Research Article Chip-based enantioselective open-tubular capillary electrochromatography using bovine serum albumin-gold nanoparticle conjugates as the stationary phase In this study, chip-based enantioselective open-tubular CEC (OT-CEC) was developed employing BSA-gold nanoparticle (GNP) conjugates as a chiral stationary phase. An immobilization procedure was realized by prederivatization of the glass microchannel with (3-mercaptopropyl)-trimethoxysilane to provide thiol groups, which linked the BSA- GNP conjugates on the inner surface of the microchannels. Incorporation of GNPs into immobilization of BSA selectors greatly increased the BSA phase ratio and favored the BSA stationary phase generated sufficient EOF. Good resolutions of FITC-labeled ephedrine and norephedrine isomers were achieved with 36 mm effective separation channel length within 250 s. The constructed OT-CEC microdevice exhibited good repeatabilities for run-to-run enantioseparations and kept an enantioselective lifetime of more than 1 month. The effects of pH values and concentrations of a running buffer on the selectivity and resolution of enantioseparations were investigated. Keywords: BSA / Chiral stationary phase / Gold nanoparticle / Microchip / Open-tubular CEC DOI 10.1002/elps.200800359 1 Introduction In the last decade, development of microchip-based analytical separation has attracted attention because of their promising many advantages, such as speed of analysis, integration of on-line sample pretreatments and introduc- tion, controlled sample injection plug, minimized sample/ reagent consumption and high efficiency [1–3]. MCE has been widely applied to the separation of compounds, DNA analysis, cell and microparticle selections [4–6]. Recently, some researches have been devoted to application of microfabricated electrophoresis devices to enantiosepara- tions [7, 8]. The developed chip-based enantioseparations mainly employed CDs and their derivatives as chiral selectors, which were commonly dissolved in the electrolyte serving as pseudostationary phases [8]. The separation efficiencies are improved relying on changing the selector concentrations, varying the pH value and extending the microchannel length. The extensive application of chip- based chiral separation for multifold enantiomers requires different chiral selectors and alternative separation strategies. Proteins have great potential in enantiomeric analysis since they are acknowledged for their different binding strengths with enantiomers in pharmacokinetics and biochemistry [9]. In recent years, many efforts have been taken to develop albumin-based chiral separations. BSA, noted model role in protein–drug interactional research, has attracted much attention in enantiomeric drug separations [10, 11]. It was shown that BSA serving as chiral buffer additives in CE as well as immobilized chiral stationary phases (CSPs) in chromatography both demonstrated excellent enantioselectivities for a wide range of enantio- mers [12–14]. Therefore, developing new and selective BSA- based chiral separation systems has important medicinal significance in terms of rapid analysis of drug enantiomers and clinical diagnosis. Capillary electrochromatographic (CEC) technique, which combines the high efficiency of CE with the high selectivity of HPLC, has extensively engaged its applications in chiral separations in recent years [15]. However, up to date, the chip-based CEC enantioseparations are rarely reported [16]. Contrasted with the packed-CEC, monolith- CEC and open-tubular CEC (OT-CEC) are much more easily performed because of eliminating of the difficulties of end-frit fabrication and packing microcolumns in Hai-Fang Li 1 Hulie Zeng 2 Zhifeng Chen 3 Jin-Ming Lin 1 1 The Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Department of Chemistry, Tsinghua University, Beijing, P. R. China 2 Faculty of Urban Environmental Sciences, Tokyo Metropolitan University, Tokyo, Japan 3 Chinese Academy of Inspection and Quarantine, Beijing, P. R. China Received June 5, 2008 Revised August 22, 2008 Accepted September 15, 2008 Abbreviations: AFM, atom force microscope; BSA-GNP, BSA-gold nanoparticle; CSPs, chiral stationary phases; MPTMS, (3-mercaptopropyl)-trimethoxysilane; OT-CEC, open-tubular CEC; TEM, transmission electron microscope Correspondence: Professor Jin-Ming Lin, Department of Chem- istry, Tsinghua University, Beijing 100084, P. R. China E-mail: jmlin@mail.tsinghua.edu.cn Fax: 186-10-62792343 & 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com Electrophoresis 2009, 30, 1022–1029 1022