色谱柱和色谱系统的故障排队幻灯片.(165.0k).ppt

HPLC Column and System Troubleshooting 色谱柱和色谱系统的故障检修 lWhat Every HPLC lUser Should Know l每个HPLC使用者应该知道什么 Date1 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。 Date2 Categories of Column Problems 色谱 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 Date3 Pressure Issues 压力问题 lColumn Observations Potential Problems l色谱柱观测 潜在的问题 lHigh pressure Plugged frit l压力高 堵塞滤头 l Column contamination l 色谱柱污染 l Piugged packing l 堵塞填料 Date4 Determining the Cause and Correcting 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 guard l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 and l 冲洗柱子 plugged packing 排除柱污染和填料堵塞 l - high molecular weight/adsorbed l compounds 高分子量被吸附化合物 l - precipitate from sample or buffer l 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 Date5 Column Cleaning 柱子清洗 lF lush with stronger solvents than your mobile phase l用比你的流动相更强的溶剂冲洗 l Reversed-Phase Solvent Choices 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 columns l 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 Date6 Column Cleaning柱子清洗 lNormal Phase Solvent Choices in Order of Increasing Strength l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 Date7 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 touch 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 Date8 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 lAppropriate column flushing l 适当的柱冲洗 lFilter buffered mobile phase l 过滤缓冲流动相 Date9 Preventing Back Pressure Problems:In-Line Devices 压力问题的预防:在线装置 lMobile Phase Pre-Column Injector Filter Guard lFrom Pump 预柱 进样器 滤头 Column l从泵来的流动相 保护柱 l Analytical l Column lFilter and Guard Column Act on Sample 分析柱 l 滤头和保护柱作用于样品 l Pre-Column Acts on Mobile Phase l 预柱作用于流动相 To Detector l 到检测器 Date10 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 Extraction(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 Date11 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 是结合在一起的-例如: l展宽和拖尾或拖尾和保留时间增加 Date12 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 Date13 Split Peaks 裂峰 Column Contamination 柱污染 lColumn: StableBond SB-C8, 4.6 250mm, 5m lMobile Phase:60%25mM Na2HPO4,pH3.0 : 40%MeOH lFlow Rate: 1.0 mL/min Temperature: 35C lDetection: UV 254 nm lSample: Filtered OTC Cold Medication: l1. Pseudoephedrine 2. APAP 3. Unknown l4. Chlorpheniramine lInjection 1 峰3 峰形较好 lInjection 30 峰3 裂峰 lInjection 1 After Column Wash with 100% CAN l经100%乙腈冲洗后 峰3 峰形极好 Date14 Split Peaks 裂峰 Injection Solvent Effects 溶剂化效应 lColumn: StableBond SB-C8, 4.6 250mm, 5m lMobile Phase: 82%H2O : 18%ACN lInjection Volume: 30 L lSample: l1. Caffeine 咖啡因 2. Salicylamide 水杨酰胺 lA. Injection Solvent B. Injection Solvent l 100% Acetonitrile Mobile Phase l 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 Date15 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 frit l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(除非使用专门的柱子) l3. Injection solvent stronger than mobile phase - likely split and broad peaks, dependent on volume l溶剂比流动相的可能性大- 裂峰和宽峰,由样品量来决定 Date16 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 contamination 柱污染 lColumn aging 柱老化 lColumn loading 柱负荷超载 lExtra-column effects 柱外效应 Date17 Peak Tailing Column “Secondary Interactions” 峰拖尾柱“次级效应” lColumn: Alkyl-C8, 4.6 150mm, 5m lMobile Phase:85%25mM Na2HPO4,pH7.0 : 15%ACN lFlow Rate: 1.0 mL/min Temperature: 35C lSample: 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. 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. 1.19 l2. 1.91 2. 1.18 l3. 1.63 3. 1.20 l4. 2.35 4. 1.26 l5. 1.57 5. 1.14 lPeak tailing eliminated with mobile phase modifier (TEA) at pH 7 l用流动相减尾剂(三乙胺)在pH 7 消除峰拖尾 Date18 Peak Tailing Column “Secondary Interactions” 峰拖尾柱“次级效应” lColumn: Alkyl-C8, 4.6 150mm, 5m lMobile Phase:85%25mM Na2HPO4,pH7.0 : 15%ACN lFlow Rate: 1.0 mL/min Temperature: 35C lSample: 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. Phenteramine 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. 2.35 lReducing the mobile phase pH reduces interactions with silanols that cause peak tailing l降低流动相的pH ,减小与硅醇基作用引起的峰拖尾 Date19 Peak Tailing 峰拖尾 Column Contamination 柱污染 lColumn: StableBond SB-C8, 4.6 250mm, 5m lMobile Phase: 20% H2O : 80% MeOH Flow Rate: 1.0 mL/min Temperature: R.T. Detection: UV 254 nm lSample: 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 TF l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 1.06 l2. 12043 1.64 2. 12443 1.21 2. 12237 1.21 l3. 13727 1.69 3. 17999 1.19 3. 15366 1.11 l4. 13355 1.32 4. 17098 1.25 4. 19067 1.17 lQC 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 Date20 Peak Tailing/Broadening Sample Load Effects 峰拖尾/变宽样品过载效应 lColumn: 4.6 150mm, 5m lMobile Phase:40%25mM Na2HPO4,pH7.0 : 60%ACN lFlow Rate: 1.5 mL/min Temperature: 40C lSample: 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. Trimipramine lTailing Eclipse XDB-C8 Broadening / Competitive C8 lUSP TF 拖尾因子 Plates 理论板数 lHigh Load / 10 Low Load High Load / 10 Low Load l高载负/10倍 低载负 高载负/10倍 低载负 l1. 1.60 1.70 850 5941 l2. 2.00 1.90 815 7842 l3. 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 Date21 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) 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 Date22 Broad Peaks Unknown “Phantom” Peaks lColumn: Extend-C18, 4.6 150 mm, 5 m lMobile Phase: 40% 10 mM TEA, pH 11 : 60% MeOH lFlow Rate: 1.0 mL/min Temperature: R.T. Detection: UV 254 lSample: 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. 9879 l3. 779 “Phantom” peak from first injection 前次进样未出完的峰 lThe extremely low plates are an indication of an extremely 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 Date23 Peak Tailing峰拖尾 Injector Seal Failure进样器密封性差 lColumn: Bonus-RP, 4.6 75mm, 3.5m lMobile Phase: 30% H2O : 70% MeOH Flow Rate: 1.0 mL/min Temperature: R.T. Detection: UV 254 nm lSample: 1. Uracil 尿嘧啶 2. Phenol 苯酚 l 3. N,N-Dimethylaniline 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 1.45 l2. 3491 1.48 2. 10457 1.09 l3. 5432 1.15 3. 10085 1.00 lBefore After replacing rotor seal and isolation seal l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 Date24 Peak Tailing 峰拖尾 Extra-Column Volume 柱外死体积效应 lColumn: StableBond SB-C18, 4.6 30mm, 3.5m lMobile Phase: 85% H2Owith 0.1% TFA : 15% CAN lFlow Rate: 1.0 mL/min Temperature: 35C lSample: 1. Phenylalanine 2. 5-benzyl-3,6-dioxo-2-piperazine l 3. Asp-phe 4. Aspartame l10 l extra-column volume 50 l extra-column volume Date25 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 technology 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 Date26 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 Date27 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 Date28 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 Phase lVolatile 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。 Date29 Column Aging/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 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 Date30 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 u