Study of zone broadening in optically gated high-speed capillary electrophoresis

Abstract

Fluorescein isothiocyanate (FITC) labeled compounds are separated by capillary zone electrophoresis (CZE) in seconds rather than minutes while high separation efficiency is maintained. These rapid analysis times are achieved through high electric fields applied over short separation distances and the use of a unique optical-gating injection system. The optical-gating injection is based on photodecomposition of FITC induced by an argon ion laser beam. A fraction of this same laser beam is also used for detection of the analyte zones by on-column laser-induced fluorescence. Four FITC-labeled amino acids and fluorescein dye were analyzed to compare system performance to that predicted by theory. To do so, the total variance for each analyte zone was measured experimentally. This was compared to the sum of the theoretically expected variance contributions from the injection and detection systems and longitudinal diffusion. The variance due to longitudinal diffusion was calculated from diffusion constants measured experimentally with a traditional CZE system. After accounting for these three known sources of variance, this method is found to achieve better than 80% of the performance predicted by theory in analysis times as short as 2-3 s.