色譜柱和色譜系統(tǒng)的故障排除.PPT

1、2020/8/9,1,HPLC Column and System Troubleshooting 色譜柱和色譜系統(tǒng)的故障檢修,What Every HPLC User Should Know 每個HPLC使用者應(yīng)該知道什么,2020/8/9,2,HPLC Components HPLC 的組成,Pump 泵 Injector/Autosampler 進(jìn)樣器/自動進(jìn)樣器 Column 色譜柱 Detector 檢測器 Data System/Integrator 數(shù)據(jù)系統(tǒng)/積分儀表 All of these components can have problems and require tr

2、oubleshooting. 所有這些部件可能出現(xiàn)故障而需要檢修。,2020/8/9,3,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,2020/8/9,4,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,

3、2020/8/9,5,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(tǒng)或保護(hù)柱的阻塞 If Column Pressure is high 如果柱壓高: Wash column - Eliminate column contamination and 沖洗柱子 pl

4、ugged 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,2020/8/9,6,Column Cleaning 柱子清洗,F lush with stronger solvents than your mobile phase

5、 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

6、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(dāng)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,2020/8/9,7,Column Cleaning柱子清洗,Normal Phase Solvent Cho

7、ices 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,2020/8/9,8,How to Change a Frit 怎樣更換濾頭,Column Inlet Frit 柱進(jìn)口濾頭 Column Body 柱身 Compression Ferrule 壓縮金屬套圈 Wear gloves 戴手套 Do not allo

8、w 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,2020/8/9,9,Preventing Back Pressure Problems壓力問題的預(yù)防,Use column protection 柱保護(hù) - Guard columns 保護(hù)柱/預(yù)柱 - In-line filters 在線過濾器 Sample

9、 Preparation 樣品預(yù)處理 Appropriate column flushing 適當(dāng)?shù)闹鶝_洗 Filter buffered mobile phase 過濾緩沖流動相,2020/8/9,10,Preventing Back Pressure Problems:In-Line Devices 壓力問題的預(yù)防:在線裝置,Mobile Phase Pre-Column Injector Filter Guard From Pump 預(yù)柱 進(jìn)樣器 濾頭 Column 從泵來的流動相 保護(hù)柱 Analytical Column Filter and Guard Column Act on

10、Sample 分析柱 濾頭和保護(hù)柱作用于樣品 Pre-Column Acts on Mobile Phase 預(yù)柱作用于流動相 To Detector 到檢測器,2020/8/9,11,Preventing Back Pressure Problems: Sample Preparation壓力問題的預(yù)防:樣品制備,Solvent/Chemical Environment 溶劑/化學(xué)環(huán)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.

11、P.E.) 固相萃取 Cartridges or Plates 薄膜或薄層板 Disks or Membranes 圓盤或生物膜,2020/8/9,12,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(jié)合在一起的-例如: 展寬和拖尾或拖尾和保留時間增加,

12、2020/8/9,13,Split Peaks 裂峰,Can be caused by: 可能的原因: Column contamination 柱污染 Partially plugged frit 部分阻塞濾片 Column void 柱頭塌陷 Injection solvent effects 溶劑效應(yīng),2020/8/9,14,Split Peaks 裂峰Column Contamination 柱污染,Column: StableBond SB-C8, 4.6 250mm, 5m Mobile Phase:60%25mM Na2HPO4,pH3.0 : 40%MeOH Flow Rate

13、: 1.0 mL/min Temperature: 35C 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(jīng)100%乙腈沖洗后 峰3 峰形極好,2020/8/9,15,Split Peaks 裂峰Injection Solvent Effects

14、 溶劑化效應(yīng),Column: StableBond SB-C8, 4.6 250mm, 5m Mobile Phase: 82%H2O : 18%ACN Injection Volume: 30 L 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,2020/8/9,16,Determining the Cause of Split Peaks 裂峰原因的確

15、定,1. Complex sample matrix or many samples analyzed- likely column contamination or partially plugged frit 復(fù)雜樣品的基質(zhì)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 tha

16、n 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,2020/8/9,17,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(yīng)” Column void 柱塌陷 Column contamination 柱污染 Column aging 柱老化 Column loadin

17、g 柱負(fù)荷超載 Extra-column effects 柱外效應(yīng),2020/8/9,18,Peak Tailing Column “Secondary Interactions”峰拖尾柱“次級效應(yīng)”,Column: Alkyl-C8, 4.6 150mm, 5m Mobile Phase:85%25mM Na2HPO4,pH7.0 : 15%ACN Flow Rate: 1.0 mL/min Temperature: 35C 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.

18、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,2020/8/9,19,Peak

19、Tailing Column “Secondary Interactions”峰拖尾柱“次級效應(yīng)”,Column: Alkyl-C8, 4.6 150mm, 5m Mobile Phase:85%25mM Na2HPO4,pH7.0 : 15%ACN Flow Rate: 1.0 mL/min Temperature: 35C 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

20、 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,2020/8/9,20,Peak Tailing 峰拖尾Column Contamination 柱污染,Column: StableBond SB-C8, 4.6 250mm, 5m Mobile Phase: 20% H2O : 80% MeOH

21、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(shù) 拖尾因子 理論板數(shù) 拖尾因子 理論板數(shù) 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. 1

22、7999 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, 35C 向前方向 相反方向 100%異丙醇沖洗后,2020/8/9,21,Peak Tailing/Broadening Sample Load Effects峰拖尾/變寬樣品過載效應(yīng),Column: 4.6 150mm, 5m Mobile Phase:40%25mM Na2HPO4,pH7.0

23、 : 60%ACN Flow Rate: 1.5 mL/min Temperature: 40C 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(shù) High Load / 10 Low Load High Load / 10 Low Load 高載負(fù)/10倍 低載

24、負(fù) 高載負(fù)/10倍 低載負(fù) 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 10725,2020/8/9,22,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

25、 30 injections Initial 開始時 進(jìn)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(shù)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(dǎo)致塌陷,2020/8/9,23,Broad Peaks Unknown “Phantom” Peaks,Column: Extend-C18, 4.6 150 mm, 5 m Mobile Phase:

26、 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(shù) 1. 5922 2. 9879 3. 779 “Phantom” peak from first injection 前次進(jìn)樣未出完的峰 The extremely low plates are an indication of an extr

27、emely late eluting peak from the preceding run. 極低的理論板數(shù)表明這是一個前面進(jìn)樣極遲洗脫出來的峰,2020/8/9,24,Peak Tailing峰拖尾 Injector Seal Failure進(jìn)樣器密封性差,Column: Bonus-RP, 4.6 75mm, 3.5m 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-

28、Dimethylaniline N,N-二甲苯胺 Plates TF Plates TF 理論板數(shù) 拖尾因子 理論板數(shù) 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(zhuǎn)動桿和隔離密封墊檢修后 Overdue instrument maintenance can cause peak shape problems. 儀器保養(yǎng)超期可能引起峰形問題/進(jìn)樣器劃痕,202

29、0/8/9,25,Peak Tailing 峰拖尾Extra-Column Volume 柱外死體積效應(yīng),Column: StableBond SB-C18, 4.6 30mm, 3.5m Mobile Phase: 85% H2Owith 0.1% TFA : 15% CAN Flow Rate: 1.0 mL/min Temperature: 35C Sample: 1. Phenylalanine 2. 5-benzyl-3,6-dioxo-2-piperazine 3. Asp-phe 4. Aspartame 10 l extra-column volume 50 l extra-c

30、olumn volume,2020/8/9,26,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(yīng)/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 te

31、chnology 評價柱選擇 - 試用高純度硅膠柱或不同鍵合技術(shù)柱 Reduce sample load 減小進(jìn)樣量 Eliminate extra-column effects 消除柱外效應(yīng) Flush column and check for aging/void 沖洗柱子檢查柱子老化/塌陷,2020/8/9,27,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,2020/8

32、/9,28,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,2020/8/

33、9,29,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(fā)的三氟乙酸從流動相中蒸發(fā)/揮發(fā)。 Replacing it solved problem. 換用新的流動相就解決問題。 使用有機揮發(fā)性酸時，流動相應(yīng)新配。,2020/8/9,30,Column Aging/Equilibration Causes Ret

34、ention/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 fl

35、ushing 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(dá)到了預(yù)期效果,2020/8/9,31,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(shù)據(jù) 4. Revie