Micellar electrokinetic chromatography separations and analyses of biological samples on a cyclic planar...

Micellar electrokinetic capillary chromatography (MECC) separations and analyses of biological samples on a planar glass microchip capillary electrophoresis device with laser-induced fluorescence solute detection are discussed. A cyclic channel system which permits dead volume free repeated column switching and thus the use of various channel lengths together with a relatively low applied separation voltage is described. It features an unbiased, dead volume free electrokinetic sample inlet system of approximately 12 pL. Because of the small cross section and favorable heat dissipation in glass microstructures, MECC separations with an electric field strength of up to 2000 V/cm achieving efficiencies of submicrometer plate heights can be performed. After a separation length of 2 cm, six fluorescein isothiocyanate labeled amino acids are shown to be separable within a few seconds and with an imprecision for peak areas (or heights) and detection times of < 2% and < 0.5%, respectively. Without application of electrokinetic solute stacking, the detection limit of fluorescein isothiocyanate labeled arginine is 3.3 nM, corresponding to approximately 40 zmol injected. Furthermore, the feasibility of directly applying human urine and serum samples onto the uncoated channel system is demonstrated and first data of the successful performance of a chip-based MECC immunoassay for serum theophylline are presented. Compared to MECC in conventional fused-silica capillaries, MECC analyses on microchips can be performed 1-2 orders of magnitude faster, with higher efficiency and at no expense of accuracy and precision. Furthermore, versatility is shown to be much increased with the use of a cyclic rather than a single-path channel system. The MECC separation efficiency of fluorescein isothiocyanate labeled amino acids is shown to be comparable to that obtained by gel electrophoresis performed in the same chip layout.