Conventional Isoelectric Focusing and Immobilized pH Gradients

Abstract

We review here some mathematical models pertaining to separations by isoelectric focusing (lEF) in carrier ampholyte (CA) buffers and in immobilized pH gradients (IPG). In CA-IEF, the steady-state distribution profile of amphoteric compounds predicts gaussian distributions only if and when the background conductivity remains constant within the focused zone. This is in practice only achieved when focusing across neutrality. In all other pH regions, the tendency is to produce asymmetric peaks, with negatively (in acidic regions) or positively (in basic gradients) skewed profiles. The transient state, in uniformly loaded samples, is seen as two discernible peaks migrating from the two ends of the column and merging into one at the pl position. The decay state seems to be generated by an isotachophoretic phenomenon of anodic, cathodic, or symmetric loss of carrier ampholytes. IPGs are characterized by indefinitely stable pH gradients and by an extremely high resolution. For producing wide pH gradients, computer algorithms are described which optimize any mixture of Immobiline chemicals for generating linear or, in general, concave exponential pH gradients.