Instrumental Methods of Analysis of an unknown protein. The elution volume is plotted against the known molecular weights of the proteins on semi log graph paper (Fig. 7.10) and a line derived. From this line and the elution volume of an unknown Protein, the molecular weight of the unknown can be estimated. 7.3.3. Applications Purification: The main application of exclusion chromatography ig in the purification of biological macromolecules by facilitating their separation from larger and smaller molecules. Viruses, enzymes, hormones, antibodies, nucleic acids and polysaccharides have all been separated and purified. Molecular mass determination: The elution volumes of globular proteins are determined by their relative molecular mass. A considerable range of relative molecular masses, the elution volume is approximately linear function of the logarithm of relative molecular mass. Determination of solution concentration Desalting: By use of column of Sephadex G-25, solutions of high molecular mass compounds may be desalted. The high molecular substances move with the void volume, whereas the low molecular components are distributed between the mobile and stationary phases and hence move slowly. This method of desalting is faster and more efficient than dialysis. Applications include removal of phenol from nucleic acid preparations, ammonium sulphate from protein preparations and salt from samples eluted from ion-exchange chromatography columns. Protein-binding studies: Exclusion chromatography is one of method used to study the reversible binding of ligand to a macromolecule such as a protein, including receptor proteins. A sample of the protein/ligand mixture is applied to a column of. suitable gel that has been equilibrated with a solution of the ligand of the same concentration as that in the mixture. The sample is eluted with buffer in the standard way and the concentration of ligand and protein in the effluent determined. The early fractions will contain unbound ligand, but the subsequent appearance of the protein will result in an increase in the total amount of ligand.er proteins including receptor chromatography. as been purified by affinity 2. The application of the technique is limite onl ilabii of immobilized ligands. ATER LS 3. The principles have been extended to nucleic acids and have made a considerable contribution to developments in molecular biology. Messenger RNA for example is isolate by selective hybridization on poly (U) Sepharose 4B by exploiting its poly (A) tail 4, Immobilized single-stranded DNA can be used to isolate complementary RNA and DNA. 5. Affinity chromatography is used for the separation of a mixture of cells into homogeneous populations. 6. The technique relies on the antigenic properties of the cell surface or the chemical nature of exposed carbohydrate residues on the cell surface or on a specific membrane receptor-ligand interaction. 7. The immobilized ligands used include protein-A, which binds to the F, region of 1,G, a lectin or the specific ligand for a membrane receptor. 1.5. MEMBRANE SEPARATION The water like solutions that pass through the membrane are referred to as permeate. The rate at which the permeate flows through the membrane is called the flux rate. Membrane processing is a technique that permits Concentration and separation without the use of heat. Particles are separated on the basis of their molecular size and shape with the use of pressure and Specially designed semi-permeable membranes. When a solution and sealer ae separated by a semi-permeable membrane the water will move into e Solution to equilibrate the system this is known as osmotic pressure 3 Mechanical force is applied to exceed the osmotic pressure UP {0 Dit the water is forced to move down the concentration gradient i.e. from lo designates the liquid passing through the to hij i Permeate . js gh concentration. trate) designates the fraction not passing ‘mbrane and retentate (concent brough the membrane (Fig. 7.18 and 7.19).