SELECTIVE POSTCOLUMN FLUORIGENIC REACTION WITH BENZAMIDINE FOR TRACE LEVEL DETECTION OF REDUCING SACCHARIDES IN LIQUID-CHROMATOGRAPHY

A liquid chromatographic post-column fluorigenic reaction with benzamidine for the trace level detection of reducing saccharides is described. The system consists of a Sugar Pak-1 gel column (300 mm x 6.5 mm I.D.) packed with a microparticulate resin in the calcium form and heated at 70-degrees-C. Elution is carried out with water containing 10(-4) M CaEDTA at 0.4 ml min-1. The post-column reaction is carried out with a mixture of 30 mM benzamidine and 1 M potassium hydroxide solution at 1 ml min-1 using a 530-mu-l reaction coil thermostated at 100-degrees-C. Fluorescence was monitored at 470 nm (emission) with an excitation wavelength of 360 nm. The system had good reproducibility (relative standard deviation 1-3%) for an injected amount of 0.9-mu-g (5 nmol). Linear calibration graphs were obtained between 16 and 500 nmol ml-1 (r = 0.999) and detection limits of 16-63 pmol at a signal-to-noise ratio of 3 were achieved. The detection of saccharides in plants is also described.