HPLC 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.SePu.net)Date1HPLC 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 require troubleshooting. l所有这些部件可能出现故障而需要检修。Date2Categories 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Date3Pressure 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Date4Determining 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 packing 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Date5Column 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 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% Isopropanol 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Date6Column Cleaning柱子清洗lNormal Phase Solvent Choices in Order of Increasing 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Date7How 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 not 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Date8Preventing 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 column flushing l 适当的柱冲洗lFilter buffered mobile phasel 过滤缓冲流动相Date9Preventing 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 Acts on Mobile Phasel 预柱作用于流动相 To Detectorl 到检测器Date10Preventing 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lDisks or Membranes 圆盘或生物膜Date11Peak 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Date12Split Peaks 裂峰lCan be caused by: l可能的原因:lColumn 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Date13Split 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: Filtered 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Date14Split 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: