Axial-beam laser-excited fluorescence detection in capillary electrophoresis

Abstract

Capillary electrophoresis (CE) has emerged as a powerful separations technique, with applications from simple atomic ions to large DNA fragments. A challenging problem is detection and monitoring of the analytes after they are separated, since sub-nanoliter volumes are involved. Laser-excited fluorescence detection so far has provided the lowest detection limits, down to the few hundred molecules range. These low detection limits are essential for applications like DNA sequencing, where the sample sizes must be kept small to avoid clogging up the gels. Laser-excited fluorescence detection typically requires careful alignment both in excitation (to couple with the small i.d. of the capillaries) and in light collection (to discriminate against stray light). Also, the standard geometry for excitation perpendicular to the axis of the capillary requires the creation of an optically clear region in the polyimide coating. This makes the capillary fragile and complicates the preparation of capillaries for use. Furthermore, the excitation path length, and hence the fluorescence signal, is restricted to the small diameter of the capillary. The purpose of this paper is to demonstrate the feasibility of axial-beam fluorometry for CE. 12 refs., 4 figs.