Modernizing HPLC Methods II Upgrading Current Methods www.interface.co.kr Why Modernize a Method? • Improved technology available • Save time • Save solvent • Improve results • Peak shape • Resolution • Reproducibility or accuracy • Accommodate new applications or formulations • Difficulty in duplicating results Page 2 1 When to Modernize a Method • Method will be used many times so changes are cost effective • Other parameters • Reference method does not meet needs • Before any new method becomes final Page 3 Common Sources of Reference Methods • USP – Pharmaceutical compounds • NIOSH – Industrial pollutants • AOAC – Food components • EPA – Environmental methods Page 4 2 USP Provides Many Reference Methods • Important criteria are specified for each method • HPLC column type – “L” designation • Resolution • Tailing factor • Relative retention times Page 5 USP Methods – Common Liquid Chromatography Column (L) Designations • L1 – Octadecyl silane chemically bonded to porous silica or ceramic microparticles, 3 – 10 ? m in diameter (C18 or ODS) • L3 – Porous silica microparticles, 3 – 10 ? m in diameter Sil) • L7 – Octyl silane chemically bonded to totally porous microsilica particles, 3 – 10 ? m in diameter (C8) • L10 – Nitrile groups chemically bonded to porous silica microparticles, 3 – 10 ? m in diameter (CN) • L11 – Phenyl groups chemically bonded to porous silica particles, 5 – 10 ? m in diameter (Phenyl) • There are more than 40 HPLC column types designated by the USP. Page 6 3 ZORBAX L1 (C18) Columns Column Carbon pH Type Load Range Pore Size SB-C18 80Å 10% 0.8 – 8.0 Eclipse XDB10% 2.0 – 9.0 C18 Extend2.0 – 12.5% C18 11.5 Silica Surface Surface Upper Type Area Coverage Temp 2.0 B 180 m 2/g 90° C ? mole/m 2 80Å B 180 m 2/g 80Å B 180 m 2/g Rx-C18 12% 2.0 – 9.0 80Å B ODS 20% 2.0 – 8.0 70Å A 3.6 60° C ? mole/m 2 3.0 40/60 ° C ? mole/m 2 3.3 180 m 2/g 60° C ? mole/m 2 2.9 300 m 2/g 60° C ? mole/m 2 Page 7 ZORBAX L7 (C8) Columns Column Carbon pH Type Load Range Pore Size Silica Type Surface Surface Upper Area Coverage Temp SB-C8 Rx-C8 5.5% 0.8 – 8.0 80Å B 180 m 2/g 2.0 90° C ? mole/m 2 Eclipse XDB-C8 7.6% 2.0 – 9.0 80Å B 180 m 2/g 3.6 60° C ? mole/m 2 C8 12% 2.0 – 8.0 70Å A 300 m 2/g 2.9 60° C ? mole/m 2 SB-C8 is identical to Rx-C8 Page 8 4 USP Analysis of Diazepam Capsules • Requirements: CH • L1 (C18) column 3 O N • 3.9 x 300 mm recommended • Resolution between ethylparaben and diazepam is > 4.5 N Cl CH 6 5 •USP requirements permit changes in column configurations up to 50%. Page 9 USP Analysis of Diazepam Capsules Column Comparison Mobile Phase: 35% Water: 65% MeOH Flow Rate: 1.2 mL/min Rx-C18 4.6 x 250 mm, 5 ? m 1 Sample: 1. Ethylparaben 2. Diazepam ? Bondapak C18 3.9 x 300 mm, 10 ? m Retention Peak Time Plates Rs 2 1 3.47 11836 0.00 2 7.01 8885 14.75 1 Retention Peak Time Plates Rs 2 1 4.64 5947 0.00 2 8.95 5216 11.24 Analysis Solvent Time Used 8 min 9.6 mL 0 5 10 Time (min) 15 0 Analysis Solvent Time Used 10 min 12 mL 5 10 15 Time (min) • The Rx-C18 column provides higher resolution and greater efficiency in a shorter analysis. Page 10 5 USP Analysis of Diazepam on C18 Columns Column: 4.6 x 250 mm Mobile Phase: 35% Water: 65% MeOH Flow Rate: 1.2 mL/min Sample: 1. Ethylparaben 2. Diazepam ODS SB-C18 1 1 2 2 Retention Peak Time Plates Rs 1 3.81 8034 0.00 2 7.97 6020 13.45 Retention Peak Time Plates Rs 1 4.17 13850 0.00 2 9.76 12154 0.28 0 5 10 15 0 Time (min) 5 10 15 Time (min) •Several C18 columns give acceptable results. •Newer C18 provides higher efficiency. Page 11 Rapid Resolution HPLC Columns Reduce Analysis Time and Solvent Waste 5 ?m 3.5 ? m 5 ?m 3.5 ? m Dimension 4.6 x 250 mm 4.6 x 150 mm 4.6 x 150 mm 4.6 x 75 mm 40% 50% Analysis 30 min. 18 min. 18 min. reduction 9 min. reduction Time (min) Solvent Waste (mL) 20,000 unchanged Resolution ? 50% 18 mL reduction 9 mL 12,00 0 10,000 } 20,000 } N 40% 18 mL reduction 30 mL 9% difference N 1/2 Page 12 6 Update Method to Save Time and Solvent Mobile Phase: 35% water: 65% methanol Sample: 1. Ethylparaben 2. Diazepam Rapid Resolution, RxC18 4.6 x 75 mm, 5 ? m Flow Rate: 1.2 mL/min Solvent Saver, Rx-C18 3.0 x 250 mm, 5 ? m Flow Rate: 0.5 mL/min 1 1 2 2 Analysis Solvent Solvent Time Used Saved 2.5 min 3.0 mL 6.6mL Analysis Solvent Solvent Time Used Saved 8 min 4.0 mL 5.6 mL Rs(1,2) = 12.05 0 Rs(1,2) = 8.62 2.5 5 Time (min) 7.5 0 2.5 5 7.5 Time (min) •Consider all column configuration options for saving time or solvent. Page 13 USP Analysis of Triamcinolone • Requirements CH 2OH • L1 (C18) column • (3.9 x 300 mm) • Resolution between triamcinolone and hydrocortisone is > 3.0 CO H CH3 HO H H CH 3 F OH OH H O Page 14 7 USP Analysis of Triamcinolone Column Comparison Mobile Phase: 40% water: 60% methanol Sample: 1. Triamcinolone 2. Hydrocortisone ? Bondapak C18 3.9 x 300 mm, 10 ? m Rx-C18 4.6 x 250 mm, 5 ? m 2 1 1 2 Retention Peak Time Plates Rs 1 2.28 8426 0.00 2 3.23 6892 4.79 Retention Peak Time Plates Rs 1 3.39 4078 0.00 2 5.22 3769 6.16 Analysis Solvent Time Used 4 min 6 mL 0 1 2 3 4 5 6 7 8 9 Analysis Solvent Time Used 6 min 9 mL 10 0 1 2 3 Time (min) 4 5 6 7 8 9 10 Time (min) Page 15 Update Method to Save Time and Solvent Mobile Phase: 40% water: 60% methanol Solvent Saver, Rx-C18 3.0 x 250 mm, 5 ? m Flow Rate: 0.6 mL/min 1 Sample: 1. Triamcinolone 2. Hydrocortisone Rapid Resolution, Rx-C18 4.6 x 75 mm, 5 ?m Flow 1 Rate: 1.5 mL/min 2 2 Analysis Solvent Solvent Time Used Saved 1.5 min 1.5 mL 4.5mL Analysis Solvent olvent Time Used Saved 4 min 2.4 mL 3.6mL Retention Peak Time Rs 1 2.40 0.00 2 3.58 2.27 0 1 Retention Peak Time Rs 1 0.61 0.00 2 0.98 2.27 2 Time (min) 3 4 5 0 1 2 Time (min) Page 16 8 Benefits of High Purity Silica for the Analysis of Basic Compounds • Improved peak shape • More accurate quantitation • Simplified mobile phases (no amine modifiers) • Note - Different silicas – Type A and Type B – can still be the same “L” designation with same kind of bonded phase Page 17 Chromatographic Benefits of Base Deactivated Silica Mobile Phase: 5% 2-Propanol in Heptane Flow Rate: 2.0 mL/min. Temperature: 35 °C High Purity, Low Acidity ZORBAX Rx-SIL Standard Silica NH 2 CHCH 2 3 CH-OH 2 NH 2 CHCH 2 3 CH-OH 2 • Improve peak shape for basic compounds with high purity, fully hydroxylated silica such as Rx-SIL Page 18 9 USP Analysis of Propranolol Mobile Phase: 75% 50 mM KH2PO4 , pH 4.4 : 25% ACN Flow Rate: 1.5 mL/min • USP Requirements Column:ODS, 4.6 x 250mm, 5? m Plates: 92 USP Tf (5%): 2.90 • L1(C18) column • 1000 plates Propranolol pKa 9.5 • Tf < 3 OH • ODS column has “Type A” silica OCHCHCHNHCH(CH) 2 2 3 2 • L1 columns available in 198485 all had Type A silica • This requires a high tailing factor specification 0 5 10 15 Time (min) •Higher tailing factors are typical of basic compounds on older silica. Page 19 Improved Analysis of Propranolol SB-C18 Rapid Resolution 4.6 x 75 mm, 3.5 ?m SB-C18 4.6 x 150 mm, 5 ? m Plates: 6371 USP Tf (5%): 1.09 Retention Time: 6.50 min Solvent Used: 12 mL 0 5 Time (min) Mobile Phase: 75% 50 mM KH2 PO4 , pH 4.4 25% ACN Flow Rate: 1.5 mL/min Sample: 1. Propranolol 10 0 Plates: 6370 USP Tf (5%): 1.14 Retention Time: 3.11 min Solvent Used: 6 mL 5 10 Time (min) • Excellent peak shape on SB-C18 columns, easily meets requirements with very low tailing. • Rapid Resolution SB-C18 reduced analysis time with equivalent results. Page 20 10 Separation of ? -Blockers on High Purity Silica 1 •Use the same ultra-pure silica based column for best peak shape when separating related compounds. Column: ZORBAX Rapid Resolution SB -C18 4.6 x 75 mm, 3.5 mm Mobile Phase: 75% 50 mM KH2 PO4 , pH 4.4 25% ACN Flow Rate: 1.5 mL/min Sample: 1. Pindolol 2. Metoprolol 3. Oxprenolol 4. Propranolol 5. Alprenolol 4 •Rapid Resolution columns reduce analysis time. 2 5 3 0 1 2 3 4 5 Time (min) Page 21 ZORBAX Eclipse XDB Technology • Features • Benefits • Dense dimethyl bonding • Long lifetime • Double endcapping • Excellent peak shape • All accessible silanols reacted • Wide usable pH range • Ultrapure fullyhydroxylated silica • Excellent all-purpose column • Applied to C18, C8 and Phenyl bonded phases • Different selectivities for optimum resolution (2 – 9) Page 22 11 Analysis of Dexamethasone • Requirements: CHOH 2 • L7 (4.0 x 250 mm) CO • Recommendations: OH 3 HO H CH • 70:30 Water:ACN, 2 mL/min • Retention time = 7 min. CH H H 3 CH 3 H F O Page 23 Analysis of Dexamethasone Mobile Phase: 70% water: 30% ACN Eclipse XDB-C8 4.6 x 75 mm, 3.5 ? mFlow Rate: 1 mL/min Eclipse XDB-C8 4.6 x 250 mm, 5 ? m Flow Rate: 2 mL/min Retention USP Peak Time Plates Tf (5%) 1 6.59 13540 1.00 Retention USP Peak Time Plates Tf (5%) 1 4.38 9069 1.00 Analysis Solvent Time Used 7 min 14 mL 0 2.5 Analysis Solvent Time Used 5 min 5 mL 5.0 Time (min) 7.5 0 1 2 3 4 5 6 7 8 9 10 Time (min) • Updating this method saves substantial solvent and reduces cost. Page 24 12 Analysis of Diphenhydramine Children’s Chewable Tablet 1 USP Method • L10 (CN) Column • Mobile Phase: 50% ACN: 50% water 0.5% TEA pH 6.5 with glacial acetic acid Column: ZORBAX SB-CN 4.6 x 150 mm, 5 ? m Mobile Phase: A: 70% 25 m M Na2 HPO4 , pH 3.0 B: 30% Methanol Flow Rate: 1.0 mL/min Temperature: 35° C UV Detection: 254 nm Sample: 1. Unknown 2. Unknown 3. Diphenhydramine 2 Modified Method • Mobile Phase: Phosphate buffer, pH 3 No TEA necessary 3 0 5 10 Time (min) •Update this method for greater simplicity and reliability. Page 25 Separation of Cold Capsule Components Retention USP Peak Time Plates Tf (5%) 1 2.67 8864 1.00 2 11.35 6113 1.36 •Chlorpheniramine Extended Release- L1 10 mm, 900 Plates, T f < 2 Column: •Phenylpropanolamine – Ion-pair chromatograpy, L1, T f < 2.5 1 •Phenylpropanolamine and Chlorpheniramine hard to analyze together 0 2 5 10 ZORBAX SB-CN 4.6 x 150 mm, 5 ? m Mobile Phase: A: 80% 25 mM Na2 HPO4 , pH 3.0 B: 20% Methanol Flow Rate: 1.0 mL/min Temperature: 35° C UV Detection: 254 nm Sample: 1. Phenylpropanolamine 2. Chlorpheniramine 15 Time (min) •Update and combine methods to save time. Page 26 13 Steps in Developing a Modern Method • Select best silica for basic compounds • Select best bonded phase • Select most time saving configuration • Plan for LC/MS compatibility • Evaluate method for robustness and ruggedness Page 27 Separation of Herbicides on Different StableBond Bonded-Phases 2 5&6 1 8 3 SB-C8 4 9 7 10 40 0 1 2 6 8 4 3 SB-Phenyl 9 7 10 40 0 6&7 2 8 1 SB-CN 9 3 4 5 0 10 T ime (min) 40 Columns: 4.6 x 250 mm, 5 ? m Mobile Phase: 65% H2O 35% Acetonitrile Flow Rate: 1.0 mL/min Temperature: room temperature UV Detection: 254 nm Sample: 1. Bentazon 2. Tebuthiuron 3. Simazine 4. Atrazine 5. Prometron 6. Diuron 7. Propazine 8. Propanil 9. Prometryne 10. Metolachlor • Short chain bonded phases can reduce analysis time for late eluting compounds, offer selectivity advantages and eliminate the need for gradient elution. Page 28 14 ZORBAX SB-C8 and SB-C18 Comparison Separation ZORBAX SB-C8 Rs USP 1. ---2. 5.60 3. 12.37 Rs USP 1. ---2. 4.81 3. 10.35 2 Column: ZORBAX Rapid Resolution 4.6 x 75 mm, 3.5 µ m Mobile Phase:90% 25 mMNa2HPO4, pH 3.0 10% ACN 1 Flow Rate: 1.5 mL/min Temperature: 40° C Sample: 1. Theobromine 2. Theophylline 3. Caffeine 0 ZORBAX SB-C8 2 TF (5%) 1.25 1.18 1.15 0.5 1.0 3 3 1 1.5 2.0 2.5 3.0 3.5 4.0 0 0.5 1.0 Time (min) 1.5 2.0 2.5 TF (5%) 1.44 1.45 1.31 Column: ZORBAX Rapid Resolution 4.6 x 75 mm, 3.5 µ m Mobile Phase: 90%50 mM Na2HPO4, pH 3.0 10% ACN Flow Rate: 1.5 mL/min Temperature: 40° C Sample: 1. Theobromine 2. Theophylline 3. Caffeine 3.0 3.5 4.0 Time (min) •More efficient interaction with the C8 bonded phase in a high aqueous mobile phase results in lower tailing factors. Page 29 Plan for LC/MS Compatibility • Is HPLC compatible with narrow-bore columns? • Do you need a capillary LC? • Review adjusting gradient and isocratic conditions for different column dimensions. • Will mobile phase be sufficiently volatile and allow efficient sample ionization? Page 30 15 Adapting Isocratic Method to Different Sized Columns tr – Vm/F k= Vm1 V = m2 = 1.5 = 0.3 F1 F2 1. F2 0 Vm/F 1.5 F2 = 0.3 F2 = 0.2 mL/min Column 1. 4.6 x 150 mm 3.0 x 150 mm 2. 2.1 x 150 mm Column Volume 1.5 mL 0.6 mL 0.3 mL Flow Rate 1.0 mL/min 0.4 mL/min 0.2 mL/min Page 31 Isocratic Separation of Antibacterials on Columns of Different Diameters 1 Column: ZORBAX SB-C18 4.6 x 150 mm, 5 ? m Flow Rate: 1.0 mL/min Injected: 3?L Detector Cell Volume: 8 ? L Solvent Waste: 31 mL 4 6 2 5 3 1 Mobile Phase: 80% Acetonitrile 20% Citrate/phosphate pH 2.6 Temperature: Ambient Sample: Antibacterials 1. Sulfamerazine 2. Furazolidone 3. Oxolinic acid 4. Sulfadimethoxine 5. Sulfaquinoxaline 6. Nalidixic acid 0 Column: Saver 4 2 5 3 1 6 3.0 x 150 mm, 5 ? m Flow Rate: 0.5 mL/min Injected: 2?L Detector Cell Volume: 8 ? L Solvent Waste: 15 mL Column: ZORBAX SB-C18 2.1 x 150 mm, 5 ? m Flow Rate: 0.25 mL/min Injected: 1?L Detector Cell Volume: 2 ? L Solvent Waste: 8 mL 4 2 3 5 Time (min) ZORBAX SB-C18 Solvent 6 40 Page 32 16 Gradient Separation of Herbicides on SB-C18 Columns with Different Configurations 4.6 x 250 mm tG= 30 min F = 1.0 mL/min • Each separation has the same profile; b (gradient steepness) is the same. 8 2 1 4 5 3 0 5 10 2.1 x 150 mm tG= 18 min F = 0.25 mL/min 15 6 7 20 25 30 8 2 1 6 4 3 0 5 7 5 10 15 20 Mobile Phase: A: 80% water + 0.1% TFA B: 20% ACN Gradient: 20 – 60%B Flow Rate: 0.25 mL/min Sample: Herbicides 1. Tebuthiuron 2. Prometryne 3. Simazine 4. Atrazine 5. Bentazon 6. Diuron 7. Propazine 8. Propanil Time (min) Page 33 Changing Gradient Retention (k*) to Improve Gradient Resolution 8 2.1 x 150 mm tG = 30 min 6 2 1 •To increase k*, decrease gradient steepness, b - use longer gradient time •Changing k* or b by increasing tG can improve resolution 4 5 7 3 0 5 10 15 Time (min) 20 25 30 Mobile Phase: A: 80% water + 0.1% TFA B: 20% ACN Gradient: 20 – 60%B Flow Rate: 0.25 mL/min Sample: Herbicides 1. Tebuthiuron 2. Prometryne 3. Simazine 4. Atrazine 5. Bentazon 6. Diuron 7. Propazine 8. Propanil Page 34 17 Adapting Gradient Methods to Different Column Dimensions To adjust gradient methods to different column dimensions keep gradient steepness (b) the same. 1/k* ? gradient S ? ? ? ? Vm = b = steepness tG ? F S = constant ? ? = change in % organic during the gradient run Vm = void volume of column F = flow rate tG = gradient time k* = k of solute at mid point of column If “b” is kept constant from run-to-run peaks will elute in the same relative pattern. Page 35 Adjusting a Gradient from a 4.6 x 250 mm Column to a 2.1 x 150 mm Column 2.1 x 150 mm 4.6 x 250 mm ΔФ= 40 (20% - 60%) Vm = 2.5 mL F = 1.0 mL/min tG = 30 min ΔФ • Vm = F • tG Using b = 3.33 3.33 = 40 • 0.30 0.2 • tG ΔФ= 40 (20% - 60%) Vm = 0.3 mL F = 0.2 mL/min tG = ? (18 min) 40 • 2.5 = 3.33 1.0 • 30 Using new dimensions Solve for tG tG = 40 • 0.3 = 18 min 0.2 • 3.33 Page 36 18 Conclusion • Improved results and lower costs may justify time spent updating methods • Evaluate Rapid Resolution and Solvent Saver configurations for increased speed and reduced solvent usage • Check for ways to save time and solvent with any method • Apply current technology when appropriate – scale down column dimensions and particle size to meet speed and sample requirements • Plan for compatibility with LC/MS Page 37 19
© Copyright 2024