CHROMATOGRAPHY Chromatography basically involves the separation of mixtures
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.
Kinds of Chromatography 1. Liquid Column Chromatography 2. Gas Liquid 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.
Diagram of Simple Liquid Column Chromatography
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 C-CH 3 - C CH 3
Water-Soluble Vitamins
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 2 nd 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.
Types of Chromatography LIQUID MOBILE PHASE Liquid-Liquid Chromatography (Partition) FORMAT STATIONARY PHASE Normal Phase Mobile Phase - Nonpolar Stationary phase Polar Liquid-Solid Chromatography (Adsorption) Solid Liquid Reverse Phase Normal Phase Mobile Phase Polar Stationary phase - Nonpolar Reverse Phase
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 p. H 2 - SO 3 Na + H 3 N + COOH Ion-exchange Resin SO 3 - H 3 N Na + + COO - p. H 4. 5
Chromatography of Amino Acids
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 Positional isomers Moderate Polarity Molecules LC Mode 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 -400 nm 254 nm 2. Reflective Index Detector Universal Detector
High Performance Liquid Chromatography
High Performance Liquid Chromatography
Retention 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 V 0 = 1. 0 (Minutes) V 1 = 5. 0, V 2 = 7. 0, V 3 = 11. 0, V 4 = 13. 0 W 1 = 1. 0, W 2 =1. 0, W 3 = 1. 0, W 4 =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
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