Characterization of a Rugged, Open-Gap Flow Cell for Confocal Laser-Induced Fluorescence Detection in Capillary Electrophoresis

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Abstract:

Construction details and performance characteristics of an opengap flow cell for fluorescence detection in capillary electrophoresis are described. The flow cell is created by separating two pieces of capillary by a small (90 mu m) gap. The gap is surrounded with buffer and grounded, and the application of electric fields to both inlet and outlet capillaries causes the material in the inlet capillary to flow across the gap. The use of a simple confocal optical arrangement for laser-induced fluorescence detection allows straightforward application of the gap flow cell to detection in capillary electrophoresis. The signal-to-noise ratio is measured to be about a factor of 2 better than that for on-column confocal detection at nanomolar concentrations over a wide range of pinhole diameters. The detection limit for fluorescein isothiocyanate is in the low picomolar range. Detection of a simple mixture of amino acids that have been derivatized with fluorescein isothiocyanate demonstrates the stability and utility of the gap. Increased tailing is observed with the gap cell, with average asymmetry of about 1.4 near the center of the gap. Dispersion characteristics as a function of position in the gap are interpreted as dilution of the analyte as it flows across the gap. Fortunately, resolution and theoretical plates, measured by using least-squares fitting, are not significantly different from on-column separations, in spite of the tailing.
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