液相色谱：色谱柱和色谱系统的故障检查

分析化学  仪器分析 

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. 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
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
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.
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
2. 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(tr)                        保留时间改变
Capacity factor (retention) changes (k´)        容量因子改变
Selectivity changes ()                           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。
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´, , and tr for suspect peaks  对怀疑峰K´, 和tr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 (tr 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 

分析化学  仪器分析