Electrophoresis 2012, 33, 1–8 1 Zhen Wang 1 Abebaw B. Jemere 2 D. Jed Harrison 1,2 1 Department of Chemistry, University of Alberta, Edmonton, AB, Canada 2 National Institute for Nanotechnology, National Research Council Canada, Edmonton, AB, Canada Received May 25, 2012 Revised June 19, 2012 Accepted June 19, 2012 Research Article Integrated electrokinetic sample fractionation and solid-phase extraction in microfluidic devices A microfluidic device that performs “in space” sample fractionation, collection, and pre- concentration for proteomics is described. Effluents from a 2.75 mm long fractionation channel, focused via sheath flow, were sequentially delivered into an array of 36-collection channels containing monolithic polymer beds for SPE. Optimum conditions for the de- vice design, and simultaneous photolytic fabrication of 36 monolithic columns in the 36 channels, as well as for their proper performance in electrokinetic sample fractionation and collection are described. A hydrophobic butyl methacrylate-based monolithic porous polymer was copolymerized with an ionizable monomer, acryloamido-methyl-propane sulfonate, to form a polymer monolith for SPE that also sustains cathodic electroosmotic flow. The SPE bed was made deep enough to greatly reduce the linear flow rate within the bed, in order to compensate for the lower electroosmotic mobility of the cationically charged SPE bed relative to the glass walled device. Under these conditions, electrokinetic fractionation of a protein sample resulted in tightly focused sample zones delivered into each of the 36-channel polymer beds with no observed crosscontamination. Monolithic columns showed reproducible performance with preconcentration factor of 30 for 2 min loading time. The ability to fractionate, collect, and preconcentrate samples on a microflu- idic platform will be especially useful for automated or continuous operation of these devices in proteomics research. Keywords: Capillary electrophoresis / Electrokinetic / Fractionation / Microfluidics / Solid- phase extraction DOI 10.1002/elps.201200286 1 Introduction CE-ESI-MS is a powerful and versatile analytical tool for pro- teomics research [1], which has proven to be a useful tech- nique in the analysis of proteins from complex “real world” samples [2–7]. Analyzing proteins from complex samples re- quires a number of analytical steps be done prior to detection. Microfluidics allows the integration of many analytical steps on a chip, with a number of functional demonstrations of real samples analyzed with an on-line CE-ESI-MS analysis [6, 7]. An off-line microfluidics system may allow the design of a higher throughput proteomics platform, in which the com- Correspondence: Professor D. Jed Harrison, Department of Chem- istry, University of Alberta, Edmonton, AB, Canada, T6G 2G2 E-mail: jed.harrison@ualberta.ca Fax: +780-492-8231 Abbreviations: AMPS, acryloamido-methyl-propane sul- fonate; BMA, butyl methacrylate; EDMA, ethylene glycol dimethacrylate ponents are separated, fractionated, and collected for further analysis, such as desalting, tryptic digestion, and ESI-MS. SPE columns are effective for desalting [7] and on-column diges- tion [8], and their integration into a large number of parallel fractionation channels will contribute to the development of automated multidimensional separation and processing sys- tems on chip. We recently reported a microfluidic 36-channel fraction collection device, driven by electrokinetic flow [9] to yield frac- tionation in space [10–14], as opposed to fractionation in time [15–21]. The device we demonstrated allows a high number of samples to be temporally separated by CE, then fraction- ated by collecting in individual channels downstream. The introduction of an SPE material in the downstream collection channels is described in this work. By adding SPE, the individ- ual samples can be physically trapped within each collection channel, and later eluted in concentrated form for analysis or further sample processing, rather than simply delivered to one of the 36 individual collection reservoirs. This SPE step is important, since the ability to select and manipulate a single fraction is a prerequisite for performing any postseparation C  2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com