Liquid Chromatography of Sugars on Diol Columns. Detection with Cuprammonium Reagent

Abstract

Post column reaction of sugars with cuprammonium reagent is discussed. Formation of the complex involves two molecules of carbohydrate and one molecule of Cu(NH₃)₄(H₂O)₂ ²⁺ with the result of a shift towards longer wavelength in UV spectrum. 1 nanomole of fructose is detected, the reaction is simple, fast allowing very short reaction coil which does not increase band broadening it thus matches most of the requirements in the analysis of sugars in food products. Diol bonded silica is much less retentive than amino bonded silica and cannot compete in the separation of complex mixtures of sugars but is well suited for the analysis of one reducing sugar as important losses occur with NH, columns. Diol phase is well adapted to the separation of oligosaccharides. The use of HPLC as a means of determining all types of sugars has greatly increased for the last years. Many types of packings (bare silica, ion exchange with Na⁺ or Ca²⁺, alkyl or amino bonded silica) are available and have been advocated (1). Classical detection can be performed with refractive index or UV detectors. With the former the sensitivity is rather low and gradient elution is impossible, the latter operated at 190 ran require high purity solvents.