Research Article Micron-sized pillars for ion-pair reversed- phase DNA separations In the present paper, the feasibility to construct micron-sized silicon pillar channels to be used in HPLC is studied. For this, a channel with flow-through pores of 1 mm and with critical sidewall dimensions below 1 mm was constructed using advanced deep-UV litho- graphic equipment. Integrating a 3-nL injection system on the chip directly in front of the separation channel and using elongated distribution structures, a very controlled and high aspect ratio sample definition across the relatively wide separation channel was accom- plished. The system was evaluated in isocratic ion-pair RP mode, allowing the separation of a mixture of two components with, respectively, 300 and 400 base pairs in 5 s only. Keywords: DNA chromatography / Ion-pair RP-HPLC / Lab on a chip / Pillar array column DOI 10.1002/jssc.201000538 1 Introduction In the last few years, the pillar array column format has come into consideration as a serious and viable alternative for both the packed bed and the monolithic column in the field of LC. Where the flow resistance in the pillar column is comparable to that of monoliths, the ordered nature of the pillars allows for theoretical plate heights that are a factor of two smaller than (disordered) particulate columns with equally sized particles [1–5]. The group of Regnier has performed a pioneering role proposing this novel concept, applying this for electrically driven systems [1]. Working in CEC mode, relatively large efficiency separations were demonstrated by Slentz et al. [6] working with PDMS-based pillar arrays columns, achieving efficiencies of 620 000 plates per meter using diamond shaped pillars (5.2 mm  5.2 mm) of 10 mm deep. As the performance of a separation column scales inversely with the ‘‘particle’’ dimensions, an important focus in the development of this format is the production of smaller and smaller pillars. A reduction in feature sizes has an important impact on several aspects of the separation column. The depth that can be achieved between etched features is, e.g. limited by the dimension of the smallest available gap; a maximum aspect ratio of 50 is the very best value that can be expected. A reduction in channel depth will also adversely impact the detection capabilities of the system because of the low amount of available material. To avoid this, a strategy can be conceived to increase the channel’s cross section by using wider channels. When using wide channels with relatively small cross-sectional areas, the injection procedure must provide a uniform distribution of sample plugs that are well defined in volume on the chip of known value and posi- tioned close to the separation channel to avoid dispersion due to interfaces that contain stagnant fluid zones. The impossibility to establish complete zero-dead-volume couplings is in fact one of the real challenges when working with capillary columns. In capillary columns, it is common to operate in gradient mode, so that peak compression effects can result in narrower plugs at the detector than what would be the case in gradient operation. Several modes are available to separate nucleic acid mixtures by HPLC; these include size-exclusion chromato- graphy, anion-exchange HPLC, mixed mode HPLC, RP- HPLC, ion-pair RP-HPLC (IP-RP-HPLC) and affinity HPLC. Of these techniques, IP-RP-HPLC and anion-exchange HPLC are most commonly used because of their excellent resolution and the ability to separate mixtures containing a few to up to several thousands of both single- and double- stranded nucleotides [7–9]. In packed beds, polystyrene-divinylbenzene particles have been used as stationary material for IP-RP-HPLC separation [10]. In contrast with electrophoresis, where the conditions are fixed during the separation, the elution power during the course of an experiment can be tailor made on a specific sample to minimize the separation time. An interesting application in partition chromatography is the ion-pair RP (IPRP) separation of relatively small DNA molecules. Having a very slow mass transfer in the mesopores that are present in porous particles, very low efficiencies are obtained in porous particle columns. Non-porous polystyrene-divinylbenzene polymer beads have been used as stationary material for this type of Wim De Malsche 1,2 Lei Zhang 2 Jeff Op De Beeck 1 Joris Vangelooven 1 Bivragh Majeed 2 Gert Desmet 1 1 Department of Chemical Engineering at the VUB (Vrije Universiteit Brussel), Brussels, Belgium 2 Imec, Leuven, Belgium Received July 20, 2010 Revised September 11, 2010 Accepted September 12, 2010 Abbreviations: IPRP, ion-pair RP; IP-RP-HPLC, ion-pair RP- HPLC; TEAA, triethylammonium acetate Correspondence: Dr. Wim De Malsche, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium E-mail: wdemalsc@vub.ac.be Fax: 132-2628-32-48 & 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com J. Sep. Sci. 2010, 33, 3613–3618 3613