Multifaceted separations approach for elucidation of the physical and chemical properties of extracellular hydrocolloids

A multifaceted separations platform that incorporates the strengths of asymmetrical flow field-flow fractionation with multi-detectors (AF4-MD), high performance anion exchange chromatography (HPAEC), and hydrophilic interaction liquid chromatography with mass spectrometry (HILIC-MS) is developed to obtain a more complete picture of the molecular weights (MW), composition, and salt-induced aggregation behavior of extracellular polymeric substances (EPS) produced by the algae Chlorella vulgaris. The absence of a stationary phase makes AF4-MD particularly well suited for characterizing polydisperse hydrocolloid polymers as well as studies that investigate the effect of ionic environments that aligns with the natural environment of C. vulgaris. Fractionation of C. vulgaris EPS revealed three distinct MW populations ranging from 4 × 10⁴ to 3 × 10⁸ Daltons. This exceeds the previously reported MW by three orders of magnitude and reports a previously unknown size subpopulation. The optimized AF4-MD technique was then used to produce two size fractions that were probed using HPAEC and LC-MS. These orthogonal methods uncovered compositional heterogeneity across fractions, with variations in monosaccharides and amino acids. AF4-MD is also well suited for studying the behavior of EPS in the presence of different salts. For each salt studied, e.g., NaNO₃, NaCl, and MgCl₂, an increase in solution ionic strength results in aggregation as corroborated by a shift to higher MWs. Each salt exhibited distinct effects on EPS aggregation, with NaCl causing the least aggregation and MgCl₂ the most. These findings highlight the need for multiple techniques when analyzing complex polymers such as EPS and the benefits of AF4-MD in elucidating complex polymer behaviors in different ionic environments.