Wavelength-resolved fluorescence detector for microchip capillary electrophoresis separations

Abstract

A wavelength-resolved fluorescence detector for microchip and capillary separations is developed. It consists of a xenon lamp as flexible excitation source, a fluorescence microscope, a spectrograph with exchangeable gratings (150 and 600 lines/mm) and an intensified CCD camera. In contrast to standard LIF-detection systems, this set-up facilitates tuning of excitation and emission wavelengths over the whole visible spectrum of light (350–800 nm). The detector allows to record on-line emission spectra with high repetition rates of up to 60 Hz, which are needed to monitor rapid on-chip separations with peak widths <0.5 s. In this work, the detector system is applied to the capillary electrophoretic microchip separation of three rhodamines and their impurities (excitation: 450–490 nm; emission: >500 nm). Complete emission spectra of submicromolar solutions are recorded on-line. Comparable with diode-array detection in UV/vis-spectroscopy, this detector set-up yields information-rich electropherograms. The additional dimension of information compared to standard fluorescence detection systems enables peak assignment by means of fluorescent properties of the analytes and the immediate detection of coelutions due to a change of signal ratios at chosen wavelengths (peak purity plots).