We describe a new device for separation of complex biological particles and structures exploiting many physical properties of the biolytes. The device adds a new longitudinal gradient feature to insulator dielectrophoresis, extending the technique to separation of complex mixtures in a single channel. The production of stronger local field gradients along a global gradient allows particles to enter, initially transported through the channel by electrophoresis and electroosmosis, and to be isolated according to their characteristic physical properties, including charge, polarizability, deformability, surface charge mobility, dielectric features, and local capacitance. In this work, the separation mechanism is described in terms of the relevant electromechanical principles, and proof-of-principle is demonstrated using various bacteria cells as model systems. The results demonstrate the selectivity of the technique and suggest that it may form the foundation for a versatile and useful tool for separating mixtures of complex biological particles and structures.