Evaluating the degree of substitution of water-insoluble acyl derivatives of hyaluronan using Raman spectroscopy: method development and comparison with gas chromatography and 1H NMR

Abstract

The properties of water-insoluble acyl derivatives of hyaluronan (HA) are strongly influenced by the degree of HA substitution (DS) and, therefore, the knowledge of exact DS values is greatly needed. The DS values of polysaccharides are usually evaluated by ¹H NMR or chromatographic methods, which require sample dissolution. However, the studied HA derivatives are only soluble in mixed solvents, which is problematic especially for ¹H NMR. To avoid solubilization issues, we developed a method for evaluating the DS of HA in the solid state using Raman spectroscopy. Calibration was performed using palmitoyl-HA oligosaccharides with the DS known from their molecular weight. The evaluation of Raman data is therefore not based on correlation with another technique. The results of Raman spectroscopy and gas chromatography agreed very well, with an average relative deviation of 5.5%. Conversely, ¹H NMR significantly overestimated the DS due to the preferential dissolution of acyl groups. This problem was overcome by additional calibration. Our results show that Raman spectroscopy is a promising tool for evaluating the DS of HA acyl derivatives.