SEC Analysis of Hyaluronic Acid

Importance of the Topic

Hyaluronic acid is a linear, high molecular weight polysaccharide widely used in pharmaceutical, cosmetic, and biomedical applications due to its viscoelastic and biocompatible properties. The performance and functionality of hyaluronic acid critically depend on its molecular weight distribution, making precise characterization vital for quality control and application development.

Objectives and Overview of the Study

This work aimed to evaluate molecular weight and distribution profiles of four commercial hyaluronic acid samples using aqueous size exclusion chromatography. By comparing distinct samples, the study sought to demonstrate the reliability and resolution of the method employing Agilent PL aquagel-OH columns.

Methodology

An aqueous SEC method was applied using an isocratic eluent of 0.2 M NaNO₃ with 0.01 M NaH₂PO₄ at pH 7. A two-column setup combining PL aquagel-OH 60 and 40 (15 µm, 300 × 7.5 mm) minimized shear degradation and covered a broad molecular weight range (10⁴–10⁷ Da). The flow rate was 1.0 mL/min, and detection was performed via refractive index. Calibration was carried out using narrow pullulan polysaccharide standards.

Used Instrumentation

• Agilent 1260 Infinity GPC/SEC System
• PL aquagel-OH 60 (p/n PL1149-6260) and PL aquagel-OH 40 (p/n PL1149-6240), 15 µm, 300 × 7.5 mm columns
• Refractive index detector

Main Results and Discussion

The chromatographic overlays revealed well-resolved polymer peaks distinct from the saline eluent disturbance. Sample weight-average molecular weights (Mw) ranged from ~1.2 to 1.68×10⁶ Da, number-average (Mn) from ~0.83 to 1.08×10⁶ Da, and polydispersity indices between 1.5 and 1.6. These results confirm the method’s ability to discern subtle differences in molecular weight distributions among samples.

Benefits and Practical Applications

This SEC approach provides accurate, reproducible molecular weight metrics and distribution profiles, essential for formulation optimization, batch-to-batch consistency, and regulatory compliance in pharmaceutical and cosmetic industries. The mechanical and chemical stability of PL aquagel-OH columns ensures extended column life and reliable performance.

Future Trends and Opportunities

• Integration with multi-angle light scattering and viscometry detectors for absolute molecular weight determination.
• Development of faster, high-throughput SEC methods for real-time process monitoring.
• Advances in column chemistries to extend molecular weight range and resolution.
• Hyphenation with mass spectrometry to characterize degradation products and impurities.

Conclusion

Aqueous SEC with Agilent PL aquagel-OH columns is a robust, high-resolution technique for characterizing hyaluronic acid molecular weight distributions and polydispersity. The method supports critical quality control and product development workflows.

Reference

• Cleaver G. Application Note: SEC Analysis of Hyaluronic Acid. Agilent Technologies, 2019. 5991-5787EN.