ChromatographyChromatography basically involves theseparation of mixtures due to differences inthe distribution coefficient of sample componentsbetween 2 different phases.One of these phases is a mobile phase andthe other is a stationary phase.
Distribution CoefficientDefinition: Concentration of component A in stationary phase Concentration of component A in mobile phaseDifferent affinity of these 2 components to stationaryphase causes the separation.
Kinds of Chromatography1. Liquid Column Chromatography2. Gas Liquid Chromatography
Liquid Column ChromatographyA sample mixture is passed through a columnpacked with solid particles which may or may not becoated with another liquid.With the proper solvents, packing conditions, somecomponents in the sample will travel the columnmore slowly than others resulting in the desiredseparation.
Four Basic Liquid ChromatographyBasic liquid chromatography modes are named according to the mechanism involved:1. Liquid/Solid Chromatography (adsorption chromatography) A. Normal Phase LSC B. Reverse Phase LSC2. Liquid/Liquid Chromatography (partition chromatography) A. Normal Phase LLC B. Reverse Phase LLC3. Ion Exchange Chromatography4. Gel Permeation Chromatography (exclusion chromatography)
Liquid Solid Chromatography Normal phase LS Reverse phase LS δ− δ+ Si - O - H 30 µ Silica GelThe separation mechanism in LSC is based on thecompetition of the components of the mixture samplefor the active sites on an absorbent such as Silica Gel.
Liquid Solid Chromatography OH HEXANE Si - OH OH CH3 CH3 CH3 - C C-CH3 CH3 CH3 CH3
Water-Soluble Vitamins1. Niacinamide 2. Pyridoxine H 3C N N HO CH 2OH CONH 2 CH 2OH3. Riboflavin CH 2OH HOCH HOCH 4. Thiamin HOCH CH 2H 3C N N O H 3C N NH 2 S CH 2CH 2OH NH ClH 3C N N N CH 2 CH 3 O
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 3The 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 componentsmore than others to effect separation.
Types of Chromatography LIQUIDMOBILE PHASE Liquid-Liquid Liquid-SolidFORMAT Chromatography (Partition) Chromatography (Adsorption) Liquid SolidSTATIONARY PHASE Normal Phase Reverse Phase Normal Phase Reverse Phase Mobile Phase - Nonpolar Mobile Phase - Polar Stationary phase - Polar Stationary phase - Nonpolar
Ion-Exchange Chromatography SO 3 Na + -Separation in Ion-exchange Chromatography is based on thecompetition of different ionic compounds of the sample for theactive 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 - COO pH4.5 + Na
Chromatography of Amino Acids Stationary Phase Mobile Phase + H3 N - SO3 Na+ COOH + Na OH - + SO3 H3 N COOH Exchange Resin - SO3 H3N+ COOH pH3.5 OH - SO3 H 3 N+ + - + - Na COO H OH = H 2 O + Na - SO3 H3 N + - + - COO H OH = H 2 O - SO3Na+ pH4.5
Gel-Permeation ChromatographyGel-Permeation Chromatography is a mechanical sorting of moleculesbased on the size of the molecules in solution.Small molecules are able to permeate more pores and are, therefore,retained longer than large molecules.
Selecting an Operation ModeSample Type LC ModePositional isomers LSC or LLCModerate Polarity Molecules LSC or LLCCompounds with Similar Functionality LSC or LLCIonizable Species IECCompounds with Differing Solubility LLCMixture of Varying Sized Molecules GCC
Height Equivalent to a Theoretical PlateLength of a column necessary for the attainment of compounddistribution equilibrium measure the efficiency of the column. X 2 Theoretical plates (N) = 16 ( ) Y X Y
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 SystemCarbohydratesAmino acids, proteinsVitamins, A, D, E, KNucleosides (purines and pyrimidines)Fatty acids, fatsAflatoxinsAntioxidantsContaminants of packaging materialsCarotenoids, chlorophyllsSaccharines