Separation And Purification Techniques In BiotechnologyDechow生物化学生物技术教案 3

Separation And Purification Techniques In Biotechnology Dechow生物化学生物技术教案 3 Ion Exchange3259.80.81.Streat,M.,J SeparProc Technol,l (3):lO (1980)82.Mallon,C.,Richter,M.,ZfI-Mitteilunnen.Leipzig,86:39 (1984)6.87.88.Slater,M.J.,Canadian JChem Enq,52:43(Feb.1974)Shirato,S.,Miyazaki,Y.,Suzuki,I.,Fermentation Industrv(Japan),45 (1):60 (1967)Wolf,F.J.,Separation Methodsin OrPanicChemistry andBicohemistrv,Academic Press,NY,p153 (1969)Nagase,T.,Hirohare,H.,Nabeshima,S.,Brit.Patent No.1.597,436(Sept.6,1981)Commerical inertresins forthis applicationinclude DowexBuffer Beads(XFS-43179),Ambersep359and DuoliteS-3TR.Higgins,I.R.,U.S.Patent No.3,580,842(May25,1971)Himsley,A.,Canadian Patent No.980,467(Dec.23,1975)Brown,H.,U.S.Patent No.3,549,526(Dec.22,1970)Cloete,F.L.D.,Streat,M.,U.S.Patent No.3,551,118(Dec.29,1970)Cloete,F.L.D.,Streat,M.,British Patent No.1,070,251 (1962)George,D.R.,Ross,J.R.,Prater,J.D.,Min Enain,I:73 (1968)Buijs,A.,Wesselingh,J.A.,Polvtech Tiidschrift Procestechniek,36 (2):70 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twoor moresolutes in the feedstreamto be separated.Beads ofnon-ionic resins,gels ormolecular sievesmay alsobe used as the solid stationary phase incolumn chromatography.The feed solutionisadded to the chromatographic column filled with the separation beads and elutedwith solvent,often waterfor fermentationproducts.The resin beads selectivelyslow somesolutes whileothers areeluted down the column(Figure4.1).As the solutes movedownthe column,they separateand theirindividual purityincreases.Eventually,the solutesappear atdifferent timesat the column outletwhere eachcan bedrawn offseparately.Tswett(1,2)was thefirst toidentify correctlythe natureof the separation ofcolored vegetablepigments inpetroleum etherwhen thesolution was passed through a columnof fineparticle calciumcarbonate.The processwas calleda chromatographic separation becauseof the separation of the pigmentsinto bandsof differentcolors.Following Tswetthave beennumerous scientistswho havedeveloped ion exchange chromatographyinto asophisticated analyticaltechnique usedin manyscientific areas.333334Separation andPurification Techniquesin BiotechnologySolutes AddedSolute NixtureSolutes AddedTo ColumnDesorbent AddedSeparation SlowOurs FastComponent ComponentRemoved RemovedFrom FromColumn ColumnFigure4.1.The stepsof chromatographic separation are:addition of the mixedsolutes to the column,elution to effect separationsand removal of theseparated solutes.The firstmercial useof ion exchange chromatographyourred duringWorld WarII atIowa StateUniversity whereSpedding andhis coworkers(3,4)isolated transuraniumelements as their contributionto theManhattan Project.Several yearslater,the mercialpreparation of individual rareearths wasperformed usingion exchangechromatography(5,6).Despite theseinitial suesseswith chromatographyin preparingpure materialsin largequantities,other mercialapplications werenot developeduntil themid tolate1960s.It wasin the1970s beforeindustrial separationproblems wereidentified whichcould notbe solvedby conventional,well-tried methodssuch asdistillation orcrystallization.At present,there areindustrial ionexchange chromatographicColumn Chromatography Processes335units whichseparate amino acids,hydrocarbon isomers,glucose fromfructose,sucrose frommolasses,monosaharides from di-and polysaharidesand ionexchange regenerantsfrom salts.A literaturesearch bySitrin andcoworkers (7),covering theperiod from1980to1983,revealed thatout of the7,000citations forliquid chromatography,only100discussed preparativework.Few of these involvedseparations largerthan100mg.That distributionremains typicalof thepublished activityin thechromatography area.There aretwo advancesthat havecontributed stronglyto therecent developmentof industrialionexchangechromatography: (1)improvements inchromatographic equipmentwith recyclecontrol systemsand pseudo-moving bedcontrol systems;and (2)improvements byresin manufacturersin developingresins withnarrow,controlled sizedistributions andimproved osmoticresilience.General resinconsiderations andmercial ionexchange resins have beendescribed inChapter3.Descriptions ofchromatographic systemswill becovered inSection7of thischapter.4.2CLASSIFICATIONS OFCHROMATOGRAPHY4.2.1Chromatographic MethodsThe literatureis repletewith variouscolumn chromatographic methods:adsorption chromatography,ionexchangechromatography,gel filtration chromatography,gel permeation chromatography and affinity chromatography.These methodsare differentiatedon the basis of the retentiveability of the stationary phase,the type of eluentemployed and the materialused as the separationphase.In adsorption chromatography,it ismainly physicalsurface forceswhich areinvolved inretaining the solute.This methodis theoldest of the chromatographic techniques.Although itdoes nothave thespecificity ofthe othermethods,adsorptionchromatography is stillone ofthe simplestand mosteffective techniquesof separatingmixtures ofnon-polar substancesand pounds of lowvolatility.Silica,alumina,activated carbon and macroporousnon-ionic polymersare the adsorbents usedmost frequentlyas the336Separation andPurification Techniquesin Biotechnologyseparation media.Low molecular weight organic solvents arethe mostmon eluents.In ionexchangechromatography,true hetero-polar chemicalbonds areformed reversiblybetween ionic ponents in the mobileand thestationaryphase.The ionexchange beadsmay haveeither hydrophobicor hydrophilicmatrices whichhave beenfunctionalized with ionizable groups,as discussedin thelast chapter.The eluentfor thechromatographic separation of fermentationproducts at the preparativeor higherlevel isusually wateror an aqueous buffer.The stationaryphase may also serveas amolecular sieveto separatesolutes on the basis of molecular size.This methodis knownas gelfiltrationchromatographyor gelpermeationchromatography.Gel filtrationchromatography originatedin Swedenin1959 (8)when columnspacked withcrosslinked polydextrangels,swollen in anaqueoussolution,were used to separatewater-soluble macromoleculeson thebasis ofsize differences.These gels are stillused extensivelyfor separatingwater solublebiological poundsfor furthercharacterization studies (9).Gel permeationchromatography wasdeveloped atDow Chemicalin1964 (10)using crosslinkedpolystyrene gelsswollen inorganicsolventsto separatesynthetic polymerson thebasis ofsize.Column chromatographywith ionexchange resinsalso canutilize sizedifferences forseparation butmore frequentlyrelies upon adsorption foraffinity differencestoeffectseparations.Partitioning of a poundbetween ahydrophobic stationaryandapolar aqueousmobile phaseis calledreverse phaseliquid chromatography.Reverse phasechromatography isusually performedon columnspacked withsilica gelto whichaC-8to C-18hydrocarbon has been covalentlyattached.For suchsystems,the strengthof aneluting solventincreases asits polaritydecreases andpoundsofsimilar structurecan be expected toelute in order ofdecreasing polarity.Polar,charged fermentationproducts,such aspeptides andglycoproteins,are wellsuited forrecovery andpurification withreverse phasechromatography.This is due Column Chromatography Processes337to theremovalofvery polar,often coloredcontaminants atthe solventfront,ahead ofthe desiredmaterial.Using thistechnique earlyinapurification schemecan simplifythe numberof stepsone mustemploy.Affinity chromatography,which originatedin1968attheNational Institutesof Health(1I),is based onaunique andfundamental propertyof biologicalmacromolecules:their selective,high-affinity recognitionof,and reversibleinteraction with,other molecules.This techniqueis sufficientlydistinct fromthe otherchromatographicmethodsthat it will bediscussed separatelyin thenext chapter.Most literaturereferences tocolumn chromatographyare concernedstrictly with analytical applications.While someoftheinformation isdirectly relatabletothepreparative andmercial recovery of fermentationproducts,one must be cautiousdue tothe differencesin rangesof operatingparameters,throughput and the additionof ponentsto aidin analyticalresolution.The lowcrosslinked polymerbeads usedin analyticalchromatographic applicationshave meandiameters of70to150microns andcan onlybe usedat lowflow ratesand pressuresless than17bars.These beadscollapse athigher pressureswhich restrictsthe flowrate,making separationsimpossible.Pilot plantand industrial chromatography uselarger meandiameter beads(200to450microns)withahigher degree of crosslinking.The necessarydegreeofseparation isachieved byremoving aspecific cutofthe chromatogram.4.2.2Types ofChromatographic SeparationsChromatographic separationscan beclassed intofour typesaording tothe typeof materialsbeing separated:affinity differences,ion exclusion,size exclusionand ionretardation chromatography.These typesof separationsmay bedescribed interms ofthe distributionofthematerials tobeseparatedbetween thephases involved.Figure4.2shows arepresentation ofthe resin-solvent-solute ponents of acolumn chromatographicsystem.The columnis filledwith resinbeads ofthe solidstationaryphasepacked togetherwith thevoids between the beadsfilledwithsolvent solution.The phasesof interestare (1)the liquid338Separation andPurification Techniquesin Biotechnologyphase betweenthe resinbeads; (2)the liquidphase heldwithin the resinbeads;and (3)the solidphase ofthe polymericmatrix ofthe resinbeads.When thefeed solutionis placed in contactwith thehydrated resinin thechromatographiolumn,the solutesdistribute themselvesbetweenthe liquid insidethe resinbeadsandthat betweenthe resinbeads.The distributionfor ponenti isdefined by the distributioncoefficient,Rd.:1Kdi=rifli(4.1)where Criis ponentis concentrationin theliquid within the resinbead andCli is the ponentis concentrationin th