Preparation and characterization of molecular weight standards of low polydispersity from oat and barley (1 → 3)(1 → 4)-β-D-glucan

Purified (1 --> 3)(1 --> 4)-beta-D-glucans (beta-glucans) from oat and barley with broad molecular weight (MW) distribution were separated into seven fractions using gradient precipitation with ammonium sulfate (NH4)(2)SO4. The MW of each fraction decreased consecutively with the concentration of (NH4)(2)SO4 at which it was precipitated. The MW distribution of each fraction was much narrower compared to the parent sample and is comparable to commercially available pullulan MW standards. To determine whether the fractionation process was separating sub-fractions of different structure, the original beta-glucan sample and each fraction were hydrolyzed by a (1 --> 3)(1 --> 4)-D-beta-glucan-4-glucanohydrolase (lichenase, E.C.3.2.1.73) and the liberated oligosaccharides were analyzed by high performance anion exchange chromatography. The analysis revealed no differences in oligosaccharide pattern (DP 2-9) derived from each fraction and the parent sample. In particular, the tri/tetra. oligosaccharide ratio remained constant for all fractions, indicating no fractionation based on structural features had taken place. The effect of starting beta-glucan concentration on the fractionation process was studied. The results showed that it was possible to achieve good separation at overlapping parameter c[eta] lower than similar to3.5. Further increase in starting beta-glucan concentration hindered clear separation of the fractions. Temperature also affected the fractionation efficiency. The higher the temperature, the lower the amount of (NH4)(2)SO4 that was necessary to precipitate the samples of same MW. A Mark Houwink relationship was derived from the measured MW and intrinsic viscosity for fractions from oat and barley, respectively. Crown Copyright (C) 2003 Published by Elsevier Ltd. All rights reserved.