Development of Selective Analysis for Individual Functional Groups by High-performance Capillary Electrophoresis (HPCE) with Laser-induced Fluorescence (LIF) Detection

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HPCE is an excellent means for separation of multi-components with high speed and separatability. This study deals with search of labeling reagents for various functional groups such as carboxyl, amine, thiol and hydroxyl, by HPCE-LIF detection. Trace analysis of biologically importances by recommended CE-LIF procedures is also carried out with the selected fluorescent labeling reagents.Fluorophors possessing fluoresceine (e.g. FITC) and 4-nitro-2, 1,3-benzoxadiazole (e.g. NBD-F) moieties were suitable for argon-ion (Ar) laser (excitation wavelength, 488 nm) detection, in terms of wavelength and sensitivity. Whereas 4-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole structures such as DBD-F were recommended for helium-cadmium (He-Cd) laser (excitation wavelength, 442 nm) detection. The detectability using the reagents with LIF detection increased more than two orders of magnitude, as comparing with UV detection. On the other hand, the detection sensitivity with He-Cd laser at 325 nm was lower than that at 442 nm, but higher than that of UV detection. Cyanine dyes were selected for diode (DIO) laser detection at 630 nm. However, the low stability of the reagents in solution and the low derivatization yields were important disadvantages. As the application with HPCE-LIF detection , chiral separation of DL-amino acids and bioactive thiols in rat hepatocytes were performed after labeling with DBD-PyNCS and ABD-F, respectively. The derivatives of DL-amino acids were completely separated by HPCE and detected sensitively (a few fmol level) with Ar-laser detection at 488 nm. Intracellar thiols (e.g. glutathione, cysteine, N-acetylcysteine) after labeling with ABD-F were also separated and detected by HPCE-LIF with He-Cd laser at 325 nm. The concentration of glutathione in the single cell was fmol level. The proposed procedure seems to be applicable to highly sensitive detection of biological substances in a cell.