色谱系统和色谱柱的故障检修34505

Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,*,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,*,*,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,2024/10/1,1,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。,2024/10/1,2,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,2024/10/1,3,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,2024/10/1,4,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,2024/10/1,5,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,2024/10/1,6,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,2024/10/1,7,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,2024/10/1,8,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,2024/10/1,9,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,2024/10/1,10,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,2024/10/1,11,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,2024/10/1,12,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,2024/10/1,13,Split Peaks,裂峰,Column Contamination,柱污染,Column:StableBond SB-C8,4.6 250mm,5m,Mobile Phase:60%25mM Na,2,HPO,4,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,2024/10/1,14,Split Peaks,裂峰,Injection Solvent Effects,溶剂化效应,Column:StableBond SB-C8,4.6 250mm,5m,Mobile Phase:82%H,2,O: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,2024/10/1,15,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,2024/10/1,16,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,Column“secondary interactions”,柱“次级效应”,Column void,柱塌陷,Column contamination,柱污染,Column aging,柱老化,Column loading,柱负荷超载,Extra-column effects,柱外效应,2024/10/1,17,Peak Tailing Column“Seco