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:   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

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)

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.

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 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

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 (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

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

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 OH OH CH CH 3 3 CH - C C-CH 3 3 CH CH 3 3 CH 3

Water-Soluble Vitamins

Water-Soluble Vitamins

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.

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 + + SO 3 Na H N 3 COOH Ion-exchange Resin - + SO H N 3 3 - COO pH4.5 + Na

Chromatography of Amino Acids

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.

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

Selectivity Selectivity

Resolution Equation

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.

Importance of Theoretical Plates (N)

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

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