CAPILLARY ELECTROPHORESIS OF ANTS LABELED OLIGOSACCHARIDE LADDERS AND COMPLEX CARBOHYDRATES

The detection limits of the ANTS (8-aminonaphthalene-1,3,6-trisulfonic acid) label and ANTS maltose as a model carbohydrate conjugate were investigated with on-column UV and laser induced fluorescence detection. Under capillary electrophoresis conditions, the concentration and mass detection limits were found to be 5 x 10(-7) mol/l or 8 femtomole with UV and 5 x 10(-8) mol/l or 400 attomole with laser induced fluorescence detection, respectively. Including the derivatization reaction, the best concentration detection limit increases to 1 x 10(-6) mol/l carbohydrate. A model calculation shows that these detection levels are still insufficient to match those of current protein sequencing protocols. Derivatization conditions for dextran and polygalacturonic acid ladders are described with subsequent fast separation in a capillary electrophoresis system under acidic pH buffer conditions. Up to 30 oligomers could be separated in less than 10 min. The application of ANTS labelled carbohydrate analysis in the food industry is demonstrated with the carbohydrate fraction of sweets and the kinetic monitoring of the hydrolysis of polygalacturonic acid. The described ANTS derivitization protocol works with as little as 5 mu g carbohydrate as demonstrated with a complex oligosaccharide labelled in a reaction volume as little as 2 mu l. To demonstrate the applicability of this approach to complex carbohydrate analysis, an oligosaccharide mixture derived from human Immunoglobuline G was labelled and separated within 5 min. Separation efficiency and speed are superior to state-of-the-art chromatographic methods. Both electrophoretic and chromatographic methods are complementary because of their different separation mechanism. The implications of using capillary electrophoresis with laser induced fluorescence and appropriate labelling strategies for structural and compositional analysis of complex carbohydrates are discussed.