1. CHROMATOGRAPHY

2. 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.

3. Distribution Coefficient
Definition:
Different affinity of these 2 components to stationary phase causes the separation.
Concentration of component A in stationary phase
Concentration of component A in mobile phase

4. History of chromatography
- 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)

5. 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 분리 기술로 인정받고 있다.

6. Classification of Chromatography
- Paper Chromatography
- LC (Liquid Chromatography) – TLC, HPLC
- GC (Gas Chromatography)

7. 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.

8. 3. Column chromatography
Stationary phase
정지상(Stationary phase) – Column, Paper, Plate
이동상(Mobile phase) –Gas, Liquid,
- 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. A C B
Mobile phase
Column, Paper, Plate
Gas, Liquid

9. 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.

10. Fundamentals of column chromatography
흡착 크로마토그래피
(액체-고체)
실리카젤, 알루미나
정시상의 silanol 그룹과 시료의 극성 작용기와의 상호작용을 이용하여 비극성 물질 분리
분배 크로마토그래피
(액체-액체)
불활성 지지체의
흡착 혹은 결합된 액체층으로 극성과 비극성 모두 된다.
시료가 이동상과 정지상 액체에 용해도 차에 따라 분배 됨으로써 분리됨
이온교환
크로마토그래피
이온 그룹을결합시킨 다공성 수지
분석하고자 하는 시료에 있는 이온종과 정지상의 전하 (시료와 반대 전하를 가짐) 와의 상호작용을 이용하여 분리
크기 배제
크로마토그래피
화학적으로 불활성인 다공성&3차원적으로 네트웍을 이룬 겔 혹은 무기 고체
시료를 크기 별로 분리한다.
크기가 작은 시료는 정지상의 작은 구멍까지 다 거쳐 나오므로 컬럼을 빠져나 오는데 시간이 오래걸린다

11. Types of Chromatography
LIQUID
MOBILE PHASE
Liquid-Solid
Liquid-Liquid
FORMAT
Chromatography
(Adsorption)
Chromatography
(Partition)
Solid
Liquid
STATIONARY
PHASE
Normal Phase
Reverse Phase
Normal Phase
Reverse Phase
Mobile Phase -
Nonpolar
Mobile Phase -
Polar
Stationary phase -
Polar
Stationary phase -
Nonpolar

12. 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)

13. 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.

14. Liquid Solid ChromatographyOH
HEXANE
Si - OH OH OH CH CH 3 3 CH
- C
C-CH 3 3 CH CH 3 3 CH 3

15. Water-Soluble Vitamins

16. Water-Soluble Vitamins

17. Liquid-Liquid Chromatography
ODPN (oxydipropionylnitrile)
Normal Phase LLC
Reverse Phase LLC
NCCH 3 CH 2
OCH
CN(Normal)
(CH ) 16
(Reverse)
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.

18. Ion-Exchange ChromatographySO 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).

19. Mechanism of Ion-Exchange Chromatography of Amino Acids+ + SO 3 Na H N 3
COOH
Ion-exchange Resin - + SO H N 3 3 -
COO
pH4.5 + Na

20. Chromatography of Amino Acids

21. 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.

22. Solvents Polar Solvents
Water > Methanol > Acetonitrile > Ethanol > Oxydipropionitrile
Non-polar Solvents
N-Decane > N-Hexane > N-Pentane > Cyclohexane

23. 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

24. Schematic Diagram of Liquid Chromatography

25. Detector
1. Ultraviolet Detector nm nm Reflective Index Detector Universal Detector

26. High Performance Liquid Chromatography

27. High Performance Liquid Chromatography

28. Retention Time
Time required for the sample to travel from the injection port through the column to the detector.

29. Selectivity Ratio of Net Retention Time of 2 components.
(Distribution Coefficient)

30. Selectivity
Selectivity

31. Resolution Equation

32. Resolution

33. Height Equivalent to a Theoretical Plate
Length of a column necessary for the attainment of compound distribution equilibrium measure the efficiency of the column.

34. Importance of Theoretical Plates (N)

35. Theoretical Plate, Selectivity and Height Equivalent to a Theoretical Plate
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

36. Chromatogram of Orange Juice Compounds

37. 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

38. 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