Microsecond electrophoresis.

Abstract

Although analysis strategies exist for probing a diverse array of molecular properties, most of these approaches are not amenable to the study of reaction intermediates and other transient species. Separations in particular can provide detailed information on attributes not readily measured by spectroscopy but typically are performed over time scales much longer than the life span of highly unstable compounds. Here we report the development of an electrophoretic strategy that dramatically extends the practical speed limit for fractionations and demonstrate its utility in examining transient hydroxyindole photoproducts. Fluorescent reaction intermediates are optically generated in femtoliter volumes within a flowing reagent stream and are differentially transported at velocities as large as 1.3 m.s(-1), thereby minimizing band variance and allowing multicomponent reaction mixtures to be resolved over separation paths as short as 9 microm. Analyte migration times and band variances do not deviate significantly from basic theory for separations performed with fields that exceed 0.1 MV.cm(-1), indicating that effects from Joule heating are minor. We demonstrate the feasibility of achieving baseline resolution of a binary mixture in <10 micros, nearly 100-fold faster than previously possible. Application of this approach to the study of a range of short-lived molecules should be feasible.