 canb e formed atx value s close toth e criticalon e (van der Schee, 1984). Itwil l be clear thatcharg einteraction scomplicat eth epic ­ ture inth ePL-late x system.Nevertheles s theresult sshow nher ear e notconflictin gwit hth eprediction so fSilberberg . Inth eabsenc eo fprecipitatio nth eresult si nfig .6. 7 suggestth e existenceo f (pseudo)plateau svalue s forth eadsorptio no fP Lo fabou t _2 1.3 and1. 5mg. m forD P19 2an dD P168 3respectively .A ssai dbefore , thehig h adsorbed amount compared withp H6 mus tb e duet oa highe r fraction of segments in loops and tails.Theoretica lprediction sfo r theadsorptio no fwea kpolyelectrolyte s froma goo dsolvent ,a sa func ­ tion ofpH ,sho wa maximu m inth ecas eo fcharg econtras tbetwee nad ­ sorbents and adsorbate (Evers, 1984). Such amaximu m isno tobserve d here,probabl ybecaus eprecipitatio noccurs . At29 4K th ea-helica lstructur ewhic hi spresen ti nsolutio nabov e pH1 0 isslightl y morestabl etha nth eß structure ,an dprecipitatio n withoutconversio n toth e ßstructur emigh toccu ri nth epresenc eo f alreadyadsorbe dPL .Ther ema yb edifferen tkineti cpathway soperativ e inth e case ofprecipitatio n from solution or inth epresenc eo fa n adsorbing interface. Inth elatte rcas eonl ygrowt ho fa nalread yad ­ sorbed layerha s tob e occur,i nth e former caseals o (homogeneous) nucleationca npla ya role .

6.6 CHARACTERIZATIONO FFRE EAN DADSORBE DPOLYLYSIN EB YPOTENTIOMETRI C PROTONTITRATION S

6. 6.1 Introduction

Potentiometrieproto ntitratio ni sa nofte nuse dmetho dfo rstudyin g the chargedependen tconformationa l transitionsi nmostl yaqueou sso ­ lutions of ionizablepolyaminoacid s (seeals o section3.1.2) .Poly - L-glutamicaci dan dpoly-L-lysin ear eth emos tstudie dones . Studies concerning the (thermodynamic) characterization of con-• formationaltransition si npoly-L-lysin edissolve d inaqueou ssolvent s bymean so fproto ntitration shav ebee nperforme dby : Hermans, J. , 1966; Puett et al., 1967; Ciferri et al., 1968; Ptitsin, 1971; Grourke and Gibbs, 1971; Pederson et al., 1971; Barskaya, 1971; Conio et al., 1974; Cosani et al. , 1974 and Tseng and Yang, 1977. Generally,comparabl e proton titration data of these authors are inreasonabl eagreement . Theintrinsi cp Kvalue sfoun db ythes eauthor si sgenerall ylowe rtha n those reported here.Thi s is inherent toth eextrapolatio nprocedur e 143 to a - 1use db y these authors,i.e . thevalue sreporte d inth eli ­ terature areno tbase d on determinations ofp K values ofa monome r o analog as is done here.Proto n titrations of atacticpoly-DL-lysin e have been reported by Chou and Scheraga (1971). Proton titrations ofpoly-L-aminoacid s in interactionwit hsolid - liquidinterface so rmembrane sar eno treporte dye ti nth eliterature . Inthi s study theproto ntitratio ntechniqu ewa suse dt ocharacteriz e theconformatio no fadsorbe dpolylysine . 6.6.2 Principle of the method

Because of thehig hcharg edensitie swhic hca nbuil du po na poly - electrolytechain ,th etitratio nbehaviou ro fa wea kpolyaci do rpoly - basei naqueou ssolutio ndeviate s fromtha to fth ecorrespondin gmonome r analogs.Th edissociatio nequilibriu mo fa nisolate dammoniu mgrou pi n PLca nb ewritte nas :

+ R- NH * îR - NH 2 +H (6.5)

Thethermodynami J cdissociatio nconstan tK ia,sthe m n

(R-NH,)(H+) Kam = ^-+ (6'6) a'm (R-NH3) whereparenthese sdenot eth eactivit yo feac hspecies . Since the activities ofth especie sR-NH ,an dR-NH -ar eno tknown , butonl ythei ranalytica lconcentration s (inbrackets )th edissociatio n constantK o,definem db y J

[R-NH](H +)

K m = ^— (6.7) °'m [R-NH+] isuse d instead ofK , .whic hi sa tru eintrinsi cconstant .Th em i n a,m the subscriptdenote s themediu mi nwhic hth egroup sare .O nth econ ­ traryK isno t atru e intrinsicK ,sinc ei ti sstil ldependen to n the ionic strength and the species concentration. Inprincipl eth e thermodynamic constantK valid for -NH,group so fP Li na certai n chemical environment canb e obtained by extrapolationo fK values r o,m to zero ionic strength and zero concentrationo fth ecomponen tunde r investigation. 144

Equation 6.7 canb ewritte nas :

pH =p K + log rr^— (6.8) o,m 3 1-a inwhic h a is the degree of dissociation of the ammonium groups defined by

[R-NH2] (6.9) [R-NH*]+[R-NH2] and PKo,m= -l09K o,m = °-434 RT*

is used when water is the solvent, pK for an aminogroup at PK o,w o,s ' •,o an interface. AG" is the standard free enthalpy change of the dis­ sociation in the medium investigated. In this derivation activity effects other then forH are ignored. In a poly-cationic acid the dissociation of each cationic group depends also on the electric field of neighbouring other cationic groups. Generally, the free energy of dissociation now consists of an electrical term AG , ienl additio n toA G .Thi s cam nb e taken into account by modifying (6.8)into :

K P app =P H - log ^ =^ (AGo _AGei ) (6-10) in which pK is the apparent dissociation constant, related to the sum of the standard free enthalpy changeA G and the additional electro­ static free enthalpy change AG ,. In (6.10) AG , is the electrical reversible isothermal work, to bring a proton from infinity to the surface of themacroion . It follows now that

PH =P Ko,m+ 1°^- ^W AGel (6-1.1) Because of the definition ofA G ,, this quantity is equal to the change of the electrostatic free enthalpy G ,, o f the macroion, when the charge of the polymer is increased by one unit.Equatio n 6.11 is only valid when all titratable groups contained in the definition of a have the same pK .Thi s is for biopolymers not the case and aswil l be shown later on also not for adsorbed homopolyelectrolytes. When Z„ is the amount of protons dissociated from the cationic 145 polyelectrolyte, AG ,ca nb ewritte na s

3Ge_l_ AG 5 (6 12) ei = - -sir-2H - -

It followsno wtha t

Gel(c,) * =S± = - 2.303 RT ƒ {pH - log T2- - pK } dor (6.13) ZH,max or 1 a °'m where a =Z„.Z H ~H,ma xi nwhic h Z„ H,maisthx emaximu m number ofionizabl e groups withth esam ep K value. One cani nprincipl e obtainp K froma plo to fp K vsa b yextra ­ polation too r= 1.Th eelectrostati c free enthalpy G, o fth epoly ­ electrolyte can be calculated from the area under the graph (pvi K -p K )v sor ^ap p *o,m ' . The total free enthalpy ofa nadsorbe do rdissolve d polyelectrolyte as a function ofp Hca nb e directly obtained from proton titration data aswa sshow nb y Pfeil and Privalov (1976). Generally this free enthalpy isa function ofcomposition , P,T ,p Han dioni c strength. From theequatio n

<*G= (!§- ) dN= u „ (polyelectrolyte)dN= n „ (solution)d N 3N pT H H

= (M£ -2.30 3 RT.pH)d N (6.14) inwhic hN i sth eaverag e charge onth epolyelectrolyt e chain, it follows after integration: N G(N)= G(N° )- 2.30 3R T ƒp Hd N (6.15) N° where G(N) i sth efre e enthalpyi nsom e reference statei nwhic hth e chargeo nth epolyelectrolyt e isFN °(se e Pfeil and Privalov, 1976an d Norde and Lyklema, 1984). Equation 6.10 is very suitable foranalyzin g thePotentiometri e titration data ofcharge d polyaminoacids in solution andi nth ead ­ sorbed state. 146

G ,(a )ca nals ob eexpresse da s

=S± = RTJ p^ da (6.16) ^H,max a Ki where t|),i sth e electrostatic potential atth eplac eo nth emacroio n whereth eproto nassociate s (e.g. Nagasawa, 1971). From equation6.1 6 it followstha tth eelectrica l freeenthalp yo f a polyelectrolyte canb e obtained by charging themacroio n fromth e uncharged state to the actual degree ofionizatio n (1-a).Whe n>|> b(a) canb e calculated correctly, equation6.1 6 and 6.13 should giveth e same results.Thi s iswha tman ypublication s onpolyelectrolyt eti ­ trationsar edealin gabou t(e.g . Nagasawa, 1971). Ina polylysin e helix or ßstructur e the amino groupsar eclose r together than inth e coil conformation.Hence ,AG , ofhelica lrod s exceeds AG, o fP L coils.Therefor e forpolyelectrolyte s exhibiting a conformational transition,a plo t ofp K vsa show stw odistinc t partswit hdifferen tslope s (seefig .6.8) :A tlo w a thecurv eapplie s toP L inth ecoi l conformation, athig h a thecurv ei stha tfo rth e helical structure.Th e transition regionbetwee n thesepart s isth e coil-toheli xtransitio nregio n (seeals osectio n3.1.2) . Among others Ziimn and Rice (1960) and Nagasawa (1971) showed that the non-electrostatic parto f the freeenthalp ychang eo fth ehelix - coiltransitio no fioni cpolypeptide s (AG° f)ca nb eobtaine d fromth e curvep K vsa as :

AGconf= 2 -303 ZH,maxRT / tpKapp-eKapp]da (6"17)

Thisequatio ni sidentica lt oequatio n6.4 .

As therequire d plots ofp K (helix)an dp K (coil)v sa canno t bemeasure d over theentir e a range anextrapolatio n proceduremus t beadopte dt ocalculat eAG ° f/ ors . In synthetic polyelectrolytes other than charged polyaminoacids conformational transitions are not oftenobserved .A n exceptiont o this isth econformationa l transition inpolymethacryli c acid (PMA) andth emethacryli caci dmethy lesthe rcopolyme rPMA-pe . Onchargin g thesepolymer s atransitio n occursfro mth ecompact ,so - called hypercoiled form (a)t oth e commonextende dconformatio n(b) . Alsoher eAG ° -ca nb ecalculate dwit h (6.17) (Leyte and Mandel, 1964). 147

Fig.6. 8 Schematictitratio ncurv efo rpoly-L-]ysine .

The transition inPMA-p e does also takeplac ewhe nth epolyelectro - lytei sadsorbe do nparaffi ndroplet s {van Vliet and Lyklema, 1978). Insectio n6.6.4. 1an dsectio n6.6.4. 2th eproto ntitration so fPL- L andPL-D Lwil lb ecompare dwit hth ecorrespondin gsolutio ntitrations , inorde rt otrac e conformational transitions inth eadsorbe dstate , pK shiftsdu et oadsorptio nan da sa consequenc e ofadsorption ,th e amounto funtitratabl egroup si na certai np Hregion . Now firstsom e other aspectso fproto ntitratio ni nheterogeneou s mediawil lb ediscussed .

6.6.2.1 Suspension effect in polyelectrolute/charged particle systems

Whenmeasure dp Hvalue s aret ob euse d inth ecalculatio n ofp K values onemus tb eawar eo fth emeanin go fth emeasure dp Hvalue . Insimpl e electrolyte solutionsth emeanin g ofth emeasure dp Hvalu e isusuall y takena sp H= -log( H )i nwhic h (H) i sth eproto nactiv ­ ityo fth esolutio n(e.g . Bates, 1964). Asth eliqui d junctionpotentia l isabou tth esam ei nth evariou s buffers used andth emeasurin gsolution ,thi sdefinitio ni sjustifie d (see Bates, 1964). However,whe nhighl ycharge dparticle sar epresen t apotentia ldifferenc eca nb eobserve dwhe nth eirreversibl ereferenc e 148 electrode ispositione d inth eparticl e suspension or inth esuper ­ natanto r dialysate of thatsuspension , irrespectiveth epositio no f the glass electrode.Th e differencebetwee n thep Hobserve dwit hth e ref.electrode inth e suspensionan dth erea lp Hi scalle dth esuspen ­ sioneffect . The effect isequivalen tt oth edifferenc ei nelectrica lpotentia l between the two saltbridge s of twoidentica lelectrode sinserte di n a suspension and itsequilibriu m liquid.Th esuspensio neffec ti sdu e tounequa l mobilities andconcentration so fcation san danion si nth e neighbourhoodo fth esal tbridg eti pan di stherefor ea liqui djunctio n potential (Honig, 1972; Overbeek, 1953). Accordingt o Overbeek (1953) theliqui djunctio npotentia l isequi ­ valentt oth epotentia lo fth eDonna ncell ,i.e .th emeasure dpotentia l differencebetwee n theparticl esuspensio nan dit sequilibriu mliqui d thus AE F PH = PHobs. - dhr <6-18> whereE n,th emeasure dpotentia lo fth eDonna ncell ,ha sth esam esig n asth echarg eo fth epolyion .Th emagnitud eo fth esuspensio neffec ti s dependento n the chargedparticl econcentratio nan dsmal lelectrolyt e concentration.Fo rhighl y chargedparticles ,th emobilit ydifference s between the microions are the dominant contribution toth e liquid junctionpotential . Intha tcas eth epotentia lo fth eDonna ncel lca n beapproximatel ycalculate d from (Overbeek, 1953).

AE =| T ln !SUS£. (6 19) d F Ksolut. inwhic hK i sth especifi cconductivity .A tsal tconcentration sappre ­ ciably higher thanth e equivalent concentration ofparticl echarges , the suspension effect isnegligibl y small.Fo r this reason andals o forpractica lones ,mos tproto ntitration so fpolyelectrolyte s inthi s study (overall polyelectrolyte concentrations usually <0.002 5rM. ) havebee nperforme di nth epresenc eo f0. 1M NaBr . More details concerning thesuspensio neffec tca nb efoun di nth eex ­ tensiverevie wo nth esubjec to f Chernoberezhskii (1982). 149

6. 6. 2. 2 pK and the distribution of fixed charges in adsorbed app poluelectrolutes

The observed pK r ofa clas so fgroup swit hth esam ep K isde - app r r- i-0 ( m pendent onth e lowM electrolyte concentration, thepolyelectrolyt e segment-segmentinteraction ,th ep Han dth evalu eo fp K .No w Iwil l pay attention to the firsttw o effectsmentioned ,i nrelatio nt oth e proton titrations of dissolved and adsorbed PL atth e sameoveral l residuean dsal tconcentration .Unde rthes econdition son emus trealiz e that the distribution of fixedpolyme r charge (i.e.th etitratabl e groups)wil lb ecompletel ydifferen ti nbot hcases . Asth epolyme rvolum e fractioni nth eadsorbe dlaye ri smuc hhighe r thantha ti nth ecorrespondin g solution,th etitratio ncurve sar eals o expected todiffer .Becaus eo fth ehighe relectrostati c segmentalin ­ teraction inth e adsorbed layerp K isexpecte d to shiftt olowe r values inth e adsorbed state.Th e abovewa s alsonotice db y Buscall and Corner (1982), whomeasure d the titrationcurve so flatice swit h agrafte dPA Alayer . The concentration effect due to adsorption canb e simulatedb y measuring thep K vs acurve s ofP La tvariou s (high)residu econ ­ centrations asBuscal l and Corner did forPAA . Itmus tb erealize d then, thata tpolyme rconcentration so fa fe w% th esuspensio neffec t mayb e substantial even at 0.1M salt.T oobtai ncorrec tresult sth e pHmus tb emeasure dwit hth esal tbridg eo fth eref .electrod ei nth e equilibrium liquid. In the case of adsorbed polylysine andP Li n solution,th e ref.electrod ewit h salt-bridge was inth e suspension and noti nth eequilibriu m liquid,bu ther eth esuspensio neffec ti s c small (CpL< < NaBr)» henceth emeasure dp Hi st oa goo dapproximatio n equalt oth ep Ho fth eequilibriu mliquid . Buscall and Corner (1982) wereawar eo fth eabov ealthoug hthe yd o notmentio n the suspensioneffec texplicitly .The ymeasure dth ep K vsa curve so fPA Aa t0.0 1M NaC lfro m0.1-5 % (w/v),wit hth ereferenc e electrodei nth epolyelectrolyt e solution.Th ecurve swer erecalculate d toobtai nth ep K vs a curvesa si fthe ywer emeasure di nth eequilib ­ riumliquid .Thi swa sachieve db ytreatin gth eequilibriu mbetwee nth e PAAsolutio nan dth eequilibriu msolutio na sa Donna nequilibriu munde r conditionssimulatin gth eadsorbe dPA Alaye r- solutio nequilibrium . Therecalculate dp K vsa curve swer efoun dt oshif tt ohighe rp K app 3 r app with increasing PAA concentration asexpecte d andno tt odecreas ea s 9 K 9c n inth e untreated measurements.Althoug h the signo f P aDD/ pAA-"- 150 the equilibrium liquid is predicted correctly by Buscall and Corner from the measurements in the PAA solutions, the magnitude of this quantity is probably inerro r ands oi sthei r estimate ofth e average volume fractionPA Ai nth eadsorbe d layer.Th emai n objectionsare : i. Theassumptio n thatth emea n residue concentration inth e adsorbed layer ismuc h larger than theadde d NaCl concentration isno tcon ­ sistent with theestimate d volume fraction inth eadsorbe d layer, ii. The assumptions made about theactivit y coefficient ofth epolyio n inth eadsorbe d layer from ioncondensatio n theory areonl y valid athig h dilution andno ta tth e fairly high concentrations inth e adsorbed layer. May be the investigation ofth einfluenc e ofth ebul k polyelectrolyte concentration onp K canb e best performed using a reversiblere ­ ference electrode without salt bridge. However when a saltbridge is necessarily, themeasurement s should bedon e inth eequilibriu m liquid.

6. 2.2. 3 Medium effect on pK o,m Thep K value ofa n-NH_ ,grou p ina nadsorbe d PL-layer will differ o,m 3 from thesolutio n value because ofth e following: i. Thenegativ e PS surface charge isals opresen twhe nP Li suncharged . This causes ashif t tohighe rp K values. = o,m ii. Thecationi c groups ofa nadsorbe d polycation inth efirs t adsorp­ tion layerwil l bei na npolystyren e environment and consequently experience a lower dielectric constant than inth ebulk . Thisre ­ sults ina lowe rvalu e ofpK „„ . o,m iii.Du e to the high volume fraction ofP Li nth eadsorbe d layerth e mean dielectric constant is also shifted tolowe r values inth e adsorbed layer. In fact there isa distributio n ofe values .Th e effecto np K isth esam e astha tmentione d under 2,bu tprobabl y o,m less important. When extrapolations tozer o adsorbed amount andzer o surface chargear e made only theeffec tmentione d under2 isleft .Therefor e strictly only that effect onp K isa mediu m effect andappear s also inth evalu e r o,m ofK a,m When titration measurements areperforme d onlatice s with adsorbed PL at 0.1M NaBr the suspension effect will be small aspointe d out earlier. The change inp K upon adsorption ofP Li sdefine da s Atr?Ko =P Ko,s- P Ko,w (6-20) 151

A.p K stilldepend so nth esurfac echarge , a andth evolum efractio n PLi nth eadsorbe dlayer .Th emediu meffec tA .p K willb eth elarges t forth epolyme rsegment si ntrain sa tth esurface .I nthi srespec tth e heterogeneity of thepolystyren e surfaceplay sals oa role :A P Lseg ­ ment inth eneighbourhoo d ofsurfac esulphat egroup swil lhav ea nen ­ hanced pK due to ionpai r formationan dP Lsegment sembedde di na PSmatri x (benzyl groups)wil lhav ea decrease dp K assai dbefore . PL groups in tails will havep K values notver y different from thosei nth esolution . Asth ep K valuea tth esurfac eca ndiffe rconsiderabl y fromtha t inth eoute radsorptio nlayers ,difficultie s arisewit hth edefinitio n ofa inth e adsorbed state.Th e slopeo f ap K vsa plo ti nwhic h =Z„/Z „ (with the averageamoun to fproton sdissociate dpe r a ti a,ma x Z„ri molecule)ha stherefor eno tth esam emeanin ga si nequatio n6.1 0wher e a =Z„/Z ii* ti,ma„„ „i xn whic hZ *„,„ „ii,mai sth xetota lamoun to fgroup so fclas sx withth esam ep K valueo,m . Animpressio r no fth eeffec to fth edielectri cconstan to nth ep K _ o,m ofcationi c acids canb e obtained from the data of Grunwald et al. (see King (1965) p.195) .Th ep K (e= 78.3)o fmethylammoniu mi s 10.70,wherea sp K in65 %methano l (e= 40.6 )i s9.58 .Thi scorres ­ ponds to adegre e ofdissociatio n a atp H11. 0o f0.6 7 and0.9 6re ­ spectively. So for adsorbed PL ata =o ther eshoul db ea markedl y higher a atp H 11.0tha n inth e solution.However ,a swil lb eshow n lateron ,th esurfac echarg e a isth edominan tfacto rwhic hdetermine s the average shift inp K ofadsorbe dP Li nexperimenta lsituations . Thisshif tresult si na lowe ra a tp H11. 0i nth ecas eo fadsorbe dPL . The theoretical description ofth e surface charge effect onth e dissociation ofe-NH 3 groups canb e donei ndifferen tways .Whe nth e effect is regarded asdu et oio npai rformatio n (i.e.discret echarg e model)which , aswa s shown inchapte r5 ,i sth e case indeed atlo w ionic strength,th eeffec tca nb e includedi np K .Th edescriptio n o,m will be difficultbecaus e except£ atth e surface also theacidit y quotientsan dio npai rdissociatio nquotient so f-OSO~...N a;'-OSO~.. . + + - .

NH3-R and -NH3...Br must be known, togetherwit h the appropriate statisticalfactors . Whenth e surface charge isconsidere d ashomogeneou s (smearedou t surface charge model)a s in the polyelectrolyte adsorption theory of van der Schee (1984) the charge effectca nb e included inth e electrostatic potential calculations andnee dno tb einclude di nth e assumedvalu eo fp K F o,m 152

6.6 . 3 Experimental

6.6.3.1 Materials

Thepolylysine.HB r samples used inth e titrationexperiment shav e beendescribe d insectio n3.1.1 . Thepolyglutami c acid sample used wasobtaine d fromth eSigm aCo . anduse dwithou tfurthe rtreatment . The PMA-peuse d (M1 0 )wa s asampl emanufacture d by Röhm,A.G. , Darmstadt, Germany and commercially available as Rohagit S, high viscositygrade . Solutions ofPMA-p eN asal twer eprepare di nth edar ka sdescribe d by van Vliet (1977). The solution usedwa sdialyze dagains twate rt o obtaina NaCl-fre ePMA-p esolution . The concentration ofPL.HB rwa sdetermine db ytitratio nwit hpoly - vinylsulphatea sdescribe dbefor e (section4.2.2) . Adsorbed or desorbed amounts ofPL.HB rwer e determined withth e depletionmetho d asdescribe d insectio n4.2. 3 andsectio n6.5.2 .Th e polystyrene latex samplesuse di nth etitratio nexperiment shav ebee n described in section3.2 .Th ewate ruse dwa sdistille donc eo ri twa s conductivitywater .Al lothe rchemical suse dwer eo fanalytica lgrade .

6.6.3.2 Potentiometrie titrations

Potentiometrie titrations of PL,PMA-p e and PGA in solutionan d adsorbed onP S latexparticle swer eperforme dunde rN _ atmospherei n awell-close d doublewalle d titrationvesse l( ~ 40c m )wit hthermo - statted waterbein g circulated through the jacketa t293.1 5± 0.05K . 3 Usually the titrationvolum e was20. 0c m .Th eN _ usedwa sCO„-fre e andsaturate dwit hwate rvapou ro f29 3K .T otha ten dth eN _wa spasse d througha sod alim ecolum nan dwas hbottle sfille dwit h30 %KOH ,dilut e H-SO.an dwate r (293.15± 0.0 5K )respectively .Th etitratio nvesse lwa s keptunde r the slightN ? overpressure ofca .3 0m mwate rdurin gth e titrations.Durin ga titratio nth epurifie dN _wa scontinuousl ypasse d over the solution and allowed toescap e through awaterloc ko rwas h bottle containing asolutio n of Ca(OH)_. The solutionswer e stirred bymean so fa magneti cstirrer . Thep Hwa smeasure dwit h anAnkersmit hA16 1 orElectrofac t3606 0 digitalp Hmeter ,usin gcombine dglass-Ag/AgC lelectrode s fromSchot t (N58o rN59 )o rElectrofac t (7GR111).Th ep Hmete rwa sstandardize d 153 before each titrationwit hTitriso l (Merck)buffer sp H7.00 ,4.0 0an d 9.00. Additions of calibrated 0.1M NaO H or 0.1M HC l (titrisol)wer e donewit hMettle rDV10/DV20 1o rMethroh m65 5Dosima tautomati cmicro - burettesvi aa rubbe rca pi nth ecove ro fth etitratio nvessel .Depen ­ dento nth ep Hrange ,addition s of5-4 0mm 3 0.1M NaO Ho r0. 1M HC l weremad e inth e titration experiments.Afte r each addition ittoo k 2-5mi nt oobtai n anequilibriu mp Hvalue .Durin gth ep Hreading sth e titrationsampl ewa sno tstirred .On esingl epolyelectrolyt etitratio n i.e.i non edirection ,too k2- 5hours .Th eamoun to ftitran tadde dwa s corrected forb ysubtractin ga blan ctitratio no fth esolven t (seeals o below).Result so ftitration si nth epresenc eo fP Swer ecorrecte dfo r thevolum eo fth epolystyren epresent .

6. 6. 3. 3 Sample preparation

Polylysine solutions and solutionso fpolyglutami caci dfo rtitra ­ tionpurpose swer eprepare d froma weighte damoun to fth epolyaminoaci d 3 dissolved in0. 1M NaB ri na 5 0c m measuringflask ,t oobtai na nac ­ 3 curatelyknow nconcentratio no fpolymer ,usuall ybetwee n300-50 0g. m PMA-pe solutions for titration experimentswer eprepare db ydilutio n ofa stoc kPMA-p esolutio n (seesectio n6.6.3.1 )wit hth edesire dNaB r solution. Before each titration,N » wasbubble d throughth esolutio nfo ra t least 20min .Fo r comparison reasons the titrationso fth epolyelec - trolytes inaqueou s solutionwer eperforme da tabou tth esam eoveral l concentration asthos e inth e adsorbed state.Tw o differentmethod s of samplepreparatio n wereuse d for thetitration so fadsorbe dpoly - electrolytes.

Method 1 Thismetho d is analogous to theon euse db y Norde (1978) forpre ­ paring titration samples ofprotein s adsorbed onP S latex.Prio rt o mixingth epolystyren elatex ,polyelectrolyt esolutio n (usuallyPL.HBr ) 3 andNaB rsolutio nwer eadjuste dt oth edesire dp Hi fnecessary .3 0c m 3 3 polystyrene latex (ca. 7%) were mixedwit h 10c m NaBr and 10c m PL.HBr solutions of known concentrations in 50c m Sovivel3flask s with leakproo f screw caps (teflon sealing).Th e finalNaB rconcen ­ trationwa s0. 1M unles sotherwis estated . The total concentration polyelectrolyte waschose n in sucha wa ya s 154 toensur eadsorptio nsaturation .The nth eflask swer erotate den dove r end for several hoursa t29 8K t oallo w foradsorptio nequilibration . After gentlecentrifugatio na t29 3K ,th epolylysin econcentratio ni n the supernatant was determined inth e usualway .Th e loosesedimen t 3 wasredisperse di n2 0c m 0.1M NaB ro fth esam ep Ha sth eequilibriu m pHafte radsorptio nan drotate den dove ren dfo r1 6hours . Thenth edispersio nwa scentrifuge dagain .Th econcentratio nP Li nth e supernatantwa s determined again and appeared alwayst ob ever ylow , i.e. corresponding to adesorbe d amounto f less than2-4% ,whic hi s withinexperimenta laccuracy . 3 3 Again the sediment( ~2 c m )wa sredisperse di n2 0c m 0.1M NaB r solution andN _ was slowlybubble d throughth esolutio nfo ron ehou r toremov e any CO- absorbed during thesampl ehandling .The n20. 0c m 3 ofth e samplewa spipette d inth e titration cell andN _ waspasse d overth esolutio nfo ranothe r2 0mi nt oallo wth ep Helectrod et ocom e into equilibrium with the suspension.Th e startingp Hwa sthe nabou t 7.0.Afte ra titratio nth edr yweigh to fth elate xwa sagai ndetermined . Blanc titrations of latex sampleswithou tP Lwer eals operformed . These titrations (corrected for theP Svolume )di dno tdiffe rsigni ­ ficantly betweenp H5 an d1 1fro mthos eo fth esam evolum e0. 1M NaB r solution. Therefore the latter type oftitration swa susuall y also usedt oobtai nblan ccorrection si nth etitration so fadsorbe dP L(se e alsobelow) .

Method 2 Purified PS latex (~6%(w/v); H+form)titrate d withNaO H toth e equivalence point pH7 , was concentrated by means of destination undervacuu m at31 3K t oca .18 %(w/v) .Polylysin esolution si n0. 1M NaBr (pH^ 6 )wer eprepare da sdescribe dbefore . Ina smal lbeake r 15.0c m PL.HB3r in0. 1M NaB ran dknow nvolume s 3 3 ofx c m NaBrsolutio nan dy c m concentratedlate xbot ha tp H^ 6 wer e mixed.Th econcentratio no fth eNaB rwa ssuc htha tth efina lNaB rcon ­ centrationi nth emixture ,correcte d forth esoli dconten to fth elatex , wasalway s0. 1M .N ,wa sbubble dthroug hth emixtur efo ron ehour .The n 20.0c m o3f th emixtur ewit hrespec tt oth eaqueou sphas e (i.e.V(total ) 3 -V(PS) = 20.0c m )wa s brought int oth e titration cell.Thu sth e aqueousvolum ewa s keptth e same forth evariou stitrations .I nthi s wayi twa spossibl et oprepar ePL/late xsample swit ha varyin gsurfac e coverage and -equilibrium concentrationPL ,bu twit hexactl yth esam e totalamoun to fPL .Th emixtur ei nth etitratio ncel lwa sstirre dunde r N„atmospher efo ranothe r2 0min ,the nth esampl ewa stitrated . 155

Titrations ofconcentrate d latexsample scontainin gn oPL.HB rwer e used forth eblan c corrections. Insom ecase sthes eblan ctitration s hada highe r (15%a tp H11.00 )NaO Hconsumptio ntha ntha to fa corres ­ pondingvolum e ofelectrolyt esolution .Fortunatel ythi sextr aamoun t NaOH cancels whenth eblan ccorrectio ni sperformed .Tha tth eresult s remainunaffecte db yth ehighe rNaO Hconsumptio no fsom elate xsample s followsals ofro mth efac ttha tth eresult sobtaine dwer eth esam ewhe n theblan ctitratio nwa sa sexpected .Furthe rth eresult sobtaine dwit h method2 a tr = r (pH6)wer ealway sconsisten twit hthos eobtaine dwit h method1 . Thesampl epreparatio na ta p Ho fadsorptio no f11.0 0wa sanalogou s asdescribe dunde rmetho d2 bu tperforme dcompletel yunde rN ,atmosphere . To thisen d theconcentrate d latexan dNaB rsolutio nwer epipette di n the titration cell, andtitrate dunde rN 2 upt op H11.00 . Ina secon d smaller titrationvesse l aPL.HB r solutionwa spipette dan dals oti ­ trated underN _ top H 11.00.The nwit h asyringe ,a know nvolum eo f theP Lsolutio nwa stake nfro mth eP Lsolutio n (pH11.00 )vi aa rubbe r cap andbrough t into the titrationvesse l containing the latexals o via arubbe r cap,whil e the latexwa scontinuousl y stirred.The nth e PL/latex system was leftunde rstirrin gfo ron ehou rt oallo wfo rad ­ sorptionequilibriu mt oestablis han dth etitratio nwit h0. 1M HC lwa s started.Th eamoun to fadde dP Lwa ssuc htha tth econcentratio ni nth e bulkwa snegligibl ysmall .

6.6.4 Results and Discussion

6. 6.4.1 Data treatment

In fig.6.9. 1 and 6.9.2 theprimar y data of atypica l titration curve forPL- L dissolved in0. 1M NaB ran dadsorbe do nP S (method1 ) areplotted .Th ecorrespondin gblan ctitration sar eals oshown . Experimental instead ofcalculate d blanc titrationswer euse di nth e experiments because of observed deviations of the glass electrode abovep H 10.5 insom ecases .A simila rargumen twa sals ouse dexplic ­ itlyb y Marini et al. (1980) fortitration so fcytochrom eC . Inth ecas e of adsorbed PL themeasure dblan ccurv ewa srecalculate d totha tcorrespondin g toth e same aqueousvolum e asth ePL/P Slate x system. Inusin g theseblan ctitration si ti simplicitl yassume dtha t the freeproto n activity coefficient isno tsignificantl yinfluence d by thepresenc e ofPL .Volum ecorrection s fordilutio neffect si nth e 156

11 .e*****'2 o-°-o^r" pH y- pH 0 ƒ y o- ' d 10 1 10 1 1 s / / / / - q ^ /> O / 4 1 T= 293.1 5K c

i 1t 01 M NaBr. »

i >

i i i i i 10 20 30 40 50 60 10 20 30 umolNaO H nmolNaO H

Fig.6.9. 1 1.Potentiometri eproto nti ­ Fig. 6.9.2 1.Potentiometri eproto nti ­ tration ofPL.HBr- L (DP 1683)c = trationo fPL.HBr- L (DP 1683)adsorbe d on -•3 _Q VL -o 586g. m (2.82rmol. m );V . = PS (latexM ;a =-7 4mC. m )initia l 20.0cm 3;c „. =0.1». aqueousvolum e 18.37c m ;c 0.1 M. NaBr NaBr , -2 cps =85.6 5kg. m rpL =0. 4mg. m 2. Blanc titrationo f20. 0c m 2.Blan c titrationo f 18.37c m 0.1 MNaBr . 0.1 MNaBr . polymeran dblan csolution sdu et oth eadditio no ftitran twer enegli ­ giblysmal lbelo wp H11.00 . Itca nb e seenfro mfig .6. 9 thatabov ep H10. 0fairl ylarg eblan c correctionsar eneeded .Th etitration swer eno textende dbeyon dp H11. 0 becauseth eblan ccorrection sbecom ethe nto olarg et oobtai na reliabl e titrationcurve .I nadditio nprecipitatio no fP Loccur sabov ethi spH . Inprincipl e the accuracy ofth emasurement sca nb eimprove dwhe n higher PL concentration canb echose nbu tthe nals ohighe rlate xcon ­ centrations areneeded .A tlate xconcentration sabov e15-20% ,n ogoo d stirring ofth e latex-PLsyste mappeare dt ob epossibl eabov ep H8-9 . Thiswa sdu et oa drasti cincreas ei nviscosit yo fth elatex-P Lsyste m abovep H8-9 .Mos ttitratio nresult so fPL- Lreporte di nth eliteratur e havebee nperforme d at3-1 0time shighe r PL-Lconcentratio ntha nha s beenuse dhere ,henc ea highe raccurac yca nthe nb eobtained . In fig.6.10. 1an d6.10. 2th etitratio ncurve so fPL- Li nsolutio n andadsorbe do nPS ,afte rsubtractio no fth eblan ctitratio nar eshown . 157

• i i i

11 1 pH

10

•

• / / 9 / / 1 1 T= 293.1 5K 1 0-1 M NaBr. 8 1 1

i i i i 10 20 30 40 10 15 20 A umol NaOH û^mo lNaO H

Fig.6.10. 1Titratio n curveo fPL.HBr- LFig .6.10. 2Titratio n curveo fPL.HBr- L (DP1683 )afte r subtractiono fth e (DP1683 )adsorbe do nP S (latexM )afte r amountso fNaO Huse d forth eblanc . subtracting ofth eamoun to fNaO H usedfo r See furtherfig .6.9.1 . theblanc .Se efurthe rfig .6.9.2 .

Inth ePL/late x systemonl yadsorbe dPL- Li stitrate dbecaus en omea ­ surable amounto fPL- Li spresen t insolutio na tth einitia lp H(se e method 1). Asth emaxima l adsorption increases inth etitratio ndirectio nn o desorption occurso ntitration .A sn osignifican tp Hchange swer eob ­ served duet oth eP Ladsorptio nproces sa tlo wpH ,ther ei sn otitra ­ tion of-NH 3 groupsdu et oadsorption .Th efac ttha tth etitratio n curveso fadsorbe d PLwer e identical with thato fth eblan cbetwee n pH3 and7 suggest the same. Therefore thestartin gpoin to fth e titrationsi nth eadsorbe dstat ei swell-defined . Asha sbee nreporte db yothe rauthors ,th emajo rexperimenta lpro ­ blem inth ePotentiometri etitratio no fioni cpolypeptide s (orothe r polyacids)i sth edeterminatio no fth eendpoint ,wher eth epolyamino - acidreache sth euncharge dstate .Fo rP Lthi spoin tlie sabov ep H12. 5 and iti sclearl yno treache d inth etitration s shownhere .Th eoc ­ currence ofprecipitatio n andver y largeblan c correctionsusuall y preventth edeterminatio no fthi sen dpoint . 158

Inorde r tocomput e a andp K ,th evalue so ftitran tadde dwher e app a =0 an d a =1 ar eneeded .Th e startingpoin twher eth etitratio n begins (a= 0 ,p H^ 7 fo rPL )ca nusuall yb eestablishe dwithi nnarro w limitsfro mth etitratio ndat a (<1 umol )se efig .6.9 . Inth eliteratur esevera lmethod sfo robtainin gth een dpoin t(i.e . thetota lamoun to ftitratabl egroups )ar eadopte db ydifferen tauthors . Insevera lpublication so nth esubjec tmatte rth emetho do fpositionin g thep K vs a curvesi sno tmentione d explicitlya tall . When ionexchange d polyelectrolytes,whic h arecompletel y inth e H orOH " form areused , the total amounto ftitrabl egroup sca nb e obtained directly from the titration curve.Whe n thetitratio ndat a (pHv sA pmolNaOH )o fa polysal tar eincomplet eo rinaccurat ei nth e region of theuncharge d polysalt,usuall y akin d of iterativepro ­ cedure has been adopted to obtain the total amounto ftitratabl e groups from the titration data.Thes emethod s arebase d onth efac t that small differences in the chosenen dpoin tgiv emarkedl ydif ­ ferent pK vs a curves.On e ormor e assumptions about the shape of thep K vsa curve (forexampl elinearit yo fth eheli xan dcoi l branches)ar ethe n invokedt oobtai na nen dpoin tsatisfyin gth econ ­ ditionschosen .Se efo rexampl e Hermans (1966) and Tseng (1975, 1977). Sucha procedur eha sals obee nuse db y van Vliet and Lyklema (1978) to positionth etitratio ncurve so fPMA-p eadsorbe do nparaffi ndroplets . Alternatively,a ca nb ecalculate d fromth etitratio ndat awhe nth e total amount of ionizable groupspresen t isknow nindependently ,fo r example from theweighte d amounto fpolyme ror ,i nth ecas eo fpoly - aminoacids, from theN-conten to fth etitratio nsample .Th eadvantag e istha tdespit eth efac ttha tn ocomplet etitratio ncurv ei savailable , noextr aassumption sar eneede dt oobtai np K vsa plots .However ,i t is implicitly assumed, thatth ep K valueo fal ltitratabl egroup s isth e same in ahomopolyacid , aconditio nwhic h isno tnecessaril y satisfied inth e caseo fa nadsorbe dpolyelectrolyte ,a sI wil lshow . Then the pK apvps a curves cannot be interpreted with thesimpl e theorypresente d insectio n6.6.2 . Especially the slopeo fth ep K _ vs a curves has thenn o clearphysica l meaning. Inthi s studyth e followingway sfo rth ecalculatio no fa an da 'ar euse dfo rth etitra ­ tionso fP Li nsolutio nan dadsorbed :

Z ,_ H _Apmo lNaO H (e 21) " ZH,max" ^ ol P ( } inwhic hApmo lNaO Hi sobtaine dfro mplot ssuc ha sgive ni nfig .6.1 0 159 andn m°lp (res)i sth e total amountP Le-amin ogroup spresen to na weightbasis .Th e apparentp K calculated from a' andth emeasure dp H a ' isdefine da spK ' =appH-lo p g •= r.Furthe 1-a' r

a=-^- (6.22) H.max

ff on on inwhic hZ^ ri,ma' =Z „x rl,ma- zlt x, witri hz£ isth rie amoun to fionizabl e groups which are virtually untitratable, when compared withP Li n solutioni nth esam ep Hregion . eff Forpolylysm e insolutio n iti s assumedtha tZ „„,„ „= Z „„,„ ii,,sma o x xi,max a =a ' i ntha tcase .Th ep K vs a curves forPMA-p ean dPG Awer e app calculated fromth e titrationdat aonly ,i na wa ysimila rt otha tde ­ scribedb y Tseng (1975) and van Vliet and Lyklema (1978). Thiswa snecessar y becauseth eweighte dand/o radsorbe damoun tPMA-p e and PGAwa s notknow n accurately enough inthes e cases.A sa conse ­ quenceo fthi sprocedur eapproximatel y a isobtaine dan dno ta' . Whenp K canb e taken as independento fth epositio ni nth ead ­ sorbed layer (i.e.th evariatio n of thedielectri c constant inth e adsorbed layer isnegligible y small)equatio n 6.10 canb eapplie dt o plotso fp K vsa fo radsorbe dpolyelectrolyte .

Aswil lb e shown later,th emagnitud e of adi po rhum p inpK aDD vs a'plot s is influenced by thechoic e ofth e total amount ofti - tratablegroups ,i.e .i ti sdependen to nth edefinitio no fa . Aspointe d outbefore ,extrapolatio n ofp K vs a curvesfo rP L insolutio nt o a =1 shoul dgiv eth ep K valueo fth es-amin ogroup . Of course thevalu efoun dwil ldepen do nth ewa yth eextrapolatio ni s made:linea ro rcurved .T oavoi dsuc ha nil ldefine dextrapolatio nth e pK value of theP Lmonome r analogs-amino-capron-amid ewa sdeter ­ mined here potentiometrically. In thiswa y extrapolations ofp K vsa curves ofP L towards higher avalue sca nb emad emor ereliable . A similarprocedur ewa sals oadopte db y Terbojevich et al. (1972) for titrationso fpoly-L-histidine . The results ofth es-aminocapronamid e titrationsar eshow ni nfig . 6.11.

At293.1 5K thepK Q ofth ee-aminogrou p is10.7 9± 0.0 2 in0. 1M NaB r and10.6 3± 0.0 3 in0.0 1M NaBr . From thedat a in fig.6.1 0 pK was calculated andplotte da sa function of a'. The curve ofPL- L insolutio n (fig.6.12.1 )show s 160

1 i ' i • i • I T= 293.15 K ö - 10.9 - - a a* . * 10.R I X 01 M NaBr. a 10.7

10.6 001 M NaBr. I

105

i.i.i i

Fig.6.1 1 pKv sa curves of£-aminocapronamide ,a monome r analogo fP L in0. 1 and 0.01 MNaBr . clearly thecharacteristi c helix-coil transition. Inadditio ni tca n be seen from this figure thatth emagnitud e ofth edi pi nth ecurv e is strongly dependento n the actually chosen endpoint . Inth ecas e ofadsorbe dP Ln otransitio ni sobserve d (fig.6.12.2) . a' WhenpK ' =p H- lo gr— , isplotte dagains t a' foradsorbe dPL ,th e maximalvalu e ofa 1 isno t 1.0bu tlowe rwhe nther ei sa fractio no f non-titratablegroups .Therefor eestimation so fp K areno ta prior i possibleb yextrapolatio no fth epK ' vsa 'curve si nthi scase . Because of the largeblan c corrections involved thepK ' vs a' app

(orpK ao_ vs a) curvesar eles saccurat ea thig h avalues . Therando merror si nth emeasure dp Han dadde d(jmo lNaO Har esmall , butth ep K vs a curvesar esubjec tt oa systemati cerror ,th emag ­ nitude ofwhic h differs ina nuncontrolabl e way in repeatedexperi ­ ments.Th econsequenc e ofthi si stha tfairl ylarg euncertaintie sar e possible inth e slopes of thep K vsa curves,an d inth ecas ea conformational transition occurs in the value ofAG . Jconf' the non electrical freeenerg y change of the transitions.Nevertheless ,th e trendsobserve di non eexperimen tar ever yreproducible . The accuracy ofA G _determine d fromtitratio ndat ai sdependen t on: 1. systematic errorsi nth etitratio nprocedure .2 .th echoic eo f theen dpoin t(se efig .6.12.1 )an d3 .th equalit yo fth eextrapolatio n procedure.Becaus e ofthes e factors theobtaine dvalu eo fA G -ma y 161

i • l ' T= 293.15 K ,'j 0.1 M NaBr. // 107

PKopp. pK 10.6 app. T= 293 15 K • / / 0.1M NaBr. / / 10.8 10.5 / / 10.6 10.4 /// - 10.4 2 10.3 / / 10.2 10.2 10.0 10.1 ' //^^^ ' 9.8 10.0 9.6 i . I . i . r . .2

Fig. 6.12.1 pK vs a plots of PL.HBr-L Fig. 6.12.2 pK' vs a' of PL.HBr-L (DP 1923) app apP _, (DP 1923) in 0.1 MNaB r for different adsorbed on PS (latex M,; a = -70 mC.m ) values of the titration end point. ° -3 -3 according to method 1. c „ = 85.6 kg.m ; e«, = 2.81 mol .m ; total weighted -2 D Tpj = 0.4 mg.m . Total adsorbed amount amount of PL.HBr: 56.0 Mmol ; 1. 56.0 PL.HBr: 33 Mm°l • Curve 1: a' calculated |jmol P(res); 2. 52.0 pmol P(res); from the total adsorbed amount and Apmol 3. 49.0 |jmol P(res). NaOH. Curve 2: a' calculated from the total adsorbed amount, minus the total amount -0S0. groups present and Apmol NaOH. vary by a factor of 2 to 3. For further information about this matter the reader is referred to Olander and Holtzer (1968), Nagasawa and Holtzer (1964) and to Tseng (1975). 162

6.6.4.2 Proton titrations of free and adsorbed polylysine

Solution behaviour Infig .6.12. 1an dfig .6.1 3 pK vs a curveso fPL- Lan dPL-D Lar e c app shown.A sexpecte dth econformationa ltransitio ni nPL- Li sclearl yvi ­ sible,bu tPL-D L showsn o transition inth e investigated pHregion . At about the samepolyme r concentration the coilpar to fth ep K vs a plot of PL-L lies above theplo t ofPL-DL .Thi smus tb ea n effect of the different stereoregularity of the twoP L forms.Suc h an effect of the stereoregularity has also been observed forth e titration curves of isotactic and syndiotactic PAA (Nagasawa, 1971). However, Chou and Scheraga (1971) found thatth ecoi lpar t ofth e PL-L curvewa s identical with that forPL-DL .A sexpecte dth eslope s of the PL-L and PL-DL curves inth e coil regionar eth esam ewithi n experimentalerror . The data in fig.6.1 3 suggest thati nPL-D L thep K of the£ - amino groups is lower than inPL-L .Thi si sexpecte do nth ebasi so f data of Ellenbogen (1952) who found thatth ep K value of thee - aminogroups of lysine peptides decreased by 0.1-0.4p K unitswhe n d-lysinewa sincorporate d inth epeptide . Inon eexperimen tth ep K vs a curveo fa mor econcentrate dPL-D L app solution wasmeasured .On e can see in fig.6.1 3 thatth ep K vs a ploto f this concentrated PL-DL solution liesabov eth ecurv eo fth e moredilut eone .A sexplaine dbefor eth eresult smus tb ecorrecte dfo r the suspension effecta thig hPL-D Lconcentratio nt oobtai nth etru e concentrationeffect .Althoug hth eobserve dPL-D Lconcentratio neffec t is asexpected , itca nno tb erule dou tcompletel ytha tth eobserve d differencei swithi nexperimenta lerror .Furthe rexperiment sar eneede d toelucidat ethi spoin tfurther .A spointe dou tearlie rth ep Hmeasure ­ mentsough tt ob eperforme d inth eequilibriu mliquid . ÛG Thenon-electrostati c freeenthalp yo fth eheli x formation( conf/ Z„ .„)a s calculated from the titration curve ofPL- L insolutio n H,max (fig.6.12.1 )i sbetee n -0.1 and -0.3kT .Th evalue s foundb yothe r authors lieals o inthi s range.Th eobtaine daccurac yi sfa rto olo w tojustif ya nunravellin go fth evariou scontribution st oAG . f- Aspointe d outbefor eth evalu eo fA G „4r/Z„ ,„i ssmal lcompare d conx ri,max with the constituent contributions of among others,H-bondin g and hydrophobic interactions which are partly internally compensating.

Inth eadsorbe dstate ,x _i softe na tleas ta slarg ea sA G f/ZH max- Sowhe n alarg efractio no fsegment si sboun dt oa surfac eth eoccur ­ renceo fa conformationa l transitionma ybecom esuppressed . 163

T= 293.1 5K 10.7 0.1M NaBr

PK,app . 10.6

10.5

10.4

10.3

10.2

10.1 -

10.0

Fig. 6.13 pK vs a curves for PL-DL in 0.1 M NaBr. SPP o 1. PL-DL (DP 240) cpL = 38.4 mol^m . 3 2. PL-DL (DP 308) cpL = 4.87 mol^nT .

Adsorbed poly-L-lysine Infia .6.12. 2pK ' vsa 'curve so fPL-L , adsorbed accordingt o app method1 ,ar eshown .A snote d already insectio n6.6.4. 1n oconfor ­ mationaltransitio nlik eth eon eobserve di nsolutio ni spresen tunde r conditionso fconstan tadsorbe damount .A sth eadsorbe dlaye ri srathe r flatthi si sno tunexpected .O fcours e ashif to fth etransitio nt o very highp Hvalue si sno texcluded ,bu ti ti simprobabl ebecaus eth e fractiono ftrain swil lremai nhig hals oi nth euncharge dstate . Alson otransitio nwa sfoun dwhe nPL- Lwa sadsorbe da tp H11. 0a t r =T (p H6 )(metho d2 )firs tan dthe n titratedwit hHC lt olo wpH . Unfortunatelyth eaccurac yo fthes eexperiment swa sno thig henoug ht o allow forconclusion s abouta differenc e inslop eo fth epK '„ v s a' app curve atp H , 6an dp H . 11.0.On ewoul dexpec ta highe rslop ewhe n atp H11.0 0P Lmolecule sadsor ba sa heli xo ra ssom eothe rsecondar y structure andsta yi ntha tconformatio no ngoin gt olowe rpH .Howeve r the 'freezing'o fa nadsorbe d helix despite thechargin gproces si s 164 very unlikely, because theheli x stabilization free energy isver y small compared with theincreasin gelectrostati c freeenerg ywit hde ­ creasing pH.Henc e iti smor elikel ytha ta tp H11. 0PL- Lhelice sun ­ fold upon adsorptionwhe nr

tob e only 0.2,whil e for freeP L a' ^ 0.85.Whe nth e-OSO~,-NH 3io n pairs at the PS surfacewit h PL adsorbed aretake n aseffectivel y untitratable,a 'ca nb erecalculate da s

_ A|jmoiNaO H ,A9 o> 01 l " Mmo lP(res)-a Q.F.AT ' inwhic ha isth esurfac echarg eo fth elatex ,F th eFarada yconstan t andA _th etota lP Ssurfac eare ai nth esystem . Usingthes ea value scurv e2 i nfig .6.12. 2i sobtained .A sno wth e degree of dissociation isrelate d to aclas so fe-aminogroup shavin g theirp K values probably not farapart ,a nanalysi s ofth ecurv e according to equation6.1 0 is justified although notrigorous .Fo r curve1 this equation iscertainl y not applicable.Extrapolatio n of curve2 (fig.6.12.2 )t o a =1 i sno wpossible .Suc ha nextrapolatio n 165 results ina p K value which isabou tth esam ea si nsolution ,in - * o,m dicatingtha te-aminogroup si nadsorbe dPL-L ,no tinvolve di nio npai r formation,behav eno tver ydifferentl y fromthos ei nsolution .Unfortu ­ nately, thereca nb ea nunknow nincreas eo f a duet oth epresenc eo f 0.1M NaBr asdiscusse d inChapte r3 .Fo rtha treaso nth e a value (determined before contactwit h excessNaBr )use di nth ecalculatio n ofcurv e2 (fig.6.12.2 )ca nb eunderestimate du pt oa facto ro ftwo . However atp H11.0 0a i ncurv e2 i s0.8 ,nea rt oth evalu e foundi n solutiona ttha tpH .Henc eth eunderestimatio no f a seemst ob eles s thana facto ro ftwo . From theabov ei tappear s thentha tth ee-aminogroup s inadsorbe d PLca nb edivide di ntw oclasses : 1.-NH,...oso rgroup shavin ga p K appreciablyhighe rtha np K and •3-D O/S O/W nottitrate da tal lunde rth eexperimenta lconditions . 2.-NH _ groupshavin ga p K valuewhic hi sabou tth esam ea sp K . O O/S O/W The comparison ofth eproto n titrations ofadsorbe d PLwit hth e solutionbehaviou rca nb edon ebette rwhe nsampl epreparatio nmetho d2 is used. In fig.6.14. 1an d6.14. 2characteristi c resultso fthes e titrations areshown .Curv e 1i sth etitratio no fPL- L(D P1683 ) in solution,curv e2 th etitratio no fPL- Ladsorbe da tconstan tadsorp ­ tion (T= r (pH6))an dcurv e3 i sth etitratio no fadsorbe dPL- L (DP1683 )wit h increasing adsorbed amountu pt op H9. 5 andconstan t adsorption above thatpH .Th eproto n charge-pHcurve si nfig .6.14. 1 show clearly thata tp H11.0 0th eamoun to fdissociate d protonsde ­ creasesi nth eorde rPL- Li nsolution ,P Ladsorbe da tr = r (pH ) and PL-L adsorbed andr = r (pH6 )= constant .Henc eth eresult sob ­ tained with sample preparation method2 sho wmuc hmor e clearlyth e fractiono faminogroup swit ha nincrease dp K value. Curves 1an d2 i nfig .6.14. 2 showth esam etrend sa sthos eobtaine d withsampl epreparatio nmetho d1 . Asexplaine dbefor eth epK ' vsa 'curve scalculate d fromth edat a infig .6.14. 1ma yno tb einterprete d accordingt oequatio n6.10 .Thi s means thatth eslope so fthes e curvescontai nals oothe reffect sbe ­ sidesth econsequence so fth edecreas ei nG, . Furthermore,th einter ­ sectionpoint sobserve di nfig .6.14. 2hav en oclea rphysica lmeaning . Incurv e3 o ffig .6.14. 2ther ei sfirs ta decreas ei npK ' until app a' =0.35 .Abov ethi svalu eth ecurv ebehave ssimila rt ocurv e2 ,bu t isdisplace d tohighe ra 'values .I ti sals o above a' =0.3 5tha tr remains constant again,i.e .th ebul kP Li sexhausted .Th equestio n 166

1 ' i ' i i T= 293.15 K 0.1 M NoBr. 10.8 2 T=293 15 K 40 01 M NoBr pKbpp. / A 1 // 10.6 1 . 1 / i- 1 3,/ / 1 1 30 10.4 1 1 i / / i / / 10.2- 1 I f / / 6/ 20 >£———L-^" ^ / / 10.0 - ' / / - / / / / / / 9.8- - - // / V / . / 9.6 "*""'— - i i i i .2 .A PH

Fig. 6.14.1 Proton titrations of PL-L Fig. 6.14.2 pK' vs a' curves for the same (DP 1683) in 0.1 MNaB r and T=293.15 K. titrations as presented in fig. 6.14.1. Total amount of PL.HBr: 47.2 umol (weight basis). Overall concentration -3 PL: 2.4 mol .m . Total amount poly- r -2 styrene (latex M.,a - -39 mCm ): 5 o 1.11 g. (curve 3), 2.22 g (curve 2). 1. PL-L in solution; 2. PL-L adsorbed. T = T max (pH 6) = const.; 3. PL-L adsorbed. T = r (pH) if pH < 9.5 and T = T (pH 9.5) = const, for pH > 9.5. now arises whether the minimum in curve 3 of fig. 6.14.2 is only caused by a coil to helix transition in the adsorbing or adsorbed PL-L molecules. To find this out we performed an titration experiment with PL-DL in solution and in the presence of PS latex under exactly the same conditions as used for the determination of curve 2 and 3 167 infig .6.14.2 .Th eresult so fthes etitration sar eshow ni nfig .6.15 . Nominimu m inth epK ' vs a'plo t appeared. Thisi sevidentia lfo r theoccurrin g of achang e inth e secondary structure ofadsorbe dP L whenr = r (pH)(curv e3 o ffig .6.14.2) .No wi tca nb econclude dtha t nocoi lt oheli xtransitio ni nth eadsorbe dstat eoccur swhe nr = ^max (pH 6), but that such atransitio n canoccu rwhe na teac hp Hmaximu m adsorptioni sreached . Curve1 i nfig .6.1 5 representsth esolutio ntitratio no fPL-DL .I t iscomparabl et oth ecorrespondin gon ei nfig .6.13 .Th ecurv e forad ­ sorbed PL-DL in fig.6.1 5 is stronger curvedtha nth ecurv efo rcon ­ stantadsorptio no fPL- Li nfig .6.14.2 .Als oi nth ecas eo fincreasin g adsorption ofPL-D L during titration, astron g rise inpK ' isob - served above a' =0.3 5 just as in thecorrespondin g casewit hPL-L . Also for the PL-DL/latex system the adsorbed amount staysconstan t above a' =0.35 . The observed difference inbehaviou r betweenPL- L adsorbeda tconstan tadsorptio n (curve2 fig .6.14.2 )an dtha to fPL-D L at increasing adsorption (fig.6.1 5 curve2 )show s thatals oth ead ­ sorptionproces s as suchma yb elooke da ta sa phas etransition ,wit h itsensuin gconsequence sfo rth etitratio ncurves . In factthi s isno tunexpected , because the adsorption ofmacro - moleculesi sa phas etransitio no fsecon dorder ,a tleas tfo rinfinit e molecularmas s (Birshtein et al., 1979; Dimarzio and Bishop, 1974). Thesigmoida lshap eo fth er v sp Hcurve so fPL- Lan dPL-D Lpoint sals o intha tdirection .

Now Ishal l returnt oth einterpretatio no fth epK ' vsa 'curve s insom edetail .Generall yth eamoun to fproton sdissociate dpe rpoly - lysinemolecul e Z„ca nb ewritte na s

l yi(2H> Z =I » 1 »i°'

o,m H+ in which there aren . groupswit h adissociatio n constantK „' ,m and l 3 r o,m potential I|J.wit h respectt oth ebul ksolution .y.;(Z H)= e t(i./kT .a„ + isth eproto n activity. From the titrationresult sI presente dabov e itbecam eclea rtha ti nadsorbe dP Li ti sa goo dapproximatio nt ocon ­ siderjus ttw okind so f-NH _ groups.Thos eformin gio npair swit h-OSO Ö groups and thosewh oar enot .Le tth ep K valueso fthes egroup sb e 168

1 ' l ' l i * •*,app . T= 293.15 K 0.1 M NaBr. 10.8 ~

10.6 i i i 10.A - i

10.2

i ^ 10.0 - - '— / / / 9.8 - ——' y f y 9.6

i i

Fig. 6.15 Proton titrations of PL-DL (DP308 ) in 0.1 M NaBr (curve 1)an d inth e presence of PS (latex H ,a = -3 9mCm~ 2)1.1 1 g, (curve 2), T= T (pH) , pH < 10 and T = const.p H > 10.Tota l amount of PL-DL: 48.8 pmol; .3 r c_ (overall): 2.4mo l. m pK; l and pK^ 'respectively . Equation 6.24 can thenb ewritte nas : O / S O ƒ S

s _ 1 O, S H+ 2 O, S H+ (6.25) H y ( y "l + K^)e ^ H;! 1 + K^) e 2

A mean potential y(Z„)ca nb e substituted for y-,(Z„) and y,(ZH)becaus e the adsorbed layer is rather flat and the effect of the surface charge onK „ is included inK * '.Togethe r with o,m o,s 3

JH ""H,ma x n1+n2 169 and (6.26)

n1+n2 equation6.2 5 canb ewritte na s

(1) y {a ] -1 (2) y {a ] -1 u u fK ' e aut (1-f)K ' e au! 5 a'= °^ y (a,M±) +- °' y (a,) M± (6.27) ( (1) -1 y (2) -1 1+ K *->o.es a„! H+1 + K *' eo, s aui H+ a' From equation6.2 7 it follows thatpK ' =p H- lo g-r—- ,i sa compil ­ aiîpP P (2 \ 1~a cated implicit functiono f a„+,pK v ;, pKl ' f, a' and y(a'). The H O, S O, S situationwoul dbecom eeve nmor ecomplicate dwhe na continuou sdistri ­ bution ofK values andy as a function of thedistanc e fromth e o,s J surfaceha st ob eintroduced . Fromfig .6.14. 1an dtabl e6. 1 itca nb esee ntha ti nadsorbe dPL- L andPL-D La tp H11. 0ther ei sa nuntitrate damoun to f-NH ,group swit h respectt oth esolutio ntitration .Th emagnitud eo fthi samoun tpe rm 2 PS surface iswithi nth erelativel ylarg eexperimenta lerro rindepen ­ dento fr an di ti si nth esam erang ea sth esurfac echarg eo fth elatex . Hence the -NH_ groupsi nion spair sar evirtuall yuntitratabl e inth e pHregio n7-1 1 assuggeste dbefor efro mth edat agive ni nfig .6.12.2 . Therefore the first term inequatio n6.2 7 canb eneglected .The nth e followingequatio napplie s

K 10 lo P app= e«" *A " = 9î^ r +pK

One nowclearl y seestha tth estron gris eo fth epK ' vs a'plot s iscause db yth efirs tter mo nth erigh tsid eo fequatio n6.2 8 andshow s thepresenc eo fgroup swit hstron gdeviatin gp K withinth edefinitio n ofa .A simila reffec tca noccu rwit hth eproto ntitration so fprotein s when the amount ofgroup sbelongin g toon eclas s istake nto olarg e (Tanford, 1962), something which canhappe n evenmor e easily inth e caseo fadsorbe dproteins .

WhenK ^ o'an, s dK * 'ar o,enos ta sfa rapar ta si nth r ecas efo radsorbe d polylysine,a transitio ni na plo to fpK ' vsa 1 causedb yth ediffer - (1) (2) PP encebetwee nl O 'an d KK ' canaccidentl yb emisinterprete d asa con ­ formationaltransition . Whenth e amounto fnon-titratabl egroup si nP Ladsorbe da tp H11. 0 istake nequa lt oth eamoun to fio npair sthe nn ,, n _an df ar eknown . 170

Table 6.1 Titration data ofPL- Lan d PL-DL adsorbed onP S (latex Mc a = -39mCm -2 2- 1 o A = 9.2 mg )an d in solution. c„_ = 0.1 kmol.m" : =293.1 5 K.Sampl e preparation accordingt o method 2.

Total Total Titr.Am. Non.Titr. a< « (

PL-DL (DP308) 48.8 - 42.6 - 0.87 0.87 - in solution

PL-L (DP1683) adsorbeda t pH 6.5 onPS . 47,2 20.42 29.5 +52 0.63 0.81 +43 r=const=0.48 mg.m-

PL-L (DP1683) adsorbed at pH 6.5 onPS . 47.2 10.21 35.5 +47 0.75 0.84 +72 r=r(pH)pH<9. 5 r=r(pH9.5)=0.96 _2 mg.m pH>9.5

PL-DL(DP308) adsorbeda t pH 6.5 onP S 48.8 10.21 36.0 +62 0.74 0.85 +82 r=r(pH),pH<10 r=r(pHio)=o.99 _2 mg.m ,pH>10

With a = ZH/n„ theplot s infig .6.14. 2an d6.1 5 canb e recalculated to obtain pK = pH - loga/l- av s a plots.Whe n iti s furthersup - aPP /o\ posed thatth edielectri c constant effecto npK v 'i ssmall ,th ep K r o,s r app vs a curves obtained (fig.6.16 )ar e related to one class ofgroup s i.e. with thesam ep K andhenc e ananalysi s according toequatio n 6.10 is warranted. A similar reasoning canb e followed for,say ,th ecar - 171 boxyl groups in aprotei n (Tanford, 1962). The fact that the a values at pH 11.00 are nearly the same as for PL in solution (see table 6.1) (2 ) suggests indeed thatpl O 'differ s notmuc h from pK . O/ s owt Itca n be seen from fig. 6.16 that thep K vs a plots of adsorbed PL-L and PL-DL have,whe n r = const., ahighe r slope and lower values of pK . Both effects are due to stronger electrostatic segment- app segment repulsion in the adsorbed state, compared with thebul k solu­ tion. The importance of these interactions was also found from the salt dependence of the adsorbed amount as described in Chapter 4. At higher a values the pK vs a curve of adsorbed PL crosses the app corresponding solution curve. The intersection point is shifted to higher a values when r increases. Above the intersection point pK is higher than in solution. Only the intersection point of curve 3 and 1 in fig. 6.16 is at sufficiently low a value tob e accurate enough for further interpretation. The change of sign ofAp K ^ (=p K (ads)- app app pK (bulk) at the intersection point means that the mean potential _ app y(a)change s signwit h respect to thepotentia l atth eplac e of a dis­ sociating group on aP L molecule in solution. From electrokinetic measurements on PS plugs with adsorbed PL-L (T = r (pH 6) = const; 0.1 M NaBr) we found that the streaming nicix potential changed sign also about pH 10.0, i.e. at about the same pH as the occurrence of the intersection point. Because at this pH the surface charge is still neutralized by -NH. groups (see table 6.1) the negative streaming potential found must be due to incorporation of Br" ions. That such an incorporation of anions can occur was also inferred from themagnitud e of the slopes of conductometric titrations ofP S latex with PL (see chapter5) . In principle the area enclosed between curves 3 and 4 in fig. 6.16 contains information about the non-electrostatic free enthalpy of ad­ sorption of PL. By the same token the area between curves 4 and 5 represents the non-electrostatic free enthalpy of the conformational transition in PL near the PS surface. It can be seen from fig. 6.16 that the adsorption free enthalpy is much greater than the non­ electrical free enthalpy of the helix-coil transition. Hence the inter- molecular interactions between PLmolecule s in the adsorbed layer and the interactions of PL with theP S surface aremuc h stronger than the intramolecular interactions responsible for the helix stability. This suggests that the hydrophobic interactionbetwee n the alipathic parts of the PL side chains with the hydrophobic PS surface is important as was concluded before from the strong rise of the adsorbed amount 172

I ' i I'll 10.8 f*. pp.

10.6 -

10.4 ....

10.2 - 1 _JA _ 10.0 " i^ J^il - •••' // 9.8 - -""^ ..••''3 1/ -~.s / 1 9.6 / /

i 1 . 1 .4

Fig.6.1 6Proto n titrations ofPL- Lan dPL-D Li nsolutio nan dadsorbe do nP S(late x M,.,a = -39raC.m"2)wit h a= Z„/zf!£f .1 .PL- L(D P1683 )i n0. 1M NaB r DO H n,max solution; 2.PL-D L (DP308 )i n0. 1M NaB r solution;3 .Adsorbe dPL- L (DP 1683)f = r (pH6 )= const ;A .Adsorbe d PL-DL (DP308 )f = T (pH ) pH < 10, r= const (pH> 10);5 .Adsorbe d PL-L(D P1683) , T =T (pH ) pH< 9.5 ,T = T (p H9.5 )= const .p H> 9.5 .Th esam e titrationsar eplotte d infig .6.1 4an d6.1 5fo r a' =Z„.Z. " ° HH,ma x withincreasin gelectrolyt econcentratio nu pt oa tleas t1 M NaB r(se e chapter 4). Inth e case of asurfac ewhic h can formH-bond swit hth esolven to r solute, such as silica, there mayb e acompetitio nbetwee ninter - molecularH-bond s andH-bondin g with the surface,whic hca nmarkedl y influenceth eheli xstabilit ya tth einterface .

6.6.4.3 Comparison with other experimental systems

For sake ofcompariso nI performe d sometitratio nexperiment swit h PMA-peadsorbe d onpositivel y andnegativel y charged PS andPG Aad ­ sorbedo npositivel ycharge dlatex .Th esampl epreparatio nwa saccord - 173 ingt ometho d1 ,excep ttha tth ep H ofadsorptio nwa s about8 .Th e titrationswer edon ewit hHCl . Infig .6.1 7 and6.1 8 thep K vs a curvescalculate d from thedat a in akin d of iterativewa y areplotte d (seesectio n6.6.4.1) .Du et o thecalculatio nprocedur eth edegre eo fdissociatio nobtaine di sabou t equalt o =Z„/Z „ e.buf tth epositio no fth ecurve so fadsorbe dPMA - a H ri,ma x peo rPG Awit hrespec tt oth esolutio ncurv eremain srathe runcertain . Neverthelessi ti sstrikin gtha ta tintermediat ea value sAp K shows the same trends as found for adsorbedPL ,a tleas twhe nth esign so f thecharge sar etake nint oconsideration .

- ' i • I ' I • I ' pKapp . / y y.1 * I 6.2 / / ' 1 J

6.0 2__ / IS

: s\i y^ —-^y y y'. 5.8

l' / / 5.6 - 1/3/ J / / ' ' 5.4 '/ / / / ƒ / / / 5.2 ƒ / 1 / ƒ / ƒ / 5.0 ' 1 1 / 4.8 -

1 . 1 . 1 . 1 .

Fig.6.1 7 Titration curves of PMA-pe (M•* >1 0) insolutio n (curve 1)an d adsorbed (pH-\ .8 ) on negatively charged PS (a =-5 0mC. m )(curv e2 )an dposi ­ tively charged latex (curve3) . curve 1:44. 2pmo l PMA-pe,V = 20. 0cm 3 curve2 : 11.3Mmo l PMA-pe,V 19.36c m ,0.82 5g P S (negatively charged). 3 curve3 :56. 0prao l PMA-pe,V 18.47c m ,1.6 5 gP S (positively charged). 174

!--••• ! ' 1 * | i 1 ' 5.8 T=293.15K 0.1 M NaBr. PKa >p. 5.6 - -

5.4

1 / ^ b.2 - y^ r / / / 1 / 1 5.0 - / 1

1 1 4.8 - 1 1 1 l / 4.6

4.4 - -

4.2 -

1 . 1 . 1 . 1 .

Fig. 6.18 Titrations of PGA-L (DP 150) in solution (curve 1) and adsorbed on positively charged PSlatex .T T (pH8 )= const , max r

The only information thatca nb e obtainedwit hcertaint y fromth e curves isth epresenc e or absence ofa conformationa ltransition .I n solutionth etransitio nfro mth ecompac tt oth eextende dfor mo fPMA-p e and the helix-coil transition inPG A areclearl yvisible .I nth ead ­ sorbed state (T = constant) only inPMA-p e adsorbed onnegativel y charged PS latex atransitio n inth esam ea regio na si nsolutio ni s observed,whil e inth ecas e ofcharg econtras tbetwee nadsorben tan d adsorbate andr = constan tdurin gth etitration ,n otransitio ni sob ­ served. These results are inlin ewit h ourobservation s onth ePL - latexsystem . Inth e literature onlya fe wstudie so nconformationa ltransition s inth eadsorbe dstat ear ereported . Casper et al. (1974) founda helix - coil transition in acopolypeptid eo fglutami caci dan dmethy lgluta - 175 mateadsorbe da tth eair-wate rinterfac eo nchangin gth ebul kpH ,whic h wasver ysimila rt oth etransitio nobserve d inPG Asolutions . Van Vliet and Lyklema (1978) reporteda conformationa l transitiona sa functio n ofp H inPMA-p eadsorbe do nemulsio ndroplets .Th epropertie so fthi s transitionwer eals over ysimila rt othos ei naqueou ssolution . Miller and Bach (1973) foundtha tnegativel ycharge dDN Apreserve s itsdoubl ehelica lstructur ewhe nadsorbe do nnegativel ycharge dmercury , butunfold swhe nadsorptio ntake splac ea tpositivel ycharge dmercury . Pefferkorn et al. (1982) concluded fromstati can ddynami cmembran e propertiestha ti nPG Amolecule sadsorbe do na porou scellulos eacetat e filtera helix-coi ltransitio nstil lexists . From the above it isobviou s thata conformationa l transitiona t constant adsorption can takeplac e when the surface charge andth e polyelectrolyte charge have both the same sign,bu twhe n acharg e contrastexis tn otransitio na tr = cons tca noccur .A transitio ni na flat adsorbed helix isonl y likely tooccu rwhe nth eadsorptio nfre e energype rsegmen ti so fth esam emagnitud ea sth ehelix-stabilizatio n freeenerg ype rsegmen tan dwhe nther ear en okineti cbarrier s forth e transition to takeplace .Th elatte rpossibilit y ismor eunlikel yfo r liquid-liquid interfacesbecaus emolecula rrearrangement sca noccu rmor e easilyi nthos ecases .A transitio ni na fla tadsorbe dheli xwil lhow ­ everb eshifte dalon gth ep Haxi swit hrespec tt oth etransitio ni nso ­ lution,becaus eth eeffectiv es valu ewil lb edifferen ti nth eadsorbe d state.However ,i nth ecase swher ea transitio ni sobserved ,th echar ­ acteristicsar eabou tth esam ea sthos eo fth etransitio ni nsolution . Therefore the observed transitions inadsorbe d polyelectrolytes are transitions in secondary structureo floop san dtail so fth eadsorbe d molecules. Such atransitio n ismos t likely to takeplac ewhe nth e averageloo pand/o rtai lsiz ei slarg eenoug ht oallo wfo ran ysecond ­ arystructure . Inth ecas eo fpolyelectrolyte s theextensio no fth eadsorbe dlaye r depends mainly on the distancebetwee n the chargeble groups onth e polymer chain and theeffectiv eadsorptio nenerg ype rsegmen tx cc- Inth ecase swher ea transitio nwa sobserve d (atconstan tr )th esurfac e chargewa szer oo rha dth esam esig na sth epolyme rcharge .Probabl yx „ isclos e toth ecritica lvalu ei nthes ecases ,becaus ethe nth epoly ­ electrolyte adsorption theorypredict s still an appreciableadsorbe d amount but the adsorbed layer ismor e extended than forhighe rx values.I nth ecas eo fcharg econtras tbetwee nadsorbat ean dadsorben t

Xs ££wil lb emuc hhighe rtha nfo ra =0 mC. m .Th econsequenc eo f 176 this isa muc hmor e flatadsorbe d layer (atr « r )no tallowin g -1 max a conformational transition tooccur .Als oth efac ttha ti nth ecas e ofa negativel y chargedsurface ,positivel ycharge dcoi lsegment sar e preferentially adsorbed plays arol e inth ecas eo fcharg econtras t (seesectio n6.6.5) . The adsorbed layero fo nparafi n adsorbedPMA-p ei smor eextende d thani susuall y found forhomopolyelectrolytes , asa consequenc eo f theemulsificatio nprocess ,i.e .ther ear elon gPMA-p etail sdanglin g insolutio n (van Vliet and Lyklema, 1978). Theobserve d conformational transitioni nadsorbe dPMA-p ei sver ylikel yt ooccu rbecaus eo fthes e tails. When theadsorbe d amounti sallowe d toincreas e with decreasing chain charge density, anadsorbe d layerwit h longer tails andloop s canb eforme di nwhic ha helix-coi ltransitio noccur sa sshow nearlie r (fig.6.16) . When PL-Li sadsorbe d atp H11.0 ,i.e .fro mth eheli x conformationi nsolution ,th efirs tadsorbin gmolecule sca nb eunfolde d butwhe nth eadsorbe damoun tincrease srearrangement soccur ,resultin g ina nadsorbe dlaye rwit hpartiall yhelica lloop san dtails . 6.6 .5 General discussion

The results concerning theconformationa l aspectso fadsorbe dPL- L areschematicall ydepicte di nfig .6.19 .Th ebehaviou ro fadsorbe dPL- L molecules is strongly determinedb yth epresenc e of-NH 3..OSO_ ion pairsove rth ewhol ep Hregion .Du et othes eio npair sther ei sa hig h adsorptionenerg ype rcoi lsegment ,preventin gth eformatio no fsecond ­ arychai n structure athig hp Ha tleas ta tlo wsurfac ecoverag e (f= r q m*,,)- Onlywhe na thig hpH ,r ^ r m=„ther ema yb ea fractio nheli x Ó ItLcLX — ItldX inadsorbe dPL- La sdepicte di nfig .6.19.b . Asmentione dbefor eth estabilit yo fa polypeptid eheli xa scompare d totha to fa rando m coil isth eresul to fa delicat ebalanc eo fman y interaction forces.A tlo wsurfac e coverage thisbalanc e isclearl y displacedi nfavou ro fth ecoil . There aresevera lway sfo rreachin gth eadsorbe dstat ea ta chose n pHvalue .Fo rexample ,th eadsorbe dstat eo fPL- Lmolecule sa tp H11. 0 depicted infig .6.19. aan d6.19. bca nb ereache db yadsorptio na t pH6 first (processI )an da subsequen t increaseo fth ep Ht o11. 0 (processII) .Alternativel y therout evi aproces s IIIan dI Vca nb e followed. Because ofth e fact thatth er-p Hcurve sar ereversibl e bothroute swil lgiv eth esam eadsorbe dlaye rstructure . 177

® ® S^*^ ,-«. III ^* ©***-^ *& ^^ 3 \ @ 't λ ,vl 'II Hl %

®S^_©*'~ II V ^^*r^J • ..•^-v- ••- ¥ - - - pH 6 pH 11 pH 6 pH 11 r-r r=r max 1 1 -' max max

Fig.6.1 9Schemati cpictur eo fth econformationa lpropertie s ofadsorbe dan dfre e PL-L.

As explained earlier the results of theproto n titrations atdif ­ ferent pH of adsorption suggest strongly that also in the case shown in fig.6.19. a the structure of the adsorbed layer is independent of the route followed. Zhulina, Birshtein and Skvortsov (1980) investigated theoretically the adsorption behaviour of uncharged polypeptides on solid surfaces and especially the effect of secondary chain structure and helix-coil transitions on these properties.Thes e authors considered the adsorp­ tion of infinitely long isolated chains.Henc e intermolecular segment interaction is not taken into account. Inmos t theories of helix-coil transitions in solution intermolecular interaction is also not consid­ ered. The effect of the polymer concentration is a sharpening of the transition (smaller a)wit h increasing polymer volume fraction (Grover and Zwanzig, 1974). For adsorbed chains the neglect of intermolecular interactions ismuc h worse than in solution inmos t experimental situa­ tions. This is the casebecaus e investigations concerning the secondary structure of apolyaminoaci d are usually performed at rather low volume fraction in solution but necessarely high volume fraction in an ad­ sorbed layer. However it is more relevant that the fraction of seg­ ments in trains is strongly overestimated intheorie s of the adsorp­ tion of single chains at the bulk volume fractions normally encoun- 178 tered (Fleer and Lyklema, 1983). Inthei r theory Zhulina et al. de­ scribe apolypeptid e chain infac ta sa copolyme ri nwhic hth echai n segmentsca nb eeithe ri na helica lo ra coi lstate . Justa si nsolutio nth ehelix-coi l transitioni sdescribe db ythe m withth ecooperativit yparamete ro an dth eheli xgrowt hparamete rs . Inth epresenc eo fa ninterfac e theeffectiv evalue s ofa an ds ar e determinedb yth evalu eo fth eadsorptio nenergy . Zhulina et al. con­ siderno wthre ecases . i. helixan dcoi lsegment shav ebot hth esam ex value. s . ii. a helix segmen tha sa large r adsorption energy \ thana coi sl c segment(x s>- iii.a heli x segmentha sa smalle radsorptio nenerg ytha na coi lseg - h „ c mentx s< X s- In this model acompletel y flat adsorbed helical polypeptideha sa fractiono fsegment s atth esurfac eo f1.0 .I na rea lhelica lchai n thisca nb e^ o r -ra tth emost .A sth efractio no fsegment si ntrain s isstrongl yoverestimate di nth etheor yo f Zhulina et al. atth eusua l values forx „o fabou t1.0 ,the yconside ri nfac tfla tadsorbe dcoil - and/orfla tadsorbe dheli xsequence san dth etransition sbetwee nthem . Henceprobabl y only theadsorbe d states depicted infig .6.19. aca n be compared with their results,becaus e $*i sver ylo wa tp H11. 0i n that case.Th esituatio n infig .6.19. ai sclearl y acas ei nwhic h h c X X _becaus S ei tsi s unfavourable tohav echarge dcoi lsegment sadsorbed .Th eformatio no f a helix isthe npromote d atth esurface ,als oi nlin ewit hth eexis ­ tenceo fa transitio ni ntha tcase .

6.7 SUMMARYAN DCONCLUSION S

Inthi schapte r theconformatio no fadsorbe dpolylysin ewa sdis ­ cussed togetherwit hth eadsorptio n andprecipitatio n propertieso f bothpoly-L-lysin ean dpoly-DL-lysine . Itwa sshow ntha tth ecoi l toheli x transition inPL- Lwhic htake s place inaqueou s solution,wa sno tpresen ti nth eadsorbe dstat eo n negativelycharge dpolystyren elatex ,a tlo wconstan tsurfac ecoverage . 179

Athig hsurfac ecoverag eadsorbe dPL- Lappeare dt ob epartl yhelical . Insectio n6.2 ,th einteractio nforce sdeterminin gth econformatio n ofcharge dpolyaminoacid s werediscusse dan di nsectio n5.3 ,existin g theories for helix-coil transitions in charged polyaminoacidswer e discussedbriefly .Als oth epossibilit y ofsecondar y structurei npoly - DL-aminoacidswa sdiscusse d inthi ssection . Becausea tlo wchai ncharg edensit ypolylysin eca nprecipitat efro m solution, theprecipitatio n properties ofPL- L andPL-D Lwer einves ­ tigated.I tappeare dtha tth eprecipitatio ncharacteristic so fPL- Lan d PL-DLwer e the same except for somemino r differences probablycon ­ nected with the absence of secondary structurei nPL-DL .I twa scon ­ cluded from the data thatPL- L shows,a tleas tabov e30 3K ,onl yon e typeo fprecipitatio nan dno ttw oa s Zimmerman and Mandelkern (1975 a) foundfo rPGA .Th eprecipitate so fPL- Lan dPL-D Lar eprobabl yamorphous . Alsoth eamoun to fP Ladsorbe da sa functio no fth ep Hwa sth esam e forPL- L andPL-DL .Henc e the secondarychai nstructur edoe sno tin ­ fluenceth eadsorptio nproperties .I nth epresenc eo fa nadsorbe dlayer , theprecipitatio n ofP L started atth esam ep Ha si nsolution .Henc e multilayer formationoccur sonl ya twors etha n6 conditions . In section 6.6 the conformation of adsorbed PLwa s investigated potentiometrically. After anexplanatio n of themethod , themeanin g of Potentiometrie data of adsorbed weak polyacids was analyzed. Special attentionwa spai d to thedefinitio n andcalculatio n ofth e degree ofdissociatio n and the implications forth e analysis ofth e titration data. For comparison reasons also titrations ofadsorbe d PMA-pean dPG Awer eperformed . Thetitratio nresult sshowe dthat ,a sa rule ,i tca nb estate dtha t at constant (low)adsorbe d amount,n oconformatio n like theon ei n solution occurs,whe nther ei sa charg econtras tbetwee nth epolyaci d adsorbate and the adsorbent.Thi si sbecaus eth eeffectiv eadsorptio n energy per segment is high inthes e cases,compare d with thenon ­ electricconformationa ltransitio nfre eenerg ype rsegment . Inth e case ofPL-L ,io npai r formationwit h PS surface groupsan d probablyals ohydrophobi cinteraction swit hth esurfac ear eresponsibl e forth eunfoldin go fadsorbin ghelica lmolecules .Onl ya thig hadsorbe d amounts,whe nappreciabl enumber so ftail san dloop sar epresen tthes e tailsan dloop sca nb epartl yhelical . The titration results show further thatonl yi nth efirs tadsorp ­ tionlaye rth edegre eo fdissociatio na ca ndeviat esignificantl yfro m thebul kvalue .Thi s deviation is largelydetermine db yth eio npai r 180 formationwit h the surface -OSOZgroups .Outsid eth efirs tlaye rth e titrationbehaviou r isroughl y the same asi ti sfo rP Lmolecule si n solution.

6.8 REFERENCES

Applequist,J . (1963).J .Chem .Phys . 38 (4),934-941 . Barskaya,T.V . andPtitsyn ,O.B . (1971).Biopolymer s 10, 2181-2197. Bates,R.G . (1964). 'Determinationo fpH' ,Wiley ,Ne wYork . Birshtein, T.M. and Ptitsyn, O.B. (1966). 'Conformationso fmacro - molecules', IntersciencePublishers ,Ne wYork . Birshtein,T.M. ,Zhulina ,E.B .an dSkvortsov ,A.M . (1979).Biopolymer s 18, 1171-1186. Buscall,R .an dCorner ,T . (1982).Colloid san dSurface s5 ,333-351 . Cantor,C.R . andSchimmel ,P.R . (1980). 'Biophysicalchemistr ypar tIII . Thebehaviou r ofbiologica lmacromolecules' ,W.H .Freema nan dcom ­ pany,Sa nFrancisco . Casper, J., Berliner, C, Ruysschaert, J.M. and Jaffe,J . (1974). J.Colloi d InterfaceSei . 49, 433-441. Chernoberezhskii,Yu.M . (1982). 'TheSuspensio neffect 'i nSurfac ean d ColloidScienc e 12, 359-453. Chou,P.Y .an dScheraga ,H.A . (1971).Biopolymer s 10, 657-680. Ciferri,A. ,Puett ,D. , Rajagh,L . andHermans ,J . Jr. (1968).Bio - polymers6 ,1019-1036 . Conio, G., Patrone, E.,Rialdi , G. and Ciferri,A . (1974).Macro - molecules7 ,654-659 . Cosani,A. ,Terbojevich ,M. ,Romanin-Jacur ,L .an dPeggion ,E .(1974 ) in 'Peptides polypeptides andproteins ' Blout,E.R . et al.eds . J.Wile y& Sons .Ne wYork . Davidson,B .an dFasman ,G.D . (1967).Biochemistr y 6, 1616-1629. Dimarzio,E.D .an dBishop ,M . (1974).Biopolymer s 13, 2331-2348. Doty, P. andGratzer ,W.B . (1961)i n 'Polyaminoacids,polypeptides , and proteins. Stahmann, M.A. ed. University of WinconsinPres s Madison. Evers,O . (1984). MSc thesisAgricultura l UniversityWageningen ,th e Netherlands. Ellenbogen,E . (1952).J .Amer .Chem .Soc . 74, 5198-5201. Fasman,G.D . (1967)i n 'poly-a-AminoAcids 'Fasman ,G.D .ed .M .Dekke r Inc.Ne wYork . Fixman,M . (1979).J .Chem .Phys . 70, 4995-5005. 181

Fleer,G.J .an dLyklema ,J . (1983)i n 'Adsorptionfro msolutio na tth e solid-liquid interface'.Parfitt ,G.D. ;Rochester ,C.H .Eds. ,Aca ­ demicPress ,Londo netc . Franks,F . (1973). 'Water',Plenu mPress ,Ne wYork/London , 2, Chapter1 . Gratzer,W.B . (1967)i n 'Poly-a-Aminoacids' , Fasman ,G.D .ed .M .Dek ­ kerInc .Ne wYork ,Chapte r5 . Grourke,M.J .an dGibbs ,J.H . (1971).Biopolymer s 10, 795-808. Grover,M.K .an dZwanzig ,R . (1974).Biopolymer s 13, 2103-2115. Hardy,P.M. , Haylock,J.C. ,Marlborough ,D.I. ,Rydon ,H.N. ,Storey ,H.T . andThompson ,R.C . (1971).Macromolecule s 4, 435-440. Heitz,F .an dSpach ,G . (1971).Macromolecule s 4, 429-432. Heitz,F. ,Lotz ,B .an dSpach ,G . (1975).J .Mol .Biol . 92, 1-13. Hermans,J .Jr . (1966).J .Phys .Chem . 70, 510-515. Hesselink,F.Th .an dScheraga ,H.A . (1972).Macromolecule s5 ,455-463 . Hesselink,F.Th. ,Ooi ,T .an dScheraga ,H.A . (1973).Macromolecule s6 , 541-552. Honig,E.P . (1972).J .Electronal .Chem . 37, 249-266. Katchalsky,A . (1971).Pur eAppl .Chem . 26, 327-373. Kauzmann,W . (1959). 'Advancesi nprotei nchemistry ' 14, 1-63. King, E.J. (1965). 'Acid-Base equilibria' in 'The Internationalen ­ cyclopedia of physical chemistry and chemicalphysics 'Topi c15 : equilibrium properties ofelectrolyt e solutions vol.4 ,Robinson , R.A.ed .Pergamo nPres sLondon-Ne wYork . Klotz,I.M .an dFranzen ,J.S . (1962).J .Amer .Chem .Soc . 84, 3461-3466. Leyte,J.C .an dMandel ,M . (1964).J .Polym .Sei .A 2, 1879-1891. Manning,G.S . (1969).J .Chem .Phys . 51, 924-933. Manning,G.S . (1972).Ann .Rev .Phys .Chem . 23, 117-139. Manning,G.S . (1978).Q .Rev .Biophys . 11, 179-246. Marini,M.A .an dMarti ,G.E . (1980).Biopolymer s 19, 885-898. Miller, I.R. and Bach, D. (1973). Surface and Colloid Science 6, 185-260.Matijevic ,E .ed . Morawetz, H. (1965). 'Macromolecules insolution' . IntersciencePu ­ blishers,Ne wYork . Nagasawa,M . (1971).Pur eAppl .Chem . 26, 519-536. Nagasawa,M .an dHoltzer ,A . (1964).J .Amer .Chem .Soc . 86, 538-543. Nagasawa,M .an dTakahashi ,A . (1972)i n 'lightscatterin g fromPolyme r Solutions',Huglin ,M.B. ,ed .Academi cPress ,Ne wYork . Nakagaki,M . andEbert ,G . (1982). Colloidan dPolym .Sei . 260, 781- 787. Némethy,G .an dScheraga ,H.A . (1962).J .Phys .Chem . 66, 1773-1789. Norde,W .an dLyklema ,J . (1978).J .Colloi d InterfaceSei . 66, 266-276. 182

Norde, W. and Lyklema, J. (1984). To be published. Olander, D.S. and Holtzer, A. (1968). J. Amer. Chem. Soc. 90, 4549-4560. Oosawa, F. (1957). J. Polym. Sei. 23, 421. Overbeek, J.Th.G. (1953). J. Colloid Sei. 8, 593-605. Painter, P.C. and Koenig, J.L. (1976). Biopolymers 15, 229-240. Pederson, D., Gabriel, D. and Hermans, J. (Jr.) (1971). Biopolymers 10, 2133-2145. Pfeil, W. and Privalov, P.L. (1976). Biophys.Chem . 4, 23-32. Poland, D. and Scheraga, H.A. (1970). 'Theory of helix-coil transitions inbiopolymers' . Academic Press NewYork . Prokopova, E. and Ciferri,A . (1972). Biopolymers 11, 1621-1626. Ptitsyn, O.B. (1971). Macromolecular microsymposia VIII & IXPrague , p. 227-244. Puett, D., Ciferri, A., Bianchi, E. and Hermans, J. (Jr.) (1967). J. Phys.Chem . 71, 4126-4128. Puett, D. and Ciferri,A . (1968). Biopolymers 6, 1213-1217. Rosenheck, K. and Doty, P. (1961) ref. 29 of Gratzer, W.B. (1967) in 'poly-a-aminoacids', Fasman, G.D. ed.M .Dekker , Inc.Ne w York. Rice, S.A. and Nagasawa, M. (1961). 'polyelectrolyte solutions', Academic Press, London,Ne w York. Stulz, J., Müller,D. , Hull,W.E . and Kricheldorf, H.R. (1983). Macro- mol. Chem. 184, 1311-1322. Schee,H . van der (1984). Doctoral thesis. Agricultural University Wa­ geningen, theNetherlands . Schellman, J.A. (1955). Compt. Rend. Trav. Lab.Carlsber g (Ser.chim) 29, 223-229. Reprint (paper 7) in Poland, D. and Scheraga, H.A. (1970). 'Theory of helix-coil transitions inbiopolymers 'Academi c Press New York. Stigter,D . (1975). J. Colloid Interface Sei. 53, 296-306. Silberberg, A. (1972). J. Colloid Interface Sei. 38, 217-226. Tanford, C. (1962). Adv. Prot.Chem . 14. Tanford, C. (1973). 'Thehydrophobi c Effect'. J. Wiley & Sons,Ne w York. Tseng, Y.W. (1975). Ph.D. thesis,Universit y of California, SanFran ­ cisco. Tseng,Y.W . andYang , J.S. (1977). Biopolymers 16, 921-935. Terbojevich, M., Cosani,A. , Peggion, E.,Quadrifoglio , F. and Crescenzi, V. (1972). Macromolecules 5,622-627 . Vliet, T. van (1977). Doctoral thesis Agricultural University Wage­ ningen, theNetherlands . Vliet, T. van and Lyklema, J. (1978). J. Colloid Interface Sei. 63, 97-105. 183

Wada,A . (1962)i n 'Polyaminoacids,Polypeptide san dProteins' ,Stah ­ mann,M.A. ,ed .Univ .Winconsi nFres sMadiso np .131-146 . Zimm,B.H .an dBragg ,J.K . (1959).J .Chem .Phys . 31, 526-535.Reprin t (paper17 ) in Poland, D. and Scheraga, H.A. (1970). 'Theoryo f helix-coiltransition si nbiopolymers 'Academi cPres sNe wYork . Zimm,B.H .an dRice ,S.A . (1960).Mol .Phys . 3, 391-407. Zimmerman,S.S .an dMandelkern ,L . (1975a).Biopolymer s 14, 567-584. Zimmerman, S.S., Clark,J.C . andMandelkern , L. (1975b).Biopolymer s 14, 585-596. Zhulina,E.B. ,Birshtein ,T.M . andSkvortsov ,A.M . (1980).Biopolymer s 19, 805-821. 185

7 POLYELECTROLYTEADSORPTIO NTHEOR YAN DTH EADSORPTIO NO FCHARGE D POLYAMINOACIDS*

7.1 INTRODUCTION

Inth epreviou schapter sth eadsorptio no fpolylysin ewa sconsidere d mainly from anexperimenta lpoin to fview .No wth echarge doligo -an d polypeptides willb e considered asmode lpolyelectrolyte s andsom eo f thepreviousl yobtaine dexperimenta lresult swil lb ecompare dwit hth e predictions of thepolyelectrolyt e adsorptiontheor yo f van der Schee (1984). Thiscompariso nserve ssevera lpurposes : i. Undersom econditions ,charge dpolypeptide sbehav ea srando mpoly ­ electrolytes,the nthe yar esuitabl emode lsubstance st otes tth e theoreticalprediction sabou tth einfluenc eo fmolecula rmass ,pH , andioni cstrengt ho nadsorption , ii. Chargedoligopeptid eadsorptio nbridge sth ega pbetwee nadsorptio n ofmonomer san dpolymers ,o rrathe rbetwee nion san dpolyelectro ­ lytes.Th e theory for the adsorption of ions is relativelyad ­ vanced and anumbe r ofequation s havebee npu tforward ,suc ha s those of Stern andFrumkin-Fowler-Guggenheim .Polyme radsorptio n theory isbase d on statistical considerations and iti susefu l tocove rth eoverla prang ebetwee nth etw ocategories , iii.Adsorptio no fcharge dpolyaminoacid scapabl eo fformin gsecondar y structuresassum ea nintermediat epositio nbetwee nth eadsorptio n of flexiblepolyelectrolyte s andproteins . Iti so finteres tt o find outth erelevanc eo fsecondar ystructur ei nth etheoretica l descriptiono fpolyaminoaci dadsorption . The substance under consideration is (mono,oligo- ,poly-)lysine . Thepolylysine s havebee ndescribe d inchapte r3 .Oligolysine su pt o DP1 6wer e synthesized by aprocedur e describedb y van der Schee and Tesser (1983). Theexperimenta lmethod suse dan dexperimenta lresult s obtainedhav ebee ndescribe di ndetai li nth epreviou schapters . The adsorbed amounto fPL.HB ro nP San dPL.H Fo nAg ii sexpresse d inm gPL.HB rpe rm inbot 2. h cases . Special attention will be paid to the effects ofp H andioni c strength (I)o nth eadsorption ,thes evariable sdeterminin gth echarg e andth escreenin go fth echarg erespectively .

* Part of this chapter has beenpublishe d incoauthorshi pwit hH.A . vande rSche e andJ .Lyklema .Croatic a Chem.Act .56 ,695-70 4 (1983). 186

7.2 THEORY

Hitherto, no suitable polyelectrolyte adsorption theoryexisted . Theonl yon eavailabl ei stha tb y Hesselink (1977) buti ti sinadequat e because,amon gothe rdefects ,i tassume sa nexponentia l segmentdensity - distancep(z )relationship ,thereb y followingHoeve' spolyme radsorptio n theory (Hoeve, 1970). Althoughfo rpolymers ,thi si sa reasonabl eas ­ sumption, it is incorrect forpolyelectrolytes .Rather ,a n ab initio theory isneede d inwhic h p (z)i scompute dfo rvariou s Ian dp Han d themos t logical approachi st oexten don eo rmor eo fth epresen tda y suitablepolyme radsorptio ntheorie swit ha nelectrica lterm .Recently , this hasbee n achievedb yon eo fus .A mor ecomplet eaccoun ti sgive n by van der Schee (1984). Weshal lno wbriefl yrevie wth eprinciple san d someimportan tresults . Asa startin gpoint ,tw opolyme radsorptio ntheorie sma yb econsid ­ ered,viz .thos eo f Roe (1974) and Scheutjens-Fleer (SF) (1979). These twotheorie shav emuc h incommon .Bot har elattic etheorie si nwhic h polymer configurations are formulated through step-weighted random walk statistics.Th e likelinesst ofin dafte rj segment si na certai n chainth e (j+ i)-t hon ei na give nlattic elaye rdepend sessentiall yo n threefactors :(i )lattic eparameters ,suc ha sth ecoordinatio nnumber , (ii)interactio nparameter s with othermolecule s thatma yalread yb e present inthi s layer,an d in firstapproximatio nquantifie dthroug h theFlory-Huggin sinteractio nparamete rx an d (iii),onl yfo rth elaye r adjacentt oth esurfac e (trainsegments )a surfac einteractio nenerg y parameter x_- The canonical partition functionQ isthe nformulate d andmaximalize dt oobtai nth eequilibriu mdistribution . Thewa y inwhic h this isachieve d isdifferen t inth eRo ean dS F theory.Ro ewrite sQ interm so fp(z )an dmaximize sth eforme rwit h respect toth e latter. Indoin g so,h emake s anapproximatio n that virtuallyamount st oth eneglec to fen deffect s (tails).Th eS Fpictur e doesno tmak e thissimplification ,Q beingwritte ni nterm so findi ­ vidual chainconformations , and alsobein gmaximize dwit hrespec tt o these conformations. Therefore, the Roe theories gives onlyp(z) , whereas SFtheor y distinguishes between loops and tails.Thi s 'fine structure'ha sprove d tob eo fgrea trelevanc efo rsteri cinteractio n because it followed from the theory thati ti sver y likely thata number of longtail swil lb epresen ti nmos tadsorbate sunde rcondi ­ tionsme ti npractice . Bothth eRo ean dS Ftheor yhav eno wbee namplifie dwit ha nelectro - 187

staticterm . Ifi ti sdon eaccordin gt oth eRo epicture ,th ecanonica l partition functionQ fora polyelectrolyt e ata give nprofil ep(z )i s

obtained fromQ bymultiplicatio nwit hexp .(- F ,(p)/kT),wer eF el(P) isth e electrical free energy oftha tprofil e p(z).Thi sfre eenerg y canb e found,usin g some chargingprocess ,base d on amode l ofth e distribution of charges and their interactions. Inth epresen tcase , alattic edistributio no fth echarge swa sassume dan dth edistributio n ofsmal lion swa stake nt oobe yBoltzman nstatistics .Severa lchargin g processeswer e considered and their equivalenceproven ,mor eo rles s asdoubl e layers around particles (Verwey and Overbeek, 1948). IfS F theoryi suse da sth estartin gpoint ,eac hsegmenta lweighin gfacto ri s multiplied by anelectrica l termexp .(-Ft| i./RT )wher ei|> .i sth epoten ­ tial in layer i;thi slatte rquantit ybein gagai nfoun db ysom emode l picture. It appears that for polyelectrolytesth e difference betweenth e ROE and SFpictur e is lesspronounce d than foruncharge dpolymers . Thereaso ni stha tth eeffec to ftail si sles simportan ti nth echarge d systems. As themathematica l elaboration ismor e easywit h theRo e picture,w eshal lgiv ebelo wonl yresult sbase do nthi stheory .A some ­ whatmor edetaile daccoun ti sgive nb y Marra et al. (1983).

log f; — — . 1 uncharged '—' I""1 1-5-, '—'

0.5 ^, i ' -

1 —Ws |—1

0.01

-15 15 layer number i

Fig. 7.1 Semilogarithmicconcentratio nprofile s fora polyelectrolyt e chaino fD P24 0

withon e chargepe rBjerrumlengt h (0.71nm )x = 0-6 ;X =4.85 ,« ff =0.78 . Lattice sitelengt h 0.5 nm.,a = -0.2charge spe rsurfac e site.Th eioni c strength Ii sindicated . 188

Fig.7. 1 givesp(z )fo ra nadsorbe dpolyelectrolyte .A sth edistri ­ butioni sbase do na lattic epicture ,p jump sstepwis ean dth edistanc e iscounte d interm so flaye rnumber s i.I nfact ,th eordinat egive si n stead of p thelogarith mo fth evolum e fraction ty•i neac hlayer .Th e semilogarithmicplo twa schose nt oaccentuat ecertai nqualitativ efea ­ tures. In fig.7. 1 theuppe rcurv eapplie st oa nuncharge d adsorbate.Th e decayi salmos tlinear ,whic hwoul dcorrespon dt oa nexponentia ldistri ­ bution p(z).Th e deviations from linearity are aconsequenc e ofth e presenceo ftail s (forthi scurv eS Ftheor ywa sused) . .--' 'Two significant new features deserve attention.Th efirs ti stha t at low I,a regio no fnegativ eadsorptio n ($.< (f ,,, )occur si nth e profiles.Obviously ,thi si sdu et oth ehig hrepulsiv epotentia lgene ­ ratedb y thepolyelectrolyt e segmentspresen t inth elayer sadjacen t to the surface (Notetha tdu e toth elogarithmi cscal eth edepth so f theminim aar eexaggerated) .Th epresenc eo fthes eminim ademonstrate s the inadequacy ofpolyelectrolyt e adsorption theories witha pre-se t p(z). Thesecon dfeatur eo ffig .7. 1 istha telectrolyte sremai neffectiv e inth erang eabov e I~ 0. 1M .Thu sth etheor yi si nagreemen twit hth e experimentalobservation si nfig .4.8 ,wher eth esal tdependenc eo fth e PLadsorptio no nAg ian dP Si sshown .I nou rapproach, vBoltzmannstatis ­ ticswa semployed ,implyin gtha tth eion swer econsidere dpoin tcharges . Notver y significantly differingresult swer eobtaine di fth evolume s of the ionswa s taken into account,usin gRoe' smulticomponen tad ­ sorption theory {Roe, 1974), sotha tth econclusio nma yb edraw ntha t thepersistenc e ofelectrolyt eeffect su pt over yhig hconcentration s is due to the facttha tenoug hvolum e is available around andbe ­ tweenth emacromolecula rchain st oaccomodat eal lions .

7.3 CHOICEO FTH EPARAMETER S

Inthi schapte rw ewan tt ocompar enumerica lresult sfro mth epoly ­ electrolyte adsorptionmode l ofva nde r Scheewit hexperimenta ldat a concerningth ePL/latex ,PL/Ag Ian dPL/silic asystems . Forth ecalculation sth efollowin gparameter sar eneeded :th elattic e type, thenumbe r of segmentspe r chain,th e (nonelectrical )energ y parameters x andx , thechai ncharg edensit yan dth epolyme rconcen ­ tration. Inorde r toconver tth eresult st oexperimentall ymeasurabl e quantities,th emonolaye rcapacit ymus tals ob eestimated . 189

7. 3. 1 Coordination number of the lattice

Themode lo fva nde rSche euse sa lattic ewher eeithe ra segmen to r a solventmolecul e canb eplace do na site .Severa llattic etype sar e possible,e.g .a hexagona lo ra cubi cone . Following Roe (1974) and Scheutjens and Fleer (1979) ahexagona l latticewa s chosen.Th e coordination number isthe ntwelve .Bot h Roe and Scheutjens and Fleer showed that theresult s are insensitivet o thisparameter .

7.3.2 Monolayer coverage, area of a lattice site a and the distance between the lattice layers r

Theparameter suse di nth epolyelectrolyt e adsorptiontheor yan dals o inth e theories foruncharge dpolymer sar eessentiall ydimensionless . Inorde rt ocompar eexperimenta l andtheoretica lresult sth esyste mha s

tob e scaled.On ewa yo fdoin gthi si st ocalculat eth eparameter sa Q and r from the specific volumeo fth elysy lgrou pan dit smolecula r mass128 . Applequist and Doty (1961) found forth e specific volume avalu e 3-1 3 of 0.782m .kg .Th evolum e of alysy l residue isthe n0^16 6n m. 2 ' When the lattice is isotropic,a is0. 3 nm andr is0.5 5 nm.Thi s leadst o asaturate dmonolaye rcoverag eo f0.6 7mg.m " basedo nlysy l residueso r1 mg. m whe-2n B r asth -ecounterio ni sincorporated . The distance between the amino groups calculated in thisway , 0.55nm , is larger than the minimal distance between the charges when thisdistanc e isdefine d as Manning (1978) does, i.e.th edis ­ tanceb betwee n theprojectio no fth esid echai ngroup so nth elinea r backboneo fth echain . Thevalu e ofb is then 0.36n m (seefig .3.1) .Th evalu eobtaine d fromth especifi cvolum ecorrespond smor et oth eactua ldistanc ebetwee n twoneighbourin g amino groups inth eP Lchain ,becaus eo fth erathe r bulkysid egroups . 7.3.3 The chain charge density

As thedistanc ebetwee nth e charges in fullyprotonate d PL(a=o ) issmalle rtha nth eBjerru mlengt hi nth ecas eo fa (1-1 )electrolyte , counterion condensation according to Manning (1978) will occur (see also section6.2.4) .Afte r condensation ane tcharg eo f£ ~ remains, 190 i.e.on echarg epe rBjerru mlength .Th eeffectiv ea i sthe n0.22 ,whe n 0.55n m is taken forth edistanc ebetwee nth eamin ogroups .Th evalu e 0.55 nmi sprobabl ybette rtha n0.3 6 nmbecaus eth eP Lchai ni sno ta n ideallythi nro da spresuppose d inth eMannin gtheory . Inth epolyelectrolyt e adsorptionmode lo fva nde rSche eth epoly - electrolytecharge sar esmeare dou ti nplane sparalle lt oth esurface . This involves the assumption thatcharge s arefurthe rawa yfro meac h other thanth e segmentlength .Henc e inthi smode lth erol eo fcoun - terion condensation will beunderestimate d atleas ti nth ebulk .A sa correction,effectiv e values of a areuse d inth e calculations,al ­ though inth e adsorbed layer thepremise s ofth eMannin gtheor yar e notsatisfied . Especially theconditio n ofa n infinite,straigh t linecharg ei s contradictory with the flexiblepolyme rconcept ,especiall ys oi nth e adsorbed layer.Further ,th epolyelectrolyt econcentratio ni nth ead ­ sorbed layer isno ta lal lver y dilute.Nevertheles s iti sassume d heretha tth eMannin gtheor yi sapproximatel y applicable.Abov ea f f= 0.22 nocondensatio noccur san da __i sidentica lt o aagain .

7. 3. 4 The polymer-solvent interaction parameter x

TheFlory-Huggin ssolven t interactionparamete rx account sfo rth e shortrang e noncoulombi c interactionsbetwee na solven tmolecul ean d apolyme rsegment . Aswa s shown insectio n6. 4 andsectio n6.5 ,PL- Lan dPL-D Lpreci ­ pitates are formed athig hpH .Fro m this effect itca nb econclude d thatx isgreate r than 0.5. Forfull ycharge dP Lth etheoretica lad ­ sorption isotherms areno tver y sensitive toth evalu e ofx chose n (van der Schee, 1984). However atlo wchai ncharg edensit ynea rth e phaseseparatio npoin tthi si sno ts o (O.Evers ,persona lcommunication) . Forcharge dpolylysin ew echos erathe rarbitraril yx = 0. 6 andindepen ­ dento fpH , assuming thatth ehydratio no fth eaminogrou pi sno tde ­ pendento nth echarg estat eo fthi sgroup .

7.3.5 The non-electrical energy of adsorption parameter xs

Inth ecas e of.Ag i asth e adsorbent thenon-electrica lenerg yo f adsorption parameter xs was estimated by van der Schee fromdat a obtained from coagulation experiments withth eP Lmonome ranalo gan d the short oligomers. To thaten dth e x parameterwa s adjustedi n 191 such away , thata tth e experimentaldetermine d adsorbateconcentra ­ tion,wher ecoagulatio ntake splace ,th etheoretica lpotentia loutsid e thefirs tlaye rwa sver ylow .Th earithmeti caverag eo fth evalue sob ­ tainedi s4.8 5 (van der Schee, 1984). Inth ecas eo fP Sa sth eadsorben t X isprobabl y lower than forAgi ,becaus eth eadsorbe damoun to fP L forcomparabl e situations islowe ri nth ecas eo fP S (seechapte r4) . Usinga x valueo f4 i twa sfoun dtha ta reasonabl efi to fth eexperi ­ mentalresult swit hP Swa sobtained .

7.4 RESULTSAN DDISCUSSIO N

7. 4. 1 Dependence on electrolyte concentration

Inchapter s4 an d5 i twa sshow ntha tther ei sa stron ginfluenc eo f theioni cstrengt ho nth eadsorptio nproperties .Thi si sno tsurprisin g because electrical interactions arestrongl ydependen to nth econcen ­ trationo findifferen telectrolyte .Therefor eth esal tconcentratio ni s animportan tvariable . In fig.7. 2 experimental andtheoretica lresult sar eshow nfo rPL , adsorbed onAgi . The theory iscapabl eo fexplainin gth eris eo fad ­ sorptionwit h increasing ionic strength and iti sobserve dtha tthi s effectcontinue swel lbeyon d 0.1M .

i r- i 1 #A - 1 (C. s// / '/ —theor . yS /

0.5 --^A^ v

1 1 1

-logc 5

Fig. 7.2 Adsorption of poly-L-lysineo nAgi .Compariso nbetwee n theoryan dexperi ­ ment.Theoretica l adsorbed amountsar eexcesse s and expressed asequivalen t monolayers. Experimental conditions: pAg = 11.0;T = 293K ; pH6 ; PL-L DP300 .Theoretica lparameters :x =0.6; x =4.85; r= 300;a = -O.le/a, 2 ' s ' 'o ' with a =are ape r lattice site= 0.3n m 192

Althoughth esal teffec tfoun dfo rP San dAgi ,whic hi ssimilar ,i s explained well by thetheory ,thi si sno ts ofo rsilic aa sadsorbent . Inthi s case ther v s logc ,.plot s havea maximu m atconstan tp H and areconve x atconstan t surface charge,thi si ncontradistinctio n withth eAg io nP Scase swher eth eexperimenta la swel la sth etheoret ­ icalcurve sar econcave . Itappeare d notpossibl e to fitth e results obtained withsilic a by adjusting x •Probabl y the theory isno tsuitabl et odescrib eth e salteffec ti nthi scas ebecaus ev isnea rx inthi scas ean dbe - As As,cr cause ion exchange processes are relatively more important, aswa s shown inchapte r5 .Discret echarg eeffect s (i.e.io npai rformation ) whichcanno tb edescribe db yth etheor yar eimportan ti nth elatter . 7. 4.2 Influence of the surface charge

Theoretically it is expected thatth e adsorbed amount increases nearlylinearl ywit hincreasin g surfacecharge .Thi slinearit yi sals o found experimentally forth e adsorption ofP Lo nAg i (van der Schee, 1984) and also inthi sstud yfo rth eadsorptio no fP Lo nsilica .How ­ ever the agreementbetwee ntheor yan dexperimen ti sonl yqualitative . Discretecharg eeffect sma yb eresponsibl e forthi sdiscrepancy .

7.4.3 Influence of the chain charge density

Although thepolyelectrolyt e adsorptionmode l of van der Schee is not suitable for thedescriptio no fth eadsorptio npropertie so fwea k polyelectrolytes,th e surface excess as a function of thedegre eo f dissociation canb e approximately calculated when iti sassume dtha t ordoe s notchang e upon adsorption. So a isthe ntake na sindependen t of the distance zt o the surface.A swa sshow ni nchapte r6 thi sas ­ sumptioni sreasonabl efo rP Lsegment si nloop san dtails . In fig.7. 3 theoretical and experimental results are shownfo r PL-LD P19 0 adsorbed onP S as afunctio no fth epH .Th eexperimenta l a was calculated from the total amountP Lpresent ,th eadde damoun t NaOH and theequilibriu mpH .I nth etheor yunderlyin g fig.7. 2 itwa s

assumed that a „ =0.22 .A consequenc ei stha tth etheoretica l# exc isconstan t forvalue s of a below 0.22.Considerin g thevariou sun ­ certainties and approximations the correspondence between thetheo ­ reticalan dexperimenta ltrend si sreasonable . 193

i i i i I i 1.4 - - e.« '¥ 1.2 S*/s* 1.0 S*

.8 " / s Iheor - / / s / / / .6 / / / / -6

9—Û—-=

2 -

i < i

8.2 8.6 8.9 9.2 9.8 10.3 pH

Fig.7. 3 Adsorptiono fpoly-L-lysine-HB ro npolystyren ea sa functio no fp H(a) - Comparisonbetwee ntheor yan dexperiment .Theoretica ladsorbe damount sar e excessesan dexpresse da sequivalen tmonolayers .Experimenta lconditions : DP190 ;0 (latex)= -4 2mC.m" 2,a , ,= 0.22 ;T = 29 3K ;c „„ =0.0 1M . o eff ' NaBr Theoreticalparameters :cubi c lattice;x =0.6;x =4 ;r = 300 ; a - -0.1e/a 2,wit ha 2= 0. 3nm 2,I = 10~ 2M .

At high a themeasurement s indicate thatprecipitatio n occurs (see fig. 6.2).N o suitable calculations are available in the region of a = 1, son o comparison with theoretical results in thephas e separa­ tion region ispossibl e at themoment . In fact it is remarkable that a theory for the adsorption of flexible polyelectrolytes predicts reasonably well the 6 (a) curves of a polymer which is very likely to be partially helical in the adsorbed state at high pH and r .Apparentl y the PL molecules max adsorbed at high pH behave as if they are more flexible than PL molecules in the bulk, in accordance with the observation that PL is only partially helical at r „ (pH 11).Thi s is also in agreement with nicix the fact that the adsorptionbehaviou r of PL-L and PL-DL is the same. The polyelectrolyte adsorption theory can be improved when a is considered to vary with z. Also thevariatio n of the dielectric con­ stantwit h z canb e taken into account (0.Ever s personalcommunication) . 194

Thegenera lconclusio ni stha tth eagreemen tbetwee ntheor yan dex ­ periment is sufficiently satisfactory toconside rth echose napproac h asa promisin gdevelopment ,warrantin g furtherelaborations .

7.5 REFERENCES

Applequist,J . andDoty ,P . (1961), in 'Polyaminoacids,Polypeptide s andProteins' ,Stahmann ,M.A. ,ed .Univ .Winconsi nPress .Madison . Hesselink,F.Th . (1977).J .Colloi d InterfaceSei . 71, 448-466. Hoeve,C.A.J . (1970).J .Polym .Sei . C30, 361. Manning,G.S . (1978).Quart .Rev .Biophys . 11, 179-246. Marra, J., Schee,H.A .va nder ,Fleer ,G.J .an dLyklema ,J . (1983)i n 'Adsorption from Solution', Ottewill, A.H.; Rochester, C.H.an d Smith,A.L. ,eds .Acad .Press .245-258 . Roe,R.J . (1974).J .Chem .Phys . 60, 4192-4207. Schee,H.A . van der (1984).Doctora l thesis.Agricultura lUniversit y Wageningen,Th eNetherlands . Schee,H.A .va nde ran dTesser ,G.I . (1983).Colloi dPolym .J . 261, 461. Scheutjens, J.M.H.M. and Fleer, G.J. (1979). J. Phys. Chem. 83, 1619-1635, 84 (1980)178-190 . Verwey,E.J.W . andOverbeek ,J.Th.G . (1948). 'Theoryo fth eStabilit y oflyophobi cColloids' ,Elsevier ,chapte rIII . 195

SUMMARY

Thepurpos eo fthi sstud ywa st oobtai ninsigh ti nth echaracteris ­ ticso fweakl ybasi c (oracidic )polyaminoacid sa tsolid-liqui dinter ­ faces. A study of polyelectrolyte properties aswel l assecondar y structurea tth einterfac ewa sintended . Inthes erespect spoly-L-lysin e (PL-L)i sa ver ysuitabl eadsorbat e because at low pH thepositivel y charged PL-Lbehave s as ahighl y charged,mor e or less flexible,polyelectrolyte .A tlo wchai ncharg e density,PL- L assumes ana-helica lo rß structur edependen to ntempe ­ rature.Poly-DL-lysin edoe sno tsho w acoil-t o helix transition and therefore,i ti ssuitabl ea sa referenc efo rPL- Li nadsorptio nstudies . An outline and general background of thisstud yi sgive ni nchap ­ ter1 . Some aspects ofpolyelectrolyt e adsorption andpolyaminoaci d adsorption arereviewe d inchapte r2 . Chapter3 present sa selectio n ofrelevan tpropertie s ofpolylysin e and theadsorbent sused .I nth e followinga novervie wo fth emos timportan tresult si sgiven . Adsorption isotherms of poly-L-lysine and poly-DL-lysine onne ­ gatively chargedpolystyren e areno t significantly different atlo w pH,bu tals o nota thig hpH .Als oth eincreas eo fth eadsorbe damoun t with increasing ionic strengtho rp Har eth esam efo rPL- Lan dPL-DL . Hence, there isn o influence ofdifference si nchai nflexibilit yan d secondary structure between PL-L and PL-DL on the adsorbed amount (chapters4 an d 6). Secondary structure plays only amino r role inth eprecipitatio n characteristics ofPL- L (chapter 6). Precipitation ofPL- Loccur si n thepresenc e ofpolystyren e latex atth e samep H as itdoe s inth e bulk.Thi sindicate stha tmultilaye rformatio noccurs ,a stheoreticall y expected,whe nth esolven tbecome spoo r (chapter6) . Information about the secondarystructur ean dconformationa ltran ­ sitions in PL-L adsorbed at polystyrene is obtained fromproton - titrations of adsorbed PL-Lan dPL-D L (chapter 6). Atconstan t(low ) adsorbed amount, no conformational transition in PL-L, comparable to thecoi l toheli x transition insolutio n occurs.Th echarg econ ­ trastbetwee n thepolyaminoaci d and the adsorbenti slargel yrespon ­ sible forth e absenceo fsuc ha transitio ni nPL- L (andals oi nothe r systems). Only athig h adsorbed amounts,whe n appreciablenumber so ftail san d loops arepresent , formationo fsecondar ystructur ei nadsorbe dP Li s possible.The nadsorbe dPL- Lca nb epartl yhelical . 196

Analysis of the titration results further shows thatonl y inth e firstadsorptio nlaye rth edegre eo fdissociatio no fth eP Lsid egroup s issignificantl y lowertha ni ti si nth ebulk .Thi sdeviatio ni slarge ­ lydetermine db yth epresenc eo f-NH ~ -0S0 ~io npair sa tth esurface . Iti sbecaus e ofth eformatio no fthes eio npair san dals obecaus eo f hydrophobic interactions thata thig hp HPL- Lhelice sunfol dupo nad ­ sorptiona tlo wsurfac ecoverag e (chapter6) .

Polyelectrolyte adsorptionpropertie so fP La tlo wp Hhav ebee nin ­ vestigatedwit hhydrophili c (silicaan dborosilicat eglass )an dhydro ­ phobic (polystyrene (PS)(latex)an dsilveriodide )adsorbents .Th eeffec t ofchai nlength ,ioni cstrengt han dsurfac echarg ehav ebee nconsidere d (chapter 4). Theeffec to fth eP Lchai ncharg edensit yo nth eadsorptio n hasbee ninvestigate dwit hP Sa sth esubstrat ei nchapte r6 . Atlo wioni cstrengt hfull ycharge dP Ladsorb si na rathe rfla tcon ­ formation.I nal lcase sth eplatea uvalu ei sfa rles stha na monolaye r ofP L segments and nopronounce d effecto fth ehydrophobicit yo fth e substrate ispresent .Unde r these conditions theadsorptio ni sinde ­ pendent of the chain length.A n increase ofth e adsorbed amounti s prohibited because the formation of loops andtail si sprohibite da s aconsequenc eo fth estron grepulsio nbetwee nth eP Lcharges . Atlo wioni cstrengt hth eadsorptio no fP Lo nsilic aan dpolystyren e particleswa sals omonitore dconductometricall y andpotentiometricall y (chapter 5). Phenomenologically,th ebindin go fP Lo nnegativel ycharge d particles ist o acertai n extent analogous to the complexformatio n betweenpolycation s andpolyanion s inhomogeneou s solution.Plot so f conductivity againstpolylys'in econcentratio n shows inbot h casesa break. Inth eP L- soli dparticl e systems theamoun tadsorbe di nth e breakpoint is always lower than the maximum adsorption determined analytically, indicating that also other interaction forces than coulombic ones are important. Inmos tcase s the adsorption ofP Li n the breakpoint is superequivalent, which isofte nno tth ecas ei n homogeneous polyanion-polycation complexes. The main cause forth e formationo f superequivalent complexes isth erigidit y ofth esoli d particles and the relative largedistanc e betweenth echarge so nth e surface. In thebreakpoin t everynegativ e charge on the surfacei s boundt oa nammoniu mgrou po fP L (formingio npairs) .However ,no tal l -NH-.group s areboun d to anegativ esurfac echarge .Titratio no fth e + adsorbedP Lwit hpolyvinylsulphat e shows thatth efractio nfre e-NH 3 groupsaroun dth ebreakpoin ti sabou t0.3-0.4 ,indicatin g flatadsorp - 197 tion. Asexpected , in thecas e of silica the charge composition in thebreakpoin t is dependento nth eequilibriu mp Han dioni cstrength . A relation between the occurrence of conductometric breakpoints and iso-electricadsorptio nseem st oexis ti nth ecas eo fsilica . The adsorbed amount increases with increasing negative surface charge (chapter 4). In the case of silica,a linea r dependence is found, as also predicted by the polyelectrolyte adsorption theory ofva nde rSche e (chapter7) . The adsorbed amount increases strongly with decreasing PLchai n charge density.Abov ep H11. 0phas e separation isfoun dt ooccur .A s the repulsionbetwee n PL segments reduces,mor e loops and tailsca n develop andhenc e higher adsorptionvalue s are reached, untilphas e separation occurs,whe n thechai n charge density is too lowt okee p theP Ldissolved .Betwee np H6 an d11. 0th eadsorption-p Hcurve so fP L arepredicte dsatisfactoril yb yth etheor yo fva nde rSchee . Inth e case ofth e hydrophobic adsorbents polystyrene and silver iodide the adsorbed amount increases progressively with increasing saltconcentratio n at lowp H at leastu p to 1.0M (chapter 4). Also the adsorption-ionic strength curves arepredicte d wellb yth epoly - electrolyt adsorption theorymentioned . Inthes ecase sloo pan dtai l formation ispossibl e because of the screening ofth eP Lcharge sb y indifferent electrolyte.Th e coagulation concentration ofP San dAg i sols inth epresenc e ofP L isstrongl ydependen to nth epolyme rcon ­ centration in the bulk. When the saturation adsorption islargel y reached (i.e. large excess PL (above the saturation level)lef ti n the bulk solution)th e coagulation concentration increaseswit hin ­ creasing PL chain length, indicating the formation of longer loops andtails . The weakening of the attractionbetwee npositivel y charged -NH_ groups and negative surface groups due to addition ofelectrolyt e does notsuppres s the adsorptionwit h increasing salt concentration because thenon-ioni c adsorption energy is largea sa consequenc eo f hydrophobic interactions. This is not the casewit h silica asth e adsorbent forPL ,wher e hydrophobic interactions between PL andth e surface are absent.Therefor ei nthi scas eth edecreas eo fth eeffec ­ tive adsorption energy can suppress the increase in adsorbed amount significantly. Indeed,n oincreas eo fth eadsorbe damount ,a tconstan t surface charge,i s found above 0.01M salt (chapter 4). Inlin ewit h these findingsproto n titrations of silica inth epresenc e ofpoly - lysinea tvariou sioni cstrength s (chapter5 )sho wtha ta thighe rioni c strengthN a ionsca ncompet ewit hP L-NH _ groupsfo rsurfac esites . 198

ACKNOWLEDGEMENTS

Most of thiswor kwa s carried outi nth elaborator y forPhysica lan d ColloidChemistr yo fth eAgricultura lUniversity ,Wageningen . Theautho rwishe st oexpres shi sthank sto : Prof.Dr.J .Lyklem afo rhi sdiscussion san dvaluabl ecriticism . Mrs. E.Rouwenda l for carefully performing a largepar t ofth e adsorptionmeasurement san dsom eo fth etitratio nexperiments . Mr.H .Klunde r forhi s assistence inth eproto n titrationsexpe ­ riments of adsorbed andfre eP Lan dMr .J.F .Bonekam p forth ehel p withth ecalculation sinvolve dwit hth ep K vs aplots . app Ir.P-J .Bouwmeiste r forperformin gpreliminar y adsorptionmeasure ­ mentsan dtest so fth eP Ltitratio nwit hPVS. Kan dMr .W .va nLeeuwe n for the adsorptionmeasurement so fP Lo nsilica ,proto ntitration s ofth ePL/silic a systeman dprecipitatio nmeasurement so fPL . - Mr. H.E. van Beek,Mr .J.L.C .Verhage n andMr .W .va nBarnevel d forth econstructio no fth epart so fth etitratio ncells . Mr.G .Buurma nfo rth epreparatio no fth efigures . Mrs.D .Neijenhui s forth etypin go fth emanuscript . The departments ofBiochemistr yan dOrgani cChemistr yo fth eAgri ­ cultural University for granting the facilities for CD and IR spectroscopy. Finally the helpfulness, suggestions and valuable comments ofth e members ofth eDepartmen t ofPhysica l andColloi dChemistr ywa smuc h appreciated. 199

LISTO FABBREVIATION SAN DSYMBOL S

Abbreviations

CD Circulardichrois m DP Degreeo fpolymerizatio n EM Electronmicroscop y i.e.p. Isoelectri cpoin t IR Infrared MB Methyleneblu e NMR Nuclearmagneti cresonanc e OD Opticaldensit y PA Polyaminoacid PAA Polyacrylicaci d PGA Polyglutamicaci d PHIS Poly-L-histidine PL.HBr Polylysinehydrobromid e PL-L Poly-L-lysine (HBr) PL-DL Poly-DL-lysine(HBr ) PMA-pe Copolymer of methacrylic acid and the methyl ester of methacrylicaci d PO Poly-L-ornithine PS Polystyreneparticl e PSS Polystyrenesulphate ,anioni cpolyelectrolyt e PVS.K Polyvinylsulphate,potassiu msal t p.z.c. pointo fzer ocharg e r.p.m. revolutionspe rminut e

Symbols

A specificsurfac eare a A_ totalsurfac eare a a areao fa lattic esit e a„+ activityo fhydroge nion s b averagespacin gbetwee nth echarge so na full y stretchedpolyelectrolyt echai n c concentration c concentrationindifferen telectrolyt e CPL concentrationpolylysine.HB r T CpL overallconcentratio nPL.HB r 200

d diameter E fieldstrengt h E_ donnanpotentia l e elementarycharg e f fractionio npair s F Faradayconstan t AF, electrical freeenerg y G Gibbsfre eenerg y (freeenthalpy ) AG changei nfre eenthalp y AG f freeenthalp ychang efo rth eincreas eo fa helica l sequencewit hon euni t (amideresidue ) AG. • freeenthalp ychang efo rth einitiatio no fa helica l sequence AG° standard freeenthalp yo fdissociatio ni nmediu mm AG, electrostaticcontributio nt oth efre eenthalp ychang eo f associationo fa polybas e H enthalpy AH changei nenthalp y i layernumber ,inde x I ionicstrengt h k Boltzmannconstan t K dissociationconstan t K thermodynamicdissociatio nconstan to fa (cationic ) acidmonome ranalo gi nmediu mm K dissociationquotien to fa (cationic )aci dmonome r analogi nmediu mm a ta certai nsal tconcentratio nan d acidconcentratio n (i.e.,th es ocalle dintrinsi cK ofa polyacid ) K apparentdissociatio nconstan t L_ totalpersistenc e lengtho fa polyelectrolyt e 1 nonelectrostati cpersistenc elengt h 1 electrostaticpersistenc elengt h M molecularmas s M numberaverag emolecula rmas s M viscosityaverag emolecula rmas s M weightaverag emolecula rmas s N numbero fcharge dgroup so na polyelectrolyt echai n P pressure p fractiono fsegment so fadsorbe dmolecule sattache dt o thesurfac e 201

R gas constant r length of a lattice site s equilibrium constant for the increase of ahelica l sequence with one unit T Kelvin temperature U uniformity coefficient V volume y dimensionless potential Z„ number of dissociated groups on acationi c polyacid Z„„„ „ maximum number of dissociable groups on a cationic ti f max polyacid ZH,ma„ x maximum number of dissociable groups of class x with the same intrinsic dissociation constant ff n n Z®ri/ma x =Z „xi ,ma x - zS°xi with z£°n is the amount of groups which are untitratable in an adsorbed polyelectrolyte, when compared with the solution titration in the same pH region a degree of dissociation: a =Z../Z, ,„^ „ ii xima, x a' degree of dissociation: a' =Z-./Z, ,„„ „ xi rifinax «_£•£• degree of dissociation taking ion condensation into account r adsorbed amountpe r unit area of adsorbent rma x adsorbed amount at theplatea r u of an adsorption isotherr m r, adsorbed amount at aconductometri c breakpoint r. adsorbed amount atth epoin t of iso-electric adsorption e dielectric constant 9 excess amount adsorbed expressed inequivalen tmono ­ layers

8M residual charge fraction after ion condensation K conductivity also reciprokal Debye length A wavelength A molar conductance M chemical potential, dipole moment t dimensionless ioncondensatio n parameter p ratiobetwee n the total numbers ofpositivel y charged and negatively charged groups in a polyion-charged particle complex Pmam=x„ this ratio atmaximu m adsorption Pv this ratio at aconductometri c breakpoint p(z) polyelectrolyte space charge density as a function of the distance z from the surface 202 a equilibrium constant for the initiation of ahelica l sequence a surface charge density a_L total polylysine charge in an adsorbed layer expressed as surface charge t turbidity of a sol in thepresenc e ofpolylysin e and salt xo turbidity of a stable solwithou t PL andwithou telectro - lyte i relative turbidity T = x/x <|> volume fraction polymer <)•> polymer volume fraction of layeri <|>a idem inth ebul k solution X Flory-Huggins solvent quality parameter X idem, critical X polymer adsorption energy parameter XAs,c r critical value ofx As XAs,ef jTJTfidem ,' effectiv e X x of ahelica l segment X° x of acoi l segment i|) potential i|)(x) potential as a function of distance t|i• potential atth e lattice center of layer i, orpoten ­ tial atth eplac e of ionizable groups i 203

DEADSORPTI EVA NPOLY-L-LYSIN EE NPOLY-DL-LYSIN E AANVAST-VLOEISTO FGRENSVLAKKE N

SAMENVATTING

Indi tproefschrif ti sd eadsorpti eva nd ezwa kbasisch epolyamino - zurenpoly-L-lysin ee npoly-PL-lysin ebestudeerd . Onderzoeknaa rd eadsorptie-eigenschappe nva ngelade npolyaminozure n kan inzichtverschaffe n overd eeigenschappe nva na-helice s enander e secondairestructuren ,di eoo ki neiwitte nvoorkomen ,aa ngrensvlakke n en dero l van ladingsinteracties hierin.Bovendie n zijn dezemacro ­ moleculen geschikte modelpolyelectrolyten voor hetonderzoe k vand e adsorptieva npolyelectrolyten . Poly-L-lysine isi ndi tverban dee nzee rinteressant emodelsto fom ­ datdi tpolyaminozuu r inoplossin gbi jlag ep Hee nster kpositie fge ­ ladenpolyelectroly t ise nbi jhog ep H (>10),afhankelij kva nd eion - sterktee ntemperatuur ,i nd ea-heli xo fß conformati evoorkomt .Poly - DL-lysinekom t alleen ind ekluwe nconformati evoor .Daaro mkan ,doo r een vergelijking vanpoly- Le npoly-DL-lysine , informatieverkrege n worden over heteffec tva nd estereoregularitei te nsecondair estruc ­ tuuri noplossin go pd eadsorptie . Inhoofdstu k1 word the tbelan gva nd eadsorpti eva ngelade nmacro ­ moleculen,me tnam e de eiwitten,voo r talva nbiologisch ee ntechno ­ logischeprocesse n aangegeven. Indi thoofdstu kword too kd eopze te n achtergrondva nhe tonderzoe kgeschetst . Hoofdstuk2 behandel t enkele algemene aspectenva n polyelectrolyt adsorptie.Teven sbeva tdi thoofdstu kee nkor toverzich tva nd elite ­ ratuur over de adsorptieva ngelade npolyaminozuren .D eadsorpti eva n geladenpolyaminozure n aanvast-vloeisto fgrensvlakke n vannie tbio ­ logischeoorspron gblijk tno gslecht sweini gonderzoch tt ezijn . Een aantal relevante eigenschappen van de gebruikte materialen staat inhoofdstu k3 .Voora lhe toplossings -e nconformatiegedra gva n poly-L-lysine en eigenschappen van de meest gebruikte adsorbentia, polystyreen latex,silic a enborosilicaatglas ,zoal sspecifie kopper ­ vlak,oppervlakt e lading enhydrofobiciteit ,worde nbeschreve naa nd e handva neige nmetinge ne nliteratuurgegevens . Adsorptie experimenten bijmaximal e keten-ladingsdichtheid worden besproken inhoofdstu k4 .D epolylysin emolecule nzij nda nhoo ggela ­ denpolyelectrolyte n enhu n adsorptiegedrag isda nvergelijkbaa rme t datva nander eflexibel epolyelectrolyten . 204

Deinvloede nva nketenlengte ,electrolytconcentrati e enoppervlakt e ladingworde nbestudeer d voor zowel hydrofiele (silica,borosilicaa t glas)al shydrofob e (polystyreen.Agi )adsorbentia . Indi thoofdstu k wordtoo kd ekolloidal estabilitei ttege ncoagulati edoo rzou tbespro ­ kenvoo rpolystyree n latexe nAg isole ni naanwezighei dva nPL . Bij lage ionsterkte (I< 0.01M )i sd egeadsorbeerd e hoeveelheid altijdvee l lager danee nmonolaa gva npolylysin e segmenten.D ead ­ sorptie neemt toeme t toenemende wandlading vanhe tadsorbens ,maa r hangtnagenoe g nietva n depolylysin e ketenlengtee nd ehydrofobici - teitva nhe tadsorben saf .Ee ntoenam eva nd eadsorpti eword tverhin ­ derd,omda td evormin gva nlusse ne nstaarte nonderdruk tword tt.g.v . de sterke repulsie tussend esegmenten .E rzij nbi jgee nenkel ezout - concentratiewaarneembar everschille ntusse nd egeadsorbeerd ehoeveel ­ heidva npoly- Le npoly-DL-lysin egevonden . De invloed van dezoutconcentrati e op de adsorptie istweeledig : enerzijds neemt de repulsie tussen deP Lsegmente na fme ttoenemend e zoutconcentratie,waardoo r de lus-e n staart-fractieka ntoeneme ne n dusoo k de adsorptie,anderzijd sword too kd eattracti etusse nd eP L segmenten ennegatiev e oppervlakte groepenminde r alsd ezoutconcen ­ tratie toeneemt,waardoo r de adsorptie kan afnemen.Bi jnegatie fge ­ ladenpolystyree n enAg i neemtd e adsorptiester kto eme ttoenemend e zoutconcentratie,minsten s tot 1.0 M zout.Bi jhe thydrofiel esilic a is,bi j constante wandlading,bove n 0.01M zout,gee ntoenam ei nad ­ sorptiemee rwaa r tenemen .Di t komtomda tbi jsilic agee nhydrofob e attractie tussenP L segmenten enhe t oppervlak aanwezig iszoda td e effectieve adsorptie energiepe r segmentvee l lageri sda nbi jP Se n Agi. De zoutconcentratie waarbij coagulatieva nlatex -o fAg isole nop ­ treedti naanwezighei dva npolylysin ei sster kafhankelij kva nd epoly ­ meerconcentratie ind ebulk .D eresultate nwijze nero pdat ,indie nd e verzadigingsadsorptie ruimschootsbereik tis ,d elus -e nstaartfracti e toeneemtme t depolylysin e ketenlengte enhoge ri sda nbi jlag eion - sterkte. Inhoofdstu k5 word td ebindin g (i.e.adsorptie )va npositie fge ­ ladenpolylysin eaa nnegatie fgelade nkolloidal edeeltje s (polystyreen en silica)bestudeer d m.b.v.conductometri ee nPotentiometrie .D ead ­ sorptieva nP Lword tvergeleke nme td ecomplex-vormin gtusse nlineair e polyanionen en lineairepolykationen .Oo kworde nproton-titratie sva n silicai naan -e nafwezighei dva nP Lbesproken .Ui tdez eproto ntitra ­ tiesblijk t datd e silanol groepen sterkerzuu rzij ni naanwezighei d 205 vanPL .Bi jhoger ezoutconcentrati e kunnenN a ionen,-NH ,groepe nver ­ dringenva n-Si-0 ~groepe naa nhe tsilic aoppervlak .Di ti si novereen ­ stemmingme the tfei tda td eadsorpti enie tmee rtoeneem tbove n0.0 1M zout. Evenalsi nhe tgeva lva nopgelost epolyanione ne npolykatione nver ­ toontd egeleidbaarhei d alsfuncti eva nd epolylysin econcentrati eee n knik.D ep H isn ahe tknikpun tconstant .D egeadsorbeerd ehoeveelhei d inhe tknikpun t is lager dand emaximal eadsorptie .D esamenstellin g vanhe t 'PL-kolloïde'comple xi nhe tknikpun ti snie tladingsstoichio - metrisch en ind emeest egevalle ni sd eP Ladsorpti esuperequivalent . Dit komtdoorda td evast edeeltje snie tflexibe lzij ne nd enegatiev e oppervlaktegroepen relatiefve rva nelkaa rzitten .I nhe tknikpun ti s elke negatieve oppervlaktegroep gebonden aan een -NH_ groep vanP L (ion-paren),maa rhe tomgekeerd egeld tniet . Uit titraties van aan polystyreen deeltjes geadsorbeerd PLme t polyvinylsulfaat,blijk tda td efracti evrij e-NH -groepe n0.3-0. 4is . Hoofdstuk6 handel t over de secondaire structuurva ngeadsorbeer d poly-L-lysinee nd einvloe dva nd esecondair estructuu ro pd eadsorpti e enprecipitati e eigenschappenva nPL .Oo kword td einvloe dva nd ep H (i.e.dissociati e graad)o p de adsorptie vanP L aanpolystyree nbe ­ sproken. Deprecipitatie-tij d enprecipitati ep Hva npoly-L-lysin ezij nbi j 303e n313 Knagenoe ggelij kaa ndi eva npoly-DL-lysine .Blijkbaa rheef t desecondair estructuu rgee ngrot einvloe do pdez eeigenschappen . Deadsorpti eva nP Lneem tster kto eme ttoenemend epH ,omda tt.g.v . deafnemend erepulsi etusse nd esegmente nd elus -e nstaartfracti etoe ­ neemt.Bi jd ep Hwaa roo ki noplossin gprecipitati eoptreedt ,gebeur t ditoo ki naanwezighei dva npolystyree nlatex . Protontitratiesva ngeadsorbeer dP Llate nzie nda ti nhe tgeva lva n constante (lage) geadsorbeerde hoeveelheid geen conformatieovergang plaatsvindt.Allee nwannee rbi jelk ep H deverzadigingsadsorpti ebe ­ reiktis ,word tee nconformatieovergan g ingeadsorbeer dP Lwaargenomen , omdatda nd e lussene n staarten langgenoe gzij nvoo rd evormin gva n secondairestructuur .Bi jhog ep He nmaximal eadsorpti ei sgeadsorbeer d PL-Lwaarschijnlij kgedeeltelij k ind ea-heli xconformatie .T.g.v .ion - paarvormin gme td enegatiev eoppervlakt egroepen ,blijk td edissociatie ­ graad van geadsorbeerdpolylysin e alleeni nd eeerst e adsorptielaag (treinfractie)behoorlij klage rt ezij nda ni noplossing .D eontvouwin g vanPL- Li nd ea-heli x conformatiet.g.v .adsorpti ebi jlag eoppervlakt e bezettingi swaarschijnlij khe tgevol gva nd evormin gva ndez eion-pare n enhydrofob einteractie sme the tpolystyree noppervlak . 206

Inhoofdstu k7 word t dep He nzoutafhankelijkhei dva nd eadsorpti e vanP L aanpolystyree ne nAg ivergeleke nme td evoorspellinge nva nd e polyelectrolytadsorptietheorie vanva nde rSchee . Indez egevalle ni s eree ngoed eovereenstemmin g tussenexperimen te ntheorie . 207

CURRICULUMVITA E

BenBonekam p isgebore no p2 5apri l195 3t eSittard .Aa nhe tBisschop ­ pelijkColleg ei ndi eplaat sbehaald ehi ji n197 1he tdiplom aH.B.S.-B . Indatzelfd e jaarbego nhi j aand e Landbouwhogeschool teWageninge n destudi eMoleculair eWetenschappen . Hierinwer d in januari 1976he t kandidaatsexamenafgeleg de ni nsep ­ tember 1978he tdoctoraalexamen .D e doctoraalstudie omvatte devak ­ ken: Biochemie (6mnd) , Kolloïdchemie (6mnd) , Virologie (6mnd )e n Wiskunde (3mnd) . Vanaf februari 1979 tot februari 1983wa shi ji n tijdelijke dienst van de Landbouwhogeschool te Wageningen. Indi e periode verrichtte hij,al swetenschappelij kmedewerke r bij devak ­ groep Fysische enKolloïdchemi eme tee ngedeeld eonderwijs -e nonder ­ zoektaak,he ti ndi tproefschrif tbeschreve nonderzoek . Vanafoktobe r 1983 ishi jvoo ree nperiod eva n1 jaa rwerkzaa mbi jd e vakgroep Levensmiddelentechnologie, Sectie Zuivel enLevensmiddelen - natuurkundeva nd eLandbouwhogeschool . 208

NAWOORD

Aanhe t eindva ndi tproefschrif twi li kgraa giederee nbedanke ndie , direct of indirect, aan detotstandkomin g ervanheef tbijgedragen . Eenaanta lmense nwi li kpersoonlij knoemen : Mijnpromotor ,Han sLyklema ,be ni kerkentelij kvoo rd enuttig edis ­ cussiesove rd eresultate nva nhe tonderzoe ke nhe tlevere nva nopbou ­ wendekritie ko pd eteks tva nd everschillend ehoofdstukken . ErnaRouwenda le nHarc oKlunde rwi li kbedanke nvoo rd enauwgezett e wijzewaaro pzi jee ngroo taanta lva nd eexperimente nhebbe nuitgevoer d enmij nbroe r Hans,voo r hetverrichte nva n een aantalberekeninge n m.b.v.zij n'micro' . Mijndan k gaatverde rui tnaa rWi mva nLeeuwe ne nPieter-Ja nBouw ­ meister, die inhe tkade rva nhu ndoctoraalstudi ehebbe nbijgedrage n aanhe texperimentel ewerk .Teven sbe ni kHenni eva nBee ke nLoui s Verhagenerkentelij kvoo rd etechnisch eassistentie . Ikdan kGer t Buurmanvoo rhe ttekene nva n devel efigure ne nhe t makenva nd e foto's enDor yNeijenhui svoo rhe ttype nva nhe tmanus ­ cript. Tenslottewi li kmij nkamergenoo tThoni eva nde nBoomgaar dbedanke n voor deprettig e sfeer en denuttig e gesprekken.Oo k dewaardevoll e discussiesme tander emedewerker sva nd evakgroe pFysisch ee nKolloïd - chemie,me tnam eRo beleven ,waardee ri kte nzeerste . Op een geheel anderewijz ehebbe nmense nui tmij nnaast eomgeving , inhe tbijzonde rMargo ,d e totstandkoming van ditproefschrif tmed e mogelijkgemaakt .