GEL FILTRATION CHROMATOGRAPHY Size Exclusion Chromatography

What is gel filtration chromatography? Separation of molecules on the basis of molecular size and shape Separation of proteins and nucleic acids Molecular sieve chromatography - Natural or synthetic zeolites of metallic alumina-silicates Controlled pore glass chromatography - Porous glass granules

Principle Separation based on size, shape and molecular weight Mechanism - Molecular sieve properties of porous materials Porous materials – Gel beads or Porous glass granules packed into a glass column and act as molecular sieve Ability of molecules to enter the pores within the beads or granules Beads are made of different materials with different porosity and form 3D network of cross-linked chains

GEL FILTRATION ASSEMBLY

Materials Gels Majority available in two particle size grades viz. coarse (300-100 m) and fine (20-80 m). Available in bead form than powder form. Cross-linked dextrans (Sephadex) Agarose (Sepharose, Bio Gel A) Polyacrylamide (Bio Gel P) Polyacryloyl morphine (Enzocryl gel) Polystyrene (Bio Beads S) Porous glass granules Bio Glass Porous silica as Porasil.

Dextran gels Cross-linked polysaccharide, epichlorohydrin Hydrophilic character Swells in aqueous media. Stable in water, salt solution, organic solvents, alkali and weak acidic solution Molecular size exclusion limit is 200 – 800 Kda

Agarose gel Agar (polysaccharide with D-galactose and 3,6 anhydro L-galactose units) Hydrophilic and absence of charged groups. Molecular size exclusion limit several million daltons. Compatible with cell aqueous buffers Stable at pH 4-10 Mostly incorporated with bacteriostatic agent (0.2% sodium azide) To study viruses, nucleic acids and polysaccharides

Polyacrylamide gels Polymerization of acrylamide and methylene bis acrylamide form this gel Stable in aqueous buffers at pH 1-10 Molecular size exclusion limit is 1.8 to 400 Kda

Porous glass gravels Manufactured from borosilicate glass. Molecular size exclusion limit is 3000 to 9 million daltons

Procedure Column Packing First gel is converted to swollen form i.e. gel in weak salt solution. Swelling time Sephadex G-25 and G-50 – Overnight Sephadex G-75 – 1 day Sephadex G-100 – 2 days Sephadex G-200 – 3 days Swelling time reduced by heating gel in boiling water bath for 1-5 h. Space between the polymer chains is increased due to swelling. Gel is then packed into column

Procedure….. Sample application Sample (mixture of larger and smaller molecules) Application depends on the column size Smaller molecules enter the gel material and their flow is retarded Larger molecules pass down rapidly because they are unable to penetrate the gel molecules Smaller molecules later fall down slowly.

Procedure…. Retardation depends upon Size of the particles Adsorption property of the gel Void volume is measured by use of completely excluded compound (Blue dextran – high MW polysaccharide) Effluent volume of particular compound enables to calculate its distribution coefficient.

Void volume Measured by use of completely excluded compound (Blue dextran – high MW polysaccharide) Effluent volume of particular compound enables to calculate its distribution coefficient

Thin layer gel filtration Separation of hydrophilic substances such as proteins, peptides and nucleic acids Swollen gel is spread on a thin plate and placed in air tight container Then connected to reservoir at either end by filter paper bridges Plates are inclined at 20o and equilibrated for minimum 12 h Sample is applied as spot or band and the plate is developed Spots are then detected.

Applications Purification of biological macromolecules Proteins, enzymes, antibodies, hormones and nucleic acids, etc. Determination of molecular weight Proteins and enzymes. Desalting Amino acids from proteins Phenol from nucleic acids Ammonium sulphate from proteins Monosaccharides from polysaccharides Study the protein binding structures Solution concentrations