PowerPoint 프레젠테이션

Size: px

Start display at page:

Download "PowerPoint 프레젠테이션"

호빈 운
5 years ago
Views:

1 GC 와 LC 의선택 이동상의극성 LC GC 분자량 Chromatography 의비교

4 HPLC 의분리방법 흡착 분배 액체 - 고체 액체 - 액체 (LSC) (LLC) 이온교환 크기배제 고정상에부착된이온기 (IC) 분자크기에의한크기배제 (GPC)

7 전통적인분배크로마토그래피 - 지지체에액체고정상을액막으로형성시켜사용 결합상크로마토그래피 - 고정상의화합물을실리카입자의표면에화학적으로결합시켜사용 1. 화합물의극성에관계없이분리가가능. 2. 분리는시료성분의고정상과이동상에대한분배계수의차이 3. 이동상으로는두가지이상의혼합용매가흔히사용된다. 이동상에사용되는용매종류, 농도, 완충액이나변형제의농도를조절하여시간에따른선택성과 elution strength 를변화시켜분리효율을증가시킬수있다. 4. 컬럼의재평형이빠른시간내에이루어지기때문에단일용매나기울기용리를모두적용.

8 극성고정상 O OC 2 H 5 O Si O H + C 2 H 5 O Si (CH 2 ) 3 NH 2 O OC 2 H 5 O OC 2 H 5 Polar functional Group O Si O Si (CH 2 ) 3 NH 2 O OC 2 H 5

9 비극성고정상 O CH 3 O Si O H + Cl Si (CH 2 ) 7 CH 3 O CH 3 O CH3 O Si O Si (CH 2 ) 7 CH 3 O CH 3

10 Partition chromatography 가장일반적형태 고정상으로사용되는고체지지체의표면액상과이동상으로사용되는액체에대한분배력의차이가분리를좌우 고정상은다공성지지체에화학적으로결합 1. Normal phase ( 순상크로마토그래피 ) 고정상은이동상보다 more polar 비극성화합물이먼저용출극성고정상 (Nitrile, Diol, Amino) 2. Reverse phase ( 역상크로마토그래피 ) 비극성고정상과극성이동상사이의분배정도차이이용극성화합물먼저용출비극성고정상 (C 8, C 18 )

11 역상 / 순상비교 역상 순상 적용화합물 : 비극성화합물극성화합물 고정상 : alkyl-silanes alkyl-amines alkyl-nitriles Solvent 강도 : 극성 비극성 ( 이동상 ) 비극성 극성 11

12 Reverse Chromatography 의장점 상당히재현성이좋으며안정하다. 주된이동상인물은값이싸고구하기쉽다. 가장자주쓰이는이동상인 methanol, acetonitrile은세계어디서나적정한순도의것을쉽게구할수있다. 분리하고자하는시료분자의소수성정도에따라용리순서의예측이용이하다.

15 분배크로마토그래피의이동상 Solvent n-pentane Carbon Tetrachloride Chloroform Dichloromethane Tetrahydrofuran Dioxane Acetonitrile Methanol Water Reverse Phase Normal Phase

20 HPLC 의구성 (p. 384) * 탈기장치 (Degasser) * 송액장치 (Solvent Delivery systems: 펌프 ) * 시료주입기 (Injectors) * 컬럼 (Columns) * 컬럼온도조절기 (Thermostatted Column Compartment) * 검출기 (Detectors) 탈기장치펌프시료주입기 검출기 컬럼및컬럼온도조절기

23 탈기 (Degassing) 1. 목적 : 이동상의용존산소, 질소및 기포등을제거한다. 2. 방법 : * He (N 2 ) sparging * Vacuum (filtration) * Ultrasonication * On-line Degassing

24 On-line degasser 작동원리 Special plastic membrane tubing Solvent reservoir To pump Vacuum chamber

25 송액장치 :Solvent delivery system(pump) 1. 역할 : 이동상을이동상저장용기에서끌어들여 시료주입기로연속적으로밀어준다. 2. 요건 : 일정한유속과압력을유지할것. 3. Isocratic 및 Gradient 다양한용매를사용할수있을것. * Isocratic - 분석시간동안이동상조성의변화가없다. * Gradient - 분석시간동안이동상조성이시간의흐름에따라변한다.

26 단일용매펌프 (Isocratic pump) Damper From solvent bottle Inlet valve To mixing chamber To waste Seal Piston Ball screw drive Gear Motor with encoder

27 2 용매펌프 (Binary Pump) Pump outlet Purge valve Mixer Dampe r To waste Mixing chamber Inlet valve Inlet valve From Solvent bottle Seal Piston Seal Piston From Solvent bottle Pump head A Pump head B

28 시료주입기 (Injectors) 1. 역할 : 분석하고자하는시료를용매의흐름에실어준다. 2. 종류 : * 수동 (Manual Injection Valve) * 자동 (Auto Injector)

29 Injector 수동시료주입기 (Manual injector) Load Position Pump Injection Position Pump Sample Loop 6 3 Sample Loop 6 3 Column 5 4 Column 5 4 Needle Port Needl e Port

30 자동시료주입기 (AutoInjector) Vialgriper Meteringdevice Samplingunit 6-portvalve Frompump Tocolumn Towaste

31 컬럼 (Columns) 1. 구성 : 관모양의용기에충진제를채워서사용. 분석하고자하는시료의종류에따라컬럼의크기및충진제의종류를선택하여사용할수있다. 2. 역할 : 혼합상태의시료를화학적 / 물리적특성에 따른머무름정도의차이에의해분리한다. 컬럼온도조절기 (TCC): 분리능향상및분석결과의재현성보장을위해컬럼온도를적절하게설정, 유지한다.

$* 가변파장검출기 (Variable Wavelength Detector) * 다이오드어레이검출기 (Diode-Array Detector) * 형광검출기 (Fluorescence) * 굴절률검출기 (Refractive Detector) * 전기화학검출기 (electrochemical Detector) * 전도도검출기 (Conductivity$

32 1. 역할 : 컬럼에서분리된시료가일정한간격으로검출기 2. 종류 : 를통과할때시료의존재및양을일정한규칙에 의해인식하여전기적인신호로바꾸어준다. * 가변파장검출기 (Variable Wavelength Detector) * 다이오드어레이검출기 (Diode-Array Detector) * 형광검출기 (Fluorescence) * 굴절률검출기 (Refractive Detector) * 전기화학검출기 (electrochemical Detector) * 전도도검출기 (Conductivity Detector) Fluorescence 10.0% DAD(UV/VIS) 22.0% Detectors Refractive Index 8.0% Electrochemical 7.0% Conductivity 5.0% Mass spec 1.0% Others 4.0% UV/VIS(VWD) 43.0%

33 UV/VIS Detector 검출원리 Energy Absorbance S 1 Excitation S 0 S 1 = excited electronics state S 0 = ground electronics state E = excitation energy E 1/Energy

34 검출원리 Detector Cell Io I Log Io Io = 입사광의세기 I = 투과된빛의세기 A = 흡광도 I = A = bc = 몰흡광도 b = 광로의길이 c = 시료농도

35 대표적인발색단 Chromophore Structure l max(nm) Amine - NH Ethylene - C = C Kethone = C = O 195 Ester - COOR 205 Aldehyde - CHO 210 Carboxyl - COOH Nitro - NO Phenyl 202, 255 Naphthyl 220, 275 l max = wavelength of absorption maximum

36 Fluorescence Detector 검출원리 Absorption(excitation) E Emission E E * E * E o Ea = E* - Eo ex) : 빛, 화학에너지 Eo Ee = E* - Eo ex) : 열, 빛

37 Refractive Index Detector 검출원리 Reference compartment 1 2 n : medium 1 에대한 medium 2 의굴절지수 n 1 n 2 : medium 1,2 의굴절지수 1 2 : medium 1,2 에투사되는광원의각도 : reference cell 과 sample cell 의굴절차이 n = n 2 n 1 = sin 1 sin 2 Sample compartment Tangent = (n 1 - n 2 ) n 1

38 RID 의기기구조 From column 일반화합물의비선택적검출 고분자 / 당 Sample To waste 3-way fitting Reference A B A = Normally closed (Flush mode) B = Normally open

39 RID 를이용한당분석 Sample Matrix sweets, corn, beverages Column 300 X 7.8mm, Bio-Rad HPX-87P (80도) Mobile phase Water Detector RID

40 Conductivity Detector 검출원리 1 R = KA R : 두전극간의전기적저항 A : 전극의면적 L/A : 검출실고유상수 K : 용액의전도도 L : 전극사이의거리 S (or µs) = R 1 = KA

41 음이온분석 1. Fluoride (2ppm) 2. Chloride (4ppm) 3. Nitrite (4ppm) 4. Bromide (4ppm) 5. Nitrate (4ppm) 6. Phosphate (ppm) 7. Sulfate (6ppm) Column : Durasep A-1, 100x4.6mm Mobile Phase : 0.68mM NaHCO2/ 0.72mM Na2CO3 in 10% MeOH Detector Suppressed Conductivity

42 Detector 별특성 Detector 종류 감도 선택성 물질에따른 적용도 (%) 장점 / 주응용 VWD ng/pg - 80 저비용, 어느정도일반적 DAD ng/pg 피크순도확인 / 물질확인 Fluorescence pg/fg 고감도 Electrochemical pg/fg + >20 고감도 Conductivity ng/pg - 10 이온분석 Refractive Index ug/ng 일반적 /Polymer, 당 Mass Specrtometer ng/pg ++ <100 MW & 구조정보

44 이온교환크로마토그래피 고정상 음이온또는양이온교환수지 폴리스티렌이나실리카겔의표면에이온성을가진물질을화학적으로결합 이동상 aqueous/buffer counter ion 대상시료 이온성화합물및무기이온류 시료분리는 ph, 이온세기, 이동상에포함된 이온종류에영향을받음. 44

46 이온교환크로마토그래피의 분리기전 Strong Cation Exchanger Polystyrene or Silica Strong Anion Exchanger R1 (CH 2 CH 2 ) SO 3 - R4 N + R2 R3 Polystyrene or Silica CH 2 CH 2 CH 2 NR OOC R * 시료의분리는 ph, 이온세기, 이동상에포함된이온종류의영향을받는다. 46

48 Size Exclusion ( 크기배제 )chromatography 일정한크기의공극을형성시킨다공성 polystyrene 이나 silica gel 을충진입자로사용한다. 공극의크기에따라분리되는시료의분자량대가다르다. 시료의물리적특성 ( 크기 ) 에따른분리이다. 이동상은고정상에대해비활성이며 수용성용매일경우 GFC (gel filtration -) 지용성용매일경우 GPC (gel permeation -)

50 THIN LAYER CHROMATOGRAPHY Thin layer chromatography (TLC) is an important technique for identification and separation of mixtures of organic compounds. It is useful in: Identification of components of a mixture (using appropriate standards) following the course of a reaction, analyzing fractions collected during purification, analyzing the purity of a compound. In TLC, components of the mixture are partitioned between an adsorbent (the stationary phase, usually silica gel, SiO 2 ) and a solvent ( the mobile phase) which flows through the adsorbent.

51 THIN LAYER CHROMATOGRAPHY In TLC, a plastic, glass or aluminum sheet is coated with a thin layer of silica gel. A very small amount of a solution of the substance to be analyzed is applied in a small spot with a capillary tube, ~1cm from the bottom of the TLC plate The TLC is developed in a chamber which contains the developing solvent (the mobile phase). A truncated filter paper placed in the chamber serves to saturate the chamber with mobile phase. A B U C D filter paper A B U C D

52 THIN LAYER CHROMATOGRAPHY As the mobile phase rises up the TLC plate by capillary action, the components dissolve in the solvent and move up the TLC plate. Individual components move up at different rates, depending on intermolecular forces between the component and the silica gel stationary phase and the component and the mobile phase. The stationary phase is SiO2 and is very polar. More polar analytes interact more strongly with the stationary phase in move very slowly up the TLC plate. By comparison, the mobile phase is relatively nonpolar. More nonpolar analytes interact less strongly with the polar silica gel and more strongly with the less polar mobile phase and move higher up the TLC plate.

THIN LAYER CHROMATOGRAPHY Once the solvent is within ~1-2 cm of the top of the TLC sheet, the TLC is removed from the developing chamber and the farthest extent of the solvent (the solvent front) is

53 THIN LAYER CHROMATOGRAPHY Once the solvent is within ~1-2 cm of the top of the TLC sheet, the TLC is removed from the developing chamber and the farthest extent of the solvent (the solvent front) is marked with a pencil. The solvent is allowed to evaporate from the TLC sheet in the hood. The spots are visualized using a UV lamp. A fluorescent compound, usually Manganeseactivated Zinc Silicate, is added to the adsorbent that allows the visualization of spots under a blacklight (UV254). The adsorbent layer will fluoresce light green by itself, but spots of analyte quench this fluorescence and appear as a dark spot.

THIN LAYER CHROMATOGRAPHY - Visualization As the chemicals being separated may be colorless, several methods exist to visualize the spots: Visualization of spots under a UV 254 lamp.

54 THIN LAYER CHROMATOGRAPHY - Visualization As the chemicals being separated may be colorless, several methods exist to visualize the spots: Visualization of spots under a UV 254 lamp. The adsorbent layer will thus fluoresce light green by itself, but spots of analyte quench this fluorescence. Iodine vapors are a general unspecific color. Chromatogram of 10 essential oils, Stained with vanillin reagent. Specific color reagents exist into which the TLC plate is dipped or which are sprayed onto the plate. Once visible, the R f value of each spot can be determined

55 THIN LAYER CHROMATOGRAPHY Calculation of Rf s R f (A) = 2.0 cm 5.0 cm = 0.40 Solvent Front Distance solvent migrated = 5.0 cm Distance A migrated = 3.0 cm 4.0 cm R f (B) = R f (C) = 3.0 cm 5.0 cm 0.8 cm 5.0 cm = 0.60 = 0.16 Distance B migrated = 2.0 cm 3.0 cm R f (D) = 4.0 cm = cm Origen Distance C migrated = 0.8 cm x x x x A B U C x D The R f is defined as the distance the center of the spot moved divided by the distance the solvent front moved (both measured from the origin) 0.8 cm R f (U 1 ) = 3.0 cm 5.0 cm 0.8 cm R f (U 2 ) = 5.0 cm = 0.60 = 0.16

56 THIN LAYER CHROMATOGRAPHY R f s R f values can be used to aid in the identification of a substance by comparison to standards. The R f value is not a physical constant, and comparison should be made only between spots on the same sheet, run at the same time. Two substances that have the same R f value may be identical; those with different R f values are not identical.

Microsoft PowerPoint - d ppt [호환 모드]

Microsoft PowerPoint - d ppt [호환 모드] HPLC BASICS Chromatography 란? 혼합된시료성분이이동상과고정상사이를흐르면서흡착, 분배, 이온교환또는분자크기배재작용등에의해각각의단일성분으로분리되는것을말한다. 분리, 정성, 정량등의분석목적과분리, 정제, 분취목적에이용된다. Injection Interaction Elution a abacbc aabcbc HPLC System Mobile Phase