Analysis of Poly [(R)-3-hydroxybutyric acid] in chloroform using GPC and universal calibration

Significance of the Topic

Poly[(R)-3-hydroxybutyric acid] (PHB) is a sustainable, biodegradable polyester whose performance hinges on accurate determination of molecular weight distributions. Precise values of number average molecular weight (Mn), weight average molecular weight (Mw) and dispersity (PDI) are critical for tailoring PHB properties in packaging, medical devices and controlled-release applications.

Objectives and Study Overview

This study evaluates gel permeation chromatography (GPC) with universal calibration in chloroform to measure Mn, Mw and PDI of two PHB standards (10 kDa and 500 kDa). The goal is to assess method reliability for low and high molecular weight PHB samples.

Methodology and Instrumentation

The analysis employed a KNAUER HPLC system with a single linear 300 × 8 mm poly(styrene-co-divinylbenzene) column at 25 °C. Calibration kits included:

• Polystyrene (PS) 16-point kit (3.7–2561 kDa),
• Polymethyl methacrylate (PMMA) 12-point kit (2380 Da–2200 kDa).

Samples were dissolved in chloroform (10 kDa overnight at room temperature; 500 kDa at 60 °C for 2 h) and filtered. Butylated hydroxytoluene (1.5 mg/mL) served as a flow marker. Chromatograms were recorded via refractive index detection (RID) at 10 Hz, flow rate 1 mL/min, injection volume 20 µL.

Instrumental Setup

• Pump: AZURA P6.1L HPG
• Autosampler: AZURA AS 6.1L
• Detector: AZURA RID 2.1L (RID)
• Column thermostat: AZURA CT 2.1
• Software: ClarityChrom 8.2.3 with SEC/GPC extension

Main Results and Discussion

For the 10 kDa PHB standard (certified Mn = 8592 Da by NMR):

• PS calibration yielded Mn = 7632 Da (–11%).
• PMMA calibration yielded Mn = 7114 Da (–17%).

These deviations are within acceptable limits. For the 500 kDa standard, both calibrations underestimated Mn by ~69 % (Mn ≈ 154 kDa) due to incomplete dissolution of high molecular weight fractions and crystallization upon cooling.

Benefits and Practical Applications

This universal calibration approach in chloroform provides reliable molecular weight data for low molecular weight PHB, which is especially relevant for drug delivery systems and biodegradable packaging where controlled polymer chain length impacts performance.

Future Trends and Applications

Advancements may include:

• Improved solvents or solvent additives to enhance solubility of high MW PHB.
• Use of multi-angle light scattering detectors for absolute molar mass determination.
• Development of universal calibration curves specific to PHB or related polyhydroxyalkanoates.

Conclusion

Universal calibration with PS and PMMA in chloroform is effective for low molecular weight PHB analysis but fails to capture high molecular weight fractions without optimized dissolution. Proper sample preparation is essential to avoid crystallization and achieve accurate molecular weight measurements.

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