Elite 624 column

GC/MS analysis was used to determine the concentration of 3-HB in the supernatant. 0.1–1 mL culture supernatant was filtered through a 0.4 μm syringe filter and lyophilized for 24 h. The dry supernatants were subjected to propanolysis as described by Riis and Mai (1988 (link)) but with modification. 1.5 mL 1,2-DCE and 1.5 mL n-propanol-HCl solution (4:1 v/v) was added to lyophilizate and incubated at 90 °C for 4 h. After cooling to room temperature the reaction mixture was extracted twice with triple distilled H₂O and the lower organic phase was taken for analysis in a Clarus^® 680 GC combined with Clarus^® SQ 8 S MS (PerkinElmer, USA) equipped with Elite-624 column (PerkinElmer, 30 m × 0.5 mm, 1.4 μm). The GC program was set up as follows: initial 60 °C for 5 min, ramping at 10 °C min⁻¹ to 200 °C and held for 2 min and then ramping at 5 °C min⁻¹ to 235 °C and held for 10 min.
Commercial (R)-3-HB (Sigma-Aldrich) was used to construct a standard curve. The analysis was conducted in triplicate and results were analyzed by TurboMass 6.1 software.

Gas chromatography/mass spectrometry analysis was used to determine the concentration of PHA in dried cells. 3–4 mg of lyophilized cells was subjected to propanolysis as described by Riis and Mai [34 (link)] but with modification. 1 mL 1,2-DCE and 1 mL n-propanol-HCl solution (4:1 v/v) was added to cells and incubated at 90 °C for 4 h. After cooling to room temperature the reaction mixture was extracted with 2 mL of triple distilled H₂O and the lower organic phase was taken for analysis in a Clarus^® 680 GC combined with Clarus^® SQ 8 S MS (PerkinElmer, USA) equipped with Elite-624 column (PerkinElmer, 30 m × 0.5 mm, 1.4 µm). The GC program was set up as follows: initial temperature 80 °C for 5 min, ramping at 10 °C min⁻¹ to 235 °C and held for 10 min.
Commercial PHB-V (Sigma-Aldrich, 12%mol of PHV) was used to construct a standard curve. The analysis was conducted in triplicate and results were analyzed by TurboMass 6.1 software.