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

高效液相色谱是一种快速、可分离性好的多组分分离方法。本研究探讨了用HPCE-LIF检测方法寻找羧基、胺基、巯基和羟基等各种官能团的标记试剂。通过推荐的CE-LIF程序对生物重要性的痕量分析也使用选定的荧光标记试剂进行。在波长和灵敏度方面，具有荧光素（如FITC）和4-硝基- 2,1,3 -苯并二唑（如ndd - f）基团的荧光团适用于氩离子（Ar）激光（激发波长为488 nm）检测。而4-（N，N-二甲氨基磺酰基）-2,1,3-苯并恶二唑结构如DBD-F被推荐用于氦镉（He-Cd）激光（激发波长442 nm）检测。与紫外检测相比，LIF检测试剂的检出率提高了两个数量级以上。另一方面，He-Cd激光在325 nm处的探测灵敏度低于442 nm处，但高于紫外探测。选择花菁染料用于二极管（DIO）激光探测630 nm波长。然而，试剂在溶液中的稳定性低，衍生率低是重要的缺点。作为HPCE-LIF检测的应用，分别用DBD-PyNCS和ABD-F标记大鼠肝细胞中的dl氨基酸和生物活性硫醇进行手性分离。dl -氨基酸衍生物经高效液相色谱（HPCE）完全分离，并在488 nm处用ar激光检测，灵敏度可达几fmol。ABD-F标记后的胞内硫醇（如谷胱甘肽、半胱氨酸、n -乙酰半胱氨酸）也通过HPCE-LIF分离，He-Cd激光在325 nm下检测。单细胞内谷胱甘肽浓度为fmol水平。所提出的方法似乎适用于细胞中生物物质的高灵敏度检测。

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.