Inter-laboratory analysis of cereal beta-glucan extracts of nutritional importance: An evaluation of different methods for determining weight-average molecular weight and molecular weight distribution

In an interlaboratory study we compare different methods to determine the weight-average molecular weight (Mw) and molecular weight distribution of six cereal beta-glucan isolates of nutritional importance. Size exclusion chromatography (SEC) with multi-angle light scattering (MALS), capillary viscometry, sedimentation velocity analytical ultracentrifugation and one asymmetric flow field-flow fractionation (AF4)-MALS method all yielded similar Mw values for mostly individual chains of dissolved beta-glucan molecules. SEC with post-column calcofluor detection underestimated the Mw of beta-glucan > 500 x 10(3) g/mol. The beta-glucan molecules analysed by these methods were primarily in a random coil conformation as evidenced from individual MarkHouwink-Kuhn-Sakurada (MHKS) scaling coefficients between 0.5 and 0.6 and Wales-Van Holde ratios between 1.4 and 1.7. In contrast, a second AF4-MALS method yielded much larger Mw values for these same samples indicating the presence and detection of beta-glucan aggregates. Storage of the six beta-glucan solutions in the dark at 4 C for 4 years revealed them to be stable. This suggests an absence of storage-induced irreversible aggregation phenomena or chain-scission. Shear forces in SEC and the viscometer capillary and hydrostatic pressure in analytical ultracentrifugation probably led to the reversable dissociation of beta-glucan aggregates into molecularly dissolved species. Thus, all these methods yield true weight-average molecular weight values not biased by the presence of aggregates as was the case in one of the AF4 based methods employed.

Automated summary

This study compared different methods for determining the weight-average molecular weight and molecular weight distribution of cereal beta-glucan isolates. The results showed that most methods yielded similar Mw values, except for one AF4 method that detected the presence of aggregates. The analysis indicated that the beta-glucan molecules were primarily in a random coil conformation and exhibited stability during storage.