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

Abstract

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 x 10(7)-6 x 10(7) and >6 x 10(7) 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 (>10(7) Da) of NaHA is likely to result from supermolecular structures formed by aggregation of large molecules.

Flow field‐flow fractionation and multiangle light scattering for ultrahigh molecular weight sodium hyaluronate characterization

“…Figure 11A shows the fractograms of the four samples (control and samples treated thermally for 10, 20, and 30 min) including both the LS and DRI signals [37]. In the figure, F3 and F4 (marked with slashes) are the time intervals for two collected fractions that will be explained later Time (min) Figure 9.…”

“…Thermal treatment is performed by heating the raw material (10 mg/mL in 0.3 M NaCl solution) at 100°C for 5–30 min in 5 min intervals, and the resulting solutions are diluted with 0.1 M NaNO 3 to 0.8–1.0 mg/mL and left overnight without stirring at 4°C. Figure 11A shows the fractograms of the four samples (control and samples treated thermally for 10, 20, and 30 min) including both the LS and DRI signals 37. In the figure, F3 and F4 (marked with slashes) are the time intervals for two collected fractions that will be explained later.…”

Extraction of hyaluronic acid (HA) from rooster comb and characterization using flow field‐flow fractionation (FlFFF) coupled with multiangle light scattering (MALS)

“…It has been shown that, in practice, FlFFF gives better resolution for MW higher than about 10 5 43. FlFFF has shown its applicability for separation of HA or HA‐like molecules 44–50, for which the frit‐inlet FlFFF, another variant of FlFFF, was mostly used.…”

Asymmetrical flow field-flow fractionation technique for separation and characterization of biopolymers and bioparticles

“…A series of papers have been published in which AsFlFFFF-MALS was applied to study the degradation of ultrahighmolecular weight hyaluronate under the influence of thermal stress [90], ultrasonic, enzymatic or pH [91]. Electron irradiation effect was applied to study scleroglucan degradation, because of its resistance to hydrolysis, temperature and electrolytes [92], and via AsFlFFF the presence of low and high molar mass fractions could be proved.…”