Wavelength-resolved fluorescence detection in capillary electrophoresis.

Abstract

A multichannel laser-induced fluorescence detector for capillary electrophoresis is described. The detection system combines yoctomole limits of detection with the simultaneous acquisition of entire fluorescence emission spectra. An Ar/Kr mixed-gas ion laser provides great flexibility in excitation wavelengths, and a holographic grating and charge-coupled device detector combination allows a 500-nm spectral window to be acquired with 2-nm resolution. The limits of detection are 5 x 10(-14) M or 80 molecules for sulforhodamine 101 and 1.5 x 10(-13) M or 220 molecules for fluorescein in a 50 micron i.d. capillary. An electropherogram of a mixture of amino acids derivatized with both Bodipy 503/512 C3 and Bodipy 576/589 C3 demonstrates that the analytes can be differentiated on the basis of both emission characteristics and migration times. With the use of organic modifiers and other complex separation media, the spectral background increases as discrete spectral features appear; the wavelength-resolved backgrounds of a variety of common CE separation conditions are presented.