Design and performance of a microchip electrophoresis instrument with sensitive variable-wavelength fluorescence detection

Abstract

A modular instrument for high‐speed microchip electrophoresis (MCE) equipped with a sensitive variable‐wavelength fluorescence detection system was developed and evaluated. The experimental setup consists mainly of a lamp‐based epifluorescence microscope for variable‐wavelength fluorescence detection and imaging and a programmable four‐channel bipolar high‐voltage source capable of delivering up to +/– 10 kV per channel. The optical unit was equipped with a high‐sensitivity photomultiplier tube and an adjustable aperture. The system was applied to MCE separations of flurescein isothiocyanate (FITC)‐labelled amines utilizing blue light (450–480 nm) for excitation as well as for the separation of rhodamines utilizing excitation light in the green spectral region (531–560 nm). At optimized conditions baseline separation of four FITC‐labelled amines could be obtained in less than 50 s at a detection limit of 460 ppt (1 nM) with a signal‐to‐noise ratio of 3:1. Three rhodamines could be baseline‐separated in less than 6 s at a detection limit of 240 ppt (500 pM). The relative standard deviations of absolute migration times determined in repetitive MCE separations of FITC‐labelled amines were below 2.5% (n= 25). By the application of cyclodextrin‐modified electrolytes, chiral separation of FITC‐labelled amines could be performed in seconds demonstrating the potential of microchip electrophoresis for chiral high‐throughput screening.