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【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【分享】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
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【分享】色谱柱和色谱系统的故障检修
【分享】色谱柱和色谱系统的故障检修
最新回复
q_r_epcnge (2010-4-06 15:33:37)
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
bing0421gs (2010-4-06 15:34:25)
ngoir (2010-4-06 15:35:53)
notrjhn (2010-4-06 15:40:54)
zxlyid (2010-4-06 15:54:20)
【分享】色谱柱和色谱系统的故障检修