色谱柱和色谱系统的故障检修.ppt

2020 1 27 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 请访问中国色谱网 www SeP 2020 1 27 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 1 27 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 1 27 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 1 27 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 1 27 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 1 27 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 1 27 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 1 27 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 1 27 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 1 27 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 1 27 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 1 27 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 1 27 14 SplitPeaks裂峰ColumnContamination柱污染 Column StableBondSB C8 4 6 250mm 5 mMobilePhase 60 25mMNa2HPO4 pH3 0 40 MeOHFlowRate 1 0mL minTemperature 35 CDetection 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 1 27 15 SplitPeaks裂峰InjectionSolventEffects溶剂化效应 Column StableBondSB C8 4 6 250mm 5 mMobilePhase 82 H2O 18 ACNInjectionVolume 30 LSample 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 1 27 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 1 27 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 1 27 18 PeakTailingColumn SecondaryInteractions 峰拖尾柱 次级效应 Column Alkyl C8 4 6 150mm 5 mMobilePhase 85 25mMNa2HPO4 pH7 0 15 ACNFlowRate 1 0mL minTemperature 35 CSample 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 1 291 1 192 1 912 1 183 1 633 1 204 2 354 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 1 27 19 PeakTailingColumn SecondaryInteractions 峰拖尾柱 次级效应 Column Alkyl C8 4 6 150mm 5 mMobilePhase 85 25mMNa2HPO4 pH7 0 15 ACNFlowRate 1 0mL minTemperature 35 CSample 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 1 27 20 PeakTailing峰拖尾ColumnContamination柱污染 Column StableBondSB C8 4 6 250mm 5 mMobilePhase 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 35 C向前方向相反方向100 异丙醇冲洗后 2020 1 27 21 PeakTailing BroadeningSampleLoadEffects峰拖尾 变宽样品过载效应 Column 4 6 150mm 5 mMobilePhase 40 25mMNa2HPO4 pH7 0 60 ACNFlowRate 1 5mL minTemperature 40 CSample 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 56277662314 2 131 70253983595 2 151 862735100226 1 251 25518910725 2020 1 27 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 1 27 23 BroadPeaksUnknown Phantom Peaks Column Extend C18 4 6 150mm 5 mMobilePhase 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 1 27 24 PeakTailing峰拖尾InjectorSealFailure进样器密封性差 Column Bonus RP 4 6 75mm 3 5 mMobilePhase 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 1 27 25 PeakTailing峰拖尾Extra ColumnVolume柱外死体积效应 Column StableBondSB C18 4 6 30mm 3 5 mMobilePhase 85 H2Owith0 1 TFA 15 CANFlowRate 1 0mL minTemperature 35 CSample 1 Phenylalanine2 5 benzyl 3 6 dioxo 2 piperazine3 Asp phe4 Aspartame10 lextra columnvolume50 lextra columnvolume 2020 1 27 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 1 27 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 1 27 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 1 27 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 1 27 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 1 27 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 Reviewcolum