色谱柱和色谱系统的故障检修.

2019/12/171HPLCColumnandSystemTroubleshooting色谱柱和色谱系统的故障检修WhatEveryHPLCUserShouldKnow每个HPLC使用者应该知道什么2019/12/172HPLCComponentsHPLC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。2019/12/173CategoriesofColumnProblems色谱柱问题的类型A.Pressure压力B.Peakshape峰形C.Retention保留时间2019/12/174PressureIssues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2019/12/175DeterminingtheCauseandCorrectingHighBackPressure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/adsorbedcompounds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2019/12/176ColumnCleaning柱子清洗Flushwithstrongersolventsthanyourmobilephase用比你的流动相更强的溶剂冲洗Reversed-PhaseSolventChoicesinOrderofIncreasingStrength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★WhenusingeitherHexaneorMethyleneChloridethecolumnmustbeflushedwithIsopropanolbeforereturningtoyourresvered-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2019/12/177ColumnCleaning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%Chloroform50%甲醇:50%氯仿100%EthylAcetate乙酸乙酯2019/12/178HowtoChangeaFrit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2019/12/179PreventingBackPressureProblems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2019/12/1710PreventingBackPressureProblems:In-LineDevices压力问题的预防:在线装置MobilePhasePre-ColumnInjectorFilterGuardFromPump预柱进样器滤头Column从泵来的流动相保护柱AnalyticalColumnFilterandGuardColumnActonSample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2019/12/1711PreventingBackPressureProblems: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2019/12/1712PeakShapeIssues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2019/12/1713SplitPeaks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2019/12/1714SplitPeaks裂峰ColumnContamination柱污染Column:StableBondSB-C8,4.6×250mm,5µmMobilePhase:60%25mMNa2HPO4,pH3.0:40%MeOHFlowRate:1.0mL/minTemperature:35ºCDetection:UV254nmSample:FilteredOTCColdMedication:1.Pseudoephedrine2.APAP3.Unknown4.ChlorpheniramineInjection1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2019/12/1715SplitPeaks裂峰InjectionSolventEffects溶剂化效应Column:StableBondSB-C8,4.6×250mm,5µmMobilePhase:82%H2O:18%ACNInjectionVolume:30µLSample:1.Caffeine咖啡因2.Salicylamide水杨酰胺A.InjectionSolventB.InjectionSolvent100%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2019/12/1716DeterminingtheCauseofSplitPeaks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2019/12/1717PeakTailing,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2019/12/1718PeakTailingColumn“SecondaryInteractions”峰拖尾柱“次级效应”Column:Alkyl-C8,4.6×150mm,5µmMobilePhase:85%25mMNa2HPO4,pH7.0:15%ACNFlowRate:1.0mL/minTemperature:35ºCSample: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1.1.291.1.192.1.912.1.183.1.633.1.204.2.354.1.265.1.575.1.14Peaktailingeliminatedwithmobilephasemodifier(TEA)atpH7用流动相减尾剂(三乙胺)在pH7消除峰拖尾2019/12/1719PeakTailingColumn“SecondaryInteractions”峰拖尾柱“次级效应”Column:Alkyl-C8,4.6×150mm,5µmMobilePhase:85%25mMNa2HPO4,pH7.0:15%ACNFlowRate:1.0mL/minTemperature:35ºCSample:1.Phenylpropanol