Separation and detection of hemicellulose and insights into chemical heterogeneity through capillary electrophoresis

Hemicelluloses represent a family of polysaccharides abundant in biomass but which valorization is limited. Their complex molecular and macromolecular structure, as well as their lack of appropriate separation and characterization, hinder this valorization. It remains unclear whether wood hemicelluloses constitute a single polysaccharide family or several distinct ones. This limited understanding and the challenges in separating and analyzing these polysaccharides constrain their use. Therefore, a reliable method to separate intact wood hemicelluloses is crucial for unlocking their full potential. There is a great need for a robust technique for separating and detecting intact, structurally diverse wood hemicelluloses. This work presents a free solution capillary electrophoresis (CE) approach for the separation of intact “neutral” and non-UV-absorbing polysaccharides at 266 nm using an alkaline (pH 12.6) electrolyte solution of 130 mM sodium hydroxide and 36 mM disodium hydrogen phosphate. Galactomannans and glucomannans “standards” and wood hemicelluloses were separated and detected. Electrophoretic mobility distributions were determined, as well as their average mobilities, dispersities and standard deviations. Wood hemicellulose extracts showed much higher weight-average mobilities (13.14-15.60)·10 -9 m 2 .V -1 .s -1 and broader distributions (( D ( W ( μ ),1,0) = 1.10-1.18) than “standard” galacto- and gluco-mannans ((5.01-7.20)·10 -9 m 2 .V -1 .s -1 and 1.003-1.009 respectively). The higher mobility for wood hemicellulose indicates the presence of polysaccharides with much high uronic acid content (at least 15 %), than determined after hydrolysis (0.5 %) using chromatography (HPAEC). This may be due to uronic acid degradation during the harsh hydrolysis. The much higher dispersity of hemicelluloses extract indicate a much greater compositional heterogeneity. This study demonstrates CE's potential for examining structural changes within polysaccharides and proposes a unique detection method without any sample preparation (no filtration, no derivatization). For the first time, “standard” and wood hemicelluloses were separated using CE and detected by UV absorption at highly alkaline pH. Additionally, the high level of molecular heterogeneity of intact wood hemicelluloses was quantified through dispersity of electrophoretic mobility distributions, revealing that a number of polysaccharides are charged and contain uronic acids. • Separation of “standard” and wood hemicelluloses using free solution capillary electrophoresis (CE). • Detection of “standard” and wood hemicelluloses by UV absorption at highly alkaline pH. • Wood hemicelluloses are heterogeneous, composed of various polysaccharides. • A number of polysaccharides in wood hemicelluloses are charged, containing uronic acids.