Separation And Purification Techniques In BiotechnologyDechow生物化学生物技术教案 2

Separation And Purification Techniques In Biotechnology Dechow生物化学生物技术教案 2 Adsorption15945.46.Rosene,M.R.,ACS Presentation,March198147.Berthier,P.,Kerlan,L.,Courty,C.,Compt Rend,246:1851 (1958)48.Blackburn,A.,Kipling,J.J.,J ChemSot,4103 (1955)49.Ores,B.,Rauber,C.,U.S.Patent No.3,741,949(June26,1973)3.54.55.McKay,G.,Chem EnpRes Des,61 (1):29 (1983)56.HSU,E.H.,Fan,L.T.,“A NovelPorous MediumFilter”,In:Proceedings ofWorld FiltrationCongress III,Philadelphia,PA,p420(September13-17,1982)57.58.59.Hutchins,R.A.,Chem EnaProg,71 (5):80 (1975)60.DeJohn,P.B.,Chem Enq,113(April28,1975)Rosene,M.R.,Lutchko,J.R.,Deithom,R.T.,et al.,Presentation atACS NationalMeeting,Miami,FL,September1981Mathews,A.P.,A ICh ESvmp Set-,79 (230):18 (1983)Patil,V.K.,Joshi,J.B.,Sharma,M.M.,Chem EnnRes&,62:247 (1984)Jordon,D.,“Carbon Decolorization”,In:Fermentation andBiochemical Ennineerinqcourse,The Centerfor ProfessionalAdvancement,New Brunswick,NJ (1983)Fornwalt,H.J.,Hutchins,R.A.,Chem 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(1972)Sargent,R.N.,Graham,D.L.,Ind EnnChem ProcDes m,l(l):56 (1962)Paleos,J.,J Co11Interf Sci,31 (1):7 (1969)Anand,P.S.,Dasare,B.D.,J ChemTech Biotechnol,31:213 (1981)White,J.D.,Schwartz,D.P.,J Chromatoq,196:303 (1980)Funakubo,E.,Matsuo,T.,Taira,T.,et al.,U.S.Patent No.3,849,298(Nov19,1974)Walsh,P.K.,Liu,C.P.,Findley,M.W.,et al.,In:Fundamentals ofAdsorption(Myers,A.L.,Belfort,G.,eds.)Engineering Foundation,NY,p667 (1984)162Separation andPurification Techniquesin Biotechnology94.Dessau,R.M.,Haag,W.O.,U.S.Patent No.4,442,210(April10,1984)95.Feldman,J.,U.S.Patent No.4,450,294(May22,1984)96.Marshall,V.P.,Little,M.S.,Johnson,L.E.,J Antibiot,34 (7):902 (1981)97.Schindler,J.,Ind EngChem ProdDes Dev,21537 (1982)98.Everett,D.H.,In:Fundamentals ofAdsorption(Myers,A.L.,Belfort,G.,eds.)Engineering Foundation,NY,p1 (1984)3Ion Exchange3.1INTRODUCTION Separation and purificationoperations withion exchange resins involvethe reversibleinterchange of ions between a functionalizedinsoluble resin(the ion exchange material)and an ionizable substancein solution.While Thompson (1)reported in1850the firstion exchangeapplications whichused naturallyourring clays,ion exchange resins have only beenused inbiochemical andfermentation productrecovery for the lastfew decades(2,3).In theseearly studies,biochemicals such as adenosiriphosphate (4),alcohols (5),alkaloids (6),amino acids (7),growth regulators (8),hormones (9),nicotine(lo),penicillin(1l),and vitaminB-12 (12)were purifiedusing ion exchange resins.Ion exchangeapplications intensifiedfollowing thework ofMoore andStein (13),which showedhow veryplex mixturesof biochemicals,in thiscase amino acids and aminoacidresidues,could beisolated fromeach otherusing the ion exchange resin asa columnchromatographic separator.Specific chromatographicapplications of ion exchange resins will be coveredin Chapter4.This chapterwill concentrateon the theory,characterization and process applications of ion exchange resins.In biotechnologyapplications,ion exchangersare importantin preparingwater of the necessaryquality toenhance thedesired microorganismactivity during163164Separation andPurification Techniquesin Biotechnologyfermentation.Downstream of the fermentation,ion exchange resins may be used to convert,isolate,purify orconcentrate thedesired productor by-products.3.2THEORY The important featuresof ion exchange reactionsare that they arestoichiometric,reversible andpossible with any ionizablepound.The reactionthat oursin aspecific lengthof timedepends on the selectivity of the resin for the ionsor moleculesinvolved and the kiics of thatreaction.The stoichiometriature of the reaction allows resinrequirements to be predictedand equipmentto besized.The reversiblenature of the reaction,illustrated asfollows:R-H+Na+Cl-r-”R-Na+H+Cl-(3.1)allows for the repeatedreuse of the resinsince there is nosubstantial changein itsstructure.The equilibriumconstant,K,for equation3.1is definedfor suchmonovalent exchangeby theequation:(3.2)In general,if K is alarger number,the reversereaction ismuch lessefficient thanthe forwardreaction andrequires alarge excessof regenerantchemical,HCl in this instance,for moderateregeneration levels.The equilibriumconstant definedin Equation3.2pares toKu,the equilibriumconstant definedin Chapter2for adsorptiveprocesses:s=Ci/CO(3.3)where K,is the ratio of the soluteconcentration within(Ci)and surrounding(c,)the adsorbentmaterial.Starobinietz andGleim (14)distinguished between the amountof molecular adsorption andion exchangethat ourredwith astrong baseanion resin andaseries of fatty acids.The overalladsorption ofcarboxylic acids on theIon Exchange165resin wasdetermined from the changein itssolution phaseconcentration and the amountof ion exchange from the amountof counterionsdisplaced fromthe resin into the solution.As Figure3.1shows,thereis a steadyincrease inmolecularadsorptionwith hydrocarbonincrease inchain length.However,the amountof ion exchange decreasesfrom formicthrough aceticto propionicacid and increases againfor the higher acids.This type of distinction is notoften measured,particularly whenbiomolecules withmultiple modesof interactionare underinvestigation.1.f j-1.220406060100/.-I I I I I1I I I I III020406080100120Figure3.1.Adsorption isothermsoffattyacidsonDowex1X2in chlorideform:(a)molecular sorption;(b)ion exchange.1.formic;2.acetic;3.propionic;4.butyric;5.valeric;6.caproic;7.heptanoic;8.caprylic;9.pelargonic acids(Reference14).166Separation andPurification Techniquesin BiotechnologyWith proper processing andregenerants,the ion exchange resinsmay beselectively andrepeatedly convertedfrom oneionic formto another.The definitionof theproperprocessingrequirements is based upon the selectivityand kiictheory of ion exchange reactions.3.2.1Selectivity Forion exchange resins,the selectivityis developedin terms of theDonnan potential,En.The Donnanpotential is the differencein the electric potentialbetween the ion exchangeresin,I$,and the solution,.This may be related to thechemical potentialor activityby theequation:1ED=Qr-+s=-ziF ai,s-llv(3.4)i a.=,r whereZi is the charge on the ion,F is the Faradayconstant,ai*and airare theactivities of ion“i”in solution and the resin,respectively,and Viis thepartial molar volume of ion“i”.The partialmolarvolumeis assumedto be the samein the liquid and resin phase.When ion“B”,which isinitially in the resin,is exchangedfor ion“A”,the selectivityis representedby:+*PB-WA)(3.5)The selectivitywhich aresin hasfor variousions is affected bymany factors.These factorsinclude thevalence andsize of the exchangeion,the ionic form of the resin,the totalionic strengthof thesolution,crosslinkage of the resin,the typeof functional group and the natureof the non-exchanging ions.The ionichydration theoryhas beenused toexplain theeffect ofsome of these factorson selectivity (15).Aording tothis theory,the ions in aqueous solution arehydrated and the degree of hydrationfor cations increases with increasing chargeand decreasingcrystallographic radius,as shown in Table3.1 (16).It is thehighdielectric constantof watermolecules thatis responsiblefor thehydration of ionsinaqueous solutions.The hydrationpotential of an iondepends on the intensityof the chargeon its surface.The degreeof hydrationof an ion increases as itsvalence increasesand decreasesas itshydrated radiusincreases.Therefore,it isexpected that the selectivityof aresin for anion is inversely proportional to theratioof thevalence/ionic radiusfor ionsof a given radius.In dilutesolution thefollowing selectivityseries arefollowed:Ion Exchange167Li (16)Crystallographic HydratedIonization IonRadius(#)Radius(I()Potential-Li0.6810.01.3Na0.987.91.0K1.335.30.75NH41.435.31_Rb1.495.090.67CS1.655.050.61Mg0.8910.82.6CaSr1.349.61.6Ba1.498.81.4As Figure3.2 (17)shows,as thevalence of the exchangeion increasesand as the hydratedradius of the exchangeion decreases,the selectivityor affinityof the ion exchangeresin forthat ion increases.As shouldbe apparent,if the resin isinanionic form“A”which has a lowerreplacing powerthan anion“B”in solution,the resin willbeconverted atequilibrium to the ionicform“B”.The selectivityof resinsin the hydrogen ionorthehydroxide ionform,however,depends on the strengthof the acid or base formedbetweenthe functional groupandthe hydrogen orhydroxide ion.The strongertheacid orbaseformed,the loweris the selectivity coefficient.It shouldbe notedthat168Separation andPurification Techniquesin Biotechnologythese seriesare notfollowed innon-aqueous highsolute concentrationsor athigh temperatures.8-I,solutions,at0.60,70080.91.01.1EQUILIBRIUM EXCHANGE(MILLIEQUIVALENTS/GRAM)Figure3.2.Effect of ionic radiuson ion exchange ina carbonaceouszeolite(Reference17).The dependenceof selectivityon the ionic strengthof thesolution wasrelated through the mean activity coefficientto beinverselyproportionalto theDebye-Huckel parametera0 (18):-A&-log Y*=1+Bafi(3.6)where r+is themeanactivity coefficient,A andB areconstants,andpis the ionic strengthof thesolution.The meanactivitycoefficientinthisinstance representsthe standardfree energyof formation(-AF”)for thesalt formedIon Exchange169by theion exchangeresin andthe exchangedion.The Debye-Huckel parameteris ameasure of the distanceof closestapproach which is alsorelatedto the ionichydration size.Figure3.3 (19)shows thisdependence as theionic concentration of thesolution is changed.As theionioncentration of thesolution increases,the differencesin the selectivityof the resin for ionsof differentvalence decreases,and insome cases,the affinitymay begreater for the lowervalence ion.00,40.81,21,G2,o MOLARITYOF SOLUTIONFigure3.3.Dependence of the activitycoefficient on theionioncentration ofaqueoussolution(Reference19).The selectivityof anion exchangeresinwillalso dependonitscrosslinking.The polymerstructure of theion exchangeresin can bethought ofas collectionsof coiledsprings which can swellor contractduring the exchange of170Separation andPurification Techniquesin Biotechnologyions (20).The crosslinkingof thepolymer limitsthe extent to which the resinmay swell:the higherthe degreeof crosslinking,the lowerthe extentto which the resincan be hydrated.This limiton theextenttowhich the resincan behydrateddetermines the relative equivalentvolumes ofhydrated ionswhichthecrosslinked polymerwork canaommodate.This isshown inTable3.2 (21)and in Figure3.4 (22).As the resins degreeof crosslinkingor itsfixed ion concentration islowered,the selectivityof the resin decreases.Table3.2:Selectivity andHydration of Cation Resinswith DifferentDegrees ofCrosslinking (21)Cation Li4%DVB KH-1.004188%DVB-I!K-1.0021116%DVB IT-II1.00130H1.304311.26xx.45136NS1.493721.88la32.23113NH41.753602.221723.07106K2.093412.631634.15106CS2.373422.911594.15102Ag4.002897.3616319.4102Tl5.202299.6611322.2a5K=Selectivity paredto LiH=Hydration(g H*O/eq resin)-The degreeof crosslinkingcan affectthe equilibriumlevel obtained,particularly asthe molecularweight of the organic ion beeslarge.With highlycrosslinked resins and largeorganic ions,the concentrationof theorganicionin theouter layersof the resin particlesismuch higherthan in the centerof the particle.This isshown in Figure3.5for the distribution ofmethylene bluein carboxylic cation exchangeresins (23).The selectivityof theresinfor a givenionis also influenced by thedissociation constantsofthe functional groupcovalently attached to theresin(the fixedion)and ofthe counterionsin solution.Since thecharge perunit volumewithin theresin particleis high,a significantpercentage ofthe functionalgroups may be un-ionized.This Ion Exchange171x7-0.6-x0.5-01.0MOLE FRACTIONNAR Figure3.4.Dependence oftheselectivitycoefficient forthe Na-H ionicexchange asa functionof resin ionic position andresincrosslinking(Reference22).01002ou TIME(HR)Figure3.5.Sorption ofmethylene blueby carboxyliation exchangein theNa form.The resinforthetop curve has2%crosslinking whilethat forthe lowercurvehas10%crosslinking(Reference23).172Separation andPurification Techniquesin Biotechnologyis particularlytrue ifthefunctionalgroup is a weakacidorbase.For cation exchange,the degreeof dissociationofthefunctionalgroupincreasesasthe pHis increased.However,the degreeof dissociationforthe ionsin solution decreaseswithincreasingpH.Therefore,if a cation resinhad weakacid functionality,it wouldexhibit little affinity at any pH fora weak basesolute.Similarly,an anion resin withweak basefunctionality exhibitslittleaffinityatanypHforaweakacid solute.The influenceof pHon thedissociation constants for resin with agiven functionalitycan beobtained bytitration in the presenceof anelectrolyte.Typical titrationcurves are shown inFigure3.6for cation resinsandinFigure3.7for anion resins (24).For sulfonicacid functionalgroups,thehydrogen ionis a veryweak replacingion andis similarto thelithium ionin itsreplacing power.However,for resinwith carboxylicacid functionality,thehydrogenion exhibitsthe highestexchanging power.Table3.3(25,26)summarizes theeffect differentanion exchangeresin functionalitieshaveonthe equilibriumexchange constants forawide seriesof organicand inorganicanions.PH0246a1012MEQ NAOHPER GRAMRESIN Figure3.6.Titration(Reference24).curves of typical cation exchangeresinsIon Exchange173Figure3.7.Titration curvesoftypicalanion exchangeresins(Reference24).Table3.3:Selectivity Coefficientsfor StronglyBasic AnionResin(25,26)Type IAnion TypeII AnionAnion KXC1Anion KXClSalicylatt!32.2Salicylate28I-a.7C&O8.7CGll505.2I7.3HSO,4.1MO,-6.1NOa-3.8NOs-3.3Br2.8nr-2.3-1.6-1.3HSOs-1.3Iwo,-1.3NOp-1.2NO*-1.3cl-1.00Cl-1.00HcoJ-0.32OH-0.65llZPOl0.25lico3-0.53HCOO-0.22112PO0.34cH3coo-0.17HCOO-0.22HzNCH2C00-0.10CHICDO-0.18Oil-0.09F-0.13F-0.09HYNCH,COO-0.10174Separation andPurification Techniquesin BiotechnologyThe selectivityisalsoinfluenced by thenon-exchanging ions(co-ions)in solutioneven thoughthese ionsare notdirectly involvedin the exchange reaction.An exampleof thisinfluence would be the exchange ofcalcium ascorbatewith ananion exchangeresininthe citrateform.Although calciumdoes nottake partintheexchange reaction,sequestering ofcitrate willprovide an additional driving force fortheexchange.This effect,of course,would havebeen diminishedhad aportion ofthe ascorbatebeen addedasthesodium ascorbateinstead ofthe calciumascorbate.For non-polar organicsolutes,association intoaggregates,perhaps evenmicelles,may depresssolution activity.These associationsmaybeinfluencedby the co-ions present.The selectivityof syntheticion exchangeresins fororganic hydrophilicsubstances is usually low.The selectivityconstantsforcarboxyliation resins(Na+form)for antibioticsofthegroup kanamycin,gentamycin,monomycin andsisomycin areintherange0.5-1.0 (27).A procedurewas developed (23)to prepareconcentrated solutionsof theseantibiotics.In a batch reactor,instead ofmerely allowingthe cationresin andthe antibioticfermentation brothfiltrate toe toequilibrium,theresinis firstwashed witha plexforming agentbefore addingthe antibioticfiltrate.Such washingresults inanadditionalsorption ofthe antibioticand higherelimination ofboth the other organionstituents andinorganic ionsfromtheion exchangeresin asshown inFigure3.8.Many fermentation and biotechnologyproducts areampholytes orzwitterions which can adsorbon ion exchangeresinsnot onlyby actualexchange butalso bydistribution ofthe zwitterionsegments and bytheDonnan exclusionofthe ampholyte fromtheresin.A theory of ampholyteinteraction withionexchangeresins hasbeen developed (23)which allowsa quantitativeevaluation ofthe equilibriumstate forthese systems.This evaluationcan be used toselect theoptimum conditionsfor adsorbingand desorbi