主题：Column

Báez-Saldaña A, Zendejas-Ruiz I, Revilla-Monsalve C, et al. Effects of biotin on pyruvate carboxylase, acetyl-CoA carboxylase, propionyl-CoA carboxylase, and markers for glucose and lipid homeostasis in type 2 diabetic patients and nondiabetic subjects. Am J Clin Nutr 2004;79:238–43.

The doses of biotin used in the study are wrongly reported in several instances. In the Objective section of the abstract, on page 240 (left column, first full paragraph; right column, paragraph 1), and in the legends for Figures 1, 2, and 3, the correct dose is 61.4 µmol/d. On page 239 (left column, third full paragraph) and on page 242 (left column, line 5), the correct dose is 20.5 µmol/d.

Purification of Demethylated Sphingomyelin

Contributor: Suprya Jayadev
Date: Feb. 20, 1991
1) Pack a Bio-Sil A column as follows:
a) Pack base of column with a small portion of glass wool.
b) Suspend 20 grams of 100-200 mesh BioSil A in 100 ml chloroform.
c) Pour silica/chloroform mixture into column eluting chloroform from base.
- Press out any air bubbles in the glass wool as the gel is initially being poured. While pouring column matrix, stir continuously with a stir bar in order to prevent bubble formation in the column.
d) Wash column through with "200 ml chloroform.
- ALWAYS maintain a fluid level above the packing silica... NEVER let column dry out!!
2) Dry pooled lower phases from the demethylation reaction using the rotovapor system.
--> DO NOT heat!
3) Resuspend dried lipid in a minimal volume of chloroform and carefully layer over packed column.
4) Allow sample to run into column while maintaining a small level of chloroform above the packed silica.
5) Sequentially elute with the following solvents:
a) 100 ml chloroform
b) 200 ml chloroform/methanol (20:1)
c) 200 ml chloroform/methanol (9:1)
d) 200 ml chloroform/methanol (5:1)
e) 200 ml chloroform/methanol (4:1)
f) 800 ml chloroform/methanol (3:1)
g) 200 ml chloroform/methanol (3:1)
6) Dry down fractions (f) & (g) separately and re-suspend in 4 ml of chloroform/methanol (1:1).
7) Spot 2 µl of each fraction and a small portion of DMSM & SM standard onto a TLC plate.
8) Run TLC in chloroform/methanol/ammonium hydroxide (60:35:8) and develop spots using iodine vapor then potassium permanganate spray.
--> Fraction (f) should contain the DMSM.
9) Completely dry down the DMSM containing fraction.
10) Determine gram weight of DMSM recovered.
11) Resuspend in 2 ml of methanol and store at -20°C until needed for labeled SM synthesis.

维生素A 
方法：  
Column：HPLC 
Gradient：None 
Flow Rate：1.0 ml/min 
Inj Vol：5 祃 
Detection：UV @ 280nm 
 
 
 
组分： 
vitamin K3 (37 礸/ml) 
vitamin A (74) 
vitamin A Acetate (222) 
vitamin D2 (111) 
vitamin D3 (37) 
alpha-tocopherol (667) 
vitamin E acetate (667) 
vitamin K1 (370) 
 
 

Capillary Column Installation Quick Reference Guide

毛细柱安装快捷指南

Precolumn installation check list
预检查清单

1. Replace oxygen, moisture, and hydrocarbon traps as needed.

如有必要，替换掉除氧、除湿、除烃的捕集阱。

2. Check gas cylinder pressure to ensure that an adequate supply of carrier, makeup, and fuel gases is available. Minimum carrier gas purity percentages: helium 99.9995, hydrogen 99.9995.

检查气瓶压力，确认载气、补充气、可燃气合适。载气纯度百分比最小应达到：氦气，99.995；氢气，99.995。

3. Clean the injector port, replace critical injection port seals, replace injection port liners, and change septa as needed.

清洗进样口，如有必要，更换密封垫，衬管，隔垫。

4. Check detector seals, and replace as necessary. Clean or replace detector jets as necessary.

检查检测器密封垫，如有必要就替换。如有必要清洗或替换检测器喷嘴。

5. Carefully inspect the column for damage or breakage.

仔细检查柱是否破损。

6. Gather the necessary installation tools: you will need a column cutter, column nuts, ferrules, a magnifying loop, and typewriter correction fluid.

收集必要的安装工具：柱切割器、柱螺母、垫圈、放大镜、打字机改正液。

Installing the column
安装柱

1. Uncoil approximately 0.5m of tubing from the column basket at both ends of the column for injector and detector installation. Avoid sharp bends in the tubing.

从柱圈两端各解开约0.5米的柱，以便进样口和检测器安装。

2. Mount the column in the oven. Use the hanging bracket if available.

在柱箱中装柱。如果可能的话，用柱悬挂架。

3. Install the column nut and Vespel or graphite ferrule at each column end; pull the nut and ferrule down the tubing approximately 5 cm.

在柱两端按装螺母和Vespel或石墨垫。将螺母和垫圈顺着柱拉下约5厘米。

4. Score the column. Use a light touch to score the column about 4 to 5 cm from each end.

切割柱。距柱每个尾端约4-5cm处轻划一下。

5. Make a clean break. Grasp the column between the thumb and forefinger as close to the score point as possible. Gently pull and bend the column. The column should part easily. If the column doesn’t break easily, don’t force it! Score the column again in different place, and try for a clean break.

确保整齐的划口。用拇指和食指抓住柱，离划痕处越近越好。轻轻地弯曲柱。柱应该很容易地折断。如果柱不能折断，不能强行用力。在不同的地方再割一次柱，尝试获是整齐地划口。

6. Use a magnifying loop to inspect the cut. Make sure the cut is square across the tubing with no polyimide or glass fragments at the end of the tube.

用放大镜检查划口。确认划口正交于柱，柱端没有聚酰亚胺或石英碎片。

7. Install the column in the inlet. Check the GC manufacturer’s instrument manual for the correct insertion distance. Mark the correct distance on the column with typewriter correction fluid. Insert the column into the injector. Finger tighten the column nut until it starts to grab the column, and then tighten the nut an additional 1/4 to 1/2 turn so that the column can’t be pulled from the fitting when gentle pressure is applied.

在进样口装柱。查阅GC生产商的仪器说明书，以便获取正确的安装距离。用打字机改正液在柱子上标识正确地距离。将柱插入进样口。用手拧紧柱螺母直至其开始夹住柱子。再拧1/4-1/2圈，这样轻轻拉动时，柱子不会脱出来。

8. Turn on the carrier gas, and establish the proper flow rate. Set head pressure, split flow, and septum purge flow to appropriate levels. See Table B for nominal head pressure. If using a split/splitless inlet, check that the purge valve is ON.

接通载气，调至合适地流速。设置相应的柱头压、分流比、隔垫吹扫。常规地柱头压参考表B。如果使用分流/不分流进样口，检查分流阀是否打开。

9. Confirm carrier gas flow through the column. Immerse the end of the column in a vial of acetone and check bubbles.

确保载气通过了柱子。将柱尾端入丙酮中检查气泡。

10. Install the column into detector. Check the instrument maufacture’s manual for the proper insertion distance.

将柱子装进检测器。查阅仪器生产商的说明书，以便获取合适的安装距离。

11. Check for leaks. This if very important. Don’t enven think about heating the column without thoroughly checking for leaks.

检漏。这非常重要。不能不检漏就加热柱子。

12. Establish proper injector and detector temprature.

给进样口和检测器设定恰当的温度。

13. Establish proper makeup and detector gas flows. Ignite or turn on the detector.

设定恰当的补充气和检测气流量。点火或打开检测器。

14. Purge the column for a minimum of 10 minutes at ambient temperature(approximately six column volumes).

在室温下冷吹柱子至少10分钟(约6倍的柱体积)。

15. Inject a nonretained substance to check for proper injector installation. Examples: butane or methane(FID),headspace vapors from acetonitrile(NPD), methylene chloride(ECD),air(TCD),argon(mass spectrometer). Proper installation is indicated by a symmetrical peak. If tailing is observed, repeat the injector installation process.

进没有保留的物质检查进样口安装是否合适。如丁烷或甲烷(FID)，乙腈顶空蒸汽(NPD)，氯仿(ECD)，空气(TCD)，氩气(MSD)。安装合适会得到对称的峰形。如果峰拖尾，重新安装进样口。

1.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：

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 problems and require troubleshooting.

所有这些部件可能出现故障而需要检修。

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

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 packing

                                          堵塞填料

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

 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

6.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

^l100% Methylene Chloride^★  二氯甲烷

^l100% Hexane ^★  已烷

^★  When using either Hexane or Methylene Chloride the column must be flushed with

lIsopropanol 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

7.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

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

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

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

Appropriate column flushing

                     适当的柱冲洗

Filter buffered mobile phase

                     过滤缓冲流动相

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  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

 

Preventing Back Pressure Problems: Sample Preparation
11.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

Peak Shape Issues
12.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

13.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

14.Split Peaks  裂峰
Column Contamination  柱污染

Column: StableBond SB-C8, 4.6 × 250mm, 5µm

Mobile Phase:60%25mM Na₂HPO₄,pH3.0 : 40%MeOH

Flow Rate: 1.0 mL/min         Temperature: 35ºC

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

15.Split Peaks  裂峰
Injection Solvent Effects 溶剂化效应

Column: StableBond SB-C8, 4.6 × 250mm, 5µm

Mobile Phase: 82%H₂O : 18%ACN

Injection Volume: 30 µL      

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

16.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

Peak Tailing, Broadening and Loss of Efficiency
峰拖尾、变宽和柱效降低

 

17.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

18.Peak Tailing Column “Secondary Interactions”
峰拖尾柱“次级效应”

Column: Alkyl-C8, 4.6 × 150mm, 5µm

Mobile Phase:85%25mM Na₂HPO₄,pH7.0 : 15%ACN

Flow Rate: 1.0 mL/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

No TEA 无三乙胺                              10 mM TEA   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.  1.19

2.  1.91                                            2.  1.18      

3.  1.63                                            3.  1.20

4.  2.35                                            4.  1.26

5.  1.57                                            5.  1.14

19.Peak 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

Peak Tailing Column “Secondary Interactions”
峰拖尾柱“次级效应”

Column: Alkyl-C8, 4.6 × 150mm, 5µm

Mobile Phase:85%25mM Na₂HPO₄,pH7.0 : 15%ACN

Flow Rate: 1.0 mL/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

 

         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.  2.35

 

Reducing the mobile phase  pH reduces interactions 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

Peak Tailing 峰拖尾
Column Contamination 柱污染

Column: StableBond SB-C8, 4.6 × 250mm, 5µm

Mobile Phase: 20% H₂O : 80% MeOH       Flow Rate: 1.0 mL/min         Temperature: R.T.                                    Detection: UV 254 nm

 

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

QC test forward direction     QC test reverse direction           QC test after cleaning

                                                                                                          100% IPA, 35ºC 

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

Peak Tailing/Broadening
                                Sample Load Effects
峰拖尾/变宽样品过载效应

Column: 4.6 × 150mm, 5µm

Mobile Phase:40%25mM Na₂HPO₄,pH7.0 : 60%ACN

Flow Rate: 1.5 mL/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  Eclipse XDB-C8              Broadening /  Competitive C8

USP 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                             5941

2.  2.00                       1.90                          815                             7842

3.  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        

Peak Broadening,Splitting Column Void
   峰变宽,裂开 柱塌陷

20.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.

21.多数峰形改变可能是同一柱问题引起的。在这个例子中，高pH溶解硅胶导致塌陷

Broad Peaks Unknown “Phantom” Peaks

Column: Extend-C18, 4.6 × 150 mm, 5 µm

Mobile Phase: 40% 10 mM TEA, pH 11 : 60% MeOH

Flow Rate: 1.0 mL/min      Temperature: R.T.      Detection:  UV 254

Sample:     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. 9879

3.  779  “Phantom” 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.

22.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

Peak Tailing峰拖尾
Injector Seal Failure进样器密封性差

Column: Bonus-RP, 4.6 × 75mm, 3.5µm

Mobile Phase: 30% H₂O : 70% MeOH       Flow Rate: 1.0 mL/min         Temperature: R.T.                                    Detection: UV 254 nm

 

Sample: 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    1.45

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 maintenance can cause peak shape problems.

仪器保养超期可能引起峰形问题/进样器划痕

Peak Tailing 峰拖尾
Extra-Column Volume  柱外死体积效应

Column: StableBond SB-C18, 4.6 × 30mm, 3.5µm

Mobile Phase: 85% H₂Owith 0.1% TFA : 15% CAN

Flow Rate: 1.0 mL/min         Temperature: 35ºC                                   

 

Sample: 1. Phenylalanine   2. 5-benzyl-3,6-dioxo-2-piperazine   

              3. Asp-phe             4. Aspartame

 

 

 

 

 

10 µl extra-column volume               50 µl extra-column volume

 

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

Evaluate 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

Retention Issues
保留时间问题

Retention time changes(t_r)                        保留时间改变

Capacity factor (retention) changes (k´)        容量因子改变

Selectivity changes (a)                           选择性改变

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  不够平衡

Poor column/mobile phase combination                                                                                                           柱/流动相组成差

Change in mobile phase  改变流动相

Change in flow rate  改变流速

other instrument issues  其他仪器问题

Mobile phase Change Causes Change in Retention
流动相改变引起保留时间改变

60%MeOH:40% 0.1% TFA       Fresh TFA Added to Mobile Phase

 

 

 

Volatile 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。

 

23.使用有机挥发性酸时，流动相应新配。

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   

 

 

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

Determining the Cause of Retention Changes on the same Column
同一柱上保留时间改变原因的确定

1. Determine K´, a, and t_r for suspect peaks  对怀疑峰K´, a和t_r的确定

2. Wash column  冲洗柱子

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

5. Make up fresh mobile phase4 and test             配制新鲜流动相再试验

6. Check instrument performance 检查仪器性能

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 流速改变

Other instrument issues其他仪器问题

Slight changes in column bed volume (t_r only)                柱内体积轻微改变

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 1

pH 4.5 - Lot 2                      pH 3.0 - Lot 2

 

 

pH 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 shows 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

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

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