Micellar Electrokinetic Chromatography Separations and Analyses of Biological Samples on a Cyclic Planar Microstructure

Abstract

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 ∼12 pL. Because of the small cross section and favorable heat dissipation in glass microstuctures, 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 ∼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.