Key Points
- Hydrocolloids are complex, though due to difficulties in characterizing molecular properties, scientists’ understandings of their ecological and biological significance are incomplete.
- A new separations platform was created for this purpose, with techniques involved including high-performance anion exchange chromatography (HPAEC) and hydrophilic interaction liquid chromatography with mass spectrometry (HILIC-MS).
- The platform was tested while analyzing extracellular polymeric substances in Chlorella vulgaris algae.
Researchers from the Colorado School of Mines and the National Renewable Energy Laboratory (Golden, Colorado) studied the properties of extracellular hydrocolloids with various chromatographic techniques. Their findings were published in the Journal of Chromatography A (1).
Hydrocolloids are macromolecular biopolymers–often used in emulsions and foams–to modify appearance, texture, stability, and taste (2). These capabilities stem from hydrocolloids’ ability to form gels or increase the viscosity of aqueous solutions. Complex hydrocolloid biopolymers are ubiquitous in nature, though scientists’ understandings of their ecological and biological significance are currently incomplete due to inherent difficulties in characterizing their molecular properties. These biopolymers often display a wide distribution of variable, high molecular weight (MW) species, form aggregates, and have highly heterogeneous chemical compositions, all of which complicate characterization. Regardless, the physical, structural, and chemical properties of biopolymers directly influence their function and performance, making knowledge of their molecular properties vital for understanding their fundamental significance and potential uses.
In this study, the researchers created a separations platform that combines the strengths of symmetrical 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). With this system, they aimed to obtain a more complete picture of the molecular weights (MW), composition, and salt-induced aggregation behavior of extracellular polymeric substances (EPS)–which are polymeric substances secreted by microbials–produced by the algae Chlorella vulgaris (3). The lack of a stationary phase is said to make AF4-MD well suited for characterizing polydisperse hydrocolloid polymers, in addition to studies that investigate the effects of ionic environments that aligns with the natural environment of C. vulgaris.
Fractionation of C. vulgaris revealed three different MW populations, ranging from 4 × 10⁴ to 3 × 10⁸ Daltons. This exceeded previously reported MW by three orders of magnitude and reported previously unknown size subpopulation. Optimized AF4-MD techniques were then used to create two size fractions probed using HPAEC and LC–MS. Altogether, the orthogonal methods uncovered compositional heterogeneity across fractions, with variations in monosaccharides and amino acids.
The scientists also noted that AF4-MD proved suitable for studying EPS behavior in the presence of different salts. For each salt studied, increases in solution ionic strength resulted in aggregation, as corroborated by a shift to higher MWs. The salts exhibited distinct effects on EPS aggregation, with sodium chloride (NaCl) causing the least aggregation, while magnesium chloride (MgCl2) caused the most. Differences in aggregation were attributed to differences in kosmotropicity of the salt, electrostatic screening, and the formation of divalent salt bridges. Isolating individual causes of aggregation in environmental hydrocolloids, such as EPS, could be challenging. However, simplifying the system with standards may fail to reflect the true complex nature of the EPS. Preliminary investigations into the influence of three salts on zeta potential, hydrodynamic radii, molecular weight (MW), and aggregation can provide key insights into how simple salts interact with EPS. Complete understanding of the salt matrix in natural systems will require using the developed analytical tools while integrating additional techniques.
While this study sheds light on the complex nature of algal EPS, including their chemical and physical properties and salt interactions, further research is needed to explore structural features. This includes polymer conformation, the biological importance of distinct fractions, and the mechanisms underlying salt-driven effects in protein-polysaccharide complexes. These findings provide groundwork for addressing these issues in future research efforts.
References
(1) Lesco, K. C.; Van Wychen, S.; Deshpande, A.; Laurens, L. M. L.; Williams, S. K. R. Multifaceted Separations Approach for Elucidation of the Physical and Chemical Properties of Extracellular Hydrocolloids. J. Chromatogr. A 2025, 1753, 465980. DOI: 10.1016/j.chroma.2025.465980
(2) Hydrocolloids. ScienceDirect 2011. https://www.sciencedirect.com/topics/immunology-and-microbiology/hydrocolloid (accessed 2025-6-17)
(3) Extracellular Polymeric Substance. ScienceDirect 2021. https://www.sciencedirect.com/topics/engineering/extracellular-polymeric-substance (accessed 2025-6-17)
