Question

briefly discuss the theory , operation and application of size eclusion chromatography with reference to the utility of lazer light scattering detector

Answer 1

Size exclusion chromatography or SEC is used for purifications of substances according to their size. This technique is mainly based on the particle size and geometry of the molecules. The molecules are separated based on their size. Unlike the ion exchange chromatography, size exclusion chromatography does not depend on the chemical interaction with biomolecules, but it is based on a physical property of the biomolecule - which have effective molecular radius.

Basic theory of Size exclusion chromatography (SEC) :

SEC is the technique to separate molecules from a mixture based on the size of the molecules. Separation is carried out by the differential exclusion or inclusion of solutes particles and the solute particles are passed through stationary phase whic have different sizes pores made by cross linked polymeric gels. The process is based on the different rate of permeation of each solute molecule into the interior of gel particles. Size exclusion chromatography posses gentle interaction with the sample, enabling high retention of macromolecular activity. To separate biomolecules from the aqueous systems, SEC is called as gel filtration chromatography (GFC), but the separation of organic polymers in non-aqueous systems is called gel permeation chromatography (GPC).

The basic principle of SEC is that a column of gel particles equilibrium with a suitable mobile phase for the molecules which have to be separated. Large size molecules are excluded from the pores will pass through the space in between the gel particles first in the effluent. Small size molecules are randomly moved within the mobile phase of in and outside the molecular sieve and after that these particles will pass through the column at a slower rate, hence shows smaller effluent rate.

There is a typical molecular mass which will be completely excluded from the gel filtration beads.

SEC can be used for :

(1) Purification of chemical components

(2) Desalting of the molecules.

(3) To study the protein ligand binding SEC can be used.

(4) SEC can also be used to study the Protein folding phenomenon.

(5) To measure the concentration of sample.

(6) For the determination of relative molecular mass SEC also can be used