Mercury plus nmr spectrometer

For cell biomass
and PHA extraction, the cultures were harvested and pelleted by centrifugation
at 10 000 rpm for 10 min. Cell pellets were processed for cell
dry mass and PHA content as described previously.^{32 (link)} The functional group composition of extracted PHA was studied
by FTIR. A thin pellet was prepared and scanned as described earlier.^{12 (link)}The chemical structure of PHA was analyzed
by ¹H NMR spectroscopy using a Varian Mercury Plus NMR
spectrometer. The extracted and dried PHA was dissolved in deuterated
chloroform (CDCl₃; chloroform is toxic and proper precaution
was taken during experiments) at a concentration of 10 mg/mL with
tetramethylsilane as an internal reference. The 400 MHz ¹H NMR spectrum was recorded at 10 330.578 Hz, with a pulse
width of 3.17 s, 45.2° pulse angle, recycle delay of 1 s, and
16 scans.
GC–MS was used to study the monomeric composition
of the
polymer. The composition of PHA was analyzed by methanolysis of dried
cells with a solution of 2 mL chloroform, 0.3 mL of 98% sulfuric acid,
and 1.7 mL methanol heated at 100 °C for 140 min to convert the
constituents into methyl esters. Then, the reaction mixture was allowed
to cool at room temperature followed by the addition of 1 mL water
to induce phase separation. The lower chloroform layer was used for
GC–MS analysis as described earlier.^{32 (link)}