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.

Fundamentals and practice for ultrasensitive laser‐induced fluorescence detection in microanalytical systems

“…Good discussions on background sources and their magnitude include [9,10,12,[16][17][18][19][20][21]. All of the background sources have shot noise associated with them.…”

“…The latter is especially problematic with certain types of glass, such as soda lime glass, and with plastics, and the problem is often more severe in the UV [14,21,[31][32][33][34]. Broadband emission from the laser can be minimized with laser line filters, as discussed earlier.…”

Capillary electrophoresis of catecholamines with laser‐induced fluorescence intensified charge‐coupled device detection

“…Sweedler et al (1991) utilized the time-delayed integration mode to detect subattomole quantity of fluorescein isothiocyanate-labeled amino acids and neuropeptides. The multi-wavelength fluorescence electropherograms detected by CCD were used for rapid DNA sequencing (Karger et al, 1991), purity analysis of fluorescence agents (Xiong et al, 1996b), determination of tryptophan and tyrosine contents of peptides (Timperman et al, 1995), and multicomponent analysis of highly overlapped capillary electrophoretic peaks (Xiong et al, 1996c). This laboratory has established a capillary electrophoresis-laser-induced fluorescence-intensified charge-coupled device (CE-LIF-ICCD) system.…”

Detection in capillary electrophoresis

“…The LODs of the best CE/laser-based systems are well below 10 ±13 M [11,31] producing mass detection limits of less than 10 molecules [32]. In fact, single molecule detection has been demonstrated in a capillary using LIF [33] and subsequently at the stochatic noise limit in CE [34].…”