Measurement of Molecular Weight Distribution of Hyaluronic Acid by Size Exclusion Chromatography Using Low-angle Laser Light Scattering

Abstract

The molecular weight distribution and average molecular weight (Mw) of hyaluronic acid (HA) powders and their injectable preparations were measured by size exclusion chromatography/low-angle laser light scattering (SEC/LALLS). The columns used were TSK gel G6000PW_XL (30cm×4) made from cross-linked hydrophilic polymers, and were eluted with 0.2M sodium sulfate at a flow rate of 1.0ml/min. A reasonable relationship between the retention time and the molecular weight of the HA was obtained under these SEC conditions. The degree of depolymerization of HA by heating at 70°C was related to the molecular weight, it was found to be more extreme the higher the original molecular weight. The measured value of Mw was in good agreement with the viscosity average molecular weight in eight kinds of cosmetic grade HA powders. The preparation from an injectable HA preparation was conveniently made by diluting with the mobile phase which was directly injected into the SEC. Among examined eight kinds of cosmetic grade HA powders and four injectable HA preparations, HA from an injection showed the lowest degree of polydispersity. It was revealed that SEC/LALLS was a useful tool for examining the molecular weight distribution of HA and its related medicines.