色谱柱和色谱系统的故障检修Word

　　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2016/3/18 1

　　Pressure Issues 压力问题

　　Column Observations 色谱柱观测

　　Potential Problems 潜在的问题

　　High pressure 压力高

　　Plugged frit 堵塞滤头 Column contamination 色谱柱污染 Piugged packing 堵塞填料

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　　Column Cleaning 柱子清洗

　　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2016/3/18 6

　　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 ml50% Methanol : 50% Chloroform 50% 甲醇 : 50% 氯仿 100% Ethyl Acetate 乙酸乙酯7

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　　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2016/3/18 8

　　Preventing Back Pressure Problems:In-Line Devices

　　压力问题的预防:在线装置Pre-Column 预柱

　　Mobile Phase From Pump 从泵来的流动相

　　Injector Filter Guard 进样器 滤头 Column 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

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　　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13

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　　Split Peaks 裂峰

　　Column Contamination 柱污染Column: StableBond SB-C8, 4.6 250mm, 5m Mobile Phase:60%25mM Na2HPO4,pH3.0 : 40%MeOH Flow Rate: 1.0 mL/min Temperature: 35 C Detection: UV 254 nm Sample: Filtered OTC Cold Medication: 1. Pseudoephedrine 2. APAP 3. Unknown 4. Chlorpheniramine

　　Injection 1 Injection 30 Injection 1

　　峰3 峰3

　　峰形较好 裂峰峰形极好14

　　After Column Wash with 100% CAN

　　经100%乙腈冲洗后 峰3

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　　Split Peaks 裂峰

　　Injection Solvent Effects 溶剂化效应Column: StableBond SB-C8, 4.6 250mm, 5m Mobile Phase: 82%H2O : 18%ACN Injection Volume: 30 L Sample: 1. Caffeine 咖啡因 2. Salicylamide 水杨酰胺A. Injection Solvent 100% Ac

　　etonitrile 峰形宽拖尾 B. Injection Solvent Mobile Phase 峰形尖锐对称

　　样品溶剂尽可能与流动相匹配2016/3/18 15

　　Determining the Cause of Split Peaks

　　裂峰原因的确定

　　1. Complex sample matrix or many samples analyzedcolumn contamination or partially plugged frit

　　likely

　　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16

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　　Peak Tailing Column “Secondary Interactions”

　　峰拖尾柱“次级效应”

　　Column: Alkyl-C8, 4.6 150mm, 5m Mobile Phase:85%25mM Na2HPO4,pH7.0 : 15%ACN Flow Rate: 1.0 mL/min Temperature: 35 C 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 USP TF(5%) 美国药典拖尾因子 1. 1.29 2. 1.91 3. 1.63 4. 2.35 5. 1.57 10 mM TEA 10 mM 三乙胺 USP TF(5%) 美国药典拖尾因子 1. 1.19 2. 1.18 3. 1.20 4. 1.26 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18

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　　Peak Tailing Column “Secondary Interactions”

　　峰拖尾柱“次级效应”

　　Column: Alkyl-C8, 4.6 150mm, 5m Mobile Phase:85%25mM Na2HPO4,pH7.0 : 15%ACN Flow Rate: 1.0 mL/min Temperature: 35 C 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 USP TF(5%) 美国药典拖尾因子 4. 1.33

　　pH 7.0 USP TF(5%) 美国药典拖尾因子 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19

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　　Peak Tailing/Broadening

　　Sample Load Effects

　　峰拖尾/变宽样品过载效应

　　Column: 4.6 150mm, 5m Mobile Phase:40%25mM Na2HPO4,pH7.0 : 60%ACN Flow Rate: 1.5 mL/min Temperature: 40 C Sample: 1. Desipramine 去郁敏/脱甲基丙米嗪 2. Nortriptyline 4. Imipramine 丙米嗪 5. Amitriptyline 阿米替林

　　3. Doxepin 6. Trimipramine

　　Tailing Eclipse XDB-C8 USP TF 拖尾因子 High Load / 10 Low Load 高载负/10倍 低载负1. 2. 3. 4. 5. 1.60 2.00 1.56 2.13 2.15 1.70 1.90 1.56 1.70 1.86 1.25

　　Broadening / Competitive C8 Plates 理论板数 High Load / 10 Low Load 高载负/10倍 低载负850 815 2776 2539 2735 5189 5941 7842 6231 8359 10022 10725

　　6. 1.25

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　　Broad Peaks Unknown “Phantom” Peaks

　　Column: Extend-C18, 4.6 150 mm, 5 m 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2016/3/18 23

　　Peak Tailing峰拖尾

　　Injector Seal Failure进样器密封性差Column: Bonus-RP, 4.6 75mm, 3.5m 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 pea

　　k shape problems.

　　仪器保养超期可能引起峰形问题/进样器划痕24

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　　柱与柱之间保留时间/选择性的改变

　　Change in Retention/Selectivity Column-to Column

　　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2016/3/18

　　Lot-to-Lot Selectivity Change (pH)

　　批与批之间选择性改变(pH)pH 3.0 - Lot 1 pH 3.0 - Lot 2

　　pH 4.5 - Lot 1 pH 4.5 - Lot 2

　　pH for pH pH

　　4.5 shows selectivity change from lot-to-lot basic compounds 4.5 显示批与批之间对碱性化合物的选择性改变 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2016/3/18 33