Enzyme Technology, Size Exclusion Chromatography - Essay Example

Download file to see previous pages Chromatography became a popular technique only after the development of partition chromatography by Archer Martin and R.L.M. Synge in 1944, who received a Nobel Prize for their contribution. (Freifelder, 1982, 216)

Size exclusion Chromatography
Size exclusion chromatography is also known as molecular sieve chromatography. Separation, in this method, is based on the molecular size of the protein. The basic principle of the size exclusion chromatography method is not very complex. It involves a column of beads that are small and inert and also have pores. Synge and Tiselius were the first to observe and suggest that small molecules which could pass through the tiny pores of zeolites were excluded based on size (Synge and Tiselius, 1950).

If a mixture of proteins of various dimensions is passed through the column, proteins larger than the pores do not enter the beads and move only in the space between the beads. Thus, the larger proteins are not retarded by the column material. However, protein molecules smaller than the pores move in and out of the column beads. The smaller the protein in size the higher the probability of diffusion and thus, slower their movement down the column. The rate of movement of proteins through the column depends on their ability to penetrate the beads. Hence, proteins are eluted from the column in the decreasing order of their molecular weight.

Size exclusion chromatography is one of the best techniques to separate proteins that differ in molecular weight. It can be carried out more or less under all conditions as the chromatographic behavior does not depend on pH, temperature, ionic strength, and buffer composition, so as not to compromise the stability of the proteins. It can be used to separate labile proteins as there is virtually no adsorption. Moreover, as the elution volume is related to the molecular weight, the estimation of the molecular weight of unknown proteins can easily be done. (Freifelder, 1982, p220)

Ion exchange chromatography
Ion exchange chromatography is another special chromatographic technique that is used to separate proteins based on charge. The principle behind this chromatographic technique is that the affinity of a protein to the ion exchanger depends on the charge of the desired protein and the relative affinity of other undesired proteins in the mixture. ...Download file to see next pages Read More