Nanoparticles and biological molecules high throughput robust separation is of significant interest in many healthcare and nanoscience industrial applications. In this work, we report an on-chip automatic efficient separation and preconcentration method of dissimilar sized particles within a microfluidic platform using integrated membrane valves controlled microfiltration. Micro-sized E. coli bacteria are sorted from nanoparticles and preconcentrated on a microfluidic chip with six integrated pneumatic valves (sub-100 nL dead volume) using hydrophilic PVDF filter with 0.45 μm pore diameter. The proposed on-chip automatic sorting sequence includes a sample filtration, dead volume washout and retentate backflush in reverse flow. We showed that pulse backflush mode and volume control can dramatically increase microparticles sorting and preconcentration efficiency. We demonstrate that at the optimal pulse backflush regime a separation efficiency of E. coli cells up to 81.33% at a separation throughput of 120.45 μL/min can be achieved. A trimmed mode when the backflush volume is twice smaller than the initial sample results in a preconcentration efficiency of E. coli cells up to 121.96% at a throughput of 80.93 μL/min. Finally, we propose a cyclic on-chip preconcentration method which demonstrates E. coli cells preconcentration efficiency of 536% at a throughput of 1.98 μL/min and 294% preconcentration efficiency at a 10.9 μL/min throughput.