Chromatofocusing
A technique which is related to ion-exchange chromatography, but which separates on a different principle, is Chromatofocusing. In Chromatofocusing, use is made of the buffering capacity of the ion- exchange substituent groups, themselves. The column is equilibrated with starting buffer, the sample applied, and immediately the finishing buffer is applied. Displacement of the starting buffer by the finishing buffer generates a moving pH gradient in the column. Proteins which fall behind their pI on this gradient will no longer bind to the column and will be swept along faster than the pH gradient. Proteins which move ahead of the pH gradient, by contrast, will bind strongly to the column and will be immobilized until overtaken by the pH gradient. The net result is that proteins will be eluted from the column at their respective pI values.
In practice it is found that simple displacement of one buffer with another in a conventional exchanger causes too sharp a change in pH. A shallower pH gradient, more suitable for Chromatofocusing separations, can be generated by using ampholytes as the eluting buffer, and a substituent group, such as polyethyleneimine, which titrates over a larger pH range. Ampholytes are mixtures of randomly substituted poly amino- poly carboxylic acids. They are also used in isoelectric focusing and in isotachophoresis. This requirement for ampholytes makes Chromatofocusing more expensive than normal ion-exchange chromatography.
References
-Dennison, C. (2002). A guide to protein isolation . School of Molecular mid Cellular Biosciences, University of Natal . Kluwer Academic Publishers new york, Boston, Dordrecht, London, Moscow .
