Molecular weight of chitosan: From structural complexity to analytical reliability

The functional properties of chitosan are intrinsically linked to its molecular weight (MW), yet accurately determining MW remains a significant analytical challenge. This review critically evaluates methods for characterizing the MW of chitosan, addressing discrepancies arising from polydispersity, aggregation, and polyelectrolyte effects. Techniques such as size-exclusion chromatography, light scattering, viscometry, and analytical ultracentrifugation are compared, with an emphasis on their principles, advantages, and limitations. The impact of structural heterogeneity - primarily degree of deacetylation, but also acetylation patterns - on the reliability of MW measurements is critically examined. The analysis reveals widespread inconsistencies in reported data due to the absence of standardized protocols for routine relative MW methods (e.g., conventional size-exclusion chromatography with column calibration using pullulan or dextran standards, and capillary viscometry). Recent methodological advances aimed at improving precision are also discussed. The review further emphasizes the urgent need for interlaboratory validation and unified standards to bridge the gap between laboratory research and commercial application. Notably, size-exclusion chromatography coupled with multi-angle laser light scattering has emerged as the "gold standard" for absolute MW determination of chitosan, as formalized in ASTM F2602-18. Robust MW characterization protocols are essential for advancing chitosan-based technologies in biomedicine and beyond.