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1 HPLC BASICS

2 Chromatography 란? 혼합된시료성분이이동상과고정상사이를흐르면서흡착, 분배, 이온교환또는분자크기배재작용등에의해각각의단일성분으로분리되는것을말한다. 분리, 정성, 정량등의분석목적과분리, 정제, 분취목적에이용된다. Injection Interaction Elution a abacbc aabcbc

3 HPLC System Mobile Phase Reservoir Pressure Gauge Data System Pump Pulse Damper Injector Column Detector Fraction Collector Gradient Device

4 Solvent Delivery System (Pump) 1. Pump a.. b.. 2. Waters Pump a. b. Pulse system (Gear Cam Pulse.) c.. d. Isocratic Gradient. e..

5 FLOW PATH DIAGRAM High sensitivity accessory Transducer Solvent revervior filter High pressure filter Pump outlet Tee Left pump head Outlet check valve Inlet check valve Mechanical drive Outlet check valve Inlet check valve Right pump head Inlet manifold assembly Solvent drawoff assembly

6 ( Isocratic Mode) ;. ( Gradient Mode) ;. - Single pump ; Low pressure - Multiple pump ; High pressure

7 일정용매조성법 pump detector solvent resorvoir injector column pump.

8 구배용매조성법 (Gradient Mode) Gradient Mode 는분석동안용매의조성을바꾸어줄수있을뿐만아니라, 시간에따른변화및그형태까지도변화시킬수있다. 1) Single pump. Low pressure solvent A pump solvent B mixer

9 2) Multiple pump High pressure pumpa solvent A mixer pumpb solvent B

10 Injector loading Column Injector 1. Rheodyne injector (Valve injector) 2. U6K injector (Syringe & Valve Mixed injector) 3. Automatic injector

11 Rheodyne injector 1. loop, loop 2. Sample loop

12 Auto Injector 1. Free injection Step 3. 4 ml vial : 48, 1 ml vial : : 5. : 6. Auto priming Auto needle washing

13 컬럼 3.5um, 4um, 5um, 6um, 7um, 10um, 37um, 55um, 75um Silica, Alumina, Silica-Bonded, Polymer(resin)- Bonded : Spherical, irregular Pore : 50 A ~ 300 A

14 충진제의특성 Pore size & Distribution : Small Pore = higher efficiency = less band broadening Spherical or Irregular : Irregular = higher Surface area = higher carbon load Spherical = lower pressures = longer life Spherical = better structural stability = longer life Particle size Smaller particle = higher operating pressure = higher efficiency

15 컬럼의종류 재질에따른분류 Steel 컬럼 PEEK 컬럼 Radial-pak 컬럼 분리기작에따른분류 순상컬럼 역상컬럼 이온컬럼 크기배재컬럼

16 재질에따른분류 Steel 컬럼 Steel 카트리지컬럼 - End fitting 장치 PEEK (polyetheretherketone) 컬럼 플라스틱 Radial-Pak 카트리지컬럼 방사압축기술

17 1. Normal Phase( 흡착작용 ) Functional group 의 polarity 에의한분리이다. 일반적으로 non-aqueous, non-polar solvent 를사용한다 ; 극성이큰물질이가장나중에용출된다. Hexane polar Si-OH O Si-OH---OH

18 순상충전컬럼의종류 Silica Phase Resolve Silica, uporasil, Nova-pak Silica Bonded Phase Nova-pak CN, NH2 u-bondapak CN, NH2 Resolve CN, NH2

19 2. Reverse Phase( 분배작용 ) Bonded compound(c18, C8, CN, ph) 와분석물질간의상호작용에의한분리 비극성이큰물질이가장나중에용출된다 CH3CN/H2O non-polar -Si-O-Si-C18 --

20 역상충전컬럼의종류 u-bondapak C18, CN, Phenyl Resolve C8, C18, CN, Phenyl Lichrosorb RP-18, RP-8 Deltapak C18, C8 Symmetry C18, C8 Symmetryshield C8

21 C18 Column 제조방법 Silica (SiO2) Acid Treatment Free Silanol (Si-OH) Chemical Bonding of C18 End-capping by TMS Element Analysis

22 End-capping 이란? TMS-Cl -Si-O-Si-C18 non-polar -Si-OH -Si-O-Si-C18 -Si-O-Si-C18 non-polar -Si-O-Si-(CH3)3 -Si-O-Si-C18

23 3. Ion Exchange( 이온교환작용 ) ; 이온화도의차이에의해분리된다 counter ion sample

24 이온교환컬럼의종류 IC-pak Anion/Cation SAX(Strong Anion Exchenger)/SCX Protein-pak DEAE/SP HR

25 D. Size Exclusion( 분자체작용 )

26 분자체 column 의종류 GPC : Styragel HR, HT, HMW Series Aqueous GPC : Ultrahydrogel Series Aqueous GFC : Protein-pak Series

27 Hanxane N MeOH N THF N IONIC < 2000 Y Y Y Y > 2000 Silica, Porasil Y CN, Ph, C8, C18 Styragel HR, HT, HMW NR3, COO, SO3 Organic Sol. N Water Y

28 컬럼효율계산 ( 이론단수 )

29 분리능 (Resolution) R=1/4(1-1/a )SQRT(N){k`/(1+k`)} a : 선택성인자 N: 이론단수 ( 컬럼의효율 ) k`: 용량인자

30 Detector 컬럼에의해분리된시료의양에비례한전기적신호를만든다 1. Signal to noise ratio (S/N ratio) 2. Noise, Drift 3. Detection limit 4. Flow rate, temperature, pressure 5. Gradient

31 Detector 의종류 Optical Detector 1. UV/Visible Detector 2. Fluorescence Detector 3. Refractive Index Detector Electrochemical Detector 1. Conductivity Detector 2. Electrochemical Detector

32 Detector RI UV/VIS Fluorescence Electrochemical Conductivity Response Sensitivity ( typical) Flow sensitivity Temperature sensitivity universal selective selective selective selective microgram nanogram picogram picogram nanogram YES NO NO YES YES YES NO NO YES YES

33 자외선 / 가시선검출기 cell,,. (A) - (C), -, spectrum( Molar Absorptivity ), - (path length, b ). photodiode,. A = Ebc ( Lambert- Beer Law )

35 3) Photodiode Array Type nm scan. Photodiode Array Detector Shutter Cell Holographic Grating D2 Lamp Diode Array 512 Diodes nm

36 1) Exitation filter sample cell excited state, ground state.,. 2) Benzene -OH, -OCH3, -H2, -NH(CH3), -N(CH3)2, -F, -CO

37 3) Emition Grating Emission Slit Photomultiplier Tube Mirror Collection Mirror Flow Cell Exitation Grating Exitation Slit Photo Diode Beam Splitter Lamp Focusing Mirror Mirror

38 용매조건 HPLC grade solvent가물인경우 18 mega ohm이상. Low Viscosity 용매간섞임성. (miscibility No. 차가 15미만 ) 이동상은고정상을변화시키면안된다. 분리코자하는시료는이동상에녹아야한다. UV cutoff, refractive index가낮은용매.

39 Buffer 용액의사용목적 1) 이온성시료일때 -이온억제및이온쌍형성 2) 생리활성물질의순수분리, 정제시 (ph조절) 3) 이온성물질분리시이온강도조절

40 이동상변형제 목적 : 역상방법에서이온성시료의분리능을높일뿐만아니라시료를비이온화시키기위하여사용한다 종류 PIC A / Low UV PIC B Series

41 이동상제조 (I) 용매준비 HPLC 용용매 초순수 ( 비저항값 ;18mega ohm) 부피대부피계량 계량시 volume 비로계량후혼합 용매성질에따라흡열, 발열반응가능 부피비가달라질수있다.

42 이동상제조 (II) Filter 수용성 FILTER(PVDF) WATER 로 ACTIVATION 지용성 FILTER(PTFE) MeOH/ 적합한용매로 ACTIVATION

43 이동상제조 (III) 탈기 이동상에용존된 air, oxygen, bubble제거 Vacuum Degassing( 진공탈기 ) 여과 + 탈기 Helium Sparging 직접이동상에 sparging Ultrasonication( 초음파파쇄 )

PowerPoint 프레젠테이션

PowerPoint 프레젠테이션 크로마토그래피의원리와분석법 HPLC 의검출기및원리 Soonchunhyang University Department of Chemical Engineering Prof. Jungkyun Im 순천향대 나노화학공학과 임정균교수 HPLC 의 detector( 검출기 ) Detector( 검출기 ) 굴절률검출기 (RI Detector) UV/Vis 검출기 형광검출기