(541a) Isoelectric Focusing: Current Limitations and Prospects

Isoelectric focusing (IEF) is an analytical and preparative technique which allows for analyzing amphoteric substances based on their isoelectric points (pIs). IEF plays an important role in protein characterization and proteomics related applications. Being discovered almost hundred years ago, IEF has gained its popularity in the sixties of the last century with the synthesis of complex amphoteric mixtures called carrier ampholytes (CAs) allowing for obtaining stable pH gradients with acceptable separation properties for different wide and narrow pH ranges. The most popular IEF applications, capillary electrophoresis and gel electrophoresis, rely essentially on CA technology. While the traditional formula for resolving power of IEF, already proposed by Kauman and Svensson more than fifty years ago, predicts resolution increase as a square root of voltage gradient, in a CA system the resolution is defined mostly by the “density” of CA preparations, a number of individual CA components per pH unit. Alternative approaches for pH gradient forming, in particular immobilized pH gradients (CA free) and thermal pH-gradients, are also analyzed and their resolving power is discussed.