HPLC Column Basic Bene Technology MKT 1 1
HPLC Column Basic Bene Technology MKT 1
내용 1. HPLC 구조 이해 2. How to Downscale 3. Downscale of Method 4. USP & FDA 방법 조정 가이드 5. Column Chemistry 이해 6. 시료 전 처리 이해 7. PREP 과정 이해 8. Isomer 이해 2
1. HPLC 구조 이해 Scale ? Speed Separation Sensitivity Reproducibility … Conventional LC Fluores. Detector LP Gradient Pump Manual/Auto Injector Column UV/Vis Detector ELS Detector Pump Mass Spec HT LC : UPLC, RRLC, … 3
b. Column의 분리 Mechanism 이해 Steric Interaction Adsorption Interaction: 컬럼 내/외 Surface GPC/GFC Steric(Entropic) Interaction Injection HPLC(RPC, NPC, IEX, . . ) Adsorption(Energetic) Interaction VE Vo VR Log M Retention Time 5
c. Column의 Surface의 특징 Silica Surface(NPC) Area m 2/g Chemistry Si-OH’s Bonding Pore Size Chemistry Particle 분포도 분자량 /Hydrodynamic Volume Size 분포도 분리 능 • Silica는 일반적으로 p. H 2 -6. 5 에서 사용하고 Bonding에 의해서 p. H 1 -11 에서 안정함 • pore size 60 -120 A은 4 -10 K 이하, 200 -300 A은 100 -500 K 이하 분리함 6
d. Resolution Theoretical Plate Selectivity Retention Factor TR 1 Response K 0 , T 0 TR 2 Time 7
e. Column의 분리 능 vs 선 속도 : Van Deemter Plot Column Downscale 8
How to Downscale K *= t g F / S f G d V t Tg : gradient time, F : 유속 Sf : 상수 Gd : gradient 변화 Vt : Column & 시스템 의 빈 공 간 80 % 10 % 0 Tg : 25분 30 0 Tg : 5분 10 10
a. Column Dimension Downscale : N/Res. Peptide Mapping 11
b. Column Dimension Downscale : Selectivity/Res. 12
3. Downscale of Method 13
a. Downscale of Method : Pressure B C A A D E A : 2 um, YMC Ultra HT Pro C 18 B C D : 3 um, YMC Ultra Pro C 18 E : 3 um, YMC Ultra Pro C 18 14
b. Downscale of Method : Chemistry Retention Factor 15
Downscale of Method : Chemistry 16
c. Downscale of Method : Robustness 17
Downscale of Method : Robustness 18
a. 조정 시 Validation 고려사항 1. 정확성 2. 정밀성 A. Ruggedness System Suitability Guide Ø Repeatability • Resolution > 2 Ø Reproducibility • USP Tf < 2 • Repeatability Area, Tf 3. Selectivity/Specificity • Retention Range 2<K>10 4. 직선성 • Relative Retention RR>1. 2 5. Range • S/N >10 B. Robustness 6. LOQ : S/N >10 7. LOD : S/N > 3 20
b. Isocratic Separation 고려사항 1. 2. 현재 방법 성능 ü Resolution ü USP Tf ü Repeatability ü S/N ratios 3. 분석조건 ü 컬럼 : Length/Internal Diameter/Particle Size/ 온도 ü 유속 기기 조건 ü 이동상점도 ü Void Volume : Mixing/Tubing/Cell Volume ü 주입량 ü 시료용매/농도 ü Data Rate ü Backpressure ü Cell Path Length ü System Pressure 21
c. Isocratic/Gradient Separation 고려사항 Isocratic Separation Gradient Separation 1. Sample Load 1. Isocratic + : Vt 의 0. 5 -1% 이하, 농도 1% 이하 2. Sample 용매 강도 고려 3. System Void Volume : Mixing/Tubing/Cell Volume 4. Injection 정밀성 : Vinj 변화 5. Data Rate : 25 -50 Points/Peak 2. Delay Volume : Backpressure변화 3. Gradient Time : K* 4. Gradient Delay Time : Gradient Volume/Column Volume Ratio 유지 5. Post Run Equilibration Time : Delay Volume x 5 이상 22
5. Column Chemistry 이해 23
Mid to Non polar Compounds 24
Polar Compounds 25
Basic Compounds 26
MS 응용 1 1% Acetic Acid Me. OH: 10 mmol NH 4 PO 4=5: 95 1% Acetic Acid 0. 01% TFA 27
MS 응용 2 Diltiazem Metabolite ASMS 2009 MPS 447 : non Phosphate buffer 28
6. 시료 전처리 의 종류 및 분리원리 시료 전 처리는 Sample/Sample Matrix을 Clean-Up하고 Target을 사 전에 농축하는 과정으로 노동력과 시간이 많이 소요됩니다. 최종 분석이나 분취를 위한 컬럼과 장비를 보호합니다. • Precipitation : Solubility • LLE : Partitioning in one of two liquid phases • SPE : Adsorption/partitioning onto solid sorbent • Dialysis/ultrafiltration : MW/size • Electrophoresis : Charge • Distillation/Evaporation : Boiling point/vapor pressure • Supercritical Fluid Extraction : Partitioning into supercritical fluid 29
시료 전 처리 과정 : SPE의 ANB 전처리 예 일반 process 시료 Acidic Water soluble Neutral Me. OH soluble Basic Water soluble SPE SAX- Hydrophobic Liphophilic. Hydrophilic SCXHydrophobic Condition Me. OH 0. 1% acetic acid Equilibrate : 1 ml/mn이하 Water 1% acetic acid Load =>dry Water Buffer(Ph 7) Internal std Water Internal std Wash 1 =>dry 10 m. M ammonium acetate in 5 -10% Me. OH 1% Acetic acid Wash 2 =>dry Me. OH Elute =>Dry reconstitution Me. OH 1% acetic acid Me. OH 0. 1% formic acid Me. OH 2 -3% NH 4 OH 32
SPE 의 종류 예 Reverse Phase • Nonpolar Interactions • Van der Waals Forces • / Interactions • Secondary Interactions Normal Phase • Polar Interactions • Hydrogen Bonding • Dipole-Dipole Interactions Ion Exchange • Cationic Interactions • Anionic Interactions Mixed Phases • Combine Ionic Non-polar and Polar Interactions -Si-O-Si -C (2/4/8/18) -Si-O-Si-Phenyl -Si-OH -Si-O-Si -C 3 -(CN/NH 2) -(NR 3)+ -SO-3 - NH 2 - COO- Si-O-C Si-O-Si-C SO 3 C 18 C 2 Phenyl Silica CN NH 2 SAX/WAX SCX/WCX SDB-RPS SDB-SCX 33
7. 1 Dynamic Axial Compression Column : YMC 35
7. 2 CPC prep 요약 36
Prep 1 : GPC_HPLC 2 D 1. Pre-Prep - GPC : 지용성 시료 - GFC : 수용성 시료 2. Fine-Prep - RPC : Acidic/Basic Polar - NPC : Non Polar, HILIC (Aqueous NPC) - IEC : Ionic 37
Prep 2 : CPC_HPLC 2 D 1. Pre-Prep - CPC : 지용성 시료, 수용성시료 2. Fine Prep - RPC : Acidic/Basic Polar - NPC : Non Polar, HILIC (Aqueous NPC) - IEC : Ionic 38
Prep 3 : Recycle 1. GPC Recycle Prep - HPLC System Recycle 2. Valve Recycle Prep - Clean Recycle : Valve + 2 Columns 1 column 2 columns 39
Prep 4 : Scale up 1. Pre-Prep을 실시한다. 2. Scouting with Analytical Column - 분리 능이 좋고 분석시간이 짧은 컬럼으로 분리한다 - Gradient, Ph , 용매 조성 등을 최적화하고 - 최소 분석시간, 최대 시료 주입량을 확인한다 3. Scale up - Scale Factor = Prep Column Volume/ Anal Column Volume - Prep mass weight = Anal mass weight X SF - 유속 : - Gradient 시간 : 4. 기타 - Particle Size 조정 40
8. Isomers 이해 R-/S-. +/-(d-/l-), D-/L- => Optical Activity or Configuration 41
8. 1 Chiral K&L CSP - Chiral Stationary Phase - Polymeric 1 -naphylethylamine bonded to a wide pore S/silica Separation Mechanism - P-P interaction Hydrogen Bonding Dipole Interaction Setric Effect Solvent - Normal Phase Separation 42
8. 2 Chiral NEA A : Chiral selector B : Polymeric Chain C : Spacer Reverse Phase Separation 43
8. 3 Chiral CD BR Reverse Phase Separation : Enantomer, Positional Isomer w/wromatics 44
8. 4 Sumichiral OA CSP - Covalent bonded CSPs : Stable, Reproducible exept Ligand Exchange Type - Amide Type/QA 2000 s : Aromatics, Estere, Carboxylic Acid, Alcohol, … - Urea Type/QA 3000 s, 4000 s : Hydrogen Bonding, Pharmaceutical Amine, Amino Alcohol, … - Ligand Type/QA 5000 s, 6000 s : Reverse Phase Mode, Amino acids, Hydroxy acid, Chelate - 21 Phases 45
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