Download Chromatography

CHROMATOGRAPHY
Chromatography
Chromatography basically involves the
separation of mixtures due to differences in
the distribution coefficient of sample components
between 2 different phases.
One of these phases is a mobile phase and
the other is a stationary phase.
Distribution Coefficient
Definition:
Concentration of component A in stationary phase
Concentration of component A in mobile phase
Different affinity of these 2 components to stationary
phase causes the separation.
History of chromatography
1) Chromatography의 기원
- 1850년 F.F.Runge가 여과지를 이용하여 염료를 분리한 것에서 유래
(Paper Chromatography)
2) Chromatography의 어원
- 1906년 M.Tswett가 흡착제를 충진시킨 유리관을 이용하여 식물의 색
소를 분리시키면서 명명
- Chromatography = Chromos(color) + graphy(write)
History of chromatography
 1941년 Martin과 Synge에 의해 발전된 액체-액체 크로마토그래피(LLC)이
다. 단 하나의 고체 흡착제 대신에 그들은 불용성 흡착제를 고정상에 결합시
킨 고정 액상을 사용했다. 용질 성분은 용해도에 따라 두 액체 (고정상과 이동
상)에서 서로 이루어진다.
 이후 크로마토그래피 기술은 발전을 거듭하여 최근에는 HPLC가 널리 각광
을 받고 있는데 이는 비휘발성 용질이나 열에 약한 시료의 신속한
기술로 인정받고 있다.
분리
Classification of Chromatography
- Paper Chromatography
- LC (Liquid Chromatography) – TLC, HPLC
- GC (Gas Chromatography)
Liquid Column Chromatography
A sample mixture is passed through a column packed
with solid particles which may or may not be coated
with another liquid.
With the proper solvents, packing conditions, some
components in the sample will travel the column
more slowly than others resulting in the desired
separation.
3. Column chromatography
- 정지상(Stationary phase) – Column, Paper, Plate
- 이동상(Mobile phase) –Gas, Liquid,
C
B
A
Mobile phase
Gas,
Liquid
-
Stationary phase
Column, Paper,
Plate
혼합시료를 이동상의 흐름에 따라 정지상을 통과시키면, 시료의 구
성성분에 따라 이동률(migration rate)이 다르다는 것을 이용하여 물질
을 분리 시키는 방법
A+B+C  A, B, C
Chromatography 기술은 혼합물의 separation, isolation, 동정, 정량에
아주 유용한 방법이다.
Column chromatography
Column 은 유리관(column)과 같은 원기둥 모양의 관에 산화알루
미늄이나 이온교환수지 등을 충전한 것이다. 칼럼의 충전제로서
산화알루미늄 ·활성탄 ·산화마그네슘 등을 사용한 것을 흡착크로
마토그래피, 녹말 ·셀룰로오스 등을 사용한 것을 분배크로마토그
래피, 이온교환수지를 사용한 것을 이온교환 크로마토그래피 및
분자크기를 이용하여 분리하는 것을 크기배제크로마토그래피라
고 한다.
Fundamentals of column chromatography
흡착 크로마토그래피 분배 크로마토그래피
(액체-고체)
(액체-액체)
이온교환
크로마토그래피
실리카젤, 알루미나
이온
그룹을결합시킨
다공성 수지
화학적으로 불활성인
다공성&3차원적으로 네
트웍을 이룬 겔 혹은
무기 고체
분석하고자 하는
시료에 있는
이온종과
정지상의
전하 (시료와 반대
전하를 가짐) 와의
상호작용을
이용하여 분리
시료를 크기 별로
분리한다.
크기가 작은 시료는
정지상의 작은 구멍까지
다 거쳐 나오므로
컬럼을 빠져나 오는데
시간이 오래걸린다
정시상의 silanol
그룹과 시료의 극성
작용기와의
상호작용을
이용하여 비극성
물질 분리
불활성 지지체의
흡착 혹은 결합된
액체층으로 극성과
비극성 모두 된다.
시료가 이동상과
정지상 액체에
용해도 차에 따라
분배 됨으로써
분리됨
크기 배제
크로마토그래피
Types of Chromatography
LIQUID
MOBILE PHASE
Liquid-Liquid
Chromatography (Partition)
FORMAT
STATIONARY PHASE
Normal Phase
Liquid-Solid
Chromatography (Adsorption)
Solid
Liquid
Reverse Phase
Normal Phase
Mobile Phase - Nonpolar
Mobile Phase - Polar
Stationary phase - Polar
Stationary phase - Nonpolar
Reverse Phase
Four Basic Liquid Chromatography
Basic liquid chromatography modes are named according to the mechanism
involved:
1. Liquid/Solid Chromatography (adsorption chromatography)
A. Normal Phase LSC
B. Reverse Phase LSC
2. Liquid/Liquid Chromatography (partition chromatography)
A. Normal Phase LLC
B. Reverse Phase LLC
3. Ion Exchange Chromatography
4. Gel Permeation Chromatography (exclusion chromatography)
Liquid Solid Chromatography
Normal phase LS
Reverse phase LS
d- d+
Si - O - H
30 m
Silica Gel
The separation mechanism in LSC is based on the
competition of the components of the mixture sample
for the active sites on an absorbent such as Silica Gel.
Liquid Solid Chromatography
OH
HEXANE
Si - OH
CH 3
OH
CH 3
C-CH
CH 3
CH 3 - C
CH 3
CH 3
3
Water-Soluble Vitamins
1.
Niacinamide
2.
Pyridoxine
H3C
N
N
HO
CH2OH
CH2OH
CONH 2
3.
Riboflavin
CH2OH
HOCH
HOCH
HOCH
CH2
H3C
N
N
H3C
4. Thiamin
O
NH
N
O
H3C
N
N
NH 2
CH2
S
N
CH2CH2OH
Cl
CH3
Water-Soluble Vitamins
2
3
Inject
1
0
4
5
10
15
20
Column: u Bondapak C18
Solvent: MeOH
Sample: Water-Soluble Vitamins
Liquid-Liquid Chromatography
ODPN (oxydipropionylnitrile)
Normal Phase LLC
Reverse Phase LLC
NCCH CH OCH CH CN(Normal)
3 2
2 2
CH (CH ) CH (Reverse)
3
2 16
3
The stationary solid surface is coated with a 2nd liquid (the Stationary Phase)
which is immiscible in the solvent (Mobile) phase.
Partitioning of the sample between 2 phases delays or retains some components
more than others to effect separation.
Ion-Exchange Chromatography
SO 3- Na +
Separation in Ion-exchange Chromatography is based on the
competition of different ionic compounds of the sample for the
active sites on the ion-exchange resin (column-packing).
Mechanism of Ion-Exchange Chromatography of Amino Acids
pH2
SO 3
-
Na
+
H3N
+
COOH
Ion-exchange Resin
SO 3
-
H 3N
Na
+
+
COO
-
pH4.5
Chromatography of Amino Acids
Stationary Phase
Mobile Phase
H3 N
-
SO3 Na+
+
COOH
+
Na
SO3
OH
-
H3 N
+
COOH
Exchange Resin
-
SO3 H3N+
COOH
SO3
pH3.5
OH
-
H3 N+
+
-
Na
COO
H
+
-
OH = H 2 O
+
Na
SO3
-
H3 N
+
-
COO
H
+
-
OH = H 2 O
-
SO3Na+
pH4.5
Gel-Permeation Chromatography
Gel-Permeation Chromatography is a mechanical sorting of molecules
based on the size of the molecules in solution.
Small molecules are able to permeate more pores and are, therefore,
retained longer than large molecules.
Solvents
• Polar Solvents
Water > Methanol > Acetonitrile > Ethanol >
Oxydipropionitrile
• Non-polar Solvents
N-Decane > N-Hexane > N-Pentane >
Cyclohexane
Selecting an Operation Mode
Sample Type
LC Mode
Positional isomers
LSC or LLC
Moderate Polarity Molecules
LSC or LLC
Compounds with Similar Functionality
LSC or LLC
Ionizable Species
IEC
Compounds with Differing Solubility
LLC
Mixture of Varying Sized Molecules
GCC
Schematic Diagram of Liquid Chromatography
Detector
1.
Ultraviolet Detector
200-400nm
254 nm
2.
Reflective Index Detector
Universal Detector
High Performance Liquid Chromatography
High Performance Liquid Chromatography
Retention Time
Time required for the sample to travel from the injection port through
the column to the detector.
Response
D
B
A
C
5
10
15
Retention Time
20
25
Selectivity
Ratio of Net Retention Time of 2 components.
(Distribution Coefficient)
 
X2
X1
-
X0
X0
Selectivity
 Selectivity
Response
X
2
X1
X0
1
3
Retention Time
6
Resolution Equation
V2 - V1
R=
1/2(W1 + W2)
Response
V2
V1
W1
W2
W1
W2
Volumes
Resolution
Height Equivalent to a Theoretical Plate
Length of a column necessary for the attainment of compound
distribution equilibrium
measure the efficiency of the column.
X 2
Theoretical plates (N) = 16 (
)
Y
X
Y
Importance of Theoretical Plates (N)
Theoretical Plate, Selectivity and Height Equivalent
to a Theoretical Plate
2
4
V2
V1
1
3
V0
W2
W1
W3
W4
V3
V4
V0 = 1.0 (Minutes) V1 = 5.0, V2 = 7.0, V3 = 11.0, V4 = 13.0
W1 = 1.0, W2 =1.0, W3 = 1.0, W4 =1.0
Chromatogram of Orange Juice Compounds
General Factors Increasing Resolution
•
•
•
•
•
•
•
•
•
•
Increase column length
Decrease column diameter
Decrease flow-rate
Pack column uniformly
Use uniform stationary phase (packing material)
Decrease sample size
Select proper stationary phase
Select proper mobile phase
Use proper pressure
Use gradient elution
LC Application in Food System
Carbohydrates
Amino acids, proteins
Vitamins, A, D, E, K
Nucleosides (purines and pyrimidines)
Fatty acids, fats
Aflatoxins
Antioxidants
Contaminants of packaging materials
Carotenoids, chlorophylls
Saccharines