β glucan standards

The Mw distribution of β-glucans in oat samples was determined by high-performance size-exclusion chromatography (HP SEC), which consisted of an Alliance 2690 separation module, using Calcofluor white (Fluorescent Brightener 28, Aldrich, Germany) and Scanning Fluorescence 474 detector (Waters Inc., Milford, MA, USA), as described in Suortti [27 (link)]. The samples were prepared by dissolving 20 mg into 10 mL of NaOH (0.1 M) and 0.1% NaBH₄ under magnetic stirring at room temperature overnight. The samples were diluted and filtered using a 0.45 µm syringe filter prior to HP SEC measurement. The linear size-exclusion calibration curve was prepared with β-glucan standards from Megazyme ranging from 33.6 to 667 kDa.

The molecular weight and homogeneity of the polysaccharide fractions were determined by size exclusion chromatography (HPSEC) with light scattering (ELSD) detection according to the procedure reported by Cheong et al. (2015) [77 (link)], with modifications. The GPC system (Shimadzu) is equipped with two HPLC pumps (Shimadzu), the GPC TSKgel SuperMultipore PW-H column, 6.0 ID × 150 mm, (TOSOH Bioscience), Guard SuperMP PW-H 4.6 mm ID × 35 mm (TOSOH Bioscience), column oven (Shimadzu) and evaporator lamp–scattering detector (ELSD LTII). Deionized water was used as the mobile phase. Column and detector temperatures were 40 °C and 50 °C, respectively, and the flow rate and injection volume were 0.4 mL/min and 20 µL, respectively. The β-glucan standards (40 kDa, 123 kDa, 183 kDa, 245 kDa, 359 kDa, Megazyme, Ireland and 500 kDa, Boc Sciences, New York USA) and α-glucan standards (401 kDa, 277 kDa, 196 kDa, 124 kDa, 43.5 kDa, 21.4 kDa, 9.9 kDa, 4.4 kDa, PSS Polymer Standards Service Gmbh) were used to plot a calibration curve. A calibration curve was plotted as the correlation between the retention time and the molecular weight (Mp) of the standards.