Heterogeneous hydrolysis of hyaluronic acid in ethanolic HCl slurry

In this work, a novel procedure for hydrolysis of hyaluronic acid (HA) is presented. HA was hydrolysed in ethanol with added HCl during heterogeneous conditions using varying temperatures (40, 60 and 80 °C) and acid concentrations (0.01, 0.05, and 0.1 M HCl). The degradation process was monitored by determination of weight average molecular weight (Mw) by size-exclusion chromatography with online multi-angle laser light, refractive index and intrinsic viscosity detectors (SEC-MALLS-RI-visc) on samples withdrawn continuously during the hydrolysis. SEC-MALLS-RI-visc showed that the degradation gave molecular weight distributions with polydispersity indexes (Mw/Mn) of 1.3–1.8. Kinetic plots of (1/Mw − 1/Mw,0) vs. time gave linear plots, showing that heterogeneous acid hydrolysis of HA is a random process and that it follows a first order kinetics making it possible to determine the kinetic rate constant (kh) from the slope. During the hydrolysis in heterogeneous conditions it was found that kh depended on the water concentration in the slurry (increases with increasing water concentration). Additionally, for heterogeneous hydrolysis in ethanol/HCl at 40, 60 and 80 °C, it was shown that kh depended linearly on the acid concentration, but not to the same extent as previously seen in hydrolysis under aqueous, homogeneous conditions. Further, the dependence of temperature on the hydrolysis in 0.05 and 0.1 M ethanol/HCl was found to follow the Arrhenius kinetics (linear plot of ln kh vs. 1/T). For hydrolysis in 0.05 and 0.1 M ethanolic HCl, the activation energy (Ea) was determined to 107 and 104 kJ/mol and the Arrhenius constant (A) was determined to 1.2 × 1010 and 8.8 × 109 h−1, respectively. Both Ea and A were significantly lower compared to those determined for hydrolysis performed under aqueous, homogeneous conditions at the same acid concentration. It was shown that the availability of water was rate limiting for the hydrolysis process in ethanol/HCl slurry. 1H NMR was used to characterize the product of extensive hydrolysis. No indication of de-N-acetylation of the GlcNAc units, indication of ethanolysis or other by-products were seen. The new heterogeneous hydrolysis procedure provides several advantages compared to homogenous hydrolysis such as lower ethanol and water consumption and the possibility to hydrolyse a larger amount of material in the same volume.