Separation Basics UHPLC_LC MSMS & Column Technology

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Separation Basics UHPLC_LC MSMS & Column Technology Bene Technology Co., Ltd. MKT

내용 1. HPLC 의 역사 2. HPLC 의 구조, Terminology 3. 컬럼 의 분리 단, 구조 4. 분리모드 5. Reversed Phase Column선택하는 법 6. 분석방법 조정 가이드 Appendix

1. HPLC 의 역사 1903 ; Mikhail Tswett Separates Plant Pigments on Chalk Columns 1970년 이전 ; few reliable chromatographic methods 1970’년대 ; open-column chromatography, paper chromatography, and thin-layer chromatography => similar compounds pressure liquid chromatography => flowthrough/purification times by column chromatogaphy. Pump Technology (constant flow rate or constant pressure) 1970년대 중반 : High pressure liquid chromatography, column packing materials and the on-line detectors => Performance I970년대 후반 reverse phase liquid chromatography => very similar compounds. 1980년대 이후 ; 대부분의 chemical compounds 분리. New techniques=> Computers and automation, typical column(250 mm L, 3-5 mm ID) => micro/capillary columns(1-2mm ID, 3 µm-300 µm ID), affinity/chiral columns and Fast HPLC(Length 3-5mm, smaller particles size10, 5, 3um), optimal analysis ; various types of columns, a variety of detectors of the compound. 2000년대 ; GLP, UHPLC(Sub 2um), MSMS,… Quantitaton/Qualification,… Prep,… 응용영역 : biotechnological, biomedical, biochemical research, pharmaceutical industry, cosmetics, energy, food, and environmental industries,…

2. HPLC 구조 Conventional LC HT LC : UPLC, RRLC, … Scale ? Speed Separation Sensitivity Reproducibility … Mass Spec UV/Vis Detector LP Gradient Pump Column ELS Manual/Auto Injector Fluores. Conventional LC HT LC : UPLC, RRLC, …

HPLC Terminology 용매 =>펌프 =>시료주입기 =>컬럼 =>검출기 ->Data System LC/LCMS용 수용성 용매 지용성 용매 휘/비 Buffer Filter Degassing Isocratic HP/LP G . 유속 압력 Run Time 수용성 용매 지용성 용매 Buffer Filter 시료주입량 Injection Start Separation Reversed P Normal P IE, SEC,… Mixed Chiral,… Preheat /cool Chromatogram Quan/Qual Purity RI, UV/Vis Fluo., PAD ELSD,… MS ; MRM/SRM DDA,… FTIR, NMR,… Automation Validation IQ/OQ/PQ Traceable 유로(시스템/Tubing/컬럼) ; 내경, 길이, Void Volume, Equilibrium Time, Injection Volume,… Teflon, SUS316, In-line filter, HDPE, Quarz,… Nut Ferrule, Hand-tight fitting,… * Compound Specific Information : m/z,…

3. 컬럼의 분리 단 분배계수 KA = [A]u phase/[A]l phase KA = [A]sol phase/[A]lliq phase

Column의 구조의 활용 Vt = Vo + Ve Tubing Frit 분리관 Vt는 GPC/GFC의 경우 V의 약 75-80% HPLC의 경우 V의 약 60-65% 안정화 부피 : Vt 의 10-20배 이상 시료 주입량 : Vt 의 0.5-10% 이하 시료 농도 : 시료 주입량의 0.I -10% 이하 충전물

Totally Porous, Superficially Porous, Surface Porous Column 충전물의 구조 Porous Beads Non-Porous Beads Totally Porous, Superficially Porous, Surface Porous

Column 충전물의 Surface의 특징 Silica Surface(NPC) Area m2/g Chemistry Si-OH’s Bonding RPC Pore Size 분포도 Particle 분자량 /Hydrodynamic Volume 분리 능 Silica는 일반적으로 pH 2-6.5 에서 사용하고 Bonding에 의해서 pH 1-11 까지 가능함 pore size 60-120A은 4-10K 이하, 200-300A은 20-100K이하 분리함

4. 분리모드 1 ; A, B, C term 이해 A. Eddy Diffusion : 경로차 B. Longitudinal Diffusion : 확산 C. Mass Transfer : Pore Surface Interaction Lateral Diffusion

Van Deemter Plot

분리모드 2 ; 그림으로 보는 분리 Steric Interaction Adsorption Interaction: 컬럼 내/외 Surface Injection Vo VE VR Log M Retention Time GPC/GFC Steric(Entropic) Interaction HPLC(RPC, NPC, IEX,..) Adsorption(Energetic) Interaction Adsorption

분리모드 3 ; Resolution TR1 TR2 K0 , T0 Theoretical Plate Selectivity Retention Factor K0 , T0 Time Response TR1 TR2

K’ : Capacity Factor /Correct Mobile Phase Strength 수식으로 보는 분리 : Resolution R > 1.5 -2.0 : Baseline Resolution K’ : Capacity Factor /Correct Mobile Phase Strength α : Selectivity Factor /Right Chemistry N : Efficiency Factor /Correct Column Dimension

A.1 Capacity/Retention Factor K 2 5 7.5 10 R ∝ ↑ 2<K<10 K’=(Ta-T0)/T0 Mobile Phase Strength ↓ K’ ↑ => R↑ RPC, NPC, IEX, …

Mobile/Stationary Phase (C18, C8, C4) Strength

∝ α > 1.2 α=KA’/KB’ How to Interact with compound A.2 Selectivity - Stationary/Mobile Phase - pH - Temperature - Additives

Chemical Selectivity : C18 end capping process TMS => - CH3 Batch Synthesis Flow Synthesis/Microreactor : Tri step end-capping

Anthocyanin/Anthocyanidin

∝ 5000<N<25000 A.3 Efficiency/Speed Clength ↑ N ↑ Dparticle↓ N ↑ Minimize Extra Column Volume Mixing Performance Optimum Velocity(Flow Rate)

Column의 분리 능 vs 선 속도 : Van Deemter Plot Downscale

Column Dimension Downscale : N/Res. Peptide Mapping

Small Particle or Large Particle의 특성 HETP 선 속도 5um 2um TR N 5um 2um HETP vs u TR vs N Column Permeability Large Peak Capacity => Large Particle Size Very Fast Separation => Small Particle Size

5. Reversed Phase Column 선택 하는 법 1

Reversed Phase Column 선택 하는 법 2

Polar, Mid to Non polar Compounds

Polar Compounds

Basic Compounds

Reversed Phase Column 선택 하는 법 3 : SRM 870

6. 분석 방법 조정가이드 ; USP & FDA Parameter Max Spec 컬럼 길이 ±70% 컬럼 ID 컬럼 선 속도 유지 유속 ±50% 주입량 최대한 적게 감도/재현성 유지 Particle Size To 50% * 주요 관공서의 표준방법을 수정할 경우 기존분석법과 수정방법을 동시에 수행하여 정확성, 재현성등 검증조건에 영향을 주지 않아야 합니다.

a. 조정 시 Validation 고려사항 정확성 정밀성 Selectivity/Specificity 직선성 Range Ruggedness Repeatability Reproducibility Robustness Selectivity/Specificity 직선성 Range LOQ : S/N >10 LOD : S/N > 3 System Suitability Guide Resolution > 2 USP Tf < 2 Repeatability Area, Tf Retention Range 2<K>10 Relative Retention RR>1.2 S/N >10

b. Isocratic Separation 고려사항 현재 방법 성능 Resolution USP Tf Repeatability S/N ratios 기기 조건 Void Volume : Mixing/Tubing/Cell Volume Data Rate Cell Path Length System Pressure 3. 분석조건 컬럼 : Length/Internal Diameter/Particle Size/온도 유속 이동상점도 주입량 시료용매/농도 Backpressure

c. Isocratic/Gradient Separation 고려사항 Isocratic Separation Sample Load : Vt 의 0.5-1% 이하, 농도 1% 이하 Sample 용매 강도 고려 System Void Volume : Mixing/Tubing/Cell Volume Injection 정밀성 : Vinj 변화 Data Rate : 25-50 Points/Peak Gradient Separation Isocratic + Delay Volume : Backpressure변화 Gradient Time : K* Gradient Delay Time : Gradient Volume/Column Volume Ratio 유지 Post Run Equilibration Time : Delay Volume x 5 이상

Appendix 1. SRM 870 Uracil : Void volume marker Quinizarin : Activity toward chelating reagent Toluene : Hydrophobic Retention Amitriptyline : activity toward bases Ethylbenzene : Methylene Selectivity

Reversed Phase Column Chemistry 이해

Reversed Phase Column Chemistry 이해

Appendix 2. DAC Column/YMC Prep LC

Prep Process