Batch and Size-Exclusion Chromatographic Characterization of Ultra-high Molar Mass Sodium Hyaluronate Containing Low Amounts of Strongly Scattering Impurities by Dual Low Angle Light Scattering/Refractometric Detection

Abstract

It is shown that very pure sodium hyaluronate (HA) contains small amounts of strongly scattering impurities not detectable on mass scale by refractometric detection, but clearly detectable using low angle light scattering detection during a static bulk light scattering experiment. Size filtration of its solutions does not remove these impurities, only reduces their amount depending on filter porosity. Complete removal of these particle impurities, independent of filter porosity, is achieved by hydrophobic adsorption on hydrophobic filter membranes. Using 0.1 M NaCl as a mobile phase, size exclusion chromatography (SEC) column removes the impurities by hydrophobic adsorption as well; molar masses obtained from both techniques thus agree when hydrophobic filters are used in bulk light scattering experiments. Diverse hydrodynamic flow retardation effects including slalom chromatography behavior are shown to substantially bias molar mass distributions obtained for ultra-high molar mass (UHM) HA, unless the flow rate in SEC analysis is reduced below 0.1 mL/min. Too high injected concentration (c _inj ) is shown to introduce the onset of HA on column degradation. Correct polydispersity indices and molar mass distributions of UHM HA are obtained from SEC at a flow rate of 0.09 mL/min and optimized c _inj .

Keywords:

• Dual low-angle light scattering/refractometric detection
• Size-exclusion chromatography
• Sodium hyaluronate
• Static light scattering
• Strongly scattering impurities

ACKNOWLEDGMENTS

The authors wish to thank the Grant Agency of the Academy of Sciences of the Czech Republic (projects No. A1005 00501 and AVOZ 4050913) for financial support.

Notes

^a 1 µm PTFE sample filter.

^b 1 µm PVDF sample filter.

^a c _inj = 17.1 µg/mL.

^b c _inj = 50.5 µg/mL.