In this article, the nanofluidic separation of non-neutral analytes using a pressure-gradient in combination with a counteracting electroosmotic flow field has been theoretically investigated. This approach takes advantage of simultaneous separation due to axial electrophoresis and solute interaction with the channel surface charges under optimal flow conditions to yield over an order of magnitude reduction in the separation length compared to that required in nanofluidic separations driven by pressure-gradients or electric-fields alone. Optimum results using this strategy are obtained under thin Debye layer and rapid transverse diffusion conditions which are both easy to realize in nanofluidic separation devices. The reported analyses further show that the separation strategy proposed here can allow the assay of charged species with a significantly greater resolving power than that possible using the capillary zone electrophoretic technique. Such an enhancement in resolving power has the potential to not only further miniaturize charge based separations but also permit their operation at reduced voltages. Interestingly, this improvement in the separation performance can be realized for analytes that differ in their electrophoretic mobilities over a wide range of values making this approach a valuable one for enhancing bioanalytical capabilities. (C) 2013 Elsevier Ltd. All rights reserved.