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○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○HiPrep16/10CMFFHiPrep16/10DEAEFFinstructionsiiiii71-5004-48EditionACp1○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○BuffersandsolventresistanceDe-gasandfilterallsolutionsthrougha0.45µmfiltertoincreasecolumnlife-time.DailyuseAllcommonlyusedaqueousbuffers(see“Columndata”forrecommendedpH)Guanidinehydrochloride,upto6MUrea,upto8MCleaningSodiumhydroxide,upto1MEthanol,upto70%Aceticacid,upto1MIsopropanol,upto30%AvoidOxidizingagentsCationicdetergentsandbuffers(CM)Anionicdetergentsandbuffers(DEAE)PhenolSamplerecommendationsNetchargeofprotein:Positive(CM),negative(DEAE)RecommendedNotmorethan10-20%ofthesampleload:dynamiccapacity(seesection“Columndata”).Preparation:Dissolvethesampleinstartbuffer,filterthrough0.45µmorcentrifugeat10000xgfor10minInstructionsHiPrep16/10CMFForHiPrep16/10DEAEFFConnectors2xUnion1/16”female/M6maleInstructionsHiPrep™16/10CMFFandHiPrep16/10DEAEFFarepre-packed,readytousecolumnsforionexchangechromatography.Theyprovidefast,preparativeseparationsofproteinsandotherbiomolecules.Seebelowforcolumncharacteristics.ColumndataMatrix6%highlycross-linkedsphericalagaroseMeanparticlesize90µmBedvolume20mlBedheight100mmi.d.16mmColumncompositionPolypropyleneConnectorsM6Recommendedflowrate12–10ml/min(60–300cm/h)Maximumflowrate110ml/min(300cm/h)Maximumpressureoverthe0.15MPa,1.5bar,22psipackedbedduringoperation,∆p3HiPrepcolumnhardware0.5MPa,5bar,73psipressurelimit3CMDEAETypeofexchangerweakcationweakanionChargedgroup-0-CH2COO--N+(C2H5)2HpHstabilityshortterm2–141–14working6–102–9longterm4–132–13Totalioniccapacity0.09–0.130.11–0.16mmolH+/mlgelmmolCl-/mlgelDynamicbindingcapacity(mg/mlgel)2HSA(Mr68000)N.D110RibonucleaseA(Mr13700)50N.D1Wateratroomtemperature.Flowrateisdeterminedbyvη≤10ml/minwherev=flowrateandη=viscosity.2Determinationofdynamicbindingcapacity:Sampleswereappliedat75cm/huntil50%breakthrough.Columns:0.5x5cm.Buffers:0.05MTris,(+2MNaClintheelutionbuffer),pH7.5(DEAE),0.1Macetate,(+2MNaClintheelutionbuffer),pH5.0(CM).3Manychromatographysystemsareequippedwithpressuregaugestomeasurethepressureataparticularpointinthesystem,usuallyjustafterthepumps.Thepressuremeasuredhereisthesumofthepre-columnpressure,thepressuredropoverthegelbed,andthepost-columnpressure.Itisalwayshigherthanthepressuredropoverthebedalone.Werecommendkeepingthepressuredropoverthebedbelow1.5bar.Settingtheupperlimitofyourpressuregaugeto1.5barwillensurethepumpshutsdownbeforethegelisoverpressured.Ifnecessary,post-columnpressureofupto3.5barcanbeaddedtothelimitwithoutexceedingthecolumnhardwarelimit.Todeterminepost-columnpressure,proceedasfollows:Toavoidbreakingthecolumn,thepost-columnpressuremustneverexceed3.5bar.1.Connectapieceoftubinginplaceofthecolumn.2.Runthepumpatthemaximumflowyouintendtouseforchromatography.Useabufferwiththesameviscosityasyouintendtouseforchromatography.Notethebackpressureastotalpressure.3.Disconnectthetubingandrunatthesameflowrateusedinstep2.Notethisbackpressureaspre-columnpressure.4.Calculatethepost-columnpressureastotalpressureminuspre-columnpressure.Ifthepost-columnpressureishigherthan3.5bar,takestepstoreduceit(shortentubing,clearcloggedtubing,orchangeflowrestrictors)andperformsteps1-4againuntilthepost-columnpressureisbelow3.5bar.Whenthepost-columnpressureissatisfactory,addthepost-columnpressureto1.5barandsetthisastheupperpressurelimitonthechromatographysystem.FirsttimeuseEnsureanappropriatepressurelimithasbeenset.Equilibratethecolumnforfirsttimeuseorafterlong-termstoragebyrunning:1.100mlstartbuffer(lowionicstrength)at5ml/minatroomtemperature(seesection“Choiceofbuffer”forbufferrecommendations).2.100mlelutionbufferat5ml/minatroomtemperature.3.100mlstartbufferat5ml/minatroomtemperature.TrytheseconditionsfirstFlowrate:5ml/minatroomtemperatureStartbuffer:Seesection“Choiceofbuffer”Elutionbuffer:Startbuffer+1MNaClGradient:0–100%elutionbufferin200ml(10CV)EquilibrationbeforeanewrunProceedaccordingtosteps2and3inthesection“Firsttimeuse”.Extendedequilibrationmaybeneededifdetergentswereincludedintheeluent.Pleasereadthebackoftheseinstructionsforinformationonoptimizingaseparation.Delivery/storageThecolumnissuppliedin20%ethanol.Ifthecolumnistobestoredformorethantwodaysafteruse,cleanthecolumnaccordingtotheproceduredescribedunder“Cleaninginplace(CIP)”.Thenequilibratewithatleast100mlof20%ethanolataflowrateof5ml/minatroomtemperature.DONOTOPENTHECOLUMN!ChoiceofbufferToavoidlocaldisturbancesinpHcausedbybufferingionsparticipatingintheionexchangeprocess,selectabufferwithbufferingionsofthesamechargeasthesubstituentgroupsontheionexchanger.ThestartbufferpHshouldbechosensothatsubstancestobeboundtotheionexchangerarecharged,thatis,atleast1pHunitabovetheisoelectricpointforanionexchangersoratleast1pHunitbelowtheisoelectricpointforcationexchangers.Figure1andFigure2listaselectionofstandardaqueousbuffers.Table1listssuggestedvolatilebuffersusedincaseswherethepurifiedsubstancehastobefreeze-dried.iiiii71-5004-48EditionACp2○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○