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1、2018/12/20,1,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.SeP),2018/12/20,2,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 p
2、roblems and require troubleshooting. 所有这些部件可能出现故障而需要检修。,2018/12/20,3,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,2018/12/20,4,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
3、色谱柱污染 Piugged packing 堵塞填料,2018/12/20,5,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 col
4、umn 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,2018/12/20,6,Column Cleaning 柱子清洗,F lush with stronger
5、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
6、 :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,2018/12/20,7,Column Clea
7、ning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,2018/12/20,8,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
8、 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,2018/12/20,9,Preventing Back Pressure Problems 压力问题的预防,Use column protection 柱保护 - Guard columns
9、 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,2018/12/20,10,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 Analytica
10、l 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,2018/12/20,11,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 Ce
11、ntrifugation 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,2018/12/20,12,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 increase
12、d 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,2018/12/20,13,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,2018/12/20,14,Split Peaks 裂峰 Column Contamination 柱污染,Column: StableBond SB-C8, 4.6 250mm, 5m Mobi
13、le Phase:60%25mM Na2HPO4,pH3.0 : 40%MeOH Flow Rate: 1.0 mL/min Temperature: 35C 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,2
14、018/12/20,15,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 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,20
15、18/12/20,16,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,2018/12/20,17,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 Co
