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1、2018/10/16,1,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,2018/10/16,2,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 problems and require trou
2、bleshooting. 所有这些部件可能出现故障而需要检修。,2018/10/16,3,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,2018/10/16,4,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色谱柱污染Piugged packing堵塞填料,201
3、8/10/16,5,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 column contamination and冲洗柱子 plug
4、ged packing 排除柱污染和填料堵塞- high molecular weight/adsorbedcompounds 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,2018/10/16,6,Column Cleaning 柱子清洗,F lush with stronger solvents than your mobile phase 用比你
5、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% I
6、sopropanol 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,2018/10/16,7,Column Cleaning柱子清洗,Normal Phase Solvent Choices
7、 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,2018/10/16,8,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 压缩金属套圈 Wear gloves 戴手套 Do not allow b
8、ed 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,2018/10/16,9,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 Pr
9、eparation 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,2018/10/16,10,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 AnalyticalColumn Filter and Guard Column Act on Sam
10、ple 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,2018/10/16,11,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 Centrifugation 离心分离 Solid Phase Extraction(S.P.E.
11、) 固相萃取 Cartridges or Plates 薄膜或薄层板 Disks or Membranes 圆盘或生物膜,2018/10/16,12,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 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/
12、10/16,13,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,2018/10/16,14,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:
13、 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,2018/10/16,15,Split Peaks 裂峰 Injection Solvent Effec
14、ts 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 Solvent100% 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,2018/10/16,16,Determining the Cause of Split Peaks 裂峰原因的确
15、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,
