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We present a new concept for on‐chip separation that leverages bidirectional flow, to tune the dispersion regime of molecules and particles. The system can be configured so that low diffusivity species experience a ballistic transport regime and are advected through the chamber, whereas high diffusivity species experience a diffusion dominated regime with zero average velocity and are retained in the chamber. We detail the means of achieving bidirectional electroosmotic flow using an array of alternating current (AC) field‐effect electrodes, experimentally demonstrate the separation of particles and antibodies from dyes, and present a theoretical analysis of the system, providing engineering guidelines for its design and operation. [ABSTRACT FROM AUTHOR]