INTEGRATION OF A GRADIENT ELUTION SYSTEM FOR PRESSURE-DRIVEN LIQUID CHROMATOGRAPHY WITH MEMS FABRICATED EFFICIENT PILLAR ARRAY COLUMNS Y. Song 1 , M. Noguchi 2 , K. Takatsuki 2 , T. Sekiguchi 2 , S. Shoji 2 , T. Funatsu 1 , J. Mizuno 2 , and M. Tsunoda 1* 1 The University of Tokyo, JAPAN and 2 Waseda University, JAPAN ABSTRACT This paper reports a gradient elution system for pressure-driven liquid chromatography (LC) on a chip. We have already reported the microfabrication of efficient pillar array columns with low dispersion turns. For faster and more efficient analysis, we fabricated a gradient elution system with a cross-Tesla mixer for the effective mixing of two solutions on a chip with a separation channel of pillar array columns and a sample injection channel. The results of the separation of two dyes under gradient elution conditions actually showed that the developed chip performed better, faster and more efficiently. KEYWORDS: Cross-Tesla Mixer, Reversed-phase, Separation, Fluorescence INTRODUCTION High-performance liquid chromatography (HPLC) is an important tool in determining the essential compounds in the biological samples. A particle-packed column is commonly used in LC. However, because of irregularities in the mobile zone, even the best packed column is known to suffer from reduced separation efficiency. With the development of precision fabrication technology, pillar array columns with near-perfect ordered structures were developed for pressure-driven LC [1]. In a previous study, a higher separation efficiency of a long pillar array column was achieved using a separation channel with low-dispersion turns [2]. However, these methods are very difficult to apply in the analysis of biological samples containing various compounds that have large differences in polarity. To solve this problem, a gradient elution system, which accelerates the elution of strongly retained solutes and is commonly used in conventional LC, was developed to minimize the time needed to separate the compounds using pillar array columns. Even though the gradient elution can be achieved using a pump with a mixer [3], there is a considerable delay when the gradient program starts. A useful way to eliminate this delay is by directly connecting the mixer and the separation channel. Therefore, in this study, the gradient elution system and the separation channel were integrated on a chip, and separation with gradient elution was investigated. EXPERIMENTAL Three types of mixer (T-Tesla mixer, cross-Tesla mixer, and cross-obstruction mixer; Figure 1) were examined. The mixing efficiencies were investigated by computational fluid dynamics (CFD) simulations and experiments. The cross-Tesla mixer, a separation channel with pillar arrays, and a sample injection channel were fabricated on a 20 mm × 20 mm silicon microchip by multistep ultraviolet photolithography and deep reactive ion etching using the Bosch process. The depth and width of the mixer were 30 and 100 μm, respectively. The depth and width of the separation channel were 30 and 400 μm, respectively, and the depth of the injection channel was 60 μm. The pillars in the separation channel were 3-μm squares with an inter-pillar distance of 2 μm. The fabricated silicon substrate was bonded to a glass plate by anodic bonding in order to seal the channel. Dimethyloctadecylchloro- silane was used to modify the surface of the separation channel for reversed-phase separation. Coumarin 525 and 545 were separated under Figure 1: Fluorescent images of the mixing of two solutions (water and fluorescein in methanol) at various positions in the three micromixers: (a) T-Tesla mixer, (b) cross-Tesla mixer, and (c) cross-obstruction mixer. 978-0-9798064-4-5/μTAS 2011/$20©11CBMS-0001 635 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences October 2-6, 2011, Seattle, Washington, USA