液相色譜：色譜柱和色譜系統的故障檢查

分析化學  儀器分析 

每個HPLC使用者應該知道什么
HPLC Components HPLC 的組成：
    Pump 泵
    Injector/Autosampler 進樣器/自動進樣器
    Column 色譜柱
    Detector 檢測器
    Data System/Integrator 數據系統/積分儀表

All of these components can have problems and require troubleshooting.
所有這些部件可能出現故障而需要檢修。
Categories of Column Problems  色譜柱問題的類型：
 
   A. Pressure 壓力
   B. Peak shape 峰形
   C. Retention 保留時間
Pressure Issues 壓力問題
  Column Observations                     Potential Problems
  色譜柱觀測 ：                           潛在的問題 ：    
  High pressure                           Plugged frit 
  壓力高                                  堵塞濾頭
                                          Column contamination
                                          色譜柱污染
                                          Piugged packing
                                          堵塞填料
  Determining the Cause and Correcting High Back Pressure  
  查出原因 糾正壓力
Check pressure with/without column - many pressure problems are due to blockages in the system or guard
有／無柱子時檢查壓力  －  許多壓力問題是由于系統或保護柱的阻塞
If Column Pressure is high 如果柱壓高:
Wash column - Eliminate column contamination and
 沖洗柱子 ：          
                     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
                     樣品或緩沖液中的沉淀物
Back flush column - Clear plugged frit  
 反沖柱子－清潔阻塞的 濾頭
Change frit - Clear plugged frit   
更換濾頭
Column Cleaning
柱 子清洗
F lush with stronger solvents than your mobile phase
用比你的流動相更強 的溶劑沖洗
  
   Reversed-Phase Solvent Choices in Order of
   Increasing Strength   
   為了增加強度選擇反相溶劑
   Use at least 25 ml of each solvent for analytical columns
   對于分析柱每種溶劑至少用25 ml 沖洗
   Mobile phase without buffer salts  
   沒有緩沖鹽的流動相
   100% Methanol
   甲醇
   100% Acetonitrile
   乙腈
   75% Acetonitrile :25% Isopropanol
   75乙腈 ： 25％異丙醇
   100% Isopropanol
   異丙醇
   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.
當用已烷蔌二氯甲烷柱子時,必須先用異丙醇沖洗,然后再用你的反相流動相
Column Cleaning柱子清洗
Normal Phase Solvent Choices in Order of Increasing Strength
為了增加強度選用正相溶劑
Use at least 50 ml of each solvent 
每種溶劑 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
怎樣更換濾頭：
Column Inlet Frit
柱進口濾頭
Column Body
柱身
Compression Ferrule
壓 縮金屬套圈
Wear gloves
戴手套
Do not allow bed to dry
不要讓底座干燥
Do not touch the column-body heat will extrude packing     
不要使柱身受熱,否則 填料會噴出
Do not over tighten
不要旋得太緊
Female End Fitting
旋好尾端螺母
Male End Fitting
旋好尾端螺頭
Preventing Back Pressure Problems
壓力問題的預 防：
Use column protection
柱保護
- Guard columns
保護柱/預柱
- In-line filters
在線過濾器
Sample Preparation
樣品預處理
Appropriate column flushing
適當的柱沖洗
Filter buffered mobile phase
過濾緩沖流動相
Preventing Back Pressure Problems:In-Line Devices
 壓力問題的預防:在線裝置
Mobile Phase    Pre-Column    Injector  Filter  Guard
From Pump            預柱             進樣器      濾頭   Column
從泵來的流動 相                                                    
保護 柱                                                       
                                                                   Analytical
                                                                    Column
Filter and Guard Column Act on Sample      分析柱
           濾頭和保護柱作用于樣品
   Pre-Column Acts on Mobile Phase
               預柱作用于流動相                           
To Detector
                                                                      到檢測器
Preventing Back Pressure Problems: Sample Preparation 壓力問題的預防:樣品制備
Solvent/Chemical Environment
 溶劑/化學環境
Particulate/Aggregate Remove 
微粒/凝聚物的除去
Filter samples  過濾樣品
Centrifugation  離心分離
Solid Phase Extraction(S.P.E.)  固相萃取
Cartridges or Plates  薄膜或薄層板
Disks or Membranes  圓盤或生物膜
Peak Shape Issues 峰形問題：
      Split peaks   裂峰
      Peak tailing  峰拖尾
      Broad peaks   寬峰
Poor efficiency   低柱效
Many peak shape issues also combinations-I.e. Broad and tailing or tailing with increased retention許多峰形問題是結合在一起的-例如:
展寬和拖 尾或拖尾和保留時間增加
Split Peaks  裂峰
Can be caused by:
可能的原因:
Column contamination  柱污染
Partially plugged frit 部分阻塞濾片
Column void  柱頭塌陷
Injection solvent effects 溶劑效應
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    峰形較好
Injection 30     峰3    裂峰
Injection  1    After Column Wash with 100% CAN
經100%乙腈沖洗后   峰3    峰形極好
Split Peaks  裂峰 Injection Solvent Effects 溶劑化效應
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  水楊酰胺
A. Injection Solvent                               B. Injection Solvent
   100% Acetonitrile                                   Mobile Phase
    峰形寬拖尾                                                峰形尖銳對稱
樣品溶劑盡可能與流動相匹配
Determining the Cause of Split Peaks   裂峰原因的確定
1. Complex sample matrix or many samples analyzed-    likely column contamination or partially plugged frit
復雜樣品的基質或分析許多樣品 - 柱污染或部分阻塞濾片
2. Mobile phase pH>=7 - likely column void due to silica dissolution (unless specialty column used)
流動相 pH>=7 - 由于硅膠溶解使柱塌陷(除非使用專門的柱子)
3. Injection solvent stronger than mobile phase - likely split and broad peaks, dependent on volume
溶劑比流動相的可能性大 - 裂峰和寬峰,由樣品量來決定
Peak Tailing, Broadening and Loss of Efficiency 峰拖尾、變寬和柱效降低
Can be caused by: 可能的原因
     
Column "secondary interactions"
柱"次級效應"
Column void  柱塌陷
Column contamination  柱污染
Column aging  柱老化
Column loading  柱負荷超載
Extra-column effects  柱外效應
Peak Tailing Column "Secondary Interactions" 峰拖尾柱"次級效應"
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／去甲麻黃堿 
2. Ephedrine  麻黃堿    3. Amphetamine   安非他明／苯丙胺
4. Methamphetamine  脫氧麻黃堿    5. Phenteramine  苯三胺
No TEA 無三乙胺                              10 mM TEA   10 mM 三乙胺
USP TF(5%) 美國藥典拖尾因子            USP TF(5%) 美國藥典拖尾因子
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
用流動相減尾劑(三乙胺)在pH 7 消除峰拖尾
Peak Tailing Column "Secondary Interactions" 峰拖尾柱"次級效應"
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／去甲麻黃堿 
2. Ephedrine  麻黃堿    3. Amphetamine   安非他明／苯丙胺
4. Methamphetamine  脫氧麻黃堿    5. Phenteramine  苯三胺
         pH 3.0                                              pH 7.0 
USP TF(5%) 美國藥典拖尾因子            USP TF(5%) 美國藥典拖尾因子
        4.  1.33                                            4.  2.35
Reducing the mobile phase  pH reduces interactions with silanols that cause peak tailing
降低流動相的pH ,減小與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
理論板 數    拖尾因子        理論板數    拖尾因子           理論板數    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%異丙醇沖洗后
Peak Tailing/Broadening                                 Sample Load Effects 峰拖尾/變寬樣品過載效應
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/脫甲基丙米嗪   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 理論板數
High Load  / 10     Low Load           High Load  / 10      Low Load
高載負/10 倍          低載負              高載負/10倍        低載負
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    峰變寬,裂開 柱塌陷
Mobile Phase:流動相：
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 開始時
             進樣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.
多數峰形改變可能是同一柱問題 引起的。在這個例子中，高pH溶解硅膠導致塌陷
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  順丁烯二酸          2. Pseudeophedrine 偽麻黃堿      3. Chlorphenniramine  氯非尼拉明
Plates  理論板數
1. 5922
2. 9879
3.  779  "Phantom" peak from first injection 前次進樣未出完的峰
The extremely low plates are an indication of an extremely late eluting peak from the preceding run.
極低的理論板數表明這是一個前面進樣極遲洗脫出來的峰
Peak Tailing峰拖尾 Injector Seal Failure進樣器密封性差
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                 
理論板數    拖尾因子                                  理論板數    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                     轉動桿和隔離密封墊檢修后
Overdue instrument maintenance can cause peak shape problems.
儀器保養超期可能引起峰形問題/進樣器劃痕
Peak Tailing 峰拖尾 Extra-Column Volume  柱外死體積效應
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 峰拖尾原因的確定
Evaluate mobile phase effects - alter mobile phase pH and additives to eliminate secondary interactions         評價流動相效果 - 改變流動相pH和添加劑消除次級效應/流動相中組份與柱子相互作用
Evaluate column choice - try column with high purity silica or different bonding technology                              評價柱選擇 - 試用高純度硅膠柱或不同鍵合技術柱
Reduce sample load   減小進樣量
Eliminate extra-column effects  消除柱外效應
Flush column and check for aging/void                            沖洗柱子檢查柱子老化/塌陷
Retention Issues 保留時間問題
Retention time changes(tr)                        保留時間改變
Capacity factor (retention) changes (k´)        容量因子改變
Selectivity changes ()                           選擇性改變
Changes in Retention Same Column, Over Time相同柱子上保留時間改變,超時
May be cause by:                                     可能的原因:
Column aging  柱老化
Column contamination  柱污染
Insufficient equilibration  不夠平衡
Poor column/mobile phase combination                                                                                                           柱/流動相組成差
Change in mobile phase  改變流動相
Change in flow rate  改變流速
other instrument issues  其他儀器問題
Mobile phase Change Causes Change in Retention 流動相改變引起保留時間改變
60%MeOH:40% 0.1% TFA       Fresh TFA Added to Mobile Phase
 
Volatile TFA evaporated/degassed from mobile phase.    易揮發的三氟乙酸從流動相中蒸發/揮發。
Replacing it solved problem.                                            換用新的流動相就解決問題。
使用有 機揮發性酸時，流動相應新配。
Column Aging/Equilibration Causes Retention/Selectivity Changes 柱老化／平衡不夠引起保留時間／選擇性改變
Column 1 - Initial    1  號柱      開始時
Column 1 - Next Day      1  號柱      第二天         
Column 1 - After Cleaning with 1% H3PO4                                       1  號柱用 1% H3PO4 清洗   

The primary analyte was sensitive to mobile phase aging of the column.     最初的分析對流動相老化柱子是靈敏的
The peak shape was a secondary issue resolved by flushing the column.     沖洗柱子第二要解決的問題是峰形
Retention and peak shape were as expected after cleaning.                       清洗后保留時間和峰形都達到了預期效果
Determining the Cause of Retention Changes on the same Column 同一柱上保留時間改變原因的確定
1. Determine K´, , and tr for suspect peaks  對懷疑峰K´, 和tr的確定
2. Wash column  沖洗柱子
3. Test new column - note lot number                  試驗新柱- 記錄一批數據
4. Review column equilibration procedures         觀察柱平衡過程
5. Make up fresh mobile phase4 and test             配制新鮮流動相再試驗
6. Check instrument performance 檢查儀器性能
Change in Retention/Selectivity Column-to Column 柱與柱之間保留時間/選擇性的改變
Different column histories (aging)                              不同的柱歷史(老化)
Insufficient/inconsistent equilibration
平衡不夠 /不一致
Poor column/mobile phase combination                        柱/流動相差
Change in mobile phase流動相改變
Change in flow rate 流速改變
Other instrument issues其他儀器問題
Slight changes in column bed volume (tr only)                柱內體積輕微改變
Lot-to-Lot Selectivity Change (pH) 批與批之間選擇性改變(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 顯示批與批之間對堿性化合物的選擇性改變
pH 3.0 shows no selectivity change from lot-to-lot, indicating silanol sensitivity at pH 4.5    pH 3.0顯示批與批之間對堿性化合物的無選擇性改變,表明在pH 4.5硅醇基的靈敏性
Conclusions    結論
HPLC column problem are evident as:  高效液相色譜柱問題明顯的為:
High pressure   高壓
Undesirable peak shape   不希望得到的峰形
Changes in retention/selectivity   保留時間/選擇性的改變                                                   
Often these problems are not associated with the column and may be caused by instrument and experimental condition issues.
經常遇到的這些問題不一定與柱有關,而與儀器和實 驗條件方面的問題有關 

分析化學  儀器分析