A new microfluidic approach for charge-based particle separation using combined hydrodynamic and electrokinetic effects is presented. A recirculating flow pattern is employed, generated through application of bi-directional flow in a narrow glass microchannel incorporating diverging or converging segments at both ends. The bi-directional flow in turn is a result of opposing pressure-driven flow and electro-osmotic flow in the device. Trapping and preconcentration of charged particles is observed in the recirculating flow, under conditions where the average net velocity of the particles themselves approaches zero. This phenomenon is termed flow-induced electrokinetic trapping (FIET). Importantly, the electrophoretic mobility (zeta potential) of the particles determines the flow conditions required for trapping. In this paper, we exploit FIET for the first time to perform particle separations. Using a non-uniform channel, one type of particle can be trapped according to its zeta-potential, while particles with higher or lower zeta-potentials are flushed away with the pressure-driven or electro-osmotic components, respectively, of the flow. This was demonstrated using simple mixtures of two polystyrene bead types having approximately the same size (3 m) but different zeta potentials (differences were in the order of 25 to 40 mV). To gain more insight into the separation mechanism, particle separations in straight, 3 cm-long microchannels with uniform cross-section were also studied under conditions of bi-directional flow without trapping. A thorough theoretical analysis confirmed that trapping occurs when electrokinetic and pressure-driven particle velocities are equal and opposite throughout the diverging segment. This makes it possible to predict the pressure and electric field conditions required to separate particles having defined zeta potentials. © 2009 The Royal Society of Chemistry.