Third generation of focusing: gel matrices with immobilized cation gradients

A novel method is reviewed here for separation of polyanions, based not on conventional zone electrophoretic means, but on a "steady-state" process by which said polyanions are driven to stationary zones along the migration path against a gradient of positive charges affixed to the neutral polyacrylamide matrix. As the total negative surface charge of such polyanions matches the surrounding charge density of the matrix, they stop migrating and remain stationary, as typical of steady-state separation techniques. This technique has been successfully applied to SDS-protein micelles, DNAs, RNAs and heparins, with remarkable separations, often much superior than those obtained in conventional techniques. Additionally, by exploiting constant plateaus of charges, rather than gradients, it is possible to amplify the separation between species having closely spaced charge densities. This technique resembles a classical IEF process, with the proviso that the polyanions cannot be applied at any position along the gel matrix, but only at the point of low (or zero) charge density. The merits and limits of the technique are assessed.