HPLC Column and System Troubleshooting 色谱柱和色谱系统的故障检修 What Every HPLC User Should Know 每个HPLC使用者应该知道什么 请访问中国色谱网(www.SePu.net) 2018/11/18

HPLC Components HPLC 的组成 Pump 泵 Injector/Autosampler 进样器/自动进样器 Column 色谱柱 Detector 检测器 Data System/Integrator 数据系统/积分仪表 All of these components can have problems and require troubleshooting. 所有这些部件可能出现故障而需要检修。 2018/11/18

Categories of Column Problems 色谱柱问题的类型 A. Pressure 压力 B. Peak shape 峰形 C. Retention 保留时间 2018/11/18

Pressure Issues 压力问题 Column Observations Potential Problems 色谱柱观测 潜在的问题 High pressure Plugged frit 压力高 堵塞滤头 Column contamination 色谱柱污染 Piugged packing 堵塞填料 2018/11/18

Determining the Cause and Correcting High Back Pressure 查出原因 纠正压力 Check pressure with/without column - many pressure problems are due to blockages in the system or guard 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 If Column Pressure is high 如果柱压高: Wash column - Eliminate column contamination and 冲洗柱子 plugged packing 排除柱污染和填料堵塞 - high molecular weight/adsorbed compounds 高分子量／被吸附化合物 - precipitate from sample or buffer 样品或缓冲液中的沉淀物 Back flush column - Clear plugged frit 反冲柱子－清洁阻塞的滤头 Change frit - Clear plugged frit 更换滤头 2018/11/18

Column Cleaning 柱子清洗 用比你的流动相更强的溶剂冲洗 F lush with stronger solvents than your mobile phase 用比你的流动相更强的溶剂冲洗 Reversed-Phase Solvent Choices in Order of Increasing Strength 为了增加强度选择反相溶剂 Use at least 25 ml of each solvent for analytical columns 对于分析柱每种溶剂至少用25 ml 冲洗 Mobile phase without buffer salts 没有缓冲盐的流动相 100% Methanol 甲醇 100% Acetonitrile 乙腈 75% Acetonitrile :25% Isopropanol 75乙腈 ： 25％异丙醇 100% Isopropanol 异丙醇 100% Methylene Chloride★ 二氯甲烷 100% Hexane ★ 已烷 ★ When using either Hexane or Methylene Chloride the column must be flushed with Isopropanol before returning to your resvered-phase mobile phase. 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 2018/11/18

Column Cleaning柱子清洗 Normal Phase Solvent Choices in Order of Increasing Strength 为了增加强度选用正相溶剂 Use at least 50 ml of each solvent 每种溶剂至少用50 ml 50% Methanol : 50% Chloroform 50% 甲醇 : 50% 氯仿 100% Ethyl Acetate 乙酸乙酯 2018/11/18

How to Change a Frit 怎样更换滤头 Column Inlet Frit 柱进口滤头 Column Body 柱身 Compression Ferrule 压缩金属套圈 Wear gloves 戴手套 Do not allow bed to dry 不要让底座干燥 Do not touch the column-body heat will extrude packing 不要使柱身受热,否则填料会喷出 Do not over tighten 不要旋得太紧 Female End Fitting 旋好尾端螺母 Male End Fitting 旋好尾端螺头 2018/11/18

Preventing Back Pressure Problems 压力问题的预防 Use column protection 柱保护 - Guard columns 保护柱/预柱 - In-line filters 在线过滤器 Sample Preparation 样品预处理 Appropriate column flushing 适当的柱冲洗 Filter buffered mobile phase 过滤缓冲流动相 2018/11/18

Preventing Back Pressure Problems:In-Line Devices 压力问题的预防:在线装置 Mobile Phase Pre-Column Injector Filter Guard From Pump 预柱 进样器 滤头 Column 从泵来的流动相 保护柱 Analytical Column Filter and Guard Column Act on Sample 分析柱 滤头和保护柱作用于样品 Pre-Column Acts on Mobile Phase 预柱作用于流动相 To Detector 到检测器 2018/11/18

Preventing Back Pressure Problems: Sample Preparation 压力问题的预防:样品制备 Solvent/Chemical Environment 溶剂/化学环境 Particulate/Aggregate Remove 微粒/凝聚物的除去 Filter samples 过滤样品 Centrifugation 离心分离 Solid Phase Extraction(S.P.E.) 固相萃取 Cartridges or Plates 薄膜或薄层板 Disks or Membranes 圆盘或生物膜 2018/11/18

Peak Shape Issues 峰形问题 Split peaks 裂峰 Peak tailing 峰拖尾 Broad peaks 宽峰 Poor efficiency 低柱效 Many 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: 展宽和拖尾或拖尾和保留时间增加 2018/11/18

Split Peaks 裂峰 Can be caused by: 可能的原因: Column contamination 柱污染 Partially plugged frit 部分阻塞滤片 Column void 柱头塌陷 Injection solvent effects 溶剂效应 2018/11/18

Split Peaks 裂峰 Column Contamination 柱污染 Column: StableBond SB-C8, 4.6 × 250mm, 5µm Mobile Phase:60%25mM Na2HPO4,pH3.0 : 40%MeOH Flow Rate: 1.0 mL/min Temperature: 35ºC Detection: UV 254 nm Sample: Filtered OTC Cold Medication: 1. Pseudoephedrine 2. APAP 3. Unknown 4. Chlorpheniramine Injection 1 峰3 峰形较好 Injection 30 峰3 裂峰 Injection 1 After Column Wash with 100% CAN 经100%乙腈冲洗后 峰3 峰形极好 2018/11/18

Split Peaks 裂峰 Injection Solvent Effects 溶剂化效应 Column: StableBond SB-C8, 4.6 × 250mm, 5µm Mobile Phase: 82%H2O : 18%ACN Injection Volume: 30 µL Sample: 1. Caffeine 咖啡因 2. Salicylamide 水杨酰胺 A. Injection Solvent B. Injection Solvent 100% Acetonitrile Mobile Phase 峰形宽拖尾 峰形尖锐对称 样品溶剂尽可能与流动相匹配 2018/11/18

Determining the Cause of Split Peaks 裂峰原因的确定 1. Complex sample matrix or many samples analyzed- likely column contamination or partially plugged frit 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. Mobile phase pH>=7 - likely column void due to silica dissolution (unless specialty column used) 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. Injection solvent stronger than mobile phase - likely split and broad peaks, dependent on volume 溶剂比流动相的可能性大- 裂峰和宽峰,由样品量来决定 2018/11/18

Peak Tailing, Broadening and Loss of Efficiency 峰拖尾、变宽和柱效降低 Can be caused by: 可能的原因 Column “secondary interactions” 柱“次级效应” Column void 柱塌陷 Column contamination 柱污染 Column aging 柱老化 Column loading 柱负荷超载 Extra-column effects 柱外效应 2018/11/18

Peak Tailing Column “Secondary Interactions” 峰拖尾柱“次级效应” Column: Alkyl-C8, 4.6 × 150mm, 5µm Mobile Phase:85%25mM Na2HPO4,pH7.0 : 15%ACN Flow Rate: 1.0 mL/min Temperature: 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 No TEA 无三乙胺 10 mM TEA 10 mM 三乙胺 USP TF(5%) 美国药典拖尾因子 USP TF(5%) 美国药典拖尾因子 1. 1.29 1. 1.19 2. 1.91 2. 1.18 3. 1.63 3. 1.20 4. 2.35 4. 1.26 5. 1.57 5. 1.14 Peak tailing eliminated with mobile phase modifier (TEA) at pH 7 用流动相减尾剂(三乙胺)在pH 7 消除峰拖尾 2018/11/18

Peak Tailing Column “Secondary Interactions” 峰拖尾柱“次级效应” Column: Alkyl-C8, 4.6 × 150mm, 5µm Mobile Phase:85%25mM Na2HPO4,pH7.0 : 15%ACN Flow Rate: 1.0 mL/min Temperature: 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 pH 3.0 pH 7.0 USP TF(5%) 美国药典拖尾因子 USP TF(5%) 美国药典拖尾因子 4. 1.33 4. 2.35 Reducing the mobile phase pH reduces interactions with silanols that cause peak tailing 降低流动相的pH ,减小与硅醇基作用引起的峰拖尾 2018/11/18

Peak Tailing 峰拖尾 Column Contamination 柱污染 Column: StableBond SB-C8, 4.6 × 250mm, 5µm Mobile Phase: 20% H2O : 80% MeOH Flow Rate: 1.0 mL/min Temperature: R.T. Detection: UV 254 nm Sample: 1. Uracil 尿嘧啶 2. Phenol 苯酚 3. 4-Chloronitrobenzene 4-氯硝基苯 4. Toluene 甲苯 Plates TF Plates TF Plates TF 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. 7629 2.08 1. 7906 1.43 1. 7448 1.06 2. 12043 1.64 2. 12443 1.21 2. 12237 1.21 3. 13727 1.69 3. 17999 1.19 3. 15366 1.11 4. 13355 1.32 4. 17098 1.25 4. 19067 1.17 QC test forward direction QC test reverse direction QC test after cleaning 100% IPA, 35ºC 向前方向 相反方向 100%异丙醇冲洗后 2018/11/18

Peak Tailing/Broadening Sample Load Effects 峰拖尾/变宽样品过载效应 Column: 4.6 × 150mm, 5µm Mobile Phase:40%25mM Na2HPO4,pH7.0 : 60%ACN Flow Rate: 1.5 mL/min Temperature: 40ºC Sample: 1. 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 Tailing Eclipse XDB-C8 Broadening / Competitive C8 USP TF 拖尾因子 Plates 理论板数 High Load / 10 Low Load High Load / 10 Low Load 高载负/10倍 低载负 高载负/10倍 低载负 1. 1.60 1.70 850 5941 2. 2.00 1.90 815 7842 3. 1.56 1.56 2776 6231 4. 2.13 1.70 2539 8359 5. 2.15 1.86 2735 10022 6. 1.25 1.25 5189 10725 2018/11/18

Peak Broadening,Splitting Column Void 峰变宽,裂开 柱塌陷 Mobile Phase:流动相： 50%CAN : 50%Water : 0.2%TEA (~pH 11) 50％乙腈 : 50％水 : 0。2％三乙胺 (~pH 11) After 30 injections Initial 开始时 进样30次后 Multiple peak shape changes can be caused by the same column problem. In the case a void resulted from silica dissolved at high pH. 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 2018/11/18

Broad Peaks Unknown “Phantom” Peaks Column: Extend-C18, 4.6 × 150 mm, 5 µm Mobile Phase: 40% 10 mM TEA, pH 11 : 60% MeOH Flow Rate: 1.0 mL/min Temperature: R.T. Detection: UV 254 Sample: 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. 5922 2. 9879 3. 779 “Phantom” peak from first injection 前次进样未出完的峰 The extremely low plates are an indication of an extremely late eluting peak from the preceding run. 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 2018/11/18

Peak Tailing峰拖尾 Injector Seal Failure进样器密封性差 Column: Bonus-RP, 4.6 × 75mm, 3.5µm Mobile Phase: 30% H2O : 70% MeOH Flow Rate: 1.0 mL/min Temperature: R.T. Detection: UV 254 nm Sample: 1. Uracil 尿嘧啶 2. Phenol 苯酚 3. N,N-Dimethylaniline N,N-二甲苯胺 Plates TF Plates TF 理论板数 拖尾因子 理论板数 拖尾因子 1. 2235 1.72 1. 3670 1.45 2. 3491 1.48 2. 10457 1.09 3. 5432 1.15 3. 10085 1.00 Before After replacing rotor seal and isolation seal 之前 转动杆和隔离密封垫检修后 Overdue instrument maintenance can cause peak shape problems. 仪器保养超期可能引起峰形问题/进样器划痕 2018/11/18

Peak Tailing 峰拖尾 Extra-Column Volume 柱外死体积效应 Column: StableBond SB-C18, 4.6 × 30mm, 3.5µm Mobile Phase: 85% H2Owith 0.1% TFA : 15% CAN Flow Rate: 1.0 mL/min Temperature: 35ºC Sample: 1. Phenylalanine 2. 5-benzyl-3,6-dioxo-2-piperazine 3. Asp-phe 4. Aspartame 10 µl extra-column volume 50 µl extra-column volume 2018/11/18

Determining the Cause of Peak Tailing 峰拖尾原因的确定 Evaluate 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 Evaluate 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 Reduce sample load 减小进样量 Eliminate extra-column effects 消除柱外效应 Flush column and check for aging/void 冲洗柱子检查柱子老化/塌陷 2018/11/18

Retention Issues 保留时间问题 Retention time changes(tr) 保留时间改变 Capacity factor (retention) changes (k´) 容量因子改变 Selectivity changes () 选择性改变 2018/11/18

Changes in Retention Same Column, Over Time相同柱子上保留时间改变,超时 May be cause by: 可能的原因: Column aging 柱老化 Column contamination 柱污染 Insufficient equilibration 不够平衡 Poor column/mobile phase combination 柱/流动相组成差 Change in mobile phase 改变流动相 Change in flow rate 改变流速 other instrument issues 其他仪器问题 2018/11/18

Mobile phase Change Causes Change in Retention 流动相改变引起保留时间改变 60%MeOH:40% 0.1% TFA Fresh TFA Added to Mobile Phase Volatile TFA evaporated/degassed from mobile phase. 易挥发的三氟乙酸从流动相中蒸发/挥发。 Replacing it solved problem. 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。 2018/11/18

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 Column 1 - Initial 1 号柱 开始时 Column 1 - Next Day 1 号柱 第二天 Column 1 - After Cleaning with 1% H3PO4 1 号柱用 1% H3PO4 清洗 The 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 The 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 Retention 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 2018/11/18

1. Determine K´, , and tr for suspect peaks 对怀疑峰K´, 和tr的确定 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 1. Determine K´, , and tr for suspect peaks 对怀疑峰K´, 和tr的确定 2. Wash column 冲洗柱子 3. Test new column - note lot number 试验新柱- 记录一批数据 4. Review column equilibration procedures 观察柱平衡过程 5. Make up fresh mobile phase4 and test 配制新鲜流动相再试验 6. Check instrument performance 检查仪器性能 2018/11/18

Change in Retention/Selectivity Column-to Column 柱与柱之间保留时间/选择性的改变 Different column histories (aging) 不同的柱历史(老化) Insufficient/inconsistent equilibration 平衡不够 /不一致 Poor column/mobile phase combination 柱/流动相差 Change in mobile phase流动相改变 Change in flow rate 流速改变 Other instrument issues其他仪器问题 Slight changes in column bed volume (tr only) 柱内体积轻微改变 2018/11/18

Lot-to-Lot Selectivity Change (pH) 批与批之间选择性改变(pH) pH 4.5 - Lot 1 pH 3.0 - Lot 1 pH 4.5 - Lot 2 pH 3.0 - Lot 2 pH 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 pH 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 2018/11/18

Conclusions 结论 HPLC column problem are evident as: 高效液相色谱柱问题明显的为: High pressure 高压 Undesirable peak shape 不希望得到的峰形 Changes in retention/selectivity 保留时间/选择性的改变 Often these problems are not associated with the column and may be caused by instrument and experimental condition issues. 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 2018/11/18