色谱柱和色谱系统的故障检修.docx

色谱柱和色谱系统的故障检修.docx

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色谱柱和色谱系统的故障检修

色谱柱和色谱系统的故障检

作者：

liuying3…    文章来源：

本站原创    点击数：

2301    更新时间：

2005-8-15

HPLCColumnand SystemTroubleshooting色谱柱和色谱系统的故障检修

WhatEveryHPLC

UserShouldKnow

每个HPLC使用者应该知道什么

HPLCComponentsHPLC的组成：

   Pump泵

   Injector/Autosampler进样器/自动进样器

   Column色谱柱

   Detector检测器

   DataSystem/Integrator数据系统/积分仪表

Allofthesecomponentscanhaveproblemsandrequiretroubleshooting.

所有这些部件可能出现故障而需要检修。

CategoriesofColumnProblems 色谱柱问题的类型：

  A.Pressure压力

  B.Peakshape峰形

  C.Retention保留时间

PressureIssues压力问题

 ColumnObservations                    PotentialProblems

 色谱柱观测：

                          潜在的问题：

 Highpressure                          Pluggedfrit 

 压力高                                 堵塞滤头

                                         Columncontamination

                                         色谱柱污染

                                         Piuggedpacking

                                         堵塞填料

 DeterminingtheCauseandCorrectingHighBackPressure  

 查出原因纠正压力

Checkpressurewith/withoutcolumn-manypressureproblemsareduetoblockagesinthesystemorguard

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

IfColumnPressureishigh如果柱压高:

Washcolumn-Eliminatecolumncontaminationand

 冲洗柱子：

                    pluggedpacking 

                    排除柱污染和填料堵塞

                    -highmolecularweight/adsorbed

                      compounds 

                    高分子量／被吸附化合物

                    -precipitatefromsampleorbuffer

                    样品或缓冲液中的沉淀物

Backflushcolumn-Clearpluggedfrit  

 反冲柱子－清洁阻塞的滤头

Changefrit-Clearpluggedfrit   

更换滤头

ColumnCleaning

柱子清洗

Flushwithstrongersolventsthanyourmobilephase

用比你的流动相更强的溶剂冲洗

  Reversed-PhaseSolventChoicesinOrderof

  IncreasingStrength   

  为了增加强度选择反相溶剂

  Useatleast25mlofeachsolventforanalyticalcolumns

  对于分析柱每种溶剂至少用25ml冲洗

  Mobilephasewithoutbuffersalts  

  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%MethyleneChloride

  ★ 二氯甲烷

  100%Hexane

  ★ 已烷

★ WhenusingeitherHexaneorMethyleneChloridethecolumnmustbeflushedwith

Isopropanolbeforereturningtoyourresvered-phasemobilephase.

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

ColumnCleaning柱子清洗

NormalPhaseSolventChoicesinOrderofIncreasingStrength

为了增加强度选用正相溶剂

Useatleast50mlofeachsolvent 

每种溶剂至少用50ml

 50%Methanol:

50%Chloroform   

 50%甲醇:

50%氯仿

 100%EthylAcetate  

 乙酸乙酯

HowtoChangeaFrit

怎样更换滤头：

ColumnInletFrit

柱进口滤头

ColumnBody

柱身

CompressionFerrule

压缩金属套圈

Weargloves

戴手套

Donotallowbedtodry

不要让底座干燥

Donottouchthecolumn-bodyheatwillextrudepacking     

不要使柱身受热,否则填料会喷出

Donotovertighten

不要旋得太紧

FemaleEndFitting

旋好尾端螺母

MaleEndFitting

旋好尾端螺头

PreventingBackPressureProblems

压力问题的预防：

Usecolumnprotection

柱保护

-Guardcolumns

保护柱/预柱

-In-linefilters

在线过滤器

SamplePreparation

样品预处理

Appropriatecolumnflushing

适当的柱冲洗

Filterbufferedmobilephase

过滤缓冲流动相

PreventingBackPressureProblems:

In-LineDevices

 压力问题的预防:

在线装置

MobilePhase   Pre-Column   Injector Filter Guard

FromPump           预柱            进样器     滤头  Column

从泵来的流动相                                                    

保护柱                                                       

                                                                  Analytical

                                                                   Column

FilterandGuardColumnActonSample     分析柱

          滤头和保护柱作用于样品

  Pre-ColumnActsonMobilePhase

              预柱作用于流动相                           

ToDetector

                                                                     到检测器

PreventingBackPressureProblems:

SamplePreparation压力问题的预防:

样品制备

Solvent/ChemicalEnvironment

 溶剂/化学环境

Particulate/AggregateRemove 

微粒/凝聚物的除去

Filtersamples 过滤样品

Centrifugation 离心分离

SolidPhaseExtraction（S.P.E.） 固相萃取

CartridgesorPlates 薄膜或薄层板

DisksorMembranes 圆盘或生物膜

PeakShapeIssues峰形问题：

     Splitpeaks  裂峰

     Peaktailing 峰拖尾

     Broadpeaks  宽峰

Poorefficiency  低柱效

Manypeakshapeissuesalsocombinations-I.e.Broadandtailingortailingwithincreasedretention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

SplitPeaks 裂峰

Canbecausedby:

可能的原因:

Columncontamination 柱污染

Partiallypluggedfrit部分阻塞滤片

Columnvoid 柱头塌陷

Injectionsolventeffects溶剂效应

SplitPeaks 裂峰ColumnContamination 柱污染

Column:

StableBondSB-C8,4.6×250mm,5µm

MobilePhase:

60%25mMNa2HPO4,pH3.0:

40%MeOH

FlowRate:

1.0mL/min        Temperature:

35ºC

Detection:

UV254nm

Sample:

FilteredOTCColdMedication:

1.Pseudoephedrine 2.APAP 3.Unknown  

4.Chlorpheniramine

Injection1      峰3   峰形较好

Injection30    峰3   裂峰

Injection 1   AfterColumnWashwith100%CAN

经100%乙腈冲洗后  峰3   峰形极好

SplitPeaks 裂峰InjectionSolventEffects溶剂化效应

Column:

StableBondSB-C8,4.6×250mm,5µm

MobilePhase:

82%H2O:

18%ACN

InjectionVolume:

30µL      

Sample:

1.Caffeine 咖啡因 2.Salicylamide 水杨酰胺

A.InjectionSolvent                              B.InjectionSolvent

  100%Acetonitrile                                  MobilePhase

   峰形宽拖尾                                               峰形尖锐对称

样品溶剂尽可能与流动相匹配

DeterminingtheCauseofSplitPeaks 裂峰原因的确定

1.Complexsamplematrixormanysamplesanalyzed-   likelycolumncontaminationorpartiallypluggedfrit

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.MobilephasepH>=7-likelycolumnvoidduetosilicadissolution（unlessspecialtycolumnused）

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.Injectionsolventstrongerthanmobilephase-likelysplitandbroadpeaks,dependentonvolume

溶剂比流动相的可能性大-裂峰和宽峰,由样品量来决定

PeakTailing,BroadeningandLossofEfficiency峰拖尾、变宽和柱效降低

Canbecausedby:

可能的原因

Column“secondaryinteractions”

柱“次级效应”

Columnvoid 柱塌陷

Columncontamination 柱污染

Columnaging 柱老化

Columnloading 柱负荷超载

Extra-columneffects 柱外效应

PeakTailingColumn“SecondaryInteractions”峰拖尾柱“次级效应”

Column:

Alkyl-C8,4.6×150mm,5µm

MobilePhase:

85%25mMNa2HPO4,pH7.0:

15%ACN

FlowRate:

1.0mL/min        Temperature:

35ºC

Sample:

 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

NoTEA无三乙胺                             10mMTEA  10mM三乙胺

USPTF（5%）美国药典拖尾因子           USPTF（5%）美国药典拖尾因子

1. 1.29                                           1. 1.19

2. 1.91                                           2. 1.18      

3. 1.63                                           3. 1.20

4. 2.35                                           4. 1.26

5. 1.57                                           5. 1.14

Peaktailingeliminatedwithmobilephasemodifier（TEA）atpH7

用流动相减尾剂（三乙胺）在pH7消除峰拖尾

PeakTailingColumn“SecondaryInteractions”峰拖尾柱“次级效应”

Column:

Alkyl-C8,4.6×150mm,5µm

MobilePhase:

85%25mMNa2HPO4,pH7.0:

15%ACN

FlowRate:

1.0mL/min        Temperature:

35ºC

Sample:

 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

        pH3.0                                             pH7.0 

USPTF（5%）美国药典拖尾因子           USPTF（5%）美国药典拖尾因子

       4. 1.33                                           4. 2.35

Reducingthemobilephase pHreducesinteractionswithsilanolsthatcausepeaktailing

降低流动相的pH,减小与硅醇基作用引起的峰拖尾

PeakTailing峰拖尾ColumnContamination柱污染

Column:

StableBondSB-C8,4.6×250mm,5µm

MobilePhase:

20%H2O:

80%MeOH      FlowRate:

1.0mL/min        Temperature:

R.T.                                   Detection:

UV254nm

Sample:

1.Uracil 尿嘧啶                                   2.Phenol 苯酚    

             3.4-Chloronitrobenzene 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    1.06

2.12043   1.64         2.12443   1.21             2.12237   1.21

3.13727   1.69         3.17999   1.19             3.15366   1.11

4.13355   1.32         4.17098   1.25             4.19067   1.17

QCtestforwarddirection    QCtestreversedirection          QCtestaftercleaning

                                                                                                         100%IPA,35ºC 

       向前方向              相反方向                 100%异丙醇冲洗后

PeakTailing/Broadening                               SampleLoadEffects峰拖尾/变宽样品过载效应

Column:

4.6×150mm,5µm

MobilePhase:

40%25mMNa2HPO4,pH7.0:

60%ACN

FlowRate:

1.5mL/min        Temperature:

40ºC

Sample:

1.Desipramine 去郁敏/脱甲基丙米嗪  2.Nortriptyline                       3.Doxepin                                       4.Imipramine   丙米嗪                     5.Amitriptyline阿米替林          6.Trimipramine

Tailing EclipseXDB-C8             Broadening/ CompetitiveC8

USPTF拖尾因子                                  Plates理论板数

HighLoad /10    LowLoad          HighLoad /10     LowLoad

高载负/10倍         低载负             高载负/10倍       低载负

1. 1.60                                   1.70              850                        5941

2. 2.00                                   1.90              815                        7842

3. 1.56                                   1.56              2776                       6231                          

4. 2.13                                   1.70              2539                       8359              

5.