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1、2023/10/4,1,HPLC Column and System Troubleshooting 色谱柱和色谱系统的故障检修,What Every HPLCUser Should Know每个HPLC使用者应该知道什么,请访问中国色谱网(www.SeP),2023/10/4,2,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 proble
2、ms and require troubleshooting.所有这些部件可能出现故障而需要检修。,2023/10/4,3,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,2023/10/4,4,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
3、 packing 堵塞填料,2023/10/4,5,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
4、 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,2023/10/4,6,Column Cleaning 柱子清洗,F lush with stronger solvents than your mobile phase用
5、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
6、 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,2023/10/4,7,Column Cleaning柱子清洗,Normal Phase Solvent Choices in Order of Inc
7、reasing 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 乙酸乙酯流动相中含有高浓度无机盐缓冲溶液时色谱柱的清洗1.不能直接使用100的甲醇或乙腈冲洗色谱柱，无机盐会结晶析出，堵塞过滤器；管道；在线过滤器；定量环；柱头滤片；色谱柱和检测器流动池。后果很严重。2.正确的冲洗方法：先用不含无机盐的流动相冲洗，再用纯水冲洗，再用不含无机盐的流动相冲洗，再用100的甲醇或乙腈冲洗色谱柱。,2023/1
8、0/4,8,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 bed to dry 不要让底座干燥(不让色谱柱中的填料干燥，柱床不能干燥)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 Fitt
9、ing 旋好尾端螺头滤片的清洗是我们可以自己做的：1.打开色谱柱头时，要固定柱头螺丝，转动柱头螺母 2.小心保护柱床，不能改变柱床结构 3.滤片清洗的工具和溶剂要提前考虑好并准备好 4.清洗工作要快，防止柱床干裂 5.有的柱床需要修整，准备一个平面刀片 6.使用台钳固定柱头螺丝，使用呆扳手拧紧柱头螺母,2023/10/4,9,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 Preparation 样品预处理Ap
10、propriate column flushing 适当的柱冲洗Filter buffered mobile phase 过滤缓冲流动相,2023/10/4,10,Preventing Back Pressure Problems:In-Line Devices 压力问题的预防:在线装置,Mobile Phase Pre-Column Injector Filter GuardFrom Pump 预柱 进样器 滤头 Column从泵来的流动相 保护柱 Analytical ColumnFilter and Guard Column Act on Sample 分析柱 滤头和保护柱作用于样品 P
11、re-Column Acts on Mobile Phase 预柱作用于流动相 To Detector 到检测器,2023/10/4,11,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.)固相萃取Cartridges or Plate
12、s 薄膜或薄层板Disks or Membranes 圆盘或生物膜,2023/10/4,12,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,2023/10/4,13,Split Peaks 裂峰,Can be
13、 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,2023/10/4,14,Split Peaks 裂峰Column Contamination 柱污染,Column:StableBond SB-C8,4.6 250mm,5mMobile Phase:60%25mM Na2HPO4,pH3.0:40%MeOHFlow Rate:1.0 mL/min Temperature:35CDetection:UV 254
14、nmSample:Filtered OTC Cold Medication:1.Pseudoephedrine 2.APAP 3.Unknown 4.ChlorpheniramineInjection 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,2023/10/4,15,Split Peaks 裂峰Injection Solvent Effects 溶剂化效应,Column:StableBond SB-C8,4.6 250mm,5mMobile Phase:82%
15、H2O:18%ACNInjection 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,2023/10/4,16,Determining the Cause of Split Peaks 裂峰原因的确定,1.Complex sample matrix or many samples analyzed-likely column contaminat
16、ion 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(一柱多用产生相互影响，有时分析方法不能重复)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)(国产色谱柱装填压力和时间的不足也可导致柱塌陷；使用较高的柱压也可能)3.Injection solvent stronger than mobile phase-likely split and
17、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,2023/10/4,17,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,2023/10/4,1
18、8,Peak Tailing Column“Secondary Interactions”峰拖尾柱“次级效应”,Column:Alkyl-C8,4.6 150mm,5mMobile Phase:85%25mM Na2HPO4,pH7.0:15%ACNFlow Rate:1.0 mL/min Temperature:35CSample:1.Phenylpropanolamine 苯丙醇胺去甲麻黄碱 2.Ephedrine 麻黄碱 3.Amphetamine 安非他明苯丙胺4.Methamphetamine 脱氧麻黄碱 5.Phenteramine 苯三胺No TEA 无三乙胺 10 mM TEA
19、 10 mM 三乙胺USP TF(5%)美国药典拖尾因子 USP TF(5%)美国药典拖尾因子1.1.29 1.1.192.1.91 2.1.18 3.1.63 3.1.204.2.35 4.1.265.1.57 5.1.14Peak tailing eliminated with mobile phase modifier(TEA)at pH 7用流动相减尾剂(三乙胺)在pH 7 消除峰拖尾,2023/10/4,19,Peak Tailing Column“Secondary Interactions”峰拖尾柱“次级效应”,Column:Alkyl-C8,4.6 150mm,5mMobile
20、 Phase:85%25mM Na2HPO4,pH7.0:15%ACNFlow Rate:1.0 mL/min Temperature:35CSample:1.Phenylpropanolamine 苯丙醇胺去甲麻黄碱 2.Ephedrine 麻黄碱 3.Amphetamine 安非他明苯丙胺4.Methamphetamine 脱氧麻黄碱 5.Phenteramine 苯三胺 pH 3.0 pH 7.0 USP TF(5%)美国药典拖尾因子 USP TF(5%)美国药典拖尾因子 4.1.33 4.2.35Reducing the mobile phase pH reduces interact
21、ions with silanols that cause peak tailing降低流动相的pH,减小与硅醇基作用引起的峰拖尾(次级效应与键合固定相残留羟基有关系),2023/10/4,20,Peak Tailing 峰拖尾Column Contamination 柱污染,Column:StableBond SB-C8,4.6 250mm,5mMobile Phase:20%H2O:80%MeOH Flow Rate:1.0 mL/min Temperature:R.T.Detection:UV 254 nmSample:1.Uracil 尿嘧啶 2.Phenol 苯酚 3.4-Chlor
22、onitrobenzene 4-氯硝基苯 4.Toluene 甲苯Plates TF Plates TF Plates TF理论板数 拖尾因子 理论板数 拖尾因子 理论板数 拖尾因子1.7629 2.08 1.7906 1.43 1.7448 1.062.12043 1.64 2.12443 1.21 2.12237 1.213.13727 1.69 3.17999 1.19 3.15366 1.114.13355 1.32 4.17098 1.25 4.19067 1.17QC test forward direction QC test reverse direction QC test
23、after cleaning 100%IPA,35C 向前方向 相反方向 100%异丙醇冲洗后,2023/10/4,21,Peak Tailing/Broadening Sample Load Effects峰拖尾/变宽样品过载效应,Column:4.6 150mm,5mMobile Phase:40%25mM Na2HPO4,pH7.0:60%ACNFlow Rate:1.5 mL/min Temperature:40CSample:1.Desipramine 去郁敏/脱甲基丙米嗪 2.Nortriptyline 3.Doxepin 4.Imipramine 丙米嗪 5.Amitriptyl
24、ine 阿米替林 6.TrimipramineTailing Eclipse XDB-C8 Broadening/Competitive C8USP TF 拖尾因子 Plates 理论板数 High Load/10 Low Load High Load/10 Low Load高载负/10倍 低载负 高载负/10倍 低载负1.1.60 1.70 850 59412.2.00 1.90 815 78423.1.56 1.56 2776 6231 4.2.13 1.70 2539 8359 5.2.15 1.86 2735 10022 6.1.25 1.25 5189 10725,2023/10/4
25、,22,Peak Broadening,Splitting Column Void 峰变宽,裂开 柱塌陷,Mobile Phase:流动相：50%CAN:50%Water:0.2%TEA(pH 11)50乙腈:50水:0。2三乙胺(pH 11)After 30 injections Initial 开始时 进样30次后Multiple peak shape changes can be caused by the same column problem.In the case a void resulted from silica dissolved at high pH.多数峰形改变可能是同
26、一柱问题引起的。在这个例子中，高pH溶解硅胶导致塌陷,2023/10/4,23,Broad Peaks Unknown“Phantom”Peaks,Column:Extend-C18,4.6 150 mm,5 mMobile Phase:40%10 mM TEA,pH 11:60%MeOHFlow Rate:1.0 mL/min Temperature:R.T.Detection:UV 254Sample:1.Maleat 顺丁烯二酸 2.Pseudeophedrine 伪麻黄碱 3.Chlorphenniramine 氯非尼拉明Plates 理论板数1.5922 2.98793.779“Ph
27、antom”peak from first injection 前次进样未出完的峰The extremely low plates are an indication of an extremely late eluting peak from the preceding run.极低的理论板数表明这是一个前面进样极迟洗脱出来的峰,2023/10/4,24,Peak Tailing峰拖尾 Injector Seal Failure进样器密封性差,Column:Bonus-RP,4.6 75mm,3.5mMobile Phase:30%H2O:70%MeOH Flow Rate:1.0 mL/m
28、in Temperature:R.T.Detection:UV 254 nmSample:1.Uracil 尿嘧啶 2.Phenol 苯酚 3.N,N-Dimethylaniline N,N-二甲苯胺Plates TF Plates TF 理论板数 拖尾因子 理论板数 拖尾因子 1.2235 1.72 1.3670 1.452.3491 1.48 2.10457 1.09 3.5432 1.15 3.10085 1.00 Before After replacing rotor seal and isolation seal之前 转动杆和隔离密封垫检修后Overdue instrument m
29、aintenance can cause peak shape problems.仪器保养超期可能引起峰形问题/进样器划痕,2023/10/4,25,Peak Tailing 峰拖尾Extra-Column Volume 柱外死体积效应,Column:StableBond SB-C18,4.6 30mm,3.5mMobile Phase:85%H2Owith 0.1%TFA:15%CANFlow Rate:1.0 mL/min Temperature:35C Sample:1.Phenylalanine 2.5-benzyl-3,6-dioxo-2-piperazine 3.Asp-phe 4
30、.Aspartame(缺图)10 l extra-column volume 50 l extra-column volume(管路连接，定量环大小，连接管路与色谱柱未达到无死体积状态等),2023/10/4,26,Determining the Cause of Peak Tailing峰拖尾原因的确定,Evaluate mobile phase effects-alter mobile phase pH and additives to eliminate secondary interactions 评价流动相效果-改变流动相pH和添加剂消除次级效应/流动相中组份与柱子相互作用Evalu
31、ate column choice-try column with high purity silica or different bonding technology 评价柱选择-试用高纯度硅胶柱或不同键合技术柱Reduce sample load 减小进样量Eliminate extra-column effects 消除柱外效应Flush column and check for aging/void 冲洗柱子检查柱子老化/塌陷,2023/10/4,27,Retention Issues保留时间问题,Retention time changes(tr)保留时间改变Capacity fac
32、tor(retention)changes(k)容量因子改变Selectivity changes()选择性改变,2023/10/4,28,Changes in Retention Same Column,Over Time相同柱子上保留时间改变,超时,May be cause by:可能的原因:Column aging 柱老化Column contamination 柱污染Insufficient equilibration 平衡不够Poor column/mobile phase combination 柱/流动相组成差(色谱条件不够完美)Change in mobile phase 改变
33、流动相Change in flow rate 改变流速other instrument issues 其他仪器问题,2023/10/4,29,Mobile phase Change Causes Change in Retention流动相改变引起保留时间改变,Mobile Phase：60%MeOH:40%0.1%TFAVolatile TFA evaporated/degassed from mobile phase.易挥发的三氟乙酸从流动相中蒸发/挥发。Replacing it solved problem.换用新的流动相就解决问题。使用有机挥发性酸时，流动相应新配。流动相中含有磷酸盐缓
34、冲溶液时1。过滤2。放在透明容器中3。过夜会出现沉淀4。梯度洗脱需要检查在高比例有机溶剂时无机盐的溶解性5。使用仪器混合器混合流动相时也需要检查无机盐的溶解性,2023/10/4,30,Column Aging/Equilibration Causes Retention/Selectivity Changes柱老化平衡不够引起保留时间选择性改变,Column 1-Initial 1 号柱 开始时Column 1-Next Day 1 号柱 第二天 Column 1-After Cleaning with 1%H3PO4 1 号柱用 1%H3PO4 清洗(清洗色谱柱的目的在于用溶解性更强的溶剂
35、，洗掉色谱柱中保留的杂质)The primary analyte was sensitive to mobile phase aging of the column.最初的分析对流动相老化柱子是灵敏的The peak shape was a secondary issue resolved by flushing the column.冲洗柱子第二要解决的问题是峰形Retention and peak shape were as expected after cleaning.清洗后保留时间和峰形都达到了预期效果,2023/10/4,31,Determining the Cause of Re
36、tention Changes on the same Column同一柱上保留时间改变原因的确定,1.Determine K,and tr for suspect peaks 对怀疑峰K,和tr的确定 2.Wash column 冲洗柱子(可以使用工作站记录洗脱曲线)3.Test new column-note lot number 试验新柱-记录一批数据4.Review column equilibration procedures 观察柱平衡过程(可以使用工作站记录平衡曲线，尤其是作离子对色谱时)5.Make up fresh mobile phase4 and test 配制新鲜流动相
37、再试验6.Check instrument performance 检查仪器性能,2023/10/4,32,Change in Retention/Selectivity Column-to Column柱与柱之间保留时间/选择性的改变,Different column histories(aging)不同的柱历史(老化)Insufficient/inconsistent equilibration平衡不够/不一致Poor column/mobile phase combination 柱/流动相差Change in mobile phase流动相改变Change in flow rate 流
38、速改变Other instrument issues其他仪器问题Slight changes in column bed volume(tr only)柱内体积轻微改变,2023/10/4,33,Lot-to-Lot Selectivity Change(pH)批与批之间选择性改变(pH),pH 4.5-Lot 1 pH 3.0-Lot 1pH 4.5-Lot 2 pH 3.0-Lot 2pH 4.5 shows selectivity change from lot-to-lot for basic compounds pH 4.5 显示批与批之间对碱性化合物的选择性改变pH 3.0 sho
39、ws no selectivity change from lot-to-lot,indicating silanol sensitivity at pH 4.5 pH 3.0显示批与批之间对碱性化合物的无选择性改变,表明在pH 4.5硅醇基的灵敏性,2023/10/4,34,Conclusions 结论,HPLC column problem are evident as:高效液相色谱柱问题明显的为:High pressure 高压(需要记录冲洗色谱柱时的流动相，流速和压力)(有条件的情况下可以在冲洗好色谱柱后，作一针标准品，记录柱效和色谱图)Undesirable peak shape 不希望得到的峰形Changes in retention/selectivity 保留时间/选择性的改变 Often these problems are not associated with the column and may be caused by instrument and experimental condition issues.经常遇到的这些问题不一定与柱有关,而与仪器和实验条件方面的问题有关,
