2.5. Determination of homogeneity of polysaccharides and their molecular weight (Mw)
The determination of the homogeneity and molar mass (Mw) of the fractions were performed on a Waters high-performance size-exclusion chromatography
(HPSEC) apparatus coupled to adifferential refractometer (RI) and a Wyatt Technology Dawn-F Multi-Angle Laser Light Scattering (MALLS) detector.
The eluent was 0.1 M NaNO3, containing 0.5 g L−1 NaN3. The polysaccharides
solutions were filtered through a membrane with 0.22 m diameter pores (Millipore). The specific refractive index increment (dn/dc) was determined using a Waters 2410 detector, the samples being dissolved in the eluent, five increasing concentrations, ranging from0.2 to 1.0 mg mL−1 being used to determine the slope of the increment.
2.6. Methylation analysis of ˇ-glucan
Per-O-methylation of the isolated polysaccharide (10 mg) was carried out using NaOH-Me2SO-MeI (Ciucanu & Kerek, 1984). The process, after isolation of the products by neutralization (HOAc),dialysis, and evaporation was repeated, and the methylation wasfound to be complete. The per-O-methylated derivatives were
hydrolyzed with 45% (v/v) . formic acid (1 mL) for 15 h at 100 ◦C,followed by NaB2H4 reduction and acetylation as in item 2.4, to givea mixture of partially O-methylated
alditol acetates, which was anayzed by GC–MS using a DB-225 capillary column
(30 m × 0.25 mm i.d.) programmed from 50 to 215 ◦C at 40 ◦C min−1, then hold, and identified from m/z profiles of their positive ions, by comparison
with standards, the results being expressed as a relative percentage of each component.