色譜柱和色譜系統(tǒng)的故障檢修和保養(yǎng)

HPLCColumnandSystemTroubleshooting

色譜柱和色譜系統(tǒng)的故障檢修WhatEveryHPLCUserShouldKnow每個HPLC使用者應(yīng)該知道什么

2023/2/31HPLCComponents

HPLC的組成

Pump泵

Injector/Autosampler進(jìn)樣器/自動進(jìn)樣器

Column色譜柱

Detector檢測器

DataSystem/Integrator數(shù)據(jù)系統(tǒng)/積分儀表Allofthesecomponentscanhaveproblemsandrequiretroubleshooting.

所有這些部件可能出現(xiàn)故障而需要檢修。2023/2/32CategoriesofColumnProblems

色譜柱問題的類型

A.Pressure壓力

B.Peakshape峰形

C.Retention保留時間2023/2/33PressureIssues

壓力問題ColumnObservationsPotentialProblems色譜柱觀測潛在的問題HighpressurePluggedfrit壓力高堵塞濾頭

Columncontamination

色譜柱污染

Piuggedpacking

堵塞填料2023/2/34DeterminingtheCauseandCorrectingHighBackPressure查出原因糾正壓力Checkpressurewith/withoutcolumn-manypressureproblemsareduetoblockagesinthesystemorguard有／無柱子時檢查壓力－許多壓力問題是由于系統(tǒng)或保護(hù)柱的阻塞IfColumnPressureishigh如果柱壓高:Washcolumn-Eliminatecolumncontaminationand

沖洗柱子pluggedpacking排除柱污染和填料堵塞-highmolecularweight/adsorbedcompounds高分子量／被吸附化合物-precipitatefromsampleorbuffer

樣品或緩沖液中的沉淀物Backflushcolumn-Clearpluggedfrit反沖柱子－清潔阻塞的濾頭Changefrit-Clearpluggedfrit更換濾頭2023/2/35ColumnCleaning柱子清洗Flushwithstrongersolventsthanyourmobilephase用比你的流動相更強(qiáng)的溶劑沖洗

Reversed-PhaseSolventChoicesinOrderofIncreasingStrength為了增加強(qiáng)度選擇反相溶劑Useatleast25mlofeachsolventforanalyticalcolumns對于分析柱每種溶劑至少用25ml沖洗Mobilephasewithoutbuffersalts沒有緩沖鹽的流動相100%Methanol甲醇100%Acetonitrile乙腈75%Acetonitrile:25%Isopropanol75乙腈：25％異丙醇100%Isopropanol異丙醇100%MethyleneChloride★二氯甲烷100%Hexane★

已烷★

WhenusingeitherHexaneorMethyleneChloridethecolumnmustbeflushedwithIsopropanolbeforereturningtoyourresvered-phasemobilephase.當(dāng)用已烷蔌二氯甲烷柱子時,必須先用異丙醇沖洗,然后再用你的反相流動相2023/2/36ColumnCleaning柱子清洗NormalPhaseSolventChoicesinOrderofIncreasingStrength為了增加強(qiáng)度選用正相溶劑Useatleast50mlofeachsolvent

每種溶劑至少用50ml

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

怎樣更換濾頭ColumnInletFrit柱進(jìn)口濾頭ColumnBody柱身CompressionFerrule壓縮金屬套圈Weargloves戴手套Donotallowbedtodry不要讓底座干燥Donottouchthecolumn-bodyheatwillextrudepacking不要使柱身受熱,否則填料會噴出Donotovertighten不要旋得太緊FemaleEndFitting旋好尾端螺母MaleEndFitting旋好尾端螺頭2023/2/38PreventingBackPressureProblems

壓力問題的預(yù)防Usecolumnprotection柱保護(hù)

-Guardcolumns保護(hù)柱/預(yù)柱-In-linefilters在線過濾器

SamplePreparation樣品預(yù)處理Appropriatecolumnflushing

適當(dāng)?shù)闹鶝_洗Filterbufferedmobilephase

過濾緩沖流動相2023/2/39PreventingBackPressureProblems:In-LineDevices

壓力問題的預(yù)防:在線裝置MobilePhasePre-ColumnInjectorFilterGuardFromPump預(yù)柱進(jìn)樣器濾頭Column從泵來的流動相保護(hù)柱

AnalyticalColumnFilterandGuardColumnActonSample分析柱濾頭和保護(hù)柱作用于樣品

Pre-ColumnActsonMobilePhase

預(yù)柱作用于流動相ToDetector

到檢測器2023/2/310PreventingBackPressureProblems:SamplePreparation

壓力問題的預(yù)防:樣品制備Solvent/ChemicalEnvironment

溶劑/化學(xué)環(huán)境Particulate/AggregateRemove微粒/凝聚物的除去Filtersamples過濾樣品Centrifugation離心分離SolidPhaseExtraction(S.P.E.)

固相萃取CartridgesorPlates薄膜或薄層板DisksorMembranes圓盤或生物膜2023/2/311PeakShapeIssues

峰形問題

Splitpeaks裂峰

Peaktailing峰拖尾

Broadpeaks寬峰Poorefficiency低柱效Manypeakshapeissuesalsocombinations-I.e.Broadandtailingortailingwithincreasedretention許多峰形問題是結(jié)合在一起的-例如:展寬和拖尾或拖尾和保留時間增加2023/2/312SplitPeaks裂峰Canbecausedby:可能的原因:Columncontamination柱污染Partiallypluggedfrit部分阻塞濾片Columnvoid柱頭塌陷Injectionsolventeffects溶劑效應(yīng)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峰形較好Injection30峰3裂峰Injection1AfterColumnWashwith100%CAN經(jīng)100%乙腈沖洗后峰3峰形極好2023/2/314SplitPeaks裂峰

InjectionSolventEffects溶劑化效應(yīng)Column:StableBondSB-C8,4.6×250mm,5μmMobilePhase:82%H2O:18%ACNInjectionVolume:30μLSample:1.Caffeine咖啡因2.Salicylamide水楊酰胺A.InjectionSolventB.InjectionSolvent100%AcetonitrileMobilePhase峰形寬拖尾峰形尖銳對稱樣品溶劑盡可能與流動相匹配

2023/2/315DeterminingtheCauseofSplitPeaks

裂峰原因的確定1.Complexsamplematrixormanysamplesanalyzed-likelycolumncontaminationorpartiallypluggedfrit復(fù)雜樣品的基質(zhì)或分析許多樣品-柱污染或部分阻塞濾片

2.MobilephasepH>=7-likelycolumnvoidduetosilicadissolution(unlessspecialtycolumnused)流動相pH>=7-由于硅膠溶解使柱塌陷(除非使用專門的柱子)

3.Injectionsolventstrongerthanmobilephase-likelysplitandbroadpeaks,dependentonvolume溶劑比流動相的可能性大-裂峰和寬峰,由樣品量來決定

2023/2/316PeakTailing,BroadeningandLossofEfficiency

峰拖尾、變寬和柱效降低Canbecausedby:可能的原因

Column“secondaryinteractions”柱“次級效應(yīng)”Columnvoid柱塌陷Columncontamination柱污染Columnaging柱老化Columnloading柱負(fù)荷超載Extra-columneffects柱外效應(yīng)2023/2/317PeakTailingColumn“SecondaryInteractions”

峰拖尾柱“次級效應(yīng)”Column:Alkyl-C8,4.6×150mm,5μmMobilePhase:85%25mMNa2HPO4,pH7.0:15%ACNFlowRate:1.0mL/minTemperature:35oCSample:

1.Phenylpropanolamine苯丙醇胺／去甲麻黃堿2.Ephedrine麻黃堿3.Amphetamine安非他明／苯丙胺4.Methamphetamine脫氧麻黃堿5.Phenteramine苯三胺NoTEA無三乙胺10mMTEA10mM三乙胺USPTF(5%)美國藥典拖尾因子USPTF(5%)美國藥典拖尾因子1.1.2.9.6.354.1.265.1.575.1.14Peaktailingeliminatedwithmobilephasemodifier(TEA)atpH7用流動相減尾劑(三乙胺)在pH7消除峰拖尾2023/2/318PeakTailingColumn“SecondaryInteractions”

峰拖尾柱“次級效應(yīng)”Column:Alkyl-C8,4.6×150mm,5μmMobilePhase:85%25mMNa2HPO4,pH7.0:15%ACNFlowRate:1.0mL/minTemperature:35oCSample:1.Phenylpropanolamine苯丙醇胺／去甲麻黃堿2.Ephedrine麻黃堿3.Amphetamine安非他明／苯丙胺4.Methamphetamine脫氧麻黃堿5.Phenteramine苯三胺

pH3.0pH7.0USPTF(5%)美國藥典拖尾因子USPTF(5%)美國藥典拖尾因子4.1.334.2.35ReducingthemobilephasepHreducesinteractionswithsilanolsthatcausepeaktailing降低流動相的pH,減小與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理論板數(shù)拖尾因子理論板數(shù)拖尾因子理論板數(shù)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%異丙醇沖洗后2023/2/320PeakTailing/Broadening

SampleLoadEffects

峰拖尾/變寬樣品過載效應(yīng)Column:4.6×150mm,5μmMobilePhase:40%25mMNa2HPO4,pH7.0:60%ACNFlowRate:1.5mL/minTemperature:40oCSample:1.Desipramine去郁敏/脫甲基丙米嗪2.Nortriptyline

3.Doxepin4.Imipramine丙米嗪5.Amitriptyline阿米替林6.TrimipramineTailingEclipseXDB-C8Broadening/CompetitiveC8USPTF拖尾因子Plates理論板數(shù)HighLoad/10LowLoadHighLoad/10LowLoad高載負(fù)/10倍低載負(fù)高載負(fù)/10倍低載負(fù)1.1.601.7085059412.2.001.9081578423.1.561.56277662302539835627351005518910725

2023/2/321PeakBroadening,Splitting

ColumnVoid

峰變寬,裂開

柱塌陷MobilePhase:流動相：50%CAN:50%Water:0.2%TEA(~pH11)50％乙腈:50％水:0。2％三乙胺(~pH11)

After30injectionsInitial開始時

進(jìn)樣30次后Multiplepeakshapechangescanbecausedbythesamecolumnproblem.InthecaseavoidresultedfromsilicadissolvedathighpH.多數(shù)峰形改變可能是同一柱問題引起的。在這個例子中，高pH溶解硅膠導(dǎo)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順丁烯二酸2.Pseudeophedrine偽麻黃堿3.Chlorphenniramine氯非尼拉明Plates理論板數(shù)1.59222.98793.779“Phantom”peakfromfirstinjection前次進(jìn)樣未出完的峰Theextremelylowplatesareanindicationofanextremelylateelutingpeakfromtheprecedingrun.極低的理論板數(shù)表明這是一個前面進(jìn)樣極遲洗脫出來的峰2023/2/323PeakTailing峰拖尾

InjectorSealFailure進(jìn)樣器密封性差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理論板數(shù)拖尾因子理論板數(shù)拖尾因子1.22351.721.36701.452.34911.482.104571.093.54321.153.100851.00BeforeAfterreplacingrotorsealandisolationseal之前轉(zhuǎn)動桿和隔離密封墊檢修后Overdueinstrumentmaintenancecancausepeakshapeproblems.儀器保養(yǎng)超期可能引起峰形問題/進(jìn)樣器劃痕2023/2/324PeakTailing峰拖尾

Extra-ColumnVolume柱外死體積效應(yīng)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

峰拖尾原因的確定Evaluatemobilephaseeffects-altermobilephasepHandadditivestoeliminatesecondaryinteractions評價流動相效果-改變流動相pH和添加劑消除次級效應(yīng)/流動相中組份與柱子相互作用Evaluatecolumnchoice-trycolumnwithhighpuritysilicaordifferentbondingtechnology評價柱選擇-試用高純度硅膠柱或不同鍵合技術(shù)柱Reducesampleload減小進(jìn)樣量Eliminateextra-columneffects消除柱外效應(yīng)Flushcolumnandcheckforaging/void沖洗柱子檢查柱子老化/塌陷2023/2/326RetentionIssues

保留時間問題Retentiontimechanges(tr)保留時間改變Capacityfactor(retention)changes(k′)

容量因子改變Selectivitychanges()選擇性改變

2023/2/327ChangesinRetentionSameColumn,OverTime相同柱子上保留時間改變,超時Maybecauseby:可能的原因:Columnaging柱老化Columncontamination柱污染Insufficientequilibration不夠平衡Poorcolumn/mobilephasecombination柱/流動相組成差Changeinmobilephase改變流動相Changeinflowrate改變流速otherinstrumentissues其他儀器問題

2023/2/328MobilephaseChangeCausesChangeinRetention

流動相改變引起保留時間改變60%MeOH:40%0.1%TFAFreshTFAAddedtoMobilePhaseVolatileTFAevaporated/degassedfrommobilephase.易揮發(fā)的三氟乙酸從流動相中蒸發(fā)/揮發(fā)。Replacingitsolvedproblem.換用新的流動相就解決問題。使用有機(jī)揮發(fā)性酸時，流動相應(yīng)新配。2023/2/329ColumnAging/EquilibrationCausesRetention/SelectivityChanges

柱老化／平衡不夠引起保留時間／選擇性改變Column1-Initial1號柱開始時Column1-NextDay1號柱第二天Column1-AfterCleaningwith1%H3PO41號柱用

1%H3PO4清洗

Theprimaryanalytewassensitivetomobilephaseagingofthecolumn.最初的分析對流動相老化柱子是靈敏的Thepeakshapewasasecondaryissueresolvedbyflushingthecolumn.沖洗柱子第二要解決的問題是峰形Retentionandpeakshapewereasexpectedaftercleaning.清洗后保留時間和峰形都達(dá)到了預(yù)期效果2023/2/330DeterminingtheCauseofRetentionChangesonthesameColumn

同一柱上保留時間改變原因的確定1.DetermineK′,,andtrforsuspectpeaks對懷疑峰K′,和tr的確定2.Washcolumn沖洗柱子3.Testnewcolumn-notelotnumber試驗新柱-記錄一批數(shù)據(jù)4.Reviewcolumnequilibrationprocedures觀察柱平衡過程5.Makeupfreshmobilephase4andtest配制新鮮流動相再試驗6.Checkinstrumentperformance檢查儀器性能2023/2/331ChangeinRetention/SelectivityColumn-toColumn

柱與柱之間保留時間/選擇性的改變Differentcolumnhistories(aging)

不同的柱歷史(老化)Insufficient/inconsistentequilibration平衡不夠/不一致Poorcolumn/mobilephasecombination

柱/流動相差Changeinmobilephase流動相改變Changeinflowrate流速改變Otherinstrumentissues其他儀器問題Slightchangesincolumnbedvolume(tronly)柱內(nèi)體積輕微改變2023/2/332Lot-to-LotSelectivityChange(pH)

批與批之間選擇性改變(pH)pH4.5-Lot1pH3.0-Lot1pH4.5-Lot2pH3.0-Lot2pH4.5showsselectivitychangefromlot-to-lotforbasiccompounds

pH4.5顯示批與批之間對堿性化合物的選擇性改變pH3.0showsnoselectivitychangefromlot-to-lot,indicatingsilanolsensitivityatpH4.5

pH3.0顯示批與批之間對堿性化合物的無選擇性改變,表明在pH4.5硅醇基的靈敏性2023/2/333Conclusions結(jié)論HPLCcolumnproblemareevidentas:高效液相色譜柱問題明顯的為:Highpressure高壓Undesirablepeakshape不希望得到的峰形Changesinretention/selectivity保留時間/選擇性的改變

Oftentheseproblemsarenotassociatedwiththecolumnandmaybecausedbyinstrumentandexperimentalconditionissues.經(jīng)常遇到的這些問題不一定與柱有關(guān),而與儀器和實驗條件方面的問題有關(guān)2023/2/334高效液相色譜儀(HPLC)的保養(yǎng)

2023/