1. Gel Filtration Gel permeation chromatography Size exclusion chromatography Separation of molecules on the basis of size (and shape)

2. Theory Column matrix Porous beads Large molecules are excluded from the pores and travel through the column fastest Small molecules are included – can diffuse into the pores and elute later

3. Theory

4. Elution Profile Ve Ve = Elution volume (volume of solvent between injection and elution). Dictated by proportion of porous matrix available to molecules (Kd).

5. Column Parameters Vs= volume of solvent held in the pores. This is normally approximated to Vt-Vo = volume of beads Vo = void volume Vt = total volume Vo = Elution volume of a large totally excluded molecule such as blue dextran Vt = Physical volume of column

6. Calculation of Ve For a molecule that can partially enter the pores: Ve = Vo + Kd (Vs) or Ve = Vo + Kav (Vt-Vo) Kav = proportion of pores available to the molecule. Totally exclude Kav = 0 and Ve = Vo Totally included Kav = 1 and Ve = Vt

7. Behaviour of Molecule on any Column Kav = Ve – Vo Vt - Vo

8. Resolution Resolution proportional to square root of column length. Also affected by rate at which column is run

9. Design of Column Column size –Analytical or preparative Solvent –Inert matrix most solvents OK Matrix –Most important consideration –Many different types Material Pore size

10. Matrix Types Material Sephacryl –dextran Sephadex –dextran Sepherose –agarose Superdex –mixture SephacrylProtein (kD) Dextrans (kD) S-1001-100NS S-2005-2501-80 S-30010-15002-400 S-40020-800010-2000 S-500NS40-20,000

11. Running the column Sample size / Fraction size –0.5 – 5% of total bed volume (Vt). –Concentration limited by viscosity Running time –Determined by trial and error –Slow rates allow efficient partitioning into pores and thus increase resolution –Slow rates increase diffusion of sample on column thus increasing peak width and reducing resolution. –Protein about 5mL cm -2. h -1

12. Types of Column Systems Liquid Chromatography High Performance Liquid Chromatography (HPLC)

13. Determination of Molecular Weight Calibrate column with known standards Plot Kav against lg Mol Wt

14. Other Types of Column Chromatography Ion-Exchange Chromatography –Separation on basis of charge DEAE- sephadex Hydrophobic Interaction Chromatography –Separation on basis of hydrophobicity Phenyl-sepherose Affinity Chromatography –Affinity of enzyme for substrate or other ligand