Dielectrophoretic (DEP) force is a useful tool for manipulating particles in microfluidic systems. It is affected by the frequency of the applied electric field, which can be varied to produce repellent and attractive forces depending on the dielectric properties of particles and the media. In this work, two electric fields with different frequency are used to simultaneously separate and trap particles as well as double-trap particles by utilizing the DEP force. Initially, a single-vial microchannel was proposed to study the impact of the frequency and voltage on three types of electrodes: concentrator, repellent, and absorbing. The goal was to examine their efficacy in trapping a group of particles within the vial while separating and ejecting another group of particles from the microchannel. Performance graphs were used to determine the optimal voltages for the electrodes. Subsequently, an additional vial is incorporated into the microchannel to enable the double-trapping of particles with varying sizes and properties. With the optimal design, particles of varying sizes and properties can be trapped in separate vials within the microchannel. For the first time, the performance cartography of the proposed system has been assessed, enabling the identification of the optimal values and intelligent separations. Validation is conducted in two steps. Firstly, numerical findings are compared to previous experimental results to verify the accuracy of the numerical approach. Secondly, a microchip is fabricated, tested, and compared to numerical results using yeast cells to assess system efficiency and enhance the reliability of the numerical technique.