Accurate molecular weight analysis of polyhydroxybutyrate (phb) a bio-derived polyester, by triple detection GPC/SEC

Significance of the Topic

Poly-3-hydroxybutyrate (PHB) is a bio-derived polyester with widespread applications in biodegradable plastics, medical implants, drug delivery, and tissue engineering. Its mechanical and physicochemical properties depend critically on molecular weight and molecular weight distribution, making accurate Mw determination essential for quality control and material performance optimization.

Objectives and Study Overview

This study aimed to evaluate the accuracy of molecular weight analysis of PHB by comparing conventional single-detector GPC/SEC calibration with a triple-detection approach combining refractive index, viscometry, and light scattering. The goals were to determine true molecular weight values independent of calibration standards and to extract additional structural parameters such as polymer conformation.

Instrumentation Used

• Agilent 1260 Infinity Quaternary Pump (G1311B)
• Agilent 1260 Infinity High Performance Autosampler (G1367E)
• Agilent 1260 Infinity Thermostatted Column Compartment (G1316A)
• Agilent 1260 Infinity GPC/SEC Multi-Detector Suite (G7800A)
• MDS Viscometer Detector (Option 032)
• MDS Light Scattering Detector (Option 033)
• MDS Refractive Index Detector (Option 031)

Methodology

Mobile phase: chloroform
Column set: two PLgel 10 µm Mixed-B 7.5 × 300 mm columns
Calibration: conventional calibration with polystyrene standards and triple-detection calibration based on direct concentration and scattering measurements
Operating conditions: temperature 50 °C, flow rate 1.0 mL/min, injection volume 100 µL
Data analysis: Agilent GPC/SEC software for molecular weight distribution, Mark–Houwink plots, and branching analysis

Key Results and Discussion

• Conventional GPC/SEC using polystyrene calibration underestimated the weight-average molecular weight (Mw) of PHB by approximately 25–50 %.
• Triple-detection analysis yielded Mw values (sample 1: 229 kDa; sample 2: 24 kDa) that matched expected ranges (200–300 kDa for sample 1).
• Mark–Houwink plots from viscometry data were linear, confirming the predominantly linear polymer structure.
• Light scattering and viscometry detectors provided robust signals for PHB, improving confidence in absolute molecular weight determination.

Advantages and Practical Applications

• Absolute molecular weight determination independent of calibration standards.
• Simultaneous access to intrinsic viscosity and branching information.
• Enhanced method reliability for QC in biopolymer production and biomedical device manufacturing.
• Applicability to a wide range of synthetic and natural polymers.

Future Trends and Potential Applications

The integration of multi-detector GPC/SEC with advanced light scattering technologies and hyphenated mass spectrometry techniques is expected to further refine polymer characterization. Emerging applications include real-time monitoring of polymerization processes and detailed structure–property correlations in novel bio-based materials.

Conclusion

Triple-detection GPC/SEC represents a powerful tool for accurate molecular weight analysis of PHB, overcoming limitations of conventional calibration methods. By combining refractive index, viscometry, and light scattering, researchers can obtain true molecular weight values and structural insights necessary for advanced biopolymer applications.

References

1. Lemoigne J. Discovery of poly-3-hydroxybutyrate in bacteria. Biochem J. 1925;19(3):259–71.
2. Dawes EA, Senior PJ. Accumulation of PHB by bacteria under nutrient limitation. Appl Microbiol. 1973;26(5):688–94.
3. Agilent Technologies. A guide to Multi-Detector Gel Permeation Chromatography. Primer 5990-7196EN. 2014.