色谱柱和色谱系统的故障检修和保养

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

2023/2/31HPLCComponents

HPLC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。2023/2/32CategoriesofColumnProblems

色谱柱问题的类型

A.Pressure压力

B.Peakshape峰形

C.Retention保留时间2023/2/33PressureIssues

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2023/2/34DeterminingtheCauseandCorrectingHighBackPressure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2023/2/35ColumnCleaning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2023/2/36ColumnCleaning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50ml

50%Methanol:50%Chloroform50%甲醇:50%氯仿100%EthylAcetate乙酸乙酯2023/2/37HowtoChangeaFrit

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2023/2/38PreventingBackPressureProblems

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2023/2/39PreventingBackPressureProblems: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2023/2/310PreventingBackPressureProblems: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2023/2/311PeakShapeIssues

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2023/2/312SplitPeaks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2023/2/313SplitPeaks裂峰

ColumnContamination柱污染Column:StableBondSB-C8,4.6×250mm,5µmMobilePhase:60%25mMNa2HPO4,pH3.0:40%MeOHFlowRate:1.0mL/minTemperature:35ºCDetection: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2023/2/314SplitPeaks裂峰

InjectionSolventEffects溶剂化效应Column:StableBondSB-C8,4.6×250mm,5µmMobilePhase:82%H2O:18%ACNInjectionVolume:30µLSample: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

2023/2/315DeterminingtheCauseofSplitPeaks

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

2023/2/316PeakTailing,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2023/2/317PeakTailingColumn“SecondaryInteractions”

峰拖尾柱“次级效应”Column:Alkyl-C8,4.6×150mm,5µmMobilePhase:85%25mMNa2HPO4,pH7.0:15%ACNFlowRate:1.0mL/minTemperature:35ºCSample:

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2023/2/318PeakTailingColumn“SecondaryInteractions”

峰拖尾柱“次级效应”Column:Alkyl-C8,4.6×150mm,5µmMobilePhase:85%25mMNa2HPO4,pH7.0:15%ACNFlowRate:1.0mL/minTemperature:35ºCSample: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2023/2/319PeakTailing峰拖尾

ColumnContamination柱污染Column:StableBondSB-C8,4.6×250mm,5µmMobilePhase: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,35ºC

向前方向相反方向

100%异丙醇冲洗后2023/2/320PeakTailing/Broadening

SampleLoadEffects

峰拖尾/变宽样品过载效应Column:4.6×150mm,5µmMobilePhase:40%25mMNa2HPO4,pH7.0:60%ACNFlowRate:1.5mL/minTemperature:40ºCSample:1.Desipramine去郁敏/脱甲基丙米嗪2.Nortriptyline

3.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

2023/2/321PeakBroadening,Splitting

ColumnVoid

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2023/2/322BroadPeaksUnknown“Phantom”PeaksColumn:Extend-C18,4.6×150mm,5µmMobilePhase: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2023/2/323PeakTailing峰拖尾

InjectorSealFailure进样器密封性差Column:Bonus-RP,4.6×75mm,3.5µmMobilePhase: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2023/2/324PeakTailing峰拖尾

Extra-ColumnVolume柱外死体积效应Column:StableBondSB-C18,4.6×30mm,3.5µmMobilePhase:85%H2Owith0.1%TFA:15%CANFlowRate:1.0mL/minTemperature:35ºCSample:1.Phenylalanine2.5-benzyl-3,6-dioxo-2-piperazine

3.Asp-phe4.Aspartame10µlextra-columnvolume50µlextra-columnvolume2023/2/325DeterminingtheCauseofPeakTailing

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2023/2/326RetentionIssues

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

2023/2/327ChangesinRetentionSameColumn,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

2023/2/328MobilephaseChangeCausesChangeinRetention

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。2023/2/329ColumnAging/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2023/2/330DeterminingtheCauseofRetentionChangesonthesameColumn

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.Reviewcolumnequilibrationprocedures观察柱平衡过程5.Makeupfreshmobilephase4andtest配制新鲜流动相再试验6.Checkinstrumentperformance检查仪器性能2023/2/331ChangeinRetention/SelectivityColumn-toColumn

柱与柱之间保留时间/选择性的改变Differentcolumnhistories(aging)

不同的柱历史(老化)Insufficient/inconsistentequilibration平衡不够/不一致Poorcolumn/mobilephasecombination

柱/流动相差Changeinmobilephase流动相改变Changeinflowrate流速改变Otherinstrumentissues其他仪器问题Slightchangesincolumnbedvolume(tronly)柱内体积轻微改变2023/2/332Lot-to-LotSelectivityChange(pH)

批与批之间选择性改变(pH)pH4.5-Lot1pH3.0-Lot1pH4.5-Lot2pH3.0-Lot2pH4.5showsselectivitychangefromlot-to-lotforbasiccompounds

pH4.5显示批与批之间对碱性化合物的选择性改变pH3.0showsnoselectivitychangefromlot-to-lot,indicatingsilanolsensitivityatpH4.5

pH3.0显示批与批之间对碱性化合物的无选择性改变,表明在pH4.5硅醇基的灵敏性2023/2/333Conclusions结论HPLCcolumnproblemareevidentas:高效液相色谱柱问题明显的为:Highpressure高压Undesirablepeakshape不希望得到的峰形Changesinretention/selectivity保留时间/选择性的改变

Oftentheseproblemsarenotassociatedwiththecolumnandmaybecausedbyinstrumentandexperimentalconditionissues.经常遇到的这些问题不一定与柱有关,而与仪器和实验条件方面的问题有关2023/2/334高效液相色谱仪(HPLC)的保养

2023/