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).