SEC Analysis of Hyaluronic Acid, Raffinose, and Rhamnose Using Advanced Polymer Chromatography

Significance of the Topic

A reliable and rapid analysis of low molecular weight species in biopolymers such as hyaluronic acid (HA) is essential for quality control and formulation of biomedical hydrogels. Size exclusion chromatography (SEC) traditionally used for this purpose can be slow and requires extensive method development. The introduction of advanced polymer chromatography (APC) offers accelerated throughput, flexible mobile phase options, and robust separation that meet the demanding needs of medical research and pharmaceutical production.

Objectives and Study Overview

This study evaluates the performance of the Waters ACQUITY Advanced Polymer Chromatography (APC) system combined with ACQUITY UPLC Protein BEH Columns for the characterization of low molecular weight HA, using raffinose and rhamnose as reference standards. The goal was to demonstrate enhanced throughput, reduced development time, and reliable resolution of small polysaccharide species.

Methodology

HA samples and reference sugars were prepared at 2 mg/mL in a 12 mM ammonium acetate buffer (pH 6.8). Analyses were carried out at 10 °C sample temperature, 45 °C column and RI detector temperature. Flow rates of 0.4, 0.6, and 0.8 mL/min were assessed to optimize speed without compromising resolution. Detection was performed using tunable UV at 232 nm and refractive index (RI) detection. Chromatographic data were processed with Empower 3 CDS software.

Instrumentation Used

• LC system: ACQUITY APC
• Column: ACQUITY UPLC Protein BEH SEC, 125 Å, 4.6 × 150 mm
• Detectors: Tunable UV (232 nm) and Refractive Index at 45 °C
• Mobile phase: 12 mM ammonium acetate, pH 6.8
• Flow rates tested: 0.4, 0.6, 0.8 mL/min
• Software: Empower 3 CDS

Main Results and Discussion

The APC system achieved clear separation of raffinose (504 Da), rhamnose (164 Da), and low molecular weight HA fragments. Resolution proved insensitive to flow rate changes, allowing selection of 0.8 mL/min for rapid runs. UV and RI detectors provided complementary sensitivity: Figure 1 demonstrated baseline separation of the two sugar standards, and Figure 2 revealed HA peaks with a detection limit below 0.002 AU. The BEH column’s inert surface minimized sample–stationary phase interactions, and APC’s buffer flexibility enabled method optimization within hours rather than days.

Benefits and Practical Applications

• Significantly reduced equilibration and development time for SEC-based assays
• High throughput analysis suitable for QA/QC environments
• Accurate molecular weight distribution profiling of HA and related biopolymers
• Flexible mobile phase selection to accommodate diverse sample chemistries

Future Trends and Applications

Advancements in low-dispersion, multi-angle light scattering and viscometry detectors promise absolute molecular weight and conformational analysis when coupled to APC. Emerging applications include real‐time monitoring of polymer degradation, automated process control in biomanufacturing, and high-throughput screening of modified polysaccharide formulations.

Conclusion

The combination of the ACQUITY APC system and UPLC Protein BEH column provides a fast, flexible, and high-resolution platform for analyzing low molecular weight HA and related saccharides. Its robust performance and reduced method development time offer significant advantages for research and industrial applications.

References

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