GEL FILTRATION CHROMATOGRAPHY Size Exclusion Chromatography What is

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 3 D 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 p. H 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 p. H 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 20 o and equilibrated for minimum 12 h • Sample is applied as spot or band 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