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1、色谱柱和色谱系统故障2022/10/241第1页，本讲稿共34页HPLC Components HPLC 的组成l Pump 泵l Injector/Autosampler 进样器/自动进样器l Column 色谱柱l Detector 检测器l Data System/Integrator 数据系统/积分仪表lAll of these components can have problems and require troubleshooting.l所有这些部件可能出现故障而需要检修。2022/10/242第2页，本讲稿共34页Categories of Column Problems 色谱
2、柱问题的类型l l A.Pressure 压力l B.Peak shape 峰形l C.Retention 保留时间2022/10/243第3页，本讲稿共34页Pressure Issues压力问题 lColumn Observations Potential Problemsl色谱柱观测 潜在的问题 lHigh pressure Plugged frit l压力高 堵塞滤头l Column contaminationl 色谱柱污染l Piugged packingl 堵塞填料2022/10/244第4页，本讲稿共34页Determining the Cause and Correcting
3、High Back Pressure 查出原因 纠正压力lCheck pressure with/without column-many pressure problems are due to blockages in the system or guardl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lIf Column Pressure is high 如果柱压高:lWash column-Eliminate column contamination andl 冲洗柱子 plugged packing 排除柱污染和填料堵塞l -high molecular weight/ad
4、sorbedl compounds 高分子量被吸附化合物l -precipitate from sample or bufferl 样品或缓冲液中的沉淀物lBack flush column-Clear plugged frit 反冲柱子清洁阻塞的滤头lChange frit-Clear plugged frit 更换滤头2022/10/245第5页，本讲稿共34页Column Cleaning 柱子清洗lF lush with stronger solvents than your mobile phasel用比你的流动相更强的溶剂冲洗l Reversed-Phase Solvent Cho
5、ices in Order of l Increasing Strength 为了增加强度选择反相溶剂lUse at least 25 ml of each solvent for analytical columnsl 对于分析柱每种溶剂至少用25 ml 冲洗lMobile phase without buffer salts 没有缓冲盐的流动相l100%Methanol 甲醇l100%Acetonitrile 乙腈l75%Acetonitrile:25%Isopropanol 75乙腈：25异丙醇l100%Isopropanol 异丙醇l100%Methylene Chloride 二氯甲
6、烷l100%Hexane 已烷l When using either Hexane or Methylene Chloride the column must be flushed with lIsopropanol before returning to your resvered-phase mobile phase.l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2022/10/246第6页，本讲稿共34页Column Cleaning柱子清洗lNormal Phase Solvent Choices in Order of Increasing Strengthl
7、为了增加强度选用正相溶剂lUse at least 50 ml of each solvent l 每种溶剂至少用50 ml l50%Methanol:50%Chloroform l 50%甲醇:50%氯仿l100%Ethyl Acetate 乙酸乙酯2022/10/247第7页，本讲稿共34页How to Change a Frit 怎样更换滤头lColumn Inlet Frit 柱进口滤头lColumn Body 柱身lCompression Ferrule 压缩金属套圈lWear gloves 戴手套lDo not allow bed to dry 不要让底座干燥lDo not tou
8、ch the column-body heat will extrude packing 不要使柱身受热,否则填料会喷出lDo not over tighten 不要旋得太紧lFemale End Fitting 旋好尾端螺母lMale End Fitting 旋好尾端螺头2022/10/248第8页，本讲稿共34页Preventing Back Pressure Problems压力问题的预防lUse column protection 柱保护l-Guard columns 保护柱/预柱l-In-line filters 在线过滤器l Sample Preparation 样品预处理lApp
9、ropriate column flushing l 适当的柱冲洗lFilter buffered mobile phasel 过滤缓冲流动相2022/10/249第9页，本讲稿共34页Preventing Back Pressure Problems:In-Line Devices 压力问题的预防:在线装置lMobile Phase Pre-Column Injector Filter GuardlFrom Pump 预柱 进样器 滤头 Columnl从泵来的流动相 保护柱 l Analyticall ColumnlFilter and Guard Column Act on Sample
10、分析柱l 滤头和保护柱作用于样品l Pre-Column Acts on Mobile Phasel 预柱作用于流动相 To Detectorl 到检测器2022/10/2410第10页，本讲稿共34页Preventing Back Pressure Problems:Sample Preparation压力问题的预防:样品制备lSolvent/Chemical Environment l 溶剂/化学环境lParticulate/Aggregate Remove l微粒/凝聚物的除去lFilter samples 过滤样品lCentrifugation 离心分离lSolid Phase Ext
11、raction(S.P.E.)固相萃取lCartridges or Plates 薄膜或薄层板lDisks or Membranes 圆盘或生物膜2022/10/2411第11页，本讲稿共34页Peak Shape Issues 峰形问题l Split peaks 裂峰l Peak tailing 峰拖尾l Broad peaks 宽峰lPoor efficiency 低柱效lMany peak shape issues also combinations-I.e.Broad and tailing or tailing with increased retention许多峰形问题是结合在一起
12、的-例如:l展宽和拖尾或拖尾和保留时间增加2022/10/2412第12页，本讲稿共34页Split Peaks 裂峰lCan be caused by:l可能的原因:lColumn contamination 柱污染lPartially plugged frit 部分阻塞滤片lColumn void 柱头塌陷lInjection solvent effects 溶剂效应2022/10/2413第13页，本讲稿共34页Split Peaks 裂峰Column Contamination 柱污染lColumn:StableBond SB-C8,4.6 250mm,5mlMobile Phase:
13、60%25mM Na2HPO4,pH3.0:40%MeOHlFlow Rate:1.0 mL/min Temperature:35ClDetection:UV 254 nmlSample:Filtered OTC Cold Medication:l1.Pseudoephedrine 2.APAP 3.Unknown l4.ChlorpheniraminelInjection 1 峰3 峰形较好lInjection 30 峰3 裂峰lInjection 1 After Column Wash with 100%CANl经100%乙腈冲洗后 峰3 峰形极好2022/10/2414第14页，本讲稿共
14、34页Split Peaks 裂峰Injection Solvent Effects 溶剂化效应lColumn:StableBond SB-C8,4.6 250mm,5mlMobile Phase:82%H2O:18%ACNlInjection Volume:30 L lSample:l1.Caffeine 咖啡因 2.Salicylamide 水杨酰胺lA.Injection Solvent B.Injection Solventl 100%Acetonitrile Mobile Phasel 峰形宽拖尾 峰形尖锐对称l样品溶剂尽可能与流动相匹配 2022/10/2415第15页，本讲稿共3
15、4页Determining the Cause of Split Peaks 裂峰原因的确定l1.Complex sample matrix or many samples analyzed-likely column contamination or partially plugged fritl复杂样品的基质或分析许多样品-柱污染或部分阻塞滤片 l2.Mobile phase pH=7-likely column void due to silica dissolution(unless specialty column used)l流动相 pH=7-由于硅胶溶解使柱塌陷(除非使用专门的柱
16、子)l3.Injection solvent stronger than mobile phase-likely split and broad peaks,dependent on volumel溶剂比流动相的可能性大-裂峰和宽峰,由样品量来决定 2022/10/2416第16页，本讲稿共34页Peak Tailing,Broadening and Loss of Efficiency峰拖尾、变宽和柱效降低lCan be caused by:可能的原因l lColumn“secondary interactions”l柱“次级效应”lColumn void 柱塌陷lColumn contam
17、ination 柱污染lColumn aging 柱老化lColumn loading 柱负荷超载lExtra-column effects 柱外效应2022/10/2417第17页，本讲稿共34页Peak Tailing Column“Secondary Interactions”峰拖尾柱“次级效应”lColumn:Alkyl-C8,4.6 150mm,5mlMobile Phase:85%25mM Na2HPO4,pH7.0:15%ACNlFlow Rate:1.0 mL/min Temperature:35ClSample:1.Phenylpropanolamine 苯丙醇胺去甲麻黄碱
18、l2.Ephedrine 麻黄碱 3.Amphetamine 安非他明苯丙胺l4.Methamphetamine 脱氧麻黄碱 5.Phenteramine 苯三胺lNo TEA 无三乙胺 10 mM TEA 10 mM 三乙胺lUSP TF(5%)美国药典拖尾因子 USP TF(5%)美国药典拖尾因子l1.1.29 1.1.19l2.1.91 2.1.18 l3.1.63 3.1.20l4.2.35 4.1.26l5.1.57 5.1.14lPeak tailing eliminated with mobile phase modifier(TEA)at pH 7l用流动相减尾剂(三乙胺)在p
19、H 7 消除峰拖尾2022/10/2418第18页，本讲稿共34页Peak Tailing Column“Secondary Interactions”峰拖尾柱“次级效应”lColumn:Alkyl-C8,4.6 150mm,5mlMobile Phase:85%25mM Na2HPO4,pH7.0:15%ACNlFlow Rate:1.0 mL/min Temperature:35ClSample:1.Phenylpropanolamine 苯丙醇胺去甲麻黄碱 l2.Ephedrine 麻黄碱 3.Amphetamine 安非他明苯丙胺l4.Methamphetamine 脱氧麻黄碱 5.P
20、henteramine 苯三胺l pH 3.0 pH 7.0 lUSP TF(5%)美国药典拖尾因子 USP TF(5%)美国药典拖尾因子l 4.1.33 4.2.35lReducing the mobile phase pH reduces interactions with silanols that cause peak tailingl降低流动相的pH,减小与硅醇基作用引起的峰拖尾2022/10/2419第19页，本讲稿共34页Peak Tailing 峰拖尾Column Contamination 柱污染lColumn:StableBond SB-C8,4.6 250mm,5mlMo
21、bile Phase:20%H2O:80%MeOH Flow Rate:1.0 mL/min Temperature:R.T.Detection:UV 254 nmlSample:1.Uracil 尿嘧啶 2.Phenol 苯酚 l 3.4-Chloronitrobenzene 4-氯硝基苯 4.Toluene 甲苯lPlates TF Plates TF Plates TFl理论板数 拖尾因子 理论板数 拖尾因子 理论板数 拖尾因子l1.7629 2.08 1.7906 1.43 1.7448 1.06l2.12043 1.64 2.12443 1.21 2.12237 1.21l3.137
22、27 1.69 3.17999 1.19 3.15366 1.11l4.13355 1.32 4.17098 1.25 4.19067 1.17lQC test forward direction QC test reverse direction QC test after cleaning l 100%IPA,35C l 向前方向 相反方向 100%异丙醇冲洗后2022/10/2420第20页，本讲稿共34页Peak Tailing/Broadening Sample Load Effects峰拖尾/变宽样品过载效应lColumn:4.6 150mm,5mlMobile Phase:40%
23、25mM Na2HPO4,pH7.0:60%ACNlFlow Rate:1.5 mL/min Temperature:40ClSample:l1.Desipramine 去郁敏/脱甲基丙米嗪 2.Nortriptyline 3.Doxepin 4.Imipramine 丙米嗪 5.Amitriptyline 阿米替林 6.TrimipraminelTailing Eclipse XDB-C8 Broadening/Competitive C8lUSP TF 拖尾因子 Plates 理论板数 lHigh Load /10 Low Load High Load /10 Low Loadl高载负/1
24、0倍 低载负 高载负/10倍 低载负l1.1.60 1.70 850 5941l2.2.00 1.90 815 7842l3.1.56 1.56 2776 6231 l4.2.13 1.70 2539 8359 l5.2.15 1.86 2735 10022 l6.1.25 1.25 5189 10725 2022/10/2421第21页，本讲稿共34页Peak Broadening,Splitting Column Void 峰变宽,裂开 柱塌陷lMobile Phase:流动相：l50%CAN:50%Water:0.2%TEA (pH 11)l50乙腈:50水:0。2三乙胺 (pH 11)
25、l After 30 injections Initial 开始时 l 进样30次后lMultiple peak shape changes can be caused by the same column problem.In the case a void resulted from silica dissolved at high pH.l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2022/10/2422第22页，本讲稿共34页Broad Peaks Unknown“Phantom”PeakslColumn:Extend-C18,4.6 150 mm,5 m
26、lMobile Phase:40%10 mM TEA,pH 11:60%MeOHlFlow Rate:1.0 mL/min Temperature:R.T.Detection:UV 254lSample:1.Maleat 顺丁烯二酸 2.Pseudeophedrine 伪麻黄碱 3.Chlorphenniramine 氯非尼拉明lPlates 理论板数l1.5922 l2.9879l3.779 “Phantom”peak from first injection 前次进样未出完的峰lThe extremely low plates are an indication of an extreme
27、ly late eluting peak from the preceding run.l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2022/10/2423第23页，本讲稿共34页Peak Tailing峰拖尾 Injector Seal Failure进样器密封性差lColumn:Bonus-RP,4.6 75mm,3.5mlMobile Phase:30%H2O:70%MeOH Flow Rate:1.0 mL/min Temperature:R.T.Detection:UV 254 nmlSample:1.Uracil 尿嘧啶 2.Phenol 苯酚 l 3.N,N-Dimeth
28、ylaniline N,N-二甲苯胺lPlates TF Plates TF l理论板数 拖尾因子 理论板数 拖尾因子 l1.2235 1.72 1.3670 1.45l2.3491 1.48 2.10457 1.09 l3.5432 1.15 3.10085 1.00 lBefore After replacing rotor seal and isolation seall之前 转动杆和隔离密封垫检修后lOverdue instrument maintenance can cause peak shape problems.l仪器保养超期可能引起峰形问题/进样器划痕2022/10/2424
29、第24页，本讲稿共34页Peak Tailing 峰拖尾Extra-Column Volume 柱外死体积效应lColumn:StableBond SB-C18,4.6 30mm,3.5mlMobile Phase:85%H2Owith 0.1%TFA:15%CANlFlow Rate:1.0 mL/min Temperature:35C lSample:1.Phenylalanine 2.5-benzyl-3,6-dioxo-2-piperazine l 3.Asp-phe 4.Aspartamel10 l extra-column volume 50 l extra-column volu
30、me 2022/10/2425第25页，本讲稿共34页Determining the Cause of Peak Tailing峰拖尾原因的确定lEvaluate mobile phase effects-alter mobile phase pH and additives to eliminate secondary interactions 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lEvaluate column choice-try column with high purity silica or different bonding techno
31、logy 评价柱选择-试用高纯度硅胶柱或不同键合技术柱lReduce sample load 减小进样量lEliminate extra-column effects 消除柱外效应lFlush column and check for aging/void 冲洗柱子检查柱子老化/塌陷2022/10/2426第26页，本讲稿共34页Retention Issues保留时间问题lRetention time changes(tr)保留时间改变lCapacity factor(retention)changes(k)容量因子改变lSelectivity changes()选择性改变 2022/10/
32、2427第27页，本讲稿共34页Changes in Retention Same Column,Over Time相同柱子上保留时间改变,超时lMay be cause by:可能的原因:lColumn aging 柱老化lColumn contamination 柱污染lInsufficient equilibration 不够平衡lPoor column/mobile phase combination 柱/流动相组成差lChange in mobile phase 改变流动相lChange in flow rate 改变流速lother instrument issues 其他仪器问题
33、 2022/10/2428第28页，本讲稿共34页Mobile phase Change Causes Change in Retention流动相改变引起保留时间改变l60%MeOH:40%0.1%TFA Fresh TFA Added to Mobile PhaselVolatile TFA evaporated/degassed from mobile phase.易挥发的三氟乙酸从流动相中蒸发/挥发。lReplacing it solved problem.换用新的流动相就解决问题。l使用有机挥发性酸时，流动相应新配。2022/10/2429第29页，本讲稿共34页Column Agi
34、ng/Equilibration Causes Retention/Selectivity Changes柱老化平衡不够引起保留时间选择性改变lColumn 1-Initial 1 号柱 开始时lColumn 1-Next Day 1 号柱 第二天 lColumn 1-After Cleaning with 1%H3PO4 1 号柱用 1%H3PO4 清洗 lThe primary analyte was sensitive to mobile phase aging of the column.最初的分析对流动相老化柱子是灵敏的lThe peak shape was a secondary
35、issue resolved by flushing the column.冲洗柱子第二要解决的问题是峰形lRetention and peak shape were as expected after cleaning.清洗后保留时间和峰形都达到了预期效果2022/10/2430第30页，本讲稿共34页Determining the Cause of Retention Changes on the same Column同一柱上保留时间改变原因的确定l1.Determine K,and tr for suspect peaks 对怀疑峰K,和tr的确定 l2.Wash column 冲洗柱
36、子l3.Test new column-note lot number 试验新柱-记录一批数据l4.Review column equilibration procedures 观察柱平衡过程l5.Make up fresh mobile phase4 and test 配制新鲜流动相再试验l6.Check instrument performance 检查仪器性能2022/10/2431第31页，本讲稿共34页Change in Retention/Selectivity Column-to Column柱与柱之间保留时间/选择性的改变lDifferent column histories(
37、aging)不同的柱历史(老化)lInsufficient/inconsistent equilibrationl平衡不够/不一致lPoor column/mobile phase combination 柱/流动相差lChange in mobile phase流动相改变lChange in flow rate 流速改变lOther instrument issues其他仪器问题lSlight changes in column bed volume(tr only)柱内体积轻微改变2022/10/2432第32页，本讲稿共34页Lot-to-Lot Selectivity Change(p
38、H)批与批之间选择性改变(pH)lpH 4.5-Lot 1 pH 3.0-Lot 1lpH 4.5-Lot 2 pH 3.0-Lot 2lpH 4.5 shows selectivity change from lot-to-lot for basic compounds pH 4.5 显示批与批之间对碱性化合物的选择性改变lpH 3.0 shows no selectivity change from lot-to-lot,indicating silanol sensitivity at pH 4.5 pH 3.0显示批与批之间对碱性化合物的无选择性改变,表明在pH 4.5硅醇基的灵敏性2
39、022/10/2433第33页，本讲稿共34页Conclusions 结论lHPLC column problem are evident as:高效液相色谱柱问题明显的为:lHigh pressure 高压lUndesirable peak shape 不希望得到的峰形lChanges in retention/selectivity 保留时间/选择性的改变 lOften these problems are not associated with the column and may be caused by instrument and experimental condition issues.l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2022/10/2434第34页，本讲稿共34页