Depolymerization study of sodium hyaluronate by flow field-flow fractionation/multiangle light scattering

Thermal depolymerization of ultrahigh-molecular-weight (UHMW) sodium hyaluronate (NaHA) was studied systematically by using frit-inlet asymmetrical flow field-flow fractionation/multiangle light scattering/differential refractive index (FI-AFlFFF/MALS/DRI). FI-AFlFFF was utilized for the size separation of NaHA samples which had been thermally degraded for varied treatment times, followed by light-scattering detection to determine MW and structural information of degraded NaHA products. Analysis of NaHA products showed time-dependent depolymerization of raw molecules into smaller-MW components, as well as unfolding of compact structures of UHMW NaHA. To determine whether the observed decrease in MW of sodium hyaluronate originated from the chain degradation of UHMW molecules or from dissociation of entangled complex particles that may have been formed by intermolecular association, narrow size fractions (1×10⁷-6×10⁷ and >6×10⁷ MW) of NaHA molecules were collected during FlFFF separation and followed by thermal treatment. Subsequent FI-AFlFFF/MALS analysis of collected fractions after thermal treatment suggested that the ultrahigh-MW region (>10⁷ Da) of NaHA is likely to result from supermolecular structures formed by aggregation of large molecules.