We report on a series of hydrodynamic chromatography separations conducted in micropillar array columns with an interpillar distance spacing of, respectively, 1.00, 0.70, and 0.47 μm. The columns have been produced using state-of-the-art deep-UV lithography and deep reactive ion etching techniques. Despite the fact that the efficiency was smaller than theoretically possible (due to fabrication limitations and significant injection and detection band broadening), it was nevertheless possible to separate mixtures of fluorescein isothiocyanate (used as the t0-marker) and 20- and 40-nm polystyrene beads. With the smallest interpillar distance, a resolution of Rs = 0.5 between the 20- and 40-nm particles could be obtained in 90s over a column length of 4 cm. The selectivity obtained in the pillar array columns was found to be very similar to that observed in packed-bed columns. By detecting the fluorescent signals in a 90-μm-deep detection groove at the end of the column, the signal-to-noise ratio could be enhanced up to 150 times.