Waters---HPLC故障检修指南(英文)

HPLCTroubleshootingGuide1HPLCTroubleshootingUweD.Neue,WatersCorporation1.ColumnLife-Time2.VariableRetentionTimes3.DriftingRetentionTimes4.Column-to-ColumnandBatch-to-BatchReproducibility5.SamplePreparationProblems6.SourcesofPeakTailing7.Normal-PhaseChromatography8.SystemVolume,DeadVolume,DwellVolume9.TransferofGradientMethods10.CloggedSystem11.ColumnPlate-Count12.ColumnBackpressure13.PeakAreaFluctuations14.GhostPeaks15.DependenceofRetentionTimesonpH16.ColumnEquilibration17.ColumnConditioning18.ComplexSampleMatrices19.HydrophobicCollapse20.BaselineNoise21.Narrow-boreColumns22.SampleSolvent23.GradientScaling24.ColumnStorage25.Paired-IonChromatography26.HydrolyticStabilityofReversed-PhasePackings27.OptimalFlowRates28.CarbonLoad29.pHControl30.MobilePhaseComposition31.ColumnContamination32.MethodVerification33.DoublePeaksinSugarSeparations34.MethodControl35.MobilePhasepH36.Signal-to-NoiseImprovements37.Overload38.ColumnDurability39.FastAnalysisandColumnBackpressure40.ColumnBackflushing41.SelectivityShift42.NewMethod43.NegativePeaks44.Tricky,Tedious,TimeConsuming45.FastSeparations46.BuffersforLC/MS47.AlkalineBuffersforRPLC48.Post-ColumnDerivatization49.GradientDwellVolume50.BufferCapacity51.FlowRateChangesandQuantitation52.AnalysisofpolarcompoundsClickonsectionsbelowtogodirectlytothatsectionoftheTroubleshootingGuide231.ColumnLife-TimeQ.:Mycolumnlastedonlyforabout100injections.Afterthattime,thepeaksbecamedistortedandtheplate-countswereverylow.What’swrong?A.:100injectionsisindeedashortlife-time.Undernormalcircumstances,onecanexpectacolumntobeinservicemuchlonger.Inordertodeterminewhatiswrong,weneedtoestablishfirst,ifshortcolumnlifeistheruleforyourapplicationornot.Therearetwofundamentalcases:1.previouslycolumnsusedforthesameassaylastedmuchlonger.2.allcolumnsusedforthisapplicationdieafteraboutthesameamountofuse.Inthefirstcase,onewouldexploreiftheassayhasremainedtrulyconstant.Hasthesamplecompositionchanged?Stronglyadsorbedcontaminantsinyoursamplecandestroycolumnperformance.Arethesealsinthefluidpathofyourinstrumentinagoodcondition?Sheddingsealscanclogcolumnfiltersandthetoplayersofthepackingandthuseffectthedistributionofthesample.Ifonecanbereasonablyassuredthattherearenochangesinthechromatographicconditions,onecansafelyassumethatthecauseoftheproblemisamechanicalweaknessofthepackedbed.Thiscanbeinducedbyroughhandlingofthecolumninyourlab(didyoudropit?)orduringshipment,oritcouldbeamanufacturingdefect.SuchadefectcannotbedetectedbystandardcolumnQCandcouldshowuponlyaftersomeuseofthecolumn.Inthiscase,columnmanufacturerswillreplaceyourcolumnfreeofcharge.Q.:Thatisniceofthesemanufacturers,butthisisnotmyproblem.Mycolumnsalwayslastonlyashorttime.Sometimesit’s100injections,sometimes200.Icouldlivewith200injections,butonly100isnotgoodenough.Thisreallyisgettingexpensive.WhatcanIdo?A.:Iagreewithyou100%.Whatweneedtodotogetheristofindthecauseofyourproblemandthensee,whatwecandoaboutit.Themostlikelycauseofyourproblemisadsorptionofsampleconstituentsonthetopofthecolumn.Theymayeitherprecipitatebecauseofalowsolubilityinthemobilephaseortheymaybestronglyadsorbed.Asyouinjectmoreandmoresamples,thesecontaminantsbuilduponthetopofthecolumnandpreventthesampletoproperlyadsorbanddistribute.Thisresultsinadistortionofthepeakprofile.Oftenthisproblemisaccompaniedbyanincreaseinbackpressure.Q.:OK,thatcouldbeit.HowdoIgetaroundthisproblem?A.:Thereareseveralwaystopreventthisfromhappening.Oneistocleanupthesamplewithasuitablesamplepreparationtechnique.SolidphaseextractionusingaSPEcartridgewithasimilarchemistryastheseparationcolumnworkswellforthisproblem.Anotherandmorepowerfulapproachistouseaguardcolumn.Theprecolumnservesasasacrificialcolumntopthatisreplacedwhentheproblemoccurs.Forbestperformance,youshoulduseaguardcolumnthatcontainsexactlythesamepackingastheanalyticalcolumnandispackedwiththesamehighperformancepackingtechniqueastheanalyticalcolumn.Ifyouuseprecolumnsmadewithadifferentbrandofpacking,youwillnotgettheoptimalperformancebothinseparationcapabilityandinprotectionofyouranalyticalcolumn.Also,donotusealargerparticlesize.Largerparticlesorbadlypackedprecolumnscanresultinadeteriorationoftheseparationduetoband-broadeningintheprecolumn.Q.:Touseaguardcolumnsoundsok.Doyouhaveanyothersolutions?A.:Well,notreally.Thereareafewotherpossibilities,buttheyallhavetheirdrawbacks.Iamnotanadvocateofcolumnwashingwithsolventsthataresupposedtodissolvethecontaminantsonthetopofthecolumn.Inmanycases,thisprocesssimplydoesnotwork.Forexample,ifthecontaminantsareproteinsthathaveprecipitatedonthecolumntop,bythetimeyoutrytowashthemofftheyhaveagedalotbydenaturationandmaybeevencross-linkingthatitmaybeimpossibletosolubilizethemagain.Furthermore,everywashingwillalsoremovehydrolyzedbondedphase,whichotherwiseremainsinalocalequilibriumatthesitewherethehydrolysisoccurred.Consequently,arepetitivewashingcanactuallyresultinanacceleratedagingofthecolumn.Also,afterthiswashingyouhavetore-equilibrateyourcolumnwiththemobilephase,whichinsomecaseslikeinion-pairchromatographymaybequitetimeconsuming.Anotherapproa