色谱柱和色谱系统的故障检修.ppt

2020/6/8,1,HPLCColumnandSystemTroubleshooting色谱柱和色谱系统的故障检修,WhatEveryHPLCUserShouldKnow每个HPLC使用者应该知道什么,2020/6/8,2,HPLCComponentsHPLC的组成,Pump泵Injector/Autosampler进样器/自动进样器Column色谱柱Detector检测器DataSystem/Integrator数据系统/积分仪表Allofthesecomponentscanhaveproblemsandrequiretroubleshooting.所有这些部件可能出现故障而需要检修。,2020/6/8,3,CategoriesofColumnProblems色谱柱问题的类型,A.Pressure压力B.Peakshape峰形C.Retention保留时间,2020/6/8,4,PressureIssues压力问题,ColumnObservationsPotentialProblems色谱柱观测潜在的问题HighpressurePluggedfrit压力高堵塞滤头Columncontamination色谱柱污染Piuggedpacking堵塞填料,2020/6/8,5,DeterminingtheCauseandCorrectingHighBackPressure查出原因纠正压力,Checkpressurewith/withoutcolumn-manypressureproblemsareduetoblockagesinthesystemorguard有无柱子时检查压力许多压力问题是由于系统或保护柱的阻塞IfColumnPressureishigh如果柱压高:Washcolumn-Eliminatecolumncontaminationand冲洗柱子pluggedpacking排除柱污染和填料堵塞-highmolecularweight/adsorbedcompounds高分子量被吸附化合物-precipitatefromsampleorbuffer样品或缓冲液中的沉淀物Backflushcolumn-Clearpluggedfrit反冲柱子清洁阻塞的滤头Changefrit-Clearpluggedfrit更换滤头,2020/6/8,6,ColumnCleaning柱子清洗,Flushwithstrongersolventsthanyourmobilephase用比你的流动相更强的溶剂冲洗Reversed-PhaseSolventChoicesinOrderofIncreasingStrength为了增加强度选择反相溶剂Useatleast25mlofeachsolventforanalyticalcolumns对于分析柱每种溶剂至少用25ml冲洗Mobilephasewithoutbuffersalts没有缓冲盐的流动相100%Methanol甲醇100%Acetonitrile乙腈75%Acetonitrile:25%Isopropanol75乙腈：25异丙醇100%Isopropanol异丙醇100%MethyleneChloride二氯甲烷100%Hexane已烷WhenusingeitherHexaneorMethyleneChloridethecolumnmustbeflushedwithIsopropanolbeforereturningtoyourresvered-phasemobilephase.当用已烷蔌二氯甲烷柱子时,必须先用异丙醇冲洗,然后再用你的反相流动相,2020/6/8,7,ColumnCleaning柱子清洗,NormalPhaseSolventChoicesinOrderofIncreasingStrength为了增加强度选用正相溶剂Useatleast50mlofeachsolvent每种溶剂至少用50ml50%Methanol:50%Chloroform50%甲醇:50%氯仿100%EthylAcetate乙酸乙酯,2020/6/8,8,HowtoChangeaFrit怎样更换滤头,ColumnInletFrit柱进口滤头ColumnBody柱身CompressionFerrule压缩金属套圈Weargloves戴手套Donotallowbedtodry不要让底座干燥Donottouchthecolumn-bodyheatwillextrudepacking不要使柱身受热,否则填料会喷出Donotovertighten不要旋得太紧FemaleEndFitting旋好尾端螺母MaleEndFitting旋好尾端螺头,2020/6/8,9,PreventingBackPressureProblems压力问题的预防,Usecolumnprotection柱保护-Guardcolumns保护柱/预柱-In-linefilters在线过滤器SamplePreparation样品预处理Appropriatecolumnflushing适当的柱冲洗Filterbufferedmobilephase过滤缓冲流动相,2020/6/8,10,PreventingBackPressureProblems:In-LineDevices压力问题的预防:在线装置,MobilePhasePre-ColumnInjectorFilterGuardFromPump预柱进样器滤头Column从泵来的流动相保护柱AnalyticalColumnFilterandGuardColumnActonSample分析柱滤头和保护柱作用于样品Pre-ColumnActsonMobilePhase预柱作用于流动相ToDetector到检测器,2020/6/8,11,PreventingBackPressureProblems:SamplePreparation压力问题的预防:样品制备,Solvent/ChemicalEnvironment溶剂/化学环境Particulate/AggregateRemove微粒/凝聚物的除去Filtersamples过滤样品Centrifugation离心分离SolidPhaseExtraction(S.P.E.)固相萃取CartridgesorPlates薄膜或薄层板DisksorMembranes圆盘或生物膜,2020/6/8,12,PeakShapeIssues峰形问题,Splitpeaks裂峰Peaktailing峰拖尾Broadpeaks宽峰Poorefficiency低柱效Manypeakshapeissuesalsocombinations-I.e.Broadandtailingortailingwithincreasedretention许多峰形问题是结合在一起的-例如:展宽和拖尾或拖尾和保留时间增加,2020/6/8,13,SplitPeaks裂峰,Canbecausedby:可能的原因:Columncontamination柱污染Partiallypluggedfrit部分阻塞滤片Columnvoid柱头塌陷Injectionsolventeffects溶剂效应,2020/6/8,14,SplitPeaks裂峰ColumnContamination柱污染,Column:StableBondSB-C8,4.6250mm,5mMobilePhase:60%25mMNa2HPO4,pH3.0:40%MeOHFlowRate:1.0mL/minTemperature:35CDetection:UV254nmSample:FilteredOTCColdMedication:1.Pseudoephedrine2.APAP3.Unknown4.ChlorpheniramineInjection1峰3峰形较好Injection30峰3裂峰Injection1AfterColumnWashwith100%CAN经100%乙腈冲洗后峰3峰形极好,2020/6/8,15,SplitPeaks裂峰InjectionSolventEffects溶剂化效应,Column:StableBondSB-C8,4.6250mm,5mMobilePhase:82%H2O:18%ACNInjectionVolume:30LSample:1.Caffeine咖啡因2.Salicylamide水杨酰胺A.InjectionSolventB.InjectionSolvent100%AcetonitrileMobilePhase峰形宽拖尾峰形尖锐对称样品溶剂尽可能与流动相匹配,2020/6/8,16,DeterminingtheCauseofSplitPeaks裂峰原因的确定,1.Complexsamplematrixormanysamplesanalyzed-likelycolumncontaminationorpartiallypluggedfrit复杂样品的基质或分析许多样品-柱污染或部分阻塞滤片2.MobilephasepH=7-likelycolumnvoidduetosilicadissolution(unlessspecialtycolumnused)流动相pH=7-由于硅胶溶解使柱塌陷(除非使用专门的柱子)3.Injectionsolventstrongerthanmobilephase-likelysplitandbroadpeaks,dependentonvolume溶剂比流动相的可能性大-裂峰和宽峰,由样品量来决定,2020/6/8,17,PeakTailing,BroadeningandLossofEfficiency峰拖尾、变宽和柱效降低,Canbecausedby:可能的原因Column“secondaryinteractions”柱“次级效应”Columnvoid柱塌陷Columncontamination柱污染Columnaging柱老化Columnloading柱负荷超载Extra-columneffects柱外效应,2020/6/8,18,PeakTailingColumn“SecondaryInteractions”峰拖尾柱“次级效应”,Column:Alkyl-C8,4.6150mm,5mMobilePhase:85%25mMNa2HPO4,pH7.0:15%ACNFlowRate:1.0mL/minTemperature:35CSample:1.Phenylpropanolamine苯丙醇胺去甲麻黄碱2.Ephedrine麻黄碱3.Amphetamine安非他明苯丙胺4.Methamphetamine脱氧麻黄碱5.Phenteramine苯三胺NoTEA无三乙胺10mMTEA10mM三乙胺USPTF(5%)美国药典拖尾因子USPTF(5%)美国药典拖尾因子1.1.2.9.6.354.1.265.1.575.1.14Peaktailingeliminatedwithmobilephasemodifier(TEA)atpH7用流动相减尾剂(三乙胺)在pH7消除峰拖尾,2020/6/8,19,PeakTailingColumn“SecondaryInteractions”峰拖尾柱“次级效应”,Column:Alkyl-C8,4.6150mm,5mMobilePhase:85%25mMNa2HPO4,pH7.0:15%ACNFlowRate:1.0mL/minTemperature:35CSample:1.Phenylpropanolamine苯丙醇胺去甲麻黄碱2.Ephedrine麻黄碱3.Amphetamine安非他明苯丙胺4.Methamphetamine脱氧麻黄碱5.Phenteramine苯三胺pH3.0pH7.0USPTF(5%)美国药典拖尾因子USPTF(5%)美国药典拖尾因子4.1.334.2.35ReducingthemobilephasepHreducesinteractionswithsilanolsthatcausepeaktailing降低流动相的pH,减小与硅醇基作用引起的峰拖尾,2020/6/8,20,PeakTailing峰拖尾ColumnContamination柱污染,Column:StableBondSB-C8,4.6250mm,5mMobilePhase:20%H2O:80%MeOHFlowRate:1.0mL/minTemperature:R.T.Detection:UV254nmSample:1.Uracil尿嘧啶2.Phenol苯酚3.4-Chloronitrobenzene4-氯硝基苯4.Toluene甲苯PlatesTFPlatesTFPlatesTF理论板数拖尾因子理论板数拖尾因子理论板数拖尾因子1.76292.081.79061.431.74481.062.120431.642.124431.212.122371.213.137271.693.179991.193.153661.114.133551.324.170981.254.190671.17QCtestforwarddirectionQCtestreversedirectionQCtestaftercleaning100%IPA,35C向前方向相反方向100%异丙醇冲洗后,2020/6/8,21,PeakTailing/BroadeningSampleLoadEffects峰拖尾/变宽样品过载效应,Column:4.6150mm,5mMobilePhase:40%25mMNa2HPO4,pH7.0:60%ACNFlowRate:1.5mL/minTemperature:40CSample:1.Desipramine去郁敏/脱甲基丙米嗪2.Nortriptyline3.Doxepin4.Imipramine丙米嗪5.Amitriptyline阿米替林6.TrimipramineTailingEclipseXDB-C8Broadening/CompetitiveC8USPTF拖尾因子Plates理论板数HighLoad/10LowLoadHighLoad/10LowLoad高载负/10倍低载负高载负/10倍低载负1.1.601.7085059412.2.001.9081578423.1.561.56277662302539835627351005518910725,2020/6/8,22,PeakBroadening,SplittingColumnVoid峰变宽,裂开柱塌陷,MobilePhase:流动相：50%CAN:50%Water:0.2%TEA(pH11)50乙腈:50水:0。2三乙胺(pH11)After30injectionsInitial开始时进样30次后Multiplepeakshapechangescanbecausedbythesamecolumnproblem.InthecaseavoidresultedfromsilicadissolvedathighpH.多数峰形改变可能是同一柱问题引起的。在这个例子中，高pH溶解硅胶导致塌陷,2020/6/8,23,BroadPeaksUnknown“Phantom”Peaks,Column:Extend-C18,4.6150mm,5mMobilePhase:40%10mMTEA,pH11:60%MeOHFlowRate:1.0mL/minTemperature:R.T.Detection:UV254Sample:1.Maleat顺丁烯二酸2.Pseudeophedrine伪麻黄碱3.Chlorphenniramine氯非尼拉明Plates理论板数1.59222.98793.779“Phantom”peakfromfirstinjection前次进样未出完的峰Theextremelylowplatesareanindicationofanextremelylateelutingpeakfromtheprecedingrun.极低的理论板数表明这是一个前面进样极迟洗脱出来的峰,2020/6/8,24,PeakTailing峰拖尾InjectorSealFailure进样器密封性差,Column:Bonus-RP,4.675mm,3.5mMobilePhase:30%H2O:70%MeOHFlowRate:1.0mL/minTemperature:R.T.Detection:UV254nmSample:1.Uracil尿嘧啶2.Phenol苯酚3.N,N-DimethylanilineN,N-二甲苯胺PlatesTFPlatesTF理论板数拖尾因子理论板数拖尾因子1.22351.721.36701.452.34911.482.104571.093.54321.153.100851.00BeforeAfterreplacingrotorsealandisolationseal之前转动杆和隔离密封垫检修后Overdueinstrumentmaintenancecancausepeakshapeproblems.仪器保养超期可能引起峰形问题/进样器划痕,2020/6/8,25,PeakTailing峰拖尾Extra-ColumnVolume柱外死体积效应,Column:StableBondSB-C18,4.630mm,3.5mMobilePhase:85%H2Owith0.1%TFA:15%CANFlowRate:1.0mL/minTemperature:35CSample:1.Phenylalanine2.5-benzyl-3,6-dioxo-2-piperazine3.Asp-phe4.Aspartame10lextra-columnvolume50lextra-columnvolume,2020/6/8,26,DeterminingtheCauseofPeakTailing峰拖尾原因的确定,Evaluatemobilephaseeffects-altermobilephasepHandadditivestoeliminatesecondaryinteractions评价流动相效果-改变流动相pH和添加剂消除次级效应/流动相中组份与柱子相互作用Evaluatecolumnchoice-trycolumnwithhighpuritysilicaordifferentbondingtechnology评价柱选择-试用高纯度硅胶柱或不同键合技术柱Reducesampleload减小进样量Eliminateextra-columneffects消除柱外效应Flushcolumnandcheckforaging/void冲洗柱子检查柱子老化/塌陷,2020/6/8,27,RetentionIssues保留时间问题,Retentiontimechanges(tr)保留时间改变Capacityfactor(retention)changes(k)容量因子改变Selectivitychanges()选择性改变,2020/6/8,28,ChangesinRetentionSameColumn,OverTime相同柱子上保留时间改变,超时,Maybecauseby:可能的原因:Columnaging柱老化Columncontamination柱污染Insufficientequilibration不够平衡Poorcolumn/mobilephasecombination柱/流动相组成差Changeinmobilephase改变流动相Changeinflowrate改变流速otherinstrumentissues其他仪器问题,2020/6/8,29,MobilephaseChangeCausesChangeinRetention流动相改变引起保留时间改变,60%MeOH:40%0.1%TFAFreshTFAAddedtoMobilePhaseVolatileTFAevaporated/degassedfrommobilephase.易挥发的三氟乙酸从流动相中蒸发/挥发。Replacingitsolvedproblem.换用新的流动相就解决问题。使用有机挥发性酸时，流动相应新配。,2020/6/8,30,ColumnAging/EquilibrationCausesRetention/SelectivityChanges柱老化平衡不够引起保留时间选择性改变,Column1-Initial1号柱开始时Column1-NextDay1号柱第二天Column1-AfterCleaningwith1%H3PO41号柱用1%H3PO4清洗Theprimaryanalytewassensitivetomobilephaseagingofthecolumn.最初的分析对流动相老化柱子是灵敏的Thepeakshapewasasecondaryissueresolvedbyflushingthecolumn.冲洗柱子第二要解决的问题是峰形Retentionandpeakshapewereasexpectedaftercleaning.清洗后保留时间和峰形都达到了预期效果,2020/6/8,31,DeterminingtheCauseofRetentionChangesonthesameColumn同一柱上保留时间改变原因的确定,1.DetermineK,andtrforsuspectpeaks对怀疑峰K,和tr的确定2.Washcolumn冲洗柱子3.Testnewcolumn-notelotnumber试验新柱-记录一批数据4.Reviewcolumnequili