1. GEL FILTRATION CHROMATOGRAPHY Size Exclusion Chromatography

2. 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

3. 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

4. GEL FILTRATION ASSEMBLY

6. Materials Gels Majority available in two particle size grades
viz. coarse ( 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.

7. 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

8. 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

9. 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

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

11. 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

12. 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.

14. 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.

15. 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

16. 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.

17. 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