Table Of Content

Editors Michael Cooke Royal Holloway, University of London, Centre for Chemical Sciences Egham Hill, Egham, Surrey TW20 0EX, UK Colin F. Poole Wayne State University, Department of Chemistry, Detroit MI 48202, USA Editor-in-Chief Ian D. Wilson AstraZeneca Pharmaceuticals Limited, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK Managing Technical Editor Edward R. Adlard Formerly of Shell Research Limited, Thornton Research Centre, PO Box 1, Chester CH1 3SH, UK vi EDITORIALADVISORYBOARD Editorial Advisory Board RichardW. Baker KennethJones MembraneTechnology&ResearchInc(MTR) AffinityChromatographyLimited 1360WillowRoad,Suite103 Freeport MenloPark Ballsalla CA94025,USA Isleof Man,UK KennethL.Busch ChrisLowe KennesawStateUniversity UniversityofCambridge OfficeofSponsoredPrograms Instituteof Biotechnology 1000ChastainRoad TennisCourtRoad Kennesaw Cambridge,UK GA30144,USA DavidPerrett HowardA.Chase StBartholomew’sandtheRoyalLondonSchool UniversityofCambridge ofMedicineandDentistry DepartmentofChemicalEngineering StBartholomew’sHospital,DepartmentofMedicine PembrokeStreet WestSmithfield CambridgeCB23RA,UK LondonEC1A7BE,UK JanCilliers DouglasE.Raynie UniversityofScienceandTechnologyin Manchester TheProcterandGambleCompany DepartmentofChemicalEngineering MiamiValleyLaboratories POBox880 POBox538707 ManchesterM601QD,UK Cincinnati OH45253-8707,USA AlanDyer UniversityofSalford PeterSchoenmakers SchoolofSciences,CockroftBuilding ShellResearchandTechnologyCentreAmsterdam SalfordM54WT,UK (SRTCA)andUniversityofAmsterdam Postbus38000 HeinzEngelhardt 1030BNAmsterdam,TheNetherlands UniversitaKt desSaarlandes InstrumentelleAnalytik/Umweltanalytik DarrellN.Taulbee Postfach151150 UniversityofKentucky-CenterforAppliedEnergy 66041Saarbruecken,Germany Research(UK-CAER) 2540ResearchParkDrive WilliamF. Furter Lexington RoyalMilitaryCollegeofCanada KY40511,USA DepartmentofChemicalEngineering Kingston GerdaM. vanRosmalen OntarioK7K5L0,Canada DelftUniversityofTechnology LaboratoryforProcessEquipment JosefJanca Leeghwaterstraat44 UniversiteH deLaRochelle 2628CADelft,TheNetherlands PoleScienceset Technologie EquipedePhysico-ChimieMacromoleculaire EdwardWoodburn(cid:1) AvenueMichelCrepeauI17042LaRochelle,France UniversityofScienceandTechnologyin Manchester DepartmentofChemicalEngineering WaltJennings POBox880 J&WScientificIncorporated ManchesterM601QD,UK 91BlueRavineRoad FolsomCA95630,USA (cid:1)deceased FOREWORD vii Foreword SeparationsciencewasRrstrecognizedasadistinctareaofphysicalandanalyticalchemistryinthe1960s.The termwasRrstcoined,I believe, bythelate J.CalvinGiddings,ResearchProfessorat theUniversityofUtah. Calvin Giddings recognized that the same basic physical principles governed a wide range of separation techniques,andthatmuchcouldbelearntbyapplyingourunderstandingofonesuchtechniquetoothers.This wasespeciallytrueforhisRrstloves,chromatographyandelectrophoresisandlatterlyReldSowfractionation. Ofcoursethereare manyseparationtechniquesotherthanchromatography,manywithahistoryatleastas long, or indeed longer, than that of chromatography:distillation, crystallization, centrifugation,extraction, Sotation and particle separation, spring to mind. Other separation techniques have emerged more recently: afRnity separations, membrane separations and mass spectrometry. Most people, a few years ago, would not have classed mass spectrometry as a separation technique at all. However, with modern ionization methods,which minimizefragmentation,mixturesof compoundscanRrstof all beseparatedandtheneach componentidentiRedthroughfragmentationbysecondaryion-moleculecollisionsandfurthermassspectro- metry.Withthescaleofmassspectrometrynowmatchingthatofmicroseparationmethodssuchascapillary electrophoresisandcapillaryelectrochromatography,combinationsoforthogonalmethodscannowprovide extremely powerful separation and identiRcation platforms for characterizing complex mixtures. Basically, all separation techniques rely on thermodynamic differences between components to dis- criminateonecomponentfromanother,whilekineticfactorsdeterminethespeedatwhichseparationcanbe achieved.Thisappliesmostobviouslytodistillation,chromatographyandelectrophoresis,butisalsoobvious in most of the other techniques; even particle size separation by sieving can be classiRed in this way. The thermodynamic aspect is, of course, trivial being represented by the different sizes of the particles, as indeed it is for the size exclusion chromatography of polymers. However, the kinetic aspects are far from trivial.Anyonewhohastriedtosieveparticleswillhaveaskedthequestion:isitbettertoRllthesievenearlyto thetopandsieveforalongtime,orisitbettertodribblethematerialslowlyintothesieveandjustremovethe heavies from time to time? One might furtherask: howdoes one devise a continuoussieving processwhere largeparticlesemergefromoneportoftheequipment,andsmallonesemergefromtheotherport?Andhow does one optimize throughputand minimize unit cost? ThepublicationofthisEncyclopediaofSeparationScienceisalandmarkforthisareaofscienceatthestart of the third millennium. It will undoubtedly be of enormous value to practitioners of separation science lookingforanoverviewandforguidanceastowhichmethodtoselectforanewproblem,aswellastothose whoare atan early stage, simply dippingtheir toes into the waters,and tryingto Rndout justwhat it is all about.Mostimportantofall,byprovidingacomprehensivepicture,itadvancesthewholeReldofseparation scienceandstimulatesfurtherworkonitsdevelopmentandapplication.Thepublishers,theireditorsandtheir authorsare to be congratulatedon a splendid effort. John H. Knox Edinburgh 8 March 2000 viii PREFACE Preface Theabilitytoperformseparationsfortheanalysis,concentrationorisolationofsubstancespresentinmixtures (ofvaryingdegreesofcomplexity)isarguablyfundamentaltothemaintenanceofourtechnologicalciviliza- tion. Separations can also be rather difRcult to deRne, and over the course of a number of debates where we tried to ‘separate the wheat from the chaff’, we deRned separations for the purposes of this work as ‘processes of any scale by which the components of a mixture are separated from each other without substantialchemical modiTcation’.Of coursesome ofthe processesthatareused in separationshavea very longhistory,andthetermsusedtodescribethem areinwidespreadgeneraluse.So, we talkabouthowit is possible to distil wisdom, precipitate an argument, extract meaning and crystallize an idea and who can predict the future uses of the word chromatography? Whilst separations have been practised as an art for millennia, the last hundredyearsor so hasseen the elucidationof the fundamentalsthatlie behindmanyof theseprocesses.Thus,althoughseparationsaremostwidelyusedforachievingsomepracticalobjective,aRrm theoreticalunderstandinghasbeenputintoplacethatdoesallowtheuseofthetermseparationscience.We have tried here to reSect the theoretical and practical aspects of the topics in this encyclopedia, and have attemptedtoachieveablendoftheory,practiceandapplicationsthatwillenablesomeoneknowledgeablein aReldtogodirectlytoarelevantarticle; whilstthenovicecanbeginwithanoverviewandgraduallyiterate towardsthe practical application. One thing is clear, separations cover such a wide range of topics that no single individual can be knowledgeable,letaloneexpert,inthemall.Itisagainstthisbackgroundthatwedecidedthatanencyclopedia designedtocoverthissciencewouldbeofvalueasasinglesourceofreferencethatwouldprovideaccesstothe whole Reld of separations. For the purposes of deRning the scope and coverage of the encyclopedia we have divided the area of separations into 12 families, or topic areas, using separation principles based on afRnity, centrifugation, chromatography,crystallization, distillation, electrophoresis,extraction,Sotation,ion exchange, mass spectrometry, membranes and particle size. Whilst there is no doubt that different editors might have grouped these slightly differently, they did not seem to us to be capable of further reduction. Taken together, we believe that they provide coverage of the whole Reld. Inpreparingthismulti-authorandmulti-volumework,alloftheeditorshavebeenconsciousofthegapsin theirownexpertiseandthedebtwhichtheyandthepublishersowetotheEditorialAdvisoryBoard,whoto alargedegreehavecompensatedforthedeRcienciesinourownknowledge.Theirhelphasbeeninvaluable,as withoutthemwewouldnothavebeenabletoachievethenecessarybalanceanditwas,therefore,asourceof particularsadnessthatoneof them, TedWoodburn,died priorto publication.WithoutTedit is quite clear thatthetopicareaofSotationwouldnothavebeensowellcovered,andwewouldliketothinkthathewould havebeenwellpleasedwiththeRnishedwork.Wealsohopethatthemasterlyoverviewwhichhecontributed totheencyclopedia,willbealastingmemorialtohim.WearegratefultoJanCilliersforsteppingintotherole ofEditorialBoardAdvisoronSotationatshortnotice.Wewouldalsoliketoacknowledgethevaluableinput ofG.J.Arkenboutat thebeginningofthisproject,whowasabletoworkwithusforashortperiodoftime before his death. Assembling this knowledgeable and enthusiastic group of experts was a difRcult task, and the editors wouldalsowishtoacknowledgetheroleoftheMajorReferenceWorksdevelopmentteamatAcademicPress inthiswholearea,aswellasthankingthemfortheirassistanceandpatiencethroughouttheproject,fromthe initial planning to its Rnal publication. Finally, of course,we mustacknowledgethe contributionsof theauthorswhose expertise constitutesthis encyclopedia.SomeofthemhavebecomeRrmfriendsintheperiodbetweentheinceptionofthisprojectand its completion. After all, separation science separatesthings but brings people together. Ian D. Wilson, Edward R. Adlard, MichaelCooke, ColinF. Poole Editors INTRODUCTION ix Introduction The need for separations as a means for performing the isolation, puriRcation or analysis of substances, at scalesrangingfromtonnagequantitiesdowntopicogramsorless,isanimportantfeatureofmodernlife.Such separationsunderpinvirtuallyallaspectsofresearchandcommerceandindeeda vastindustryhasarisento provide the equipment and instrumentation to perform and control these essential processes; indeed it is impossibletoenvisageourworldwithoutreadyaccess to separations.Toprovidethesecapabilities awhole family of techniques has evolved to exploit differences in the physical or chemical properties of the compounds of interest, and to accommodate the scales on which the separations are performed. After all, aseparationthatworksonthepicogramscalebasedperhapsoncapillaryelectrophoresis,maynoteasily be transferred to the gram scale and will be utterly impossible on the kilogram scale. In such instances an alternative type of separation, based on a totally different principle must be sought. And therein lies the problem}mostscientistsarespecialistsandwhilehavinganexcellentknowledgeoftheirown,oftennarrow, sphere of expertise are generally possessed of a much more hazy view of the capabilities and attributes of techniques outside that area. Even worse, such ignorance may persuade them to adopt an approach that is quite unsuitedto the solutionof the problem in hand. The large number of articles in this encyclopedia, and the wide variety of the subject matter described in them, represents an attempt to provide separation scientists with a single authoritative source covering the broad range of separation methods currently available. Inevitably there is some overlap in places between articlesdealingwithcloselyrelatedtopics.However,inaworksuchasthiswhereeacharticleismeanttobe aself-containedsourceofinformation,someoverlapisunavoidableandnotwhollyundesirableinthecontext of ensuringfull coverage of a topic. ThearticlesintheEncyclopediaofSeparationSciencefallintothreecategoriesasfollows:‘LevelI’,which providesoverviewsofa particularseparationarea, e.g. Sotation,distillation,crystallization,etc., writtenby acknowledgedexperts in the particular Relds.These articles are presentedwith the aim of providinga wide rangingintroductiontothetopicfromwhichthereadercanthen,ifitseemsappropriate,moveonto‘LevelII’ articles. Level II articles cover the theory, development, instrumentation and practice of the various techniques containedwithineachbroadclassiRcation.ForexampletheLevelIarticleonchromatographyissupportedin Level II by descriptionsof gas, liquid and supercritical Suid chromatography,together with informationon instrumentation.Separationsare,however,oftenofinteresttopractitionersbecauseoftheirapplicationsand Level II serves as an introductionto ‘Level III’. Level III provides detailed descriptions of the use of the various methods described in Levels I and II for solvingrealproblems.Thesemightincludearticlesonthevariousmethodsforextractionofpesticidesordrugs fromamatrix,withotherarticlesonthechromatographicorelectrophoretictechniquesthatcouldbeusedfor theirsubsequentanalysis.Theextensivecross-referencingandexhaustiveindexingforallofthearticlesinthe workshouldenablethereadertoobtaineasilyandrapidlyalltherelevantinformationintheencyclopedia.In addition,eacharticleatwhateverlevelcontainsabriefbutcarefullyselectedbibliographyofkeybooks,review articlesandimportantpapers.Inthiswaytheencyclopediaprovidesthereaderwithaninvaluable‘gateway’ into the separation science literature on a topic. Lastly, because the importance of separation science lies in its value as an applied technique, we have commissionedanumberof‘essential guides’tomethod development,ina limitednumberof keyareas such as the isolation and puriRcation of proteins and enzymes, etc., or the development of chromatographic separations. While the techniques described in these volumes can in many cases be used as the basis of analytical methods,theemphasisofthisworkisonthemethodsofseparationofmixtures,ratherthantheirdetermina- tion. For a treatise devoted to Analytical Science the reader is referred to the Encyclopedia of Analytical Science,alsopublishedbyAcademicPress,whichcanbeconsideredtobecomplementarytothepresentwork, in that it deals in depth with analysis rather than the application of separations. Thereaderfacedwiththeneedtoperformaseparation,requiringinformationonatypeofdetector,orthe use of a technique for a particular class of compound, etc., should therefore be able to look into theencyclopediaforinformationonthattopic.Evenwherethereisnoinformationthatisdirectlyrelevantto x INTRODUCTION theproblem,itshouldstillbepossibletobeginatLevelIinordertodeterminethepotentialofatechniqueto solve the problem and then progress down through the levels until a solution begins to emerge. The editors believe that, taken as a whole, this encyclopedia and its electronic version should provide a valuable source of knowledge and expertise for both those already skilled in the art of some aspect of separationsand also for the novice. That, at least, is our hope. This encyclopedia is a guide providing general information concerning its subject matter; it is not a procedure manual. The readers should consult current procedural manuals for state-of-the-art instructions andapplicablegovernmentsafety regulations.Thepublisherandthe authorsdonotaccept responsibility for any misuse of this encyclopedia, including its use as a procedural manual or as a source of speciRc instructions. Sepsci*1*TSK*Venkatachala=BG I/AFFINITYSEPARATION 3 AFFINITY SEPARATION K.Jones,AffinityChromatographyLtd,Freeport, SeparationandpuriRcationmethodsforbiological Ballsalla,IsleofMan,UK macromolecules vary from the very simple to the esoteric. The type of technique adopted is basically Copyright^ 2000AcademicPress afunctionofsource,thefragilityofthemoleculeand thepurityrequired.Traditionally,highpurityprotein Introduction pharmaceuticalshaveusedmultistageprocessing,but this is very inefRcient as measured by the well- Of the collection of separation technologies known documented fact that 50}80% of total production as ‘afRnity’, afRnity chromatography is by far the costsareincurredattheseparation/puriRcationstage. mostwidelyusedvariant.AfRnitychromatographyis Incontrast,thehighlyselectiveindigenousproperties becoming increasingly important as the speed of the of the afRnity method offer the alternative of revolution taking place in biotechnology processing very elegant single-step puriRcation strategies. The increases. The concept of an ‘afRnity’ separation re- inherentsimplicityanduniversalityofthemethodhas sultsfromanaturallyoccurringphenomenonexisting already generated a wide range of separation tech- withinallbiologicalmacromolecules.Eachbiological nologies, mostly based upon immobilized naturally macromoleculecontainsauniquesetofintermolecu- occurring proteinaceous ligands. By comparing the lar binding forces, existing throughout its internal ‘old’ technologies of ‘natural’ ligands or multistage and external structure. When alignment occurs be- processing with the ‘new’, exempliRed by synthetic tween a speciRc site of these forces in one molecule designed ligands, the most recent advances in af- with the site of a set of forces existing in another Rnity processing can be described. (different) molecule, an interaction can take place between them. This recognition is highly speciRc to Biological Recognition thepairofmoleculesinvolved.Theinteractivemech- anismcanbeconvertedintoauniversalmutualbind- Asnatureevolved,lifeformshadtodevelopaprotec- ingsystem,whereoneofthebindingpairisattached tive mechanism against invading microorganisms if to an inert matrix, packed into a column and used they were to survive. Thus there is a constant battle exclusively to capture the other matching molecule. betweenthecell’sdefencemechanismandtheattack- When used in this (afRnity) mode, the technique is ing microorganisms, a battle resolved by the cells probably the simplest of all chromatographic generating antibodies(the immunoglobulins)able to methods.Itis,however,restrictedalmostexclusively recognize the protein coat of attacking microorgan- to the separationand puriRcationof biological mac- isms and signal killer cells to destroy the invaders romolecules, and is unsuitable for small molecules. beforetheycauseharmtothehost.Equally,ifmicro- AfRnity chromatography or bioselective adsorp- organisms were to survive, they had continually to tionchromatographywasRrstusedin1910,butitwas mutateandchangetheirproteincoatstoavoiddetec- only in the 1960s that afRnity chromatography as tion by existing antibodies. The ‘attack and destroy’ practised today was developed as a puriRcation tech- process is a function of changes in the molecular nique.Bythelate1970stheemergenceofrecombinant structure in a speciRc part of the protein, with only DNAtechnologyforthemanufactureofproteinphar- themostminuteofchangesoccurringatthesurfaceof maceuticals provided a new impetus for this highly the protein. Evolution has thus designed a system speciRcchromatographicmethod,implementedbythe where every protein hasa very precise structure,but demand for ever-increasing product purity implicit in onewhichwillalwaysberecognizedbyanother.One regulatory frameworks devised by (amongst others) element of the interacting pair can be covalently the USA’s Food and Drug Administration (FDA). Fi- bondedontoaninertmatrix.Theresultingchromato- nally, the need to reduce the cost of drugs is under graphic medium can then be packed into a column, constant scrutiny by many Governments, particularly andusedtoseparateexclusivelyitsmatchingpartner those with controlled health schemes funded by rev- fromanimpuremixturewhenaddedasasolutionto enue raisedbytaxation.Thesemutuallyincompatible the top of the column. This fact can be stated as pressures indicate the need for more efRcient sep- follows} forevery protein separationproblemthere aration systems; the afRnity technique provides the is always an afTnity solution. The process of promise of meeting all necessary requirements. producing a satisfactory medium is quite difRcult. 4 I/AFFINITYSEPARATION/Derivatization The matching pair must be identiRed, and one afRnity method is not restricted to protein separ- of them isolated in a pure form. Covalent bond- ations; nucleic acids and whole cells can also be ing onto an inert matrix in a stable manner must separated. always allow the ‘docking’ surface of the protein to The simplicity of the chromatographic process is be positioned to make it available to the target pro- shownin Figure 1. The ligandof interest, covalently tein.Thewholealsohastobeachievedatanaccept- bonded onto the inert matrix, is contained in the able cost. column, and a solution containing the target (the Thistechniquehasresultedinmanysuccessfulap- ligate) is passed through the bed. The ligand recog- plications, often using antibodies as the afRnity nizestheligatetotheexclusionofallothermolecules, medium (immunoafRnity chromatography), but withtheunwantedmaterialspassingthroughthecol- large scale separations using these ‘natural’ ligands umn packing while the ligate is retained. Once the are largely restricted by cost and regulatoryreasons. bed is saturated with the target molecule (as mea- Although immunoafRnity chromatography is still suredbythebreakthroughpoint),contaminatingspe- widely practised, in recent years the evolution of ciesarewashedthrough,followedbycollectionofthe design technologies has provided powerful new ap- target molecule as a very pure fraction using an proachestomimicproteinstructures,resultinginthe eluting buffer solution. Finally, the column is development of synthetic ligands able to work in cleansedfromanystronglyadsorbedtracematerials, harsh operational environmentsand at low cost. usually by regeneration with a strong alkali or acid, making it available for many more repeat runs. An The Af\nity Process outstanding advantage of the afRnity process is an ability to concentrate very dilute solutions while The afRnity method is critically dependent upon stabilizing the captured protein once adsorbed onto the ‘biological recognition’ existing between species. the column. Many of the in-demand proteins manu- Bypermanentlybondingontoaninertmatrixamol- factured by genetically engineered microorganisms ecule(theligand)thatspeciRcallyrecognizesthemol- arelabile,allowingonlyminutequantitiestobepres- ecule of interest, the target molecule (the ligate) can ent in the fermentation mix before they begin to be separated. The technique can be applied to any deteriorate. An ability to capture these very small biological entity capable of forming a dissociable quantities while stabilizing them in the adsorbant complex with another species. The dissociation con- phaseresultsinmaximizationofyield,makingmass- stant (K ) for the interaction reSects the comp- ive savings in total productioncosts. d lementarity between ligand and ligate. The optimal Although the technical processing advantages are range of K for afRnity chromatography lies be- clear there is a major difRculty in the appli- d tween 10(cid:1)4 and 10(cid:1)8molL(cid:1)1. Most biological cation of afRnity chromatography as understood ligands can be used for afRnity purposes provid- by most practitioners today. Most ligands described ing they can be immobilized, and once immobilized in Table 1 suffer from two primary disadvan- continuetointeractsuccessfullywiththeirrespective tages: a lack of stability during use; and high ligates. The ligand can be naturally occurring, an cost.Fortunatelytheseproblemshavenowbeenover- engineered macromolecule or a synthetic molecule. come, and afRnity chromatography is now accep- Table 1 provides some examples of immobili- ted as the major separations technology for zed ligands used to purify classiRed proteins. The proteins. Table1 Affinityligandsandpurifiedproteins Immobilizedligand Purifiedprotein Divalentandtrivalentmetalion Proteinswithanabudanceofhis,trypandcysresidues Lectins Glycoproteins,cells Carbohydrates Lectins Reactivedyes Mostproteins,particularlynucleotide-bindingproteins Nucleicacids Exoandendonucleases,polymerases,othernucleicacid-bindingproteins Aminoacids(e.g.lys,arg) Proteases Nucleotides,cofactorssubstratesandinhibitors Enzymes ProteinsAandG Immunoglobulins Hormones,drugs Receptors Antibodies Antigens Antigens Antibodies Sepsci*1*TSK*Venkatachala=BG I/AFFINITYSEPARATION 5 The beaded agaroses have captured over 85% of thetotalmarketforbiologicalmacromoleculesepar- ations, and are regarded as the industry standard to which all other supports are compared. They have achievedthispositionbyprovidingmanyofthedesir- able characteristics needed, and are also relatively inexpensive.Beadedagarosesdohaveoneseverelim- itation } poor mechanical stability. For analytical applications speed and sensitivity are essential, de- manding mechanically strong, very small particles. Beaded agaroses are thus of limited use analytically, Figure1 Schematicdiagramofaffinitychromatography. agapRlledbyhighperformanceliquidchromatogra- phy(HPLC)usingsilicamatrices.Forpreparativeand large scale operations other factors are more impor- Matrices tant than speed and sensitivity. For example, mass BydeRnitionmatricesmustbeinertandplaynopart transferbetweenstationaryphaseandmobilephaseis in the separation. In practice most play a (usually) much less important when compared to the contri- negative role in the separation process.To minimize bution from the chemical kinetics of the binding thesedisadvantagesmatriceshavetobeselectedwith reaction between stationary phase and protein. great care. There is a theoretically perfect matrix, Band spreading is also not a serious problem. When deRned as consisting of monodispersed perfectly combined with the highly selective nature of the shaped spheres ranging from 5 to 500(cid:1)m in dia- afRnity mechanism, these factors favour the com- meter, of high mechanical strength, zero nonspeciRc monuseoflargesized,lowmechanicalstrengthpar- adsorptionand with a range of selectable pore sizes ticles. from10}500nm,averynarrowporesizedistribution In recent years synthetic polymeric matrices have and low cost. This idealized matrix would then pro- been marketed as alternatives. Although nonbiodeg- vide the most efRcient separation under all ex- radable, physically and chemically stable, with good perimentalconditions.As always, a compromisehas permeabilities up to molecular weights greater than tobereached,theusualapproachbeingtoaccentuate 107Da, the advantages provided are generally off- the most attractive characteristics while minimizing set by otherquite seriousdisadvantages,exempliRed the limitations, usually by manipulating the experi- by high nonspeciRc adsorption. Inorganic matrices mentalconditionsmostlikelytoprovidetheoptimum have also been used for large scale protein separ- result. ations, notably reversed-phase silica for large scale The relative molecular masses of proteins vary recombinant human insulin manufacture (molecular from the low thousands to tens of millions, making weight approximately 6000Da), but are generally pore size the most important single characteristic of not preferred for larger molecular weight pro- the selected matrix. Very large molecules need very ducts. A very slow adoption of synthetic matrices is openandhighlyporousnetworkstoallowrapidand easy penetration into the core of the particle. Struc- turesofthistypemustthereforehaveverylargepores, Table2 Supportmatrices but this in turn indicates low surface areas per unit volume,suggestingrelativelylownumbersofsurface Supportmatrix Operational pHrange groups to which ligands can be covalently attached. Thematrix must also be biologically and chemically Agarose 2}14 inert. A special characteristic demanded from biolo- Cellulose 1}14 gical macromolecularseparationsmedia is anability Dextran 2}14 to be sanitized on a routine basis without damage. Silica (8 Glass (8 This requires resistance to attack by cleansing re- Polyacrylamides 3}10 agentssuchasmolarconcentrationsofstrongalkali, Polyhydroxymethacrylates 2}12 acidsandchaotropes.Incontrasttoanalyticalsepar- Oxirane}acryliccopolymers 0}12 ations, where silica-based supports are inevitably Styrene}divinylbenzenecopolymers 1}13 used, silica cannot meet these requirements and is Polyvinylalcohols 1}14 N-Acryloyl-2-amino-2-hydroxy-1,2-propane 1}11 generally not favoured for protein separations. PTFE Unaffected Table 2 contains examples of support matrices used in afRnity separations. PTFE,polytetrafluoroethylene.