Design and performance of a microchip electrophoresis instrument with sensitive variable-wavelength fluorescence detection.
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.
Belder
Detlev
D
Abteilung für Chromatographie, Max-Planck-Institut für Kohlenforschung, Mülheim an der Ruhr, Germany. belder@mpi-mulheim.mpg.de
Deege
Alfred
A
Maass
Martin
M
Ludwig
Martin
M
D003505:Cyclodextrins; D004586:Electrophoresis / Q000295:instrumentation* / Q000379:methods / Q000592:standards; D004867:Equipment Design; D016650:Fluorescein-5-isothiocyanate; D005453:Fluorescence; D008832:Microchemistry / Q000295:instrumentation* / Q000592:standards; D008856:Microscopy, Fluorescence; D008904:Miniaturization; D015203:Reproducibility of Results; D012235:Rhodamines; D012680:Sensitivity and Specificity; D013237:Stereoisomerism
D003505:Cyclodextrins; D012235:Rhodamines; D016650:Fluorescein-5-isothiocyanate