High Speed Separations of Complex Mixtures using nano-Liquid Chromatography Coupled with micro Free Flow Electrophoresis

Abstract

Micro free flow electrophoresis (µFFE) is a separation technique which can be used for unique applications due to its continuous nature. Separations are performed in space, as opposed to time, as laminar flow drives analytes down the separation chamber and are separated laterally by an electric field. This continuous nature makes it an attractive option to be used as a second dimension in multidimensional separations. The major focus of this work will be the development of a 2D separation platform coupling a commercial nano-liquid chromatography (nLC) instrument with an all glass µFFE device followed by investigating factors which could affect the efficiency of the technique. A new µFFE device was designed and fabricated for coupling with nLC. High peak capacity separations of tryptic peptides of BSA demonstrated the power of the technique. Broadening in temporal and spatial dimensions were investigated since peak capacity is calculated using analyte peak width. The observation that the adsorption of analytes only affects broadening in the temporal dimension is critical for maximizing peak capacity. Finally, the effect of using fluorescent labels in 2D nLC × µFFE separations will be demonstrated. The impact of label choice can be seen in the peak capacity and orthogonality of separations of amino acids and peptides.