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This paper presents a sequential dielectrophoretic field-flow separation method for particle populations using a chip with a 3-D electrode structure. A unique characteristic of our chip is that the walls of the microfluidic channels also constitute the device's electrodes. This property confers the opportunity to use the electrodes' shape to generate not only the electric field gradient required for dielectrophoretic force but also a fluid velocity gradient. This interesting combination gives rise to a new solution for the dielectrophoretic separation of two particle populations. The proposed sequential field-flow separation method consists of four steps. First, the microchannel is filled with the mixture of the two populations of particle. Second, the particle populations are trapped in different locations of the microfluidic channels. The population, which exhibits positive dielectrophoresis (DEP), is trapped in the area where the distance between the electrodes is the minimum, while the other population that exhibits negative DEP is trapped in locations of maximum distance between electrodes. In the next step, increasing the flow in the microchannels will result in an increased hydrodynamic force that sweeps the cell population trapped by positive DEP out of the chip. In the last step, the electric field is removed, and the second population is swept out and collected at the outlet. For theoretical and experimental exemplification of the separation method, a population of viable and nonviable yeast cells was considered.