色譜柱和色譜系統(tǒng)的故障檢修

2017/11/3,1,HPLC Column and System Troubleshooting 色譜柱和色譜系統(tǒng)的故障檢修,What Every HPLCUser Should Know每個(gè)HPLC使用者應(yīng)該知道什么,請(qǐng)?jiān)L問中國(guó)色譜網(wǎng)(www.SeP),2017/11/3,2,HPLC Components HPLC 的組成,Pump 泵 Injector/Autosampler 進(jìn)樣器/自動(dòng)進(jìn)樣器 Column 色譜柱 Detector 檢測(cè)器 Data System/Integrator 數(shù)據(jù)系統(tǒng)/積分儀表All of these components can have problems and require troubleshooting. 所有這些部件可能出現(xiàn)故障而需要檢修。,2017/11/3,3,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(shí)間,2017/11/3,4,Pressure Issues壓力問題,Column Observations Potential Problems色譜柱觀測(cè) 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,2017/11/3,5,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(shí)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 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,2017/11/3,6,Column Cleaning 柱子清洗,F lush with stronger solvents than your mobile phase用比你的流動(dòng)相更強(qiáng)的溶劑沖洗 Reversed-Phase Solvent Choices in Order of Increasing Strength 為了增加強(qiáng)度選擇反相溶劑Use at least 25 ml of each solvent for analytical columns 對(duì)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(dòng)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(dāng)用已烷蔌二氯甲烷柱子時(shí),必須先用異丙醇沖洗,然后再用你的反相流動(dòng)相,2017/11/3,7,Column Cleaning柱子清洗,Normal Phase Solvent Choices in Order of Increasing Strength為了增加強(qiáng)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,2017/11/3,8,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 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(huì)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,2017/11/3,9,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 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(dòng)相,2017/11/3,10,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 GuardFrom Pump 預(yù)柱 進(jìn)樣器 濾頭 Column從泵來的流動(dòng)相 保護(hù)柱 Analytical ColumnFilter and Guard Column Act on 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(dòng)相 To Detector 到檢測(cè)器,2017/11/3,11,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.P.E.) 固相萃取Cartridges or Plates 薄膜或薄層板Disks or Membranes 圓盤或生物膜,2017/11/3,12,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(shí)間增加,2017/11/3,13,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),2017/11/3,14,Split Peaks 裂峰Column Contamination 柱污染,Column: StableBond SB-C8, 4.6 250mm, 5mMobile Phase:60%25mM Na2HPO4,pH3.0 : 40%MeOHFlow Rate: 1.0 mL/min Temperature: 35CDetection: UV 254 nmSample: Filtered OTC Cold Medication: 1. Pseudoephedrine 2. APAP 3. Unknown 4. ChlorpheniramineInjection 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,2017/11/3,15,Split Peaks 裂峰Injection Solvent Effects 溶劑化效應(yīng),Column: StableBond SB-C8, 4.6 250mm, 5mMobile Phase: 82%H2O : 18%ACNInjection 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(duì)稱樣品溶劑盡可能與流動(dòng)相匹配,2017/11/3,16,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(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(dòng)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(dòng)相的可能性大- 裂峰和寬峰,由樣品量來決定,2017/11/3,17,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(jí)效應(yīng)”Column void 柱塌陷Column contamination 柱污染Column aging 柱老化Column loading 柱負(fù)荷超載Extra-column effects 柱外效應(yīng),2017/11/3,18,Peak Tailing Column “Secondary Interactions”峰拖尾柱“次級(jí)效應(yīng)”,Column: Alkyl-C8, 4.6 150mm, 5mMobile Phase:85%25mM Na2HPO4,pH7.0 : 15%ACNFlow Rate: 1.0 mL/min Temperature: 35CSample: 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(guó)藥典拖尾因子 USP TF(5%) 美國(guó)藥典拖尾因子1. 1.29 1. 1.192. 1.91 2. 1.18 3. 1.63 3. 1.204. 2.35 4. 1.265. 1.57 5. 1.14Peak tailing eliminated with mobile phase modifier (TEA) at pH 7用流動(dòng)相減尾劑(三乙胺)在pH 7 消除峰拖尾,2017/11/3,19,Peak Tailing Column “Secondary Interactions”峰拖尾柱“次級(jí)效應(yīng)”,Column: Alkyl-C8, 4.6 150mm, 5mMobile Phase:85%25mM Na2HPO4,pH7.0 : 15%ACNFlow Rate: 1.0 mL/min Temperature: 35CSample: 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(guó)藥典拖尾因子 USP TF(5%) 美國(guó)藥典拖尾因子 4. 1.33 4. 2.35Reducing the mobile phase pH reduces interactions with silanols that cause peak tailing降低流動(dòng)相的pH ,減小與硅醇基作用引起的峰拖尾,2017/11/3,20,Peak Tailing 峰拖尾Column Contamination 柱污染,Column: StableBond SB-C8, 4.6 250mm, 5mMobile Phase: 20% H2O : 80% MeOH Flow Rate: 1.0 mL/min Temperature: R.T. Detection: UV 254 nmSample: 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 1.062. 12043 1.64 2. 12443 1.21 2. 12237 1.213. 13727 1.69 3. 17999 1.19 3. 15366 1.114. 13355 1.32 4. 17098 1.25 4. 19067 1.17QC 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,2017/11/3,21,Peak Tailing/Broadening Sample Load Effects峰拖尾/變寬樣品過載效應(yīng),Column: 4.6 150mm, 5mMobile Phase:40%25mM Na2HPO4,pH7.0 : 60%ACNFlow Rate: 1.5 mL/min Temperature: 40CSample: 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. TrimipramineTailing Eclipse XDB-C8 Broadening / Competitive C8USP 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 低載負(fù) 高載負(fù)/10倍 低載負(fù)1. 1.60 1.70 850 59412. 2.00 1.90 815 78423. 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 10725,2017/11/3,22,Peak Broadening,Splitting Column Void 峰變寬,裂開 柱塌陷,Mobile Phase:流動(dòng)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(shí) 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(gè)例子中，高pH溶解硅膠導(dǎo)致塌陷,2017/11/3,23,Broad Peaks Unknown “Phantom” Peaks,Column: Extend-C18, 4.6 150 mm, 5 mMobile Phase: 40% 10 mM TEA, pH 11 : 60% MeOHFlow Rate: 1.0 mL/min Temperature: R.T. Detection: UV 254Sample: 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. 98793. 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 extremely late eluting peak from the preceding run.極低的理論板數(shù)表明這是一個(gè)前面進(jìn)樣極遲洗脫出來的峰,2017/11/3,24,Peak Tailing峰拖尾 Injector Seal Failure進(jìn)樣器密封性差,Column: Bonus-RP, 4.6 75mm, 3.5mMobile Phase: 30% H2O : 70% MeOH Flow Rate: 1.0 mL/min Temperature: R.T. Detection: UV 254 nmSample: 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(shù) 拖尾因子 理論板數(shù) 拖尾因子 1. 2235 1.72 1. 3670 1.452. 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(dòng)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,2017/11/3,25,Peak Tailing 峰拖尾Extra-Column Volume 柱外死體積效應(yīng),Column: StableBond SB-C18, 4.6 30mm, 3.5mMobile Phase: 85% H2Owith 0.1% TFA : 15% CANFlow Rate: 1.0 mL/min Temperature: 35C Sample: 1. Phenylalanine 2. 5-benzyl-3,6-dioxo-2-piperazine 3. Asp-phe 4. Aspartame10 l extra-column volume 50 l extra-column volume,2017/11/3,26,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(píng)價(jià)流動(dòng)相效果 - 改變流動(dòng)相pH和添加劑消除次級(jí)效應(yīng)/流動(dòng)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(píng)價(jià)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,2017/11/3,27,Retention Issues保留時(shí)間問題,Retention time changes(tr) 保留時(shí)間改變Capacity factor (retention) changes (k) 容量因子改變Selectivity changes () 選擇性改變,2017/11/3,28,Changes in Retention Same Column, Over Time相同柱子上保留時(shí)間改變,超時(shí),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(dòng)相組成差Change in mobile phase 改變流動(dòng)相Change in flow rate 改變流速other instrument issues 其他儀器問題,2017/11/3,29,Mobile phase Change Causes Change in Retention流動(dòng)相改變引起保留時(shí)間改變,60%MeOH:40% 0.1% TFA Fresh TFA Added to Mobile PhaseVolatile TFA evaporated/degassed from mobile phase. 易揮發(fā)的三氟乙酸從流動(dòng)相中蒸發(fā)/揮發(fā)。Replacing it solved problem. 換用新的流動(dòng)相就解決問題。使用有機(jī)揮發(fā)性酸時(shí)，流動(dòng)相應(yīng)新配。,2017/11/3,30,Column Aging/Equilibration Causes Retention/Selectivity Changes柱老化平衡不夠引起保留時(shí)間選擇性改變,Column 1 - Initial 1 號(hào)柱 開始時(shí)Column 1 - Next Day 1 號(hào)柱 第二天 Column 1 - After Cleaning with 1% H3PO4 1 號(hào)柱用 1% H3PO4 清洗 The primary analyte was sensitive to mobile phase aging of the column. 最初的分析對(duì)流動(dòng)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(shí)間和峰形都達(dá)到了預(yù)期效果,2017/11/3,31,Determining the Cause of Retention Changes on the same Column同一柱上保留時(shí)間改變?cè)虻拇_定,1. Determine K, , and tr for suspect peaks 對(duì)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(yàn)新柱- 記錄一批數(shù)據(jù)4. Rev