Gel Filtration Chromatography


GEL FILTRATION CHROMATOGRAPHY

PRINCIPLE

       A column of gel particles or porous glass granules is in equilibrium with a suitable solvent for the molecules to be separated.
       Large molecules which are completely excluded from the pores will pass through the interstitial spaces.
       Smaller molecules will be distributed between the solvent inside and outside the molecular sieve and will pass through the column at a slower rate.
       Thus the distribution of a solute in a column of swollen gel is determined by the total volume of solvent both inside and outside the gel particles.

PROCEDURE

       The gel medium packed into the column is a porous matrix that consists of spherical beads, which have stable physical and chemical properties such as non-reactivity and lack of adsorption.
       The small molecules can enter the beads but the larger one cannot. The small molecules are distributed in the aqueous solution both inside and between the beads where as the large molecules are located in the solution between the beads.
       The pores and space between the particles is filled with a liquid buffer, which fills the entire column.
       The liquid filling the pore space is called a stationary phase and the liquid in the space between particles is a called mobile phase.
       Once the sample has been applied to the top of the column, it passes through the column along with the mobile phase from the top of the column to the bottom.
       Smaller molecules are able to cross and go through these polymer beads but large ones are not able to.
       Therefore, small molecules in the column are both inside the polymer beads and between them, whereas large molecules can only travel between the polymer beads.
       Since less traveling space is allowed for the larger beads, they tend to move faster down the column and they emerge first at the end of the column. Think of it this way.
        The molecules traveling down the column represent a faucet. If the faucet has a smaller volume of space to allow the water to travel, the water will come out faster and with greater force.
       The same concept applies here as well. Since less volume is accessible to the bigger molecules, they move much faster through the column than smaller molecules do.
        So, since the small molecules are stuck inside the beads, they tend to move slower.
       Theoretically, molecules that have the same size should elute simultaneously.
       An elution diagram, or a chromatogram, can be constructed to verify complete separation.
       Before separation of unknown sample, solutions with known biomolecules can be run in order to make a calibration curve, which later can be used as a reference for identifying of unknown molecules.

APPLICATIONS

       Fractionation and molecular weight determination of proteins.
       Nucleic acid separation.
       Plasmid purification.
       Polysaccharide fractionation.

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