主题：【分享】色谱柱和色谱系统的故障检修

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
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。
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
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
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
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
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
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
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 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
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
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
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
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

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
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. Methamp