SEC Analysis of Chitosan

Significance of the Topic

Chitosan is a versatile cationic polysaccharide derived from the alkaline deacetylation of chitin, one of the most abundant natural biopolymers. Its positive charge in acidic environments gives it distinctive functional properties compared to neutral or negatively charged polysaccharides. This makes chitosan attractive for applications in biomedical fields (e.g., wound healing, hemostatic agents), industrial processes (paper, textile finishes), and environmental remediation (heavy metal chelation).

Objectives and Study Overview

This application note aims to demonstrate the effective use of aqueous size-exclusion chromatography (SEC/GPC) for the determination of molecular weight (MW) and molecular weight distribution (MWD) of three different grades of chitosan. The method provides a reliable quality control tool to characterize polymer samples over a broad MW range.

Methodology

• Sample Preparation: Three chitosan samples were dissolved in strong acid (0.5 M NaNO3 + 0.01 M NaH2PO4, pH 2) and allowed to stand overnight to ensure complete dissolution.
• Chromatographic Conditions: Separation was performed at a flow rate of 1.0 mL/min using two Agilent PL aquagel-OH MIXED-H 8 µm columns (300 × 7.5 mm).
• Detection and Calibration: Refractive index detection was used, and the system was calibrated with narrow pullulan standards covering MWs up to 10 000 000 Da.

Used Instrumentation

• Agilent 1260 Infinity GPC/SEC System
• PL aquagel-OH MIXED-H 8 µm columns (2 × 300 × 7.5 mm, part number PL1149-6800)

Main Results and Discussion

The analysis yielded weight-average molecular weights (Mw) of approximately 367 600, 385 800, and 449 350 Da for samples A, B, and C, respectively. Chromatograms indicated slight imbalances in peak shapes, attributed to sample preparation in strong acid. The method effectively resolved differences in MWD, revealing sample polydispersity and distribution profiles.

Practical Benefits and Applications

This SEC method offers a robust quality control approach for chitosan production, enabling batch-to-batch consistency checks and detailed profiling of polymer characteristics. Such data support the optimization of biomedical formulations, industrial processing, and environmental applications.

Future Trends and Opportunities

Advances may include coupling SEC with multi-angle light scattering (MALS) for absolute MW determination, integrating mass spectrometry for end-group analysis, and implementing process analytical technology (PAT) for real-time monitoring. Expanding the method to other polysaccharides and biopolymers could further enhance industrial and research capabilities.

Conclusion

Aqueous SEC using PL aquagel-OH MIXED-H columns on an Agilent 1260 Infinity system provides a reliable, repeatable method for determining the molecular weight and distribution of chitosan. The approach combines high resolution with robust column stability, making it suitable for routine quality control in diverse applications.

References

• Graham Cleaver. SEC Analysis of Chitosan. Agilent Technologies Application Note, 2015.