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.