GPC/SEC-MALLS Analysis of Hyaluronic Acid

Significance of the Topic

Hyaluronic acid (HA) is a high-molecular-weight polysaccharide widely used in medical, pharmaceutical and cosmetic applications. Accurate determination of its molar mass distribution is critical for quality control, product performance and regulatory compliance. Conventional gel permeation/size exclusion chromatography (GPC/SEC) with concentration detectors yields only relative molar masses, potentially leading to misinterpretation of polymer properties.

Objectives and Study Overview

This study aims to demonstrate a robust GPC/SEC method coupled with multi-angle light scattering (MALLS) and refractive index (RI) detection to obtain absolute molar masses of hyaluronic acid. Key goals include optimizing chromatographic conditions for high-molecular-weight samples and comparing results with conventional pullulan calibration.

Methodology and Instrumentation

The analysis employed Agilent SUPREMA columns with 10 µm particles and large pore sizes to minimize shear and interaction effects. Chromatographic conditions were:

• Mobile phase: phosphate buffered saline (PBS), pH 7.4
• Flow rate: 0.5 mL/min
• Injection volume: 100 µL; sample concentration: 0.5 mg/mL
• Columns: Agilent SUPREMA precolumn (8×50 mm), 30 Å (8×300 mm), and two 10 000 Å columns (8×300 mm)
• Detectors: RI detector and MALLS at λ = 638 nm; online dn/dc set at 0.165
• Software: Agilent WinGPC for data acquisition and analysis

Main Results and Discussion

Elugram data revealed complete sample recovery and a declining molar mass with elution volume, confirming size-based separation. Comparison of molecular weight averages showed:

• Pullulan calibration (apparent): Mw ≈ 3.82 MDa, Mn ≈ 1.22 MDa, Mp ≈ 1.40 MDa
• MALLS (absolute): Mw ≈ 1.20 MDa, Mn ≈ 0.93 MDa, Mp ≈ 0.93 MDa

The conventional pullulan-based calibration overestimated the weight-average molar mass by approximately threefold due to differences in hydrodynamic size between pullulan and HA chains.

Benefits and Practical Applications of the Method

The combined GPC/SEC-MALLS approach delivers true absolute molar masses, enhancing reliability in polymer characterization. Using large-pore, large-particle columns avoids shear degradation and assures interaction-free separations. This method supports quality control in biopolymer manufacturing, formulation development and regulatory reporting.

Future Trends and Opportunities

Advancements may include:

• Integration of inline dn/dc measurement for automated data processing
• Application to other high-molecular-weight biopolymers and complex formulations
• Development of microfluidic SEC platforms for high-throughput screening
• Enhanced detector sensitivity for low-concentration samples

Conclusion

GPC/SEC-MALLS using optimized SUPREMA columns provides accurate, absolute molar mass distributions of hyaluronic acid. This technique overcomes the limitations of conventional calibration, ensuring robust and reliable polymer characterization.

Reference

1. Gupta RC et al. Veterinary Pharmacology and Toxicology, Vol. 6, 2019.
2. Lavrenko PN, Linow K‐J, Gornitz E. Analytical Ultracentrifugation in Biochemistry and Polymer Science; Royal Society of Chemistry, 1992; pp 517–531.