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. Currently, the most popular IEF applications, capillary electrophoresis and gel electrophoresis, rely essentially on CA technology. Traditionally, for estimating the resolving power of IEF, one applies the classical formula initially used by Kauman and Svensson more than fifty years ago. The formula predicts resolution increase (in terms of minimal pI-difference of the two compounds to be detected) as a square root of a voltage gradient and pH curve slope. Such a formula represents an overestimation and proved to be impractical for today’s levels of current densities delivered in modern micro-devises.
For evaluation of the resolving power of IEF in natural multi-component carrier ampholyte pH gradients, the system of one-dimensional differential equations describing steady state IEF is analyzed. An estimation for peak overlapping between the neighboring CAs shows that at certain electric current densities the concentration distribution becomes essentially trapezoidal with very low degree of overlapping. In such a system the resolution that can be potentially achieved is defined mostly by such an important parameter as a “density” of CA preparations, a number of individual CA components per pH unit. Alternative approaches for pH gradient forming, in particular immobilyzed pH gradients (IPGs) and thermal pH-gradients, are also analyzed and the appropriate estimations for the resolving power are discussed.