Representative UV-Vis absorption spectra of (a) nanoparticle and (b) microparticle suspensions retrieved from the microchip at a flow rate of 3 μl/min. The 100 nm polystyrene nanoparticles were labeled with Firefli™ Fluorescent Red (Ex 542/Em 612 nm) and 5 μm polystyrene microparticles were labeled by Firefli™ Fluorescent Green (Ex 468/Em 508 nm). Suspensions containing a single type of synthetic particles were injected into the top inlet of at 3 μl/min. The outflow from the top and bottom outlets were analyzed by UV-Vis spectroscopy and repetitive spectra are presented. The absorbance at 545 nm for nanoparticles and 470 nm for microparticles were used to quantify their concentrations since they correlated linearly with the particle concentration. Similar spectra were acquired for other flow conditions. (c) Experimental data (solid dots and solid squares) showing the ratio of particles retrieved from the top outlet at different residence time points. PBS/SDS solutions containing either 5 μm or 100 nm polystyrene particles were injected into the top inlet and particle free PBS/SDS solution was injected into the bottom inlet. The two layers had identical flow rates of 20, 15, 10, 6, and 3 μl/min, and the particle residence time in the devices were calculated for the plot. Each data point was repeated in at least 3 devices, and error bars represent standard deviations from these repeats. The experimental measurements closely matched results from COMSOL simulation (blue and red lines).