HPLC short course Bene Technology Internal Use Only

HPLC 기본이론 : short course Bene Technology Internal Use Only 1

내용 1. HPLC 요약 - 3 -19 page 2. HPLC 작동원리 - 20 -24 page 3. Resolution, Method Development - 25 -42 page 2

HPLC 시스템의 종류 1. High Performance Liquid Chromatography (HPLC) High Pressure Liquid Chromatography High Price Liquid Chromatography => 400 bar system max pressure 10/5/3 um particle size, Column ID 4. 6 mm-Length 15 -30 cm 0. 8 -1. 2 ml/mn flow rate, 0. 009 inch tubing ID 2. Ultra PLC (UPLC) => 1000 -1500 bar system max pressure 2 um 이하 particle size, Column ID 2. 1 mm-Length 5 -10 cm 0. 2 -0. 5 ml/mn flow rate, 0. 005 inch tubing ID 3. Ultra HPLC (UHPLC) => 600 -800 bar system max pressure 2 -3. 5 um particle size, Column ID 3 -4. 6 mm-Length 10 -15 cm 0. 2 -1. 0 ml/mn flow rate, 0. 005 inch tubing ID * Cap/Nano LC, MPLC, Fast LC, 2 D-LC, Recycle LC, Pre/Post Column Reaction, … 3

HPLC의 구성 Scale ? Speed Separation Sensitivity Reproducibility … 이 동 상 Conventional LC Fraction or Waste Cap/Nano LC ? Fluores. Detector LP/HP Gradient Pump Manual/Auto Injector Column UV/Vis Detector ELS Detector Isocratic Pump Liquid Handling System Mass Spec HT LC : UPLC, RRLC, … 컬럼ID/Length : 유속/분석시간 4

HPLC의 유로 펌프 1 High-pressure gradient 펌프2 Low-pressure gradient Solenoid Valve Tee Mixer 펌프 * 시스템 Dead Volume < 1/3 -1/4 컬럼 Void Volume 6

HPLC의 분석결과 : Chromatogram, Pressure Injection(Data Acquisition Start) Data Acquisition End Mobile phase 에테르 Data Baseline Drift/Noise Pressure Drift/Noise Sample 클로로필 Void Volume ( Vt=Vo+Ve) Peak Area/Height, Retention Time Relative RA/PT, Tailing Factor Resolution -Capacity, Selectivity, Plate Number HETP - Optimal Linear Velocity Quan/Qual Stationary phase Ca. CO 3 Colour 기록 Fraction and/or Detector Signal System Suitability, IQ/OQ/PQ, Validation Computer System Validation Data Integration and Pump Pressure 7

분리 모드 : Polarity, 이온교환, SEC, … Normal Phase Si. OH Reverse Phase Silica Si. OH End Capping Site Silica Si. OH Si R Polymer Ion Exchange Phase N+ R Size Exclusion Pore Size/Volume R Polymer SO 3 - Hydrophobic Interaction Chromatography HILIC, Mixed Mode Column, Chiral Column, Affinity Column, … 8

Starting 컬럼 : Reverse Phase 9

Column Selection : Silica, C 18, C 4, … Type I or A : 중금속10 -20 ppm 이상 Type II or B : 5 -10 ppm 이하 Ligand Density, Surface Coverage End Capping : C 4 Si- => C 1 Si- Reverse Phase Mode 선택 10

Column Selection : Reverse Phase Non-Polar 11

Polarity : Solvent-Target Interaction 3 4 12

검출기 선택성 감도 Gradient 가능 UV/Vis chromophore Mg-ng Possible Fluorescent substances Ng-pg Possible RID (Differential refractive index) RI 차이 Mg-µg Impossible ELSD (Evaporative light scattering) 비 휘발성 µg Possible ECD (Electrical conductivity) 이온 물질 Mg-ng Possible w/suppressor 산화/환원 물질 Ng-pg Possible w/pulsed function UV/Vis D (VW, MW, PDA) FLD (Fluorescence) Electrochemical * Mass Spectrometer, ICP-MS, Corona Charged Aerosol Detector(CAD), Circular Dichroism(CD), … 13

UV/VIS Absorbance 검출기 : Baseline vs Peak 14

Report w/System Suitability 15

System Suitability s/w : USP/ICH/FDA Retention, Resolution(two peaks), RRT(relative retention time), Response(Impurities), Void volume, LOD, LLOQ and S/N, Peak Tailing(Asymmetry, Skew), Precision, Accuracy, Pressure(Max/Recommended), Blank(Carry Over), Prime Injections(Before Test), Quality Control Samples, Baseline Noise/Drift, … Parameter Limit Resolution Rs > 2 Tailing Factor Tf < 2 Theoretical Plate N > 2000 -3000 Capacity Kf > 2 -5 Precision RSD < 1% at n>5 16

분석 방법 조정가이드 ; USP & FDA Allowed HPLC Adjustment Column Length Internal Diameter Particle Size Flow Rate Column Temperature Injection Volume PH UV wavelength Conc. Salts in Buffer mobile phase USP (Ref: General Chapter <621>) EP (Ref: General Chapter 2. 2. 46) ± 70% Can be adjusted if linear velocity is kept constant Reduction of 50%, no increase ± 50% or more as long as the linear velocity is kept contant ± 10 C Change allowed as long as SST criteria are met ± 0. 2 Units <± 3 nm ± 10% ± 70% ± 10% Max 60 C May be decreased (if LOD and repeatability ok) ± 0. 2 Units (± 1% for neutral substances) <± 3 nm ± 10% Minor Components (<50%) ± 30% or ± 10% absolute whichever is smaller Minor Components ± 30% or ± 2% absolute whichever is larger ± 25% Reduction of 50%, no increase ± 50% http: //apps. usp. org/app/USPNF/columns. html 17

Method Validation - Accuracy : Authenticated values 정확성, 회수율 - Precision : 정밀성, 재현성 - Specificity : no Signal Addition/Subtraction, Purity Test - Detection limit (LOD) : DL = 3. 3 /slope, S/N = 3 - Quantitation limit (LOQ) : QL = 10 /slope, S/N = 10 - Linearity : 3 -5 Standards - Range : , target 농도 대비 80 -120% 범위 이상 - Ruggedness/Robustness : 분석조건의 작은 변수에 영향을 받지 않음 => Qb. D(Quality by Design) 18

Data Integrity : CSV, PIC/S, … Raw Chromatogram Data 시간, 관계형 기록 실시간 alarm추적성 분석/오류대응등 SOP , … 19

HPLC Terminology 용매 LC/LCMS용 수용성 용매 지용성 용매 휘/비 Buffer Miscibility PI<4 -6 Filter Degassing =>펌프 Isocratic HP/LP G. 유속 압력 Run. Time =>시료주입기 수용성 용매 지용성 용매 Buffer Solubility Filter 시료주입량 Inject_Start => 컬럼 Separation Reversed P Normal P IE, SEC, … Mixed Chiral, … Pre-heat /cool =>검출기 Chromatogram RI, UV/Vis Fluo. , PAD ELSD, … Spectrum MS ; SRM(MRM) DDA, … =>Data System Automation Validation IQ/OQ/PQ Data Validation Traceable, … Quan/Qual Purity FTIR, NMR, … 유로(시스템/Tubing/컬럼) ; 내경, 길이, Void Volume(w/column) => Equilibrium Time, Injection Volume, … Teflon, SUS 316, In-line filter, HDPE, Quarz, … => 이동상 Nut Ferrule, Hand-tight fitting, … => Easy Handling * Compound Specific Information : m/z, formula, structure, Melting/Boiling Point, … 20

시스템및 분석방법 검증_ Signal, Pressure 1. 용매 A/B -> Pump -> Injector -> Union -> Detector -> Waste 2. 용매 baseline 확인 기록 용매 A 용매 B Autozero 3. 용매 A/B -> Pump -> Injector -> Column -> Detector -> Waste 컬럼을 연결하고 초기화 세척(Vt의 5 -20배)을 진행함 21

Column특성, Sample Matrix에 따른 재생방법 23

Column 충전물의 구조 Porous Beads Totally Porous, Non-Porous Beads Superficially Porous, Surface Porous * Perfusion column : Protein/Peptide등 고분자 26

Column 충전물의 Surface/Ligand 의 특징 Electron Configuration - Zirconium : [Kr] 5 s 2 4 d 2 , (2, 8, 10, 2 : 40) - Titanium : [Ar] 3 d 2 4 s 2 , (2, 8, 10, 2 : 22) - Silicon : [Ne] 3 s 2 3 p 2, (2, 8, 4 : 14) Silica Surface : Area m 2/g Chemistry Si-OH’s Lewis Acid/Base Bronsted Acid/Base NPC Pore Bonding Particle RPC Mixed Mode IEX Size/Volume SEC concept Chemistry 분포도 Size 분포도 Peak Width 분자량 분리 능 Hydrodynamic Volume • Silica는 일반적으로 p. H 2 -6. 5 에서 사용하고 Bonding에 의해서 p. H 1 -12 까지 가능함 • pore size 60 -120 A은 4 -10 K 이하, 200 -300 A은 20 -100 K이하 분리함 27

분리모드 1 ; A, B, C term 이해 A. Eddy Diffusion : 경로차 B. Longitudinal Diffusion : 확산 C. Mass Transfer : Pore Surface Interaction Lateral(수평) Diffusion 28

Van Deemter Plot 10 5 3 2 um - 최적 선 속도 : Particle Size, Pore Size - 최적 유속 : 컬럼 ID - Linear Dynamic Range 29

분리모드 2 ; 그림으로 보는 분리 Steric Interaction Adsorption Interaction in 컬럼 내/외 Surface HPLC(RPC, NPC, IEX, . . ) Adsorption(Energetic) Interaction GPC/GFC Steric(Entropic) Interaction Injection VE VR Vo Log M Adsorption 0<KGPC<1 1<KHPLC Critical Adsorption Point, K=1 Retention Time 30

분리모드 3 ; Resolution Theoretical Plate Selectivity Retention Factor TR 1 Response K 0 , T 0 TR 2 Time 31

수식으로 보는 분리 : Resolution R > 1. 5 -2. 0 : Baseline Resolution K’ : Retention/Capacity Factor Correct Mobile Phase Strength α : Selectivity Factor Right Chemistry N : Efficiency Factor Correct Column Dimension 32

A. 1 Capacity/Retention Factor ∝ 2<K<10 K’=(Ta-T 0)/T 0 R ↑ K 2 5 7. 5 10 • Mobile Phase Strength ↓ K’ ↑ => R↑ • RPC, NPC, IEX, … 33

Mobile/Stationary Phase (C 18, C 4) Strength 3 4 7 Colum Volume 4. 6 x 150 mm ; 1. 75 ml/70% 분석시간 < Vt * (5 -10) 34

A. 2 Selectivity ∝ α > 1. 2 α=KA’/KB’ How to Interact with compound - Stationary Phase : C 18, Silica, … - Mobile Phase 1. p. H 2. Temperature 3. Additives, … 35

NPM, RPM, HILIC, Mixed Mode and IEX 36

Versatile Wide p. H and Temperature Stability 37

Versatile Wide p. H Stability Acidic Basic Amphoteric Neutral 38

Erythromycin유도체 Separation at High p. H & Temp. 39

Efficiency/Speed ∝ 5000<N<25000 Column Length Particle size ↑N↑ ↓N↑ R↑ Minimize Extra Column Volume Mixing Performance Optimum Velocity(Flow Rate) System Pressure 40

Column의 분리 능 vs 선 속도 : Van Deemter Plot Optimal Linear Velocity Column Downscale Fast LC 41

Column Dimension Downscale : N/Res. Peptide Mapping 42