A novel method for separation of low-molecular-mass heparins is reported here, on the basis of migrating the polyanionic heparins in a polycationic polyacrylamide gel, made by incorporating a gradient of positively charged monomers (the Immobilines used for creating immobilized pH gradients) into the neutral polyacrylamide backbone. Separations can be operated either in linear or nonlinear gradients of positive charges, thus modulating at whim the separation power. This allows the polydisperse heparins to reach a steady-state position along the migration path and condense (focus) in an environment inducing charge neutralization. It is shown that the separations obtained are a complex function of both size and charge distribution along the oligosaccharide chains. This novel methodology represents a marked improvement over existing techniques and appears to hold promise for applications in screening of commercial lots of heparins, also in view of possible presence of contaminants, such as those recently detected in imported heparins.