You will prepare and submit a term paper on Enzyme Technology. Your paper should be a minimum of 6000 words in length. 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 that 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 the diffusion and thus, slower is 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 which 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 with 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, estimation of the molecular weight of unknown proteins can easily be done. (Freifelder, 1982, p220)
Ion exchange chromatography is another special chromatographic technique which is used to separate proteins on the basis of 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.
The ion-exchanger is a matrix containing covalently linked charged groups. The matrix may be made of agarose, dextran, cellulose, and copolymers of styrene and vinylbenzene. If the charged group is negative, it exchanges positively charged proteins and hence is called the cation exchanger. Examples of cation exchanger groups are sulphonic acid (strongly acidic cationic exchanger), phenolic hydroxyl, and carboxyl (weakly acidic cation exchanger). Similarly, if the charged group is positive then it will exchange negatively charged proteins and are thus known as an anion exchanger. .