PHA Composition Analysis by GC-MS

GC-MS was applied for the determination of the PHA composition. Analysis of standard poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) and extracted PHA-polyester was performed using a previously reported method [8 (link)]. Briefly, 5 mg polyester was simultaneously transesterified and hydrolyzed in sealed headspace vials using 4 mL 1,2-dichloroethane, 2 mL 4:1 (vol/vol) propanol HCl- mixture, and 100 µL of 20 g L⁻¹ benzoic acid as internal standard. Propanolysis was carried out at 120 °C for 4 h. Afterward, 4 mL water was added, mixed, and maintained until phase reseparation. Then, 1000 µL of the lower (organic) phase was transferred for GC-MS analysis. Separation occurred on a Stabilwax column (Restek, Bad Homburg, Germany) using He as carrier at a flow rate of 1.44 mL min⁻¹, and a gradient of 120 °C (3 min), 140 °C at 3 °C min⁻¹, 230 °C at a heating ramp of 50 °C min⁻¹, and 240 °C at 10 °C min⁻¹. A standard curve of PHBHV (12 wt% 3HV, Sigma-Aldrich/Merck, Darmstadt, Germany) was used for the qualification of propionyl-3HB and propionyl-3HV monomers. The resulting chromatograms and mass to charge ratio (m/z) were analyzed to identify monomer composition.