What is chromatography? Chromatography is the separation of an analyte from a complicated mixture of similar constituents for qualitative or quantitative identification Applies to both organic and inorganic compounds
Chromatography separation method partitioning behavior 원리 : 시료성분 ( 용질 ) 고정상통과 ( 이동상 ) 분배 (partition), 용질성분에따른분배비율차이 ( 이동속도가달라짐 ) 에따라분리가일어남. 고정상혹은정지상 (stationary) - 관속혹은관판에고정 이동상 (mobile phase, eluent) - 액체, 기체, ( 초임계유체 ) 로서시료를이동시킴 이동상종류에따라분류 개스 (gas chromatography), 액체 (liquid chromatography)
종류 (1) 정지상과이동상을접촉시키는방법에근거 * 관 chromatography 정지상 ( 좁은관에고정 ), 이동상 ( 관을통해압력이나중력의힘으로흐름 ) * 평면 (planar-): 정지상 ( 유리판, 다공성종이에정지상도포 ), 이동상 ( 모세관운동, 중력의영향을받아정지상통해움직임 ) (2) 관크로마토그래피법의용리 elution( 용리 ) - 연속적으로새로운이동상을첨가함으로써관을통하여화학종이동 ( 분배 ), 시료중의용질 ( 정지상 이동상 ), 이동속도의차 (3) 크로마토그램 (chromatogram) 검출기를관끝에설치, 신호를시간의함수로표시 - 봉우리위치 ( 시료의성분확인 ) - 봉우리면적 ( 성분의양 )
분류 : 일반적 two phases 의종류에따라명칭을붙인다. GC: 정지상상태 GSC ( 분리 mechanism - 흡착 ) GLC ( 분리 mechanism - 분배 ) GSC ( 컬럼충진물실리카겔, 분자체, charcoal, porapak 등 ) GLC ( 정지상은불활성인고체지지체에얇은막입힌액체상 ) LC: 컬럼 ( 성능, 분리우수 ), plane method (TLC, paper chro.) 정지상인액체상이지지체에어떻게붙어있느냐? LLC ( 고체지지체에액체정지상 ) LBC ( 정지상이화학결합 ) LSC ( 정지상상태에따라 ) 정지상이흡착제인경우 ( 흡착 ; LSC) 정지상이이온교환수지 (IEC) 이온교환, 발달 (IC) 양 / 음이온분리정지상이다공성 gel ( 분자량크기분리 ) SEC GFC( 수용성 ) GPC ( 비수용성 )
Classification of column chromatography methods
분류 이동상종류분리기구 ( 고정상종류 ) 고정상형식 크로마토그래피 Gas Chromatography (GC) gas-liquid chromatography (GLC) gas-solid chromatography (GSC) Liquid Chromatography (LC) liquid-liquid chromatography (LLC) liquid-solid chromatography (LSC) 분배 (partition chromatography) 흡착 (adsorption chromatography) 이온교환 (ion exchange chromatography) 겔침투 (gel permeation chromatography) 종이 (paper chromatography) 박층 (thin-layer chromatography) 칼럼 (column chromatography)
- 정성분석 ( 용질이나타나는시간 ) - 정량분석 ( 피크면적 )
Abundance The Chromatogram E A D B C Sample Mixture Chromatograph The time that a peak appears (it s retention time) is diagnostic for a given compound Chromatogram A B C D E The relative size of a peak (area or height) is proportional to the relative abundance of the compound in the mixture 0 5 10 15 20 Time (minutes)
기본적분리원리 Introduce two solutes (A & B) onto a packed column through which a mobile phase (i.e. solvent) or eluent is continuously pumped
Basic separation principles Separations depend on the extent to which solutes partition between the mobile and stationary phases We define this interaction with a partition coefficient ( 분배계수 ): K = C s /C m C s = stationary phase concentration C m = mobile phase concentration
넓힘현상 : Zone broadening or band broadening Refers to the shape of a peak as a solute migrates through a chromatographic column; it will spread out and shorten in height with distance migrated 마라톤
10.3. 크로마토그래피의이론 1940 년대 (Martin, Synge) 단 ( 층 ) 이론 = 관은수많은연속적인얇은층 ( 이론단 ). 각단에서이동상과정지상사이에용질의평형발생. 분석물이동은평형된이동상이한단에서다음단층으로단계적이동 관효율 = 분리와넓힘고려 단층이론과속도이론이해
단층이론 분리관 = 이론단 ( 얇은단 ) 이연속적으로연결되어있는것 각단에서용질평형 ( 이동상과정지상 ) 평형은연속적으로일어남 Gauss 곡선형태와이동속도성공적설명, 넓힘현상은설명하지못함 ( 분리효율 ) 컬럼효율척도이론단수 N = L/H
단층이론 (plate theory) 분리관이얇은단들이연속적으로싸여있다고가정. Plate Height (H) and Theoretical Plates (N) 컬럼의분리효율을나타낸다 column efficiency increases as N increases N = L/H N ( 이론단수 ), H ( 이론단높이 HETP) N = the number of interactions (i.e. transitions between mobile and stationary phases) that a solute has during its residence in the column H= the distance through the column a solute travels between interactions (typically given in cm)
속도이론 ; 두가지변수설명 용리띠가분리관의끝에나타날시간에영향주는변수 용리된봉우리너비에영향을주는변수 봉우리 = 대표적인입자평균속도값 / 주위각입자속도 띠너비 (w) = 관에머무르는시간에정비례이동상흐르는속도에반비례 (1) 표준편차 관효율척도 - 봉우리는 Gauss 곡선 - 효율을표준편차혹은평방표준편차 ( 분산 ) 로정의
속도이론 rate theory (or kinetic theory) used to explain (in quantitative terms) the shapes of chromatographic peaks and the effects of chromatographic variables on peaks as a solute migrates through a column The Gaussian shape of an peak can be attributed to the additive combination of the random motions of individual solute molecules The migration of an individual molecule through a column is irregular (based on random walk mechanism) Results in a symmetric spread of velocities around the mean
크로마토그래피의띠는원칙적으로 Gaussian shape 의 peak 을형성하기때문에, 정규오차곡선의폭은측정의표준편차 (σ) 또는 평방표준편차 (σ 2 ) 와직접적관계가있다. H = 2 L 관길이당분산정도 N = L 2 2
N 과 H 의실험적측정 ( 크로마토그램을이용하여측정 ) W = magnitude of the base of the triangle (in units of time) t M = retention time of an unretained solute t R = retention time of the solute - 용질입자움직이는평균속도 (L/t r ) - 시간으로나타낸표준편차는 cm 로나타낸표준편차를이동속도로나눔
W ( 띠넓힘현상의정량적척도 ) 정규곡선에서대략 96% 면적은 ±2σ 그림에서 W=4τ 2 N = t 16 r W 실험결과 tr 과 W N H
띠넓힘현상원인 (band broadening) p. 168/330 A. Eddy diffusion( 소용돌이확산 ) 다중경로효과 (multi-path) B. Longitudinal (molecular) diffusion ( 종축확산 ) ( 용질, 운반기체확산도현상 ) N 2, Ar 분자량 > H 2, He C. 비평형질량이동순간적불완전한평행, 고체지지체입혀진액상. 정도가낮은액상 / 얇고균일한막 따라서피크폭이넓어지는이유 띠넓힘 + 관에머무르는시간에 ( 정 ) 비례 + 이동상이흐르는속도에반비례 + 요인들 : 입자직경 ( 작고균일 ), 유속, 운반기체, 액상종류, 액상양, 압력, 온도등
한개의이론적컬럼층에해당하는높이 (HETP) 에의해정량적나타냄 HETP= 시료분자인용질이이동상과고정상사이에서평형에도달되는데필요한컬럼길이 van Deemter HETP = L/N = A + B/u + C*u
컬럼효율영향인자 Liquid Chromatography Mobile phase flow rate optimum flow rate corresponds to minimum H H is much smaller for HPLC(more efficient) than for GC, but in practice GC columns are much longer than for HPLC - hence greater N for GC. Gas Chromatography
컬럼효율영향인자 Particle size of column packing. the smaller the packing particles, the greater the column efficiency.
크로마토중요용어들 retention time (t r ) = the time it takes after injection for a solute to reach the detector migration time (t m ) = the time for an unretained species to reach the detector mobile phase velocity 이동상이동속도 (u) = L/t m ; the average linear rate of movement of molecules in the mobile phase (L = 컬럼길이 ) Linear rate of solute migration 용질이동속도 (v) = L/t r
머무름비 (retention ratio) R=t m /t r = V m /V r 시료용질머무른부피 V r = t r F ( 용질체류시간 )( 이동상유속 ) 용질이동속도 v = u*( 용질이이동상에머무는시간분율 ) m s m m s s s s m m m m V V K u V C C V u C V V C V C u u v = = = = 1 1 1 1 용질의전체몰수이동상에있는용질의몰수
Retention factor or Capacity factor (k A 용량인자 ) = a term used to describe the migration rate of solutes (analytes) on columns k ' = K V V m Where: K = is the distribution constant for species A V s = volume of stationary phase V m = volume of the mobile phase s
The retention factor can be determined directly from a chromatogram using: k ' = t r - t m t m Interpreting the retention factor If k < 1; the elution is too rapid for accurate determination of tr. If k > approx. 10; the elution is too slow to be practical The preferred range for k is between 1 and 5
관분리능 Column resolution (R S ) is a quantitative measure of the ability of a column to separate two analytes: R S = 2[( tr ) W A B - ( t W B R ) A ] To increase resolution increase column length (L) Limitation: longer time and broader bands Usually compromise between resolution and speed of analysis
N = t 16 W 2 r 4 ==== W = t r N selectivity factor (a) = k B /k A Where k B is the distribution constant of the more strongly retained species (so that a >1) R S = [( tr ) B - W B ( t r ) A ] The selectivity factor can also be defined in terms of retention factors and retention times: α = K K B A = k' k' B A = (t (t r r ) ) B A - - t t m m
따라서 A 와 B 를분리하는데필요한단수를구할수있다 (N 과 H 의실험적측정 ). 2 ' ' 2 2 ' ' ) 1 ( ) 1 ( 16 ) 1 )( 1 ( 4 B B S B B S k k R N k k N R - = - -- - = a a a a
In general, separation (or Column) efficiency, as N and H 1. Particle size of packing (as size, H ) 2. Immobilized film thickness (as film thickness, H ) - due to faster diffusion rates in film 3. Viscosity of mobile phase (as viscosity, H ) 4. Temperature (as T 증가, k', but a remains approximately constant) 5. Linear velocity of mobile phase; u = L/t M (u is proportional to 1/t M because L = constant) 6. Column length (as L, N, but H = constant, and separation efficiency )
The general elution problem It is often difficult to find a set of conditions which will resolve all peaks to the same degree and also permit reliable quantification Solutions Multiple runs at different conditions Programmed elution (i.e. change conditions during the run)
Important terms