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 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。 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 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 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 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 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 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 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
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 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 Split Peaks 裂峰 Can be caused by: 可能的原因:
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 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 Peak Tailing, Broadening and Loss of Efficiency 峰拖尾、变宽和柱效降低
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