Residue-Based Thermogravimetric Analysis: A Novel Method to Quantify Carboxylate Group Modifications in Macromolecules

Quantifying the degree of modification (DoM) of hyaluronic acid (HA) is crucial for biomaterials development. This has remained a challenge, as diverse functional groups hinder precise spectroscopic quantification. Here, we present a method employing thermogravimetric analysis (TGA) by comparing residues of sodium hyaluronate (NaHA) and carboxylate-modified HA derivatives. Thermal decomposition enabled quantification of the inorganic residue (Na₂CO₃) that was obtained as the final product. Validation on four diverse HA derivatives, namely aldehyde, furan, thiol, and cyanoacetate, was performed. The first three matched ¹H NMR/UV-vis data, while the cyanoacetate sample, previously unquantifiable, was determined for the first time. Because the residue arises solely from Na⁺, the assay is independent of the attached pendant group and potentially transferable to any carboxylate-bearing polymer beyond HA. Residue-based TGA closes an analytical gap, providing a label-free tool for quantifying carboxylate modification, applicable irrespective of chemical structure, and able to characterize "silent groups," relevant for biomaterials.