Inter-laboratory evaluation of SEC-post-column calcofluor for determination of the weight-average molar mass of cereal β-glucan

•Inter-laboratory comparison of β-glucan molar mass standards with SEC-calcofluor.•The calcofluor method accurately determines molar masses between 10 and 500×103g/mol.•The calcofluor method underestimates β-glucan molar masses above 500×103g/mol.•Proposed delayed transport of β-glucan-calcofluor complexes to detector. Even though size exclusion chromatography (SEC) with post column addition of calcofluor (SEC-calcofluor) has been used for the determination of cereal β-glucan molar mass in foods for many years, there is a lack of systematic evaluation of the method. To address this issue a set of suitable β-glucan standards were generated by preparative SEC and their molar mass characteristics were determined by analytical multi-detection SEC (refractive index (RI), light scattering). Each standard was then analysed by SEC-calcofluor at three different labs. As a direct comparison, the analyses were repeated with a RI detector. For SEC-calcofluor accurate measurements of weight average molar mass (Mw) can be made for β-glucan populations within 10–500×103g/mol. Above this molar mass threshold there is an increasing tendency for underestimation of Mw. Precipitation of some β-glucan-calcofluor complexes may have delayed their transport into the detector.