High performance liquid chromatography of disaccharides on a porous graphitic carbon column applying post-column derivatization with benzamidine

A sensitive post-column fluorescence method based on the reaction of reducing sugars with a fluorigenic reagent (benzamidine) under alkaline conditions and elevated temperature is described. Optimum reaction conditions suitable for the detection of fluorescent reaction products have been investigated. A porous graphitized carbon column (Shandon Hypercarb) was employed as the chromatographic solid phase and elution was isocratic by an aqueous acetonitrile mixture. A special problem in sugar analysis is the tendency of anomer formation giving doublet peaks. This tendency varies largely among the different sugars, and systematic studies on conditions that eliminate this problem were performed. Efficient and selective separations of a series of disaccharides were obtained using high temperatures and a low concentration of acetonitrile in the mobile phase. The detection limits (S/N = 3) with an injection volume of 20 mu L ranged from 10-60 picomoles. Linear calibration graphs were obtained in the 2-20 mu M concentration range (r(2) > 0.99). A relative standard deviation between 1-3 % (n = 6) demonstrated good precision of the developed system.