INVESTIGATION OF MICELLES AND ANIONIC CYCLODEXTRINS AS PSEUDOSTATIONARY PHASES FOR THE CAPILLARY-ELECTROPHORESIS SEPARATION OF OLIGOSACCHARIDES DERIVATIZED WITH 2-AMINO-BENZAMIDE

Oligomers of glucose and oligosaccharides released from glycoproteins were derivatized with 2-aminobenzamide. As this fluorophore imparts no charge to the oligosaccharides, several strategies were investigated to achieve capillary electrophoresis (CE) separation of both neutral a nd charged derivatized glycans. Micellar electrokinetic capillary chro matography (MEKC) with the addition of anionic surfactants was evaluat ed as a first approach: sodium dodecyl sulfate (SDS) produced the best separation of the oligoglucose fragments, where the migration was inv ersely related to their degree of polymerization. To demonstrate the a pplicability of this method for complex carbohydrate analysis, oligosa ccharide mixtures derived from ribonuclease B (RNase B) and a-acid gly coprotein (alpha-AGP) were analyzed. A satisfactory separation for the high-mannose structures found in RNase B could be obtained, whereas c harged oligosaccharides from alpha-AGP were poorly resolved. Cyclodext rin-modified CE was chosen as the second approach: the effect of the a ddition of sulfobutylether-beta-cyclodextrin (SBE-beta-CD) or sulfobut ylether-gamma-cyclodextrin (SBE-gamma-CD) on the electrophoretic mobil ities and resolution of neutral and charged oligosaccharides was then studied. Selectivity of sialylated structures could be further improve d by using anionic cyclodextrins (CDs) instead of micelles. However, t his latter approach failed to baseline-resolve the different high-mann ose structures of RNase B. A successful separation of the complex mixt ure of oligosaccharides from alpha-AGP was obtained with the addition of 4% of SBE-gamma-CD and triethylamine (TEA) in a phosphate buffer, p H 6.7.