Jnm excalibur 270

PHA extraction and purification was carried out according to the method reported in (Brandl et al., 1989 (link)). PHAs were extracted from 428 mg lyophilized cells using about 100 mL of chloroform (JIS Special Grade, Wako Chemicals, Osaka, Japan). The chloroform extracts were filtered and concentrated using a rotary vacuum evaporator (NA-1, AS ONE, Osaka, Japan), and the chloroform-extracted PHAs were purified by precipitation with hexane (JIS Special Grade, Wako Chemicals, Osaka, Japan) of more than 10 times the volume of the solvent. The precipitate was filtered and then air-dried without vacuum as a convenient way overnight at room temperature. The polymer solution was concentrated again using a rotary vacuum evaporator and purified by precipitation with cold methanol (JIS Special Grade, Wako Chemicals, Osaka, Japan) of more than 10 times the volume of the solvent. The purified PHA precipitate was air-dried overnight at room temperature.
The purified PHAs were analyzed by proton nuclear magnetic resonance (¹H NMR; JNM-Excalibur 270; JEOL, Ltd., Tokyo, Japan) to determine their chemical structures and compositions. The measuring frequency was 499.87 MHz. The sample for NMR analysis was dissolved at a concentration of 4 mg/mL in CDCl₃ with 0.05% (v/v) tetramethylsilane (TMS; Wako Pure Chemical Industries Ltd., Osaka, Japan).

The purified PHAs were analyzed by proton nuclear magnetic resonance (¹H NMR) (JNM-Excalibur 270; JEOL, Ltd., Tokyo, Japan) to determine their chemical structures and compositions. The sample for NMR analysis was dissolved in CDCl₃ with 0.05% (v/v) tetramethylsilane (TMS) (Wako Pure Chemical Industries Ltd., Osaka, Japan) at concentration of 4 mg/mL. The molecular weight of the PHAs was determined by gel-permeation chromatography (GPC) (RI-2031, PU-2086, AS-2055, CO-2056; JASCO, Tokyo, Japan) using Shodex K-806M, K802 and K-G columns at 40°C, according to a previously described method [46 (link)]. Chloroform was used as the mobile phase at a flow rate of 0.8 mL/min. The concentration of the purified PHAs was approximately 1 mg/mL. The molecular weight and polydispersity index (PDI) of the PHAs were estimated by comparison with polystyrene standards of the following molecular weights: 1.32 × 10³, 3.25 × 10³, 1.01 × 10⁴, 2.85 × 10⁴, 6.60 × 10⁴, 1.56 × 10⁵, 4.60 × 10⁵, 1.07 × 10⁶, and 3.15 × 10⁶ g/mol, respectively.