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1、*1HPLC Column and System Troubleshooting 色谱柱和色谱系统的故障检修lWhat Every HPLClUser Should Knowl每个HPLC使用者应该知道什么 请访问中国色谱网(www.SeP)*2HPLC Components HPLC 的组成l Pump 泵l Injector/Autosampler 进样器/自动进样器l Column 色谱柱l Detector 检测器l Data System/Integrator 数据系统/积分仪表lAll of these components can have problems and requir
2、e troubleshooting. l所有这些部件可能出现故障而需要检修。*3Categories of Column Problems 色谱 柱问题的类型l l A. Pressure 压力l B. Peak shape 峰形l C. Retention 保留时间*4Pressure Issues 压力问题 lColumn Observations Potential Problemsl色谱柱观测 潜在的问题 lHigh pressure Plugged frit l压力高 堵塞滤头l Column contaminationl 色谱柱污染l Piugged packingl 堵塞填料*5
3、Determining the Cause and Correcting High Back Pressure 查出原因 纠正压力lCheck pressure with/without column - many pressure problems are due to blockages in the system or guardl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lIf Column Pressure is high 如果柱压高:lWash column - Eliminate column contamination andl 冲洗柱子 plugged pack
4、ing 排除柱污染和填料堵塞l - high molecular weight/adsorbedl compounds 高分子量被吸附化合物l - precipitate from sample or bufferl 样品或缓冲液中的沉淀物lBack flush column - Clear plugged frit 反冲柱子清洁阻塞的滤头lChange frit - Clear plugged frit 更换滤头*6Column Cleaning 柱子清洗lF lush with stronger solvents than your mobile phasel用比你的流动相更强的溶剂冲洗l
5、 Reversed-Phase Solvent Choices in Order of l Increasing Strength 为了增加强度选择反相溶剂lUse at least 25 ml of each solvent for analytical columnsl 对于分析柱每种溶剂至少用25 ml 冲洗lMobile phase without buffer salts 没有缓冲盐的流动相l100% Methanol 甲醇l100% Acetonitrile 乙腈l75% Acetonitrile :25% Isopropanol 75乙腈 ： 25异丙醇l100% Isoprop
6、anol 异丙醇l100% Methylene Chloride 二氯甲烷l100% Hexane 已烷l When using either Hexane or Methylene Chloride the column must be flushed with lIsopropanol before returning to your resvered-phase mobile phase.l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*7Column Cleaning柱子清洗lNormal Phase Solvent Choices in Order of Inc
7、reasing Strengthl为了增加强度选用正相溶剂lUse at least 50 ml of each solvent l 每种溶剂至少用50 ml l50% Methanol : 50% Chloroform l 50% 甲醇 : 50% 氯仿l100% Ethyl Acetate 乙酸乙酯*8How to Change a Frit 怎样更换滤头lColumn Inlet Frit 柱进口滤头lColumn Body 柱身lCompression Ferrule 压缩金属套圈lWear gloves 戴手套lDo not allow bed to dry 不要让底座干燥lDo n
8、ot touch the column-body heat will extrude packing 不要使柱身受热,否则填料会喷出lDo not over tighten 不要旋得太紧lFemale End Fitting 旋好尾端螺母lMale End Fitting 旋好尾端螺头*9Preventing Back Pressure Problems 压力问题的预防lUse column protection 柱保护l - Guard columns 保护柱/预柱l - In-line filters 在线过滤器l Sample Preparation 样品预处理lAppropriate
9、column flushing l 适当的柱冲洗lFilter buffered mobile phasel 过滤缓冲流动相*10Preventing Back Pressure Problems:In-Line Devices压力问题的预防:在线装置lMobile Phase Pre-Column Injector Filter GuardlFrom Pump 预柱 进样器 滤头 Columnl从泵来的流动相 保护柱 l Analyticall ColumnlFilter and Guard Column Act on Sample 分析柱l 滤头和保护柱作用于样品l Pre-Column
10、Acts on Mobile Phasel 预柱作用于流动相 To Detectorl 到检测器*11Preventing Back Pressure Problems: Sample Preparation 压力问题的预防:样品制备lSolvent/Chemical Environment l 溶剂/化学环境lParticulate/Aggregate Remove l微粒/凝聚物的除去lFilter samples 过滤样品lCentrifugation 离心分离lSolid Phase Extraction(S.P.E.) 固相萃取lCartridges or Plates 薄膜或薄层板
11、lDisks or Membranes 圆盘或生物膜*12Peak Shape Issues 峰形问题l Split peaks 裂峰l Peak tailing 峰拖尾l Broad peaks 宽峰lPoor efficiency 低柱效lMany 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:l展宽和拖尾或拖尾和保留时间增加*13Split Peaks 裂峰lCan be caused by: l可能的原因:lC
12、olumn contamination 柱污染lPartially plugged frit 部分阻塞滤片lColumn void 柱头塌陷lInjection solvent effects 溶剂效应*14Split Peaks 裂峰 Column Contamination 柱污染lColumn: StableBond SB-C8, 4.6 250mm, 5mlMobile Phase:60%25mM Na2HPO4,pH3.0 : 40%MeOHlFlow Rate: 1.0 mL/min Temperature: 35ClDetection: UV 254 nmlSample: Fil
13、tered OTC Cold Medication: l1. Pseudoephedrine 2. APAP 3. Unknown l4. ChlorpheniraminelInjection 1 峰3 峰形较好lInjection 30 峰3 裂峰lInjection 1 After Column Wash with 100% CANl经100%乙腈冲洗后 峰3 峰形极好*15Split Peaks 裂峰 Injection Solvent Effects 溶剂化效应lColumn: StableBond SB-C8, 4.6 250mm, 5mlMobile Phase: 82%H2O : 18%ACNlInjection Volume: 30 L lSample: l1. Caffeine 咖啡因 2. Salicylamide 水杨
