1989 Volume 38 Issue 11 Pages 568-572
A fluorometric detection system based on on-line postcolumn reacton with 4-methoxybenzamidine (MBA), a fluorogenic reagent selective for reducing sugars was investigated for analysis of reducing sugars by HPLC. The sugars were separated on a silica gel column (LiChrosorb SI-60, particle size 5 μm) by isocratic elution of acetonitrile, 10 mM tetraethylenepentamine (TEPA) and 50 mM taurine (17:2:1, v/v). The column eluate was mixed with 40 mM MBA and 1.0 M potassium hydroxide and then heated at 100°C for ca. 19 s in a reaction coil (5 m × 0.4 mm i.d., stainless steel tube). The fluorescence from each reducing sugar in the last eluate was monitored at 470 nm (emission) with excitation at 310 nm. The postcolumn fluorescence reaction with MBA was slightly inhibited by TEPA in the mobile phase, but not by acetonitrile or taurine. The proposed reactor system permitted sensitive fluorescence detection for reducing sugars sush as L-rhamnose, L-fucose, D-fructose and D-glucose. Their lower limits of detection (S/N=3) were 2.320 pmol per 100 μl injected. This system was also applicable to other HPLC elution systems using an acetonitrile-water mixture or 0.050.5 M borate buffer (pH 711) as the mobile phase.
