色谱系统和色谱柱的故障检修

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,2024/11/28,1,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。,2024/11/28,2,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,2024/11/28,3,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,2024/11/28,4,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,2024/11/28,5,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,2024/11/28,6,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,2024/11/28,7,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,2024/11/28,8,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,2024/11/28,9,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,2024/11/28,10,PreventingBackPressureProblems:Sample Preparation,压力问题的,预,预防:样品,制,制备,Solvent/Chemical Environment,溶剂/化学,环,环境,Particulate/Aggregate Remove,微粒/凝聚,物,物的除去,Filter samples,过滤样品,Centrifugation,离心分离,SolidPhase Extraction(S.P.E.),固相萃取,Cartridgesor Plates,薄膜或薄层,板,板,Disksor Membranes,圆盘或生物,膜,膜,2022/12/31,11,PeakShapeIssues 峰,形,形问题,Splitpeaks,裂峰,Peaktailing,峰拖尾,Broadpeaks,宽峰,Poorefficiency,低柱效,Manypeakshapeissues also combinations-I.e.Broad andtailing or tailingwithincreasedretention,许多峰形问,题,题是结合在,一,一起的-例,如,如:,展宽和拖尾,或,或拖尾和保,留,留时间增加,2022/12/31,12,SplitPeaks,裂峰,Can be caused by:,可能的原因:,Column contamination,柱污染,Partiallyplugged frit,部分阻塞滤,片,片,Column void,柱头塌陷,Injectionsolvent effects,溶剂效应,2022/12/31,13,SplitPeaks,裂峰,Column Contamination,柱污染,Column:StableBond SB-C8,4.6,250mm,5,m,Mobile Phase:60%25mMNa,2,HPO,4,pH3.0:40%MeOH,FlowRate:1.0mL/minTemperature:35,C,Detection:UV 254 nm,Sample:Filtered OTCColdMedication:,1.Pseudoephedrine2.APAP 3.Unknown,4.Chlorpheniramine,Injection1,峰3,峰形较好,Injection30,峰3,裂峰,Injection1,AfterColumn Wash with 100%CAN,经100%,乙,乙腈冲洗后,峰3,峰形极好,2022/12/31,14,SplitPeaks,裂峰,InjectionSolventEffects,溶剂,化,化效,应,应,Column:StableBondSB-C8,4.6,250mm,5,m,MobilePhase:82%H,2,O:18%ACN,InjectionVolume:30,L,Sample:,1.Caffeine,咖啡,因,因,2.,Salicylamide,水杨,酰,酰胺,100%AcetonitrileMobilePhase,峰形,宽,宽拖,尾,尾,峰,峰,形,形尖,锐,锐对,称,称,样品,溶,溶剂,尽,尽可,能,能与,流,流动,相,相匹,配,配,2022/12/31,15,DeterminingtheCauseofSplitPeaks,裂,峰,峰,原,原,因,因,的,的,确,确,定,定,1.,Complexsamplematrixormanysamplesanalyzed-likelycolumncontaminationorpartiallypluggedfrit,复,杂,杂,样,样,品,品,的,的,基,基,质,质,或,或,分,分,析,析,许,许,多,多,样,样,品,品-,柱,柱,污,污,染,染,或,或,部,部,分,分,阻,阻,塞,塞,滤,滤,片,片,2.,MobilephasepH=7-likelycolumnvoidduetosilicadissolution(unlessspecialtycolumnused),流,动,动,相,相,pH=7-,由,于,于,硅,硅,胶,胶,溶,溶,解,解,使,使,柱,柱,塌,塌,陷,陷(,除,除,非,非,使,使,用,用,专,专,门,门,的,的,柱,柱,子,子),3.,Injectionsolventstrongerthanmobilephase-likelysplitandbroadpeaks,dependentonvolume,溶,剂,剂,比,比,流,流,动,动,相,相,的,的,可,可,能,能,性,性,大,大-,裂,裂,峰,峰,和,和,宽,宽,峰,峰,由,由,样,样,品,品,量,量,来,来,决,决,定,定,2022/12/31,16,PeakTailing,BroadeningandLossofEfficiency,峰,拖,拖,尾,尾,、,、,变,变,宽,宽,和