Capillary Electrophoresis−Fluorescence Line-Narrowing System for On-Line Structural Characterization of Molecular Analytes

Abstract

We have demonstrated, for the first time, that capillary electrophoresis (CE) can be interfaced with low-temperature fluorescence line-narrowing (FLN) spectroscopy for on-line structural characterization. Detection by laser-induced fluorescence spectroscopy, under fluorescence non-line-narrowing and line-narrowing conditions, provides three-dimensional electropherograms and FLN spectra, which lead to significantly improved overall resolution and allow for structural characterization ("fingerprinting") of molecular analytes. This novel CE-FLN system consists of a modular CE system, instrumentation for FLN spectroscopy, and a specially designed capillary cryostat (CC). An absorbance detector serves to determine the migration rates of analytes. After the 77 K fluorescence-based electropherogram is generated, the temperature of the capillary is lowered to 4.2 K for high-resolution FLN characterization. Automated translation of the CC and capillary in the direction of the capillary axis allows the separated analytes to be sequentially characterized by fluorescence spectroscopy as the capillary is translated through the laser excitation region. Detection of fluorescence from stationary CE-separated analytes significantly improves the accuracy of quantitation and structural characterization. We believe that this interfacing represents an exciting addition to the rapidly evolving field of CE, providing a new and powerful tool for chemical analysis. The first application of the CE-FLN system to a mixture of polycyclic aromatic hydrocarbons is presented; prospects and future applications of CE-FLN are briefly addressed.

Capillary electrophoresis with laser-induced fluorescence detection for natively fluorescent analytes

“…FLNS has been applied as a stand-alone technique, for the off-line identification of HPLC fractions, or for the off-line identification of TLC-separated spots [31,32]. Recently, however, Jankowiak and coworkers managed to apply the method to CE-separated analytes [33,34].…”

Fluorescence line-narrowing detection in chromatography and electrophoresis

“…With these changes the LOD of the modified CE-FLNS system is at the low-attomole level for moderately fluorescent chromophores [30]. When the fluorescence analysis is complete, efficient warming of the CC and capillary can be achieved by closing the valve of the helium transfer line and introducing (warm) helium gas into the CC [26,27]. We emphasize that CE-FLNS, in addition to being highly selective and sensitive, is a practical technique.…”

Advances in capillary electrophoresis

“…In regard to selectivity, it has been shown that various stereoisomers and different conformations of polyaromatic hydrocarbon-DNA adducts can be resolved by this technique [125,126]. Concerning versatility, fluorescence line narrowing spectroscopy has been applied to polyaromatic hydrocarbons [127,128], and nucleoside / nucleotide DNA adducts in vivo and in vitro [129][130][131][132]. A variety of other fluorescent analytes including thiazole dyes [133], squaraine dyes [134], chlorophylls [135,136], heme proteins [137] and cytochromes [138] have also been characterized by this spectroscopic method, though the combination with capillary electrophoresis has not been explored in the past.…”