SELECTIVE DETERMINATION OF β-GLUCAN OF DIFFERING MOLECULAR SIZE, USING THE CALCOFLUOR-FLUORIMETRIC FLOW-INJECTION-ANALYSIS (FIA) METHOD

Abstract

When using the calcofluor-fluorimetric flow-injection-analysis (FIA) method to determine β-glucan in wort and beer, the actual range of β-glucan molecular weights effectively complexed by the calcofluor, and consequently the total amount detected, is dependent on the ionic strength of the eluent. In the base eluent of very low ionic strength (20 ppm of calcofluor in aqueous 0.1 M TRIS, pH = 11), only β-glucan molecules of molecular weight greater than approximately 200, 000 daltons are fully complexed by calcofluor, and consequently fully detected. As the molecular weight of β-glucan molecules decreases, β-glucan is only partially complexed by calcofluor, and hence partially detected. By increasing the ionic strength of the base eluent through adding successive amounts of NaCl up to a maximum of 1%, the molecular weight of β-glucan which can be detected by the method decreases down to about 65, 000 daltons, whereas the molecular weights below about 10,000 daltons are not significantly complexed nor detected. Different size ranges of β-glucan have been isolated and characterized by enzymatic degradation of high molecular weight standard β-glucan, followed by classical gel permeation chromatography (GPC), using Sephacryl S-400 HR as stationary phase, pure water as eluent, and dextrans as molecular weight markers.