This abstract reports an electrokinetic manipulation to separate polystyrene(PS)microspheres in high-conductivity media,although most electrokinetic operation only focusing in low-conductivity buffers.A PDMS/glass microfluidic chip with a designed of a three-layer sandwich,and three-parallel micro-electrode was fabricated [1,2] for the different size of PS microspheres separation.With optimized AC voltage and frequency,we demonstrate that 25 μm microspheres can be isolated from 5 μm or/and 10 μm microspheres and concentrated onto the inner electrode in dozen sec.The design of the microfluidic chip is shown in Fig.1.The microchannel was fabricated by molding polydimethylsiloxane(PDMS)with laser machined mold.The electrodes were fabricated by sputtering Ti-Au-Ti on a glass slide.Then the glass substrate with microelectrodes was sealed with the PDMS microchannel by plasma treatment.There three sizes of PS microspheres(5 μm,10 μm and 25 μm respectively)were used which were evenly dispersed into the conductive solution(10 ×TAE,~1.59 S/m)before experiment.To separate two or three sizes of microspheres,a series of electric field were applied.It was visible that,the 5 μm,10 μm and 25 μm microspheres were positioned into different equilibrium locations.In particular,almost all of the 25 μm microspheres migrated to the inner electrode,while the majority of 5 μm and/or 10 μm microspheres moved into the areas between the neighboring electrodes(Fig.2).Fig.3 shows that the results of the separation,in which,at the frequency range of 100 kHz-10 MHz,5 μm and 25 μm,10 μm and 25 μm polystyrene microspheres can be separated.By optimized frequency and AC voltage,we demonstrated that the better of frequency for the separation of two sizes of microspheres(10 μm and 25 μm,5 μm and 25 μm)(Fig.4)and three sizes of microspheres(5 μm,10 μm and 25 μm)were 100 kHz,2 MHz and 1 MHz separately.Meanwhile,the better AC voltage conditions for the two or three sizes of microspheres separation were 14 V,10 V and 11V respectively.