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1 크로마토그래피의원리와분석법 HPLC 의검출기및원리 Soonchunhyang University Department of Chemical Engineering Prof. Jungkyun Im 순천향대 나노화학공학과 임정균교수

2 HPLC 의 detector( 검출기 )

3 Detector( 검출기 ) 굴절률검출기 (RI Detector) UV/Vis 검출기 형광검출기

4 Detector( 검출기 )

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

6 여러가지 Detector 의특징 RI UV/VIS Fluorescence Electrochemical Conductivity Response universal selective selective selective selective Sensitivity ( typical) Flow sensitivity Temperature sensitivity microgram nanogram picogram picogram nanogram YES NO NO YES YES YES NO NO YES YES Evaporative light Scattering(ELSD): uniform response, nonvolatile to semivolatile compoudns, ng sensitivity

7 UV/Vis 검출기 사용범위 HPLC 검출기중가장많이사용. 분자구조내에 Aromatic Ring, Double Bond, Triple Bond, UV 유도체를갖고있는화합물. 사용이간편하고, Baseline 안정화시간이짧다. Functional Group Chromophore Wavelength (nm) Functional Group Chromophore Wavelength (nm) Ether -O- 185 Ketone >C=O Olefins -C=C- 185 Thioketone >C=S 205 Thioether -S- 194 Esters -COOR Aldehyde -CHO Amine Thiol -NH 2 -SH Carboxyl Nitrite -COOH -ONO Disulfide -S-S Bromide -Br Oxalic Acid Azo HOOC-COOH -H=N Iodide Azide -I >C=N Nitroso Nitrate -N=O -ONO

8 자외선 / 가시선검출기 광원에서특정파장의빛이광이나장치를거쳐 cell 내의시료에투사되면일부는흡수되고, 일부는시료를통과하게되는데, 특정한시료는특정파장의빛에대한흡광도가높아서시료를투과하는빛의강도는상대적으로 작아지게된다. 이때흡수되는빛의양 (A) 은 - 용액내의흡광시료의농도 (C) 에비례하고, - 빛의파장과, 시료의특징적인흡광 spectrum( Molar Absorptivity ), - 빛이시료를통과하는거리 (path length, b ) 와관계가있다. 따라서 Photodiode 에도달하는특정파장의빛의양과, 시료의농도사이의 관계를나타내면다음과같다. A = εbc ( Lambert- Beer s Law )

9 종류와구조 1) Fixed type - 광원 (Lamp) 에따라정해진몇개의파장만을사용할수있다 - Wavelength : 214, 229, 254, 265, 280, 313, 340, 405, 436, 546 nm 2) Variable type nm 사이의모든파장중에서원하는파장을지정하여사용한다 - 가격은 fixed type 보다매우비싸다

$굴절율검출기 (Refractive Index Detector)$ 에포함된시료와용매의굴절율차이에의하여검출 가장덜민감하다.( 가장싸다.

11 굴절율검출기 (Refractive Index Detector) 원리 Reference Cell 과 Sample Cell 에포함된시료와용매의굴절율차이에의하여검출 가장덜민감하다.( 가장싸다.) 온도, 밀도, 유속, 이동상의비율, 청결상태등에영향을받는다. 이동상의 RI의변화를감지한다. UV-Vis을잘흡수하지않는샘플의검출에용이하다. ( 예 : 탄수화물 ) (baseline 일때 )( 샘플이있으면 )

13 형광검출기 (Fluorescence Detector) 원리 시료의분자구조가형광성을띠거나, 형광유도체를만들었을때이용된다. 시료는분자구조가형광성을띠거나형광유도체를만들었을때이용하며, 시료에서발광하는빛의양은시료의농도에비례하며발광량에따른전기적신호의크기가정량의척도가된다. UV lamp 가 excitation radiation 을담당한다. 사용범위 Polycyclic Aromatic, Aflatoxin 류, Amino Acid 생화학, 의학, 공중보건, 약리학, 석유화학분야 UV 검출기보다 100 배 ~ 1000 배선택성이있다. ( 감도좋다.)

15 Evaporative Light Scattering Detector (ELSD) Traditional HPLC detectors such as UV and RI have limited capabilities UV and RI are not compatible with a wide range of solvents RI detection is not gradient compatible Different analytes produce different UV responses depending on their extinction co-efficient ELSDs can detect anything that is less volatile than the mobile phase ELSD is universal and compatible with a wide range of solvents Destructive

16 Unique Method of Detection Three steps: Nebulization Mobile Phase Evaporation Detection

17 Schematic representation of ELS Analytes are de-solvated in the detector. The reduction in light intensity detected (due to scattering by the analytes) is measured.

Step 1: Nebulization Column effluent passes through nebulizer needle

Evaporation Droplets pass through a heated zone (Drift tube) Mobile phase

Detection Sample particles pass through an optical cell Sample particles

18 Step 1: Nebulization Column effluent passes through nebulizer needle Mixes with nitrogen gas Forms dispersion of droplet Step 2: Mobile Phase Evaporation Droplets pass through a heated zone (Drift tube) Mobile phase evaporates from the sample particle Dried sample particles remain Step 3: Detection Sample particles pass through an optical cell Sample particles interrupt laser beam and scatter light Photodiode detects the scattered light

19 ELSD 또는 MS 와같은장비로분석할경우용매와 additive 가모두쉽게 evaporation 되는용매인지미리체크할필요가있다.

20 ELSD A Powerful Detector for HPLC 1. Better sensitivity 2. Gradient compatible 3. Stable baselines 4. No solvent front peaks AN ELSD IS AN EFFECTIVE REPLACEMENT OR A PERFECT COMPLEMENT TO EXISTING LC DETECTORS RI UV Fluorescence

21 The End.

36 Chap 20 : Conjugated Systems 20.1 Stability of Conjugated Dienes Diene : 2 개의 C=C 이중결합을가진화합물 C 1,4-Pentadiene 1,3-Pentadiene 1,2-Pentadiene (unconj

36 Chap 20 : Conjugated Systems 20.1 Stability of Conjugated Dienes Diene : 2 개의 C=C 이중결합을가진화합물 C 1,4-Pentadiene 1,3-Pentadiene 1,2-Pentadiene (unconjugated diene) (conjugated diene) (cumulated diene)