【正文】 法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 K180。Replacing it solved problem. 換用新的流動(dòng)相就解決問題。l extracolumn 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(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 extracolumn 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Retention Issues保留時(shí)間問題Retention time changes(tr) 保留時(shí)間改變Capacity factor (retention) changes (k180。C Sample: 1. Phenylalanine 2. 5benzyl3,6dioxo2piperazine 3. Aspphe 4. Aspartame10 181。在這個(gè)例子中，高pH溶解硅膠導(dǎo)致塌陷Broad Peaks Unknown “Phantom” PeaksColumn: ExtendC18, 150 mm, 5 181。CSample: 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 XDBC8 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. 850 59412. 815 78423. 2776 6231 4. 2539 8359 5. 2735 10022 6. 5189 10725 Peak Broadening,Splitting Column Void 峰變寬,裂開 柱塌陷 Phase:流動(dòng)相：50%CAN : 50%Water : %TEA (~pH 11)50％乙腈 : 50％水 : 0。C 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(yīng)Column: 150mm, 5181。CSample: 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 pH 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. 4. Reducing 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Peak Tailing 峰拖尾Column Contamination 柱污染Column: StableBond SBC8, 250mm, 5181。CSample: 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. 2. 2. 3. 3. 4. 4. 5. 5. 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Peak Tailing Column “Secondary Interactions”峰拖尾柱“次級(jí)效應(yīng)”Column: AlkylC8, 150mm, 5181。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 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 Peak Tailing, Broadening and Loss of Efficiency峰拖尾、變寬和柱效降低 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Extracolumn effects 柱外效應(yīng) Tailing Column “Secondary Interactions”峰拖尾柱“次級(jí)效應(yīng)”Column: AlkylC8, 150mm, 5181。CDetection: 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Injectio