Molecular weight and ionic strength dependence of fluorescence intensity of the Calcofluor/β-glucan complex in flow-injection analysis

The flow-injection analysis (FIA) method has long been used for the quantitative determination of β-glucans. FIA makes use of the enhanced fluorescence produced when β-glucan forms complexes with Calcofluor. For successful application of this analysis method, the lowest molecular weight (MW) in the sample has to be higher than a critical MW below which the fluorescence intensity of the complex becomes weaker.Fluorescence measurements reveal that the fluorescence intensity of the Calcofluor/β-glucan complex is strongly dependent on the ionic strength of the solution. At high ionic strength, fluorescence intensity is high and critical MW is low. At low ionic strength, on the other hand, fluorescence intensity is low and critical MW is high. Turbidity measurement shows that Calcofluor/β-glucan complex forms aggregates in the solution and the rate of aggregate formation depends on the MW of β-glucan and concentration of Calcofluor. This study indicates that the FIA method has to be used with solutions of high ionic strength and a high Calcofluor to β-glucan ratio to broaden the applicable MW range of β-glucan analysis. Application of FIA method to the analysis of β-glucan in oat products turns out to be successful as confirmed by comparison with conventional enzymatic analysis.