Practice and mechanism of HPLC oligosaccharide separation with a cyclodextrin bonded phase

A series of six oligosaccharides: linear arabinosides, cellosides, isomaltosides, mannosides, maltosides and xylosides containing up to 15 sugar units, as well as alpha, beta and gamma-cyclodextrins, were separated without derivatization with acetonitrile-water mobile phases and a beta-cyclodextrin bonded phase column using both UV (195 nm) and refractive index detection. The mobile phases contained more than 60%v/v acetonitrile to separate the oligosaccharides with baseline resolution with practical retention times. The chromatographic mode is intermediate between normal-phase chromatography and hydrophilic interaction chromatography. Sugar retention occurs through partition exchange and polar interactions. The saccharide-stationary phase interaction was studied through retention behavior and peak efficiency. It is shown that partitioning interactions and hydrogen bonding interactions between the hydroxyl groups of the stationary phase and those of the sugars are the two possible mechanisms responsible for sugar retention. The sugar retention times are dramatically reduced when the mobile phase water content is increased. The conformation of the oligosaccharides also affects retention. Excellent and rapid separation of the saccharide components of commercial corn syrup and dextrin are shown. The plots of the log retention factor versus degree of polymerization (DP) were linear. The slope of these plots was related to the solute-stationary phase interaction energy. This energy is directly related to the mobile phase water content. The kinetics of the interaction is slow. At 1 ml min(-1), the peak efficiencies were in the 10 000-15 000 plate m(-1) range (70-100 d(p)). The efficiency maximum was not reached at 50 mul min(-1) indicating slow interactions between large molecules.