Several of the components that participate in this reaction including soy oil, soy biodiesel, glycerol, water and methanol were sampled separately using H LF-NMR. Data was analyzed using the WinDXP ILT software package, and the combined relaxation time distributions are shown in Figure 5.
100 µl Methanol was added, samples were centrifuged (12,300 g, 10 min) and analyzed by HPLC.
Analytical grade acetic acid from Merck India was used in the mobile phase whereas HPLC grade methanol was used for sample preparation.
Then, 1150 μL of ice-cold methanol was added and samples were incubated for 2 h at −20 °C.
For samples containing FBS, 2 v methanol was added and the samples were held at −20°C to precipitate proteins before centrifugation, concentrated to ca 170 μl in a centrifugal concentrator (Savant Instruments, Farmingdale, NY, USA), centrifuged again and assayed immediately.
The sample was evaporated to dryness to ensure that all methanol was removed from the sample before it was redissolved in 0.5 mL n-hexane.
Each sample was made in triplicate. 1 mL of 0.5 M KOH in anhydrous methanol was added to each sample, which was shaken well and incubated for 30 min at 70°C.
Blank wells were treated similarly except 100 μl of methanol was used instead of sample, and the absorbances of the blank wells were subtracted from those of the other wells.
To this end, uniformly 13C-labeled cell extract from M. extorquens AM1 cells grown on methanol was added to the sample as internal standard when starting metabolite extraction.
Internal standard (heneicosanoic acid, 5 μg in methanol) was added to the samples before lipid extraction.
