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Size Exclusion Chromatography Introduction Introduction The separation of various constituents by taking advantage of the difference in size and geometry of the molecule constitute the basis of size exclusion chromatography. By virtue of a difference in size, certain particles move faster while others move slower thereby creating differential migration front in a column packed with gels. Types of exclusion chromatography 1. Gel permeation or gel filtration technique 2. Ion exclusion and retardation 3. Inorganic molecular sieves or (molecular sieve filtration) Gel chromatography Gel chromatography is a technique in which separation is based upon the molecular size and shape of the species in the sample. The stationary phase consists of beads of porous polymeric material, a gel. The liquid percolating through the bed is called the mobile phase. Theory The technique involves inclusion and exclusion of solutes through a stationary phase consisting of a heteroporous cross linked polymeric gels. The process of separation is based upon the different permeation rates of each solute molecule into the interior of the gel particles. By flow of liquid, molecules diffuse in all parts of the gel. Larger molecules appear first in the effluent followed by entrapped smaller molecules. Mechanism of Gel permeation chromatography Gel chromatography is essentially a case of liquid-liquid partition chromatography process in which the solute molecules are distributed between two liquid phases, liquid in the gel pores and the liquid outside the gel. Three mechanisms have been proposed to explain the technique of gas chromatography: 1. Steric exclusion mechanism This effect is more prominent where the major portion of a solute is larger than many pores of the gel. It is presumed that, i) Because the gel particles contain a distribution of pore sizes, small molecules can enter large number of pores while the large molecules can enter small number of pores. ii) The total volume (V) of a column packed with a gel that t has been swelled by the solvent is given by V = V + V +V -(1) t g I 0 Where, Vg is the volume occupied by the solid matrix of the gel, VI is the volume of solvent held in the pores or interstices, and V0 is the free volume outside the gel particles.
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