Pyrolysis gas chromatography of poly(beta-hydroxyalkanoates) and gas chromatography-mass spectrometry of 1,3-dioxaphospholanes

Gas chromatography (GC) was investigated for analysis of bacterially produced poly($\beta$-hydroxyalkanoate) (PHA) and 1,3-dioxaphospholanes. Pyrolysis-gas chromatography (Py-GC) was investigated as a method of analysis for an enteral food product (Vivonex T.E.N.) and PHAs. A Redox Chemiluminescence Detector (RCD) for gas chromatography was evaluated as a selective detector. Poly($\beta$-hydroxyalkanoate) investigations included mass spectrometry (MS) and Fourier transform infrared (FTIR) in conjunction with gas chromatography (Py-GC/MS, Py-GC/IR) to provide additional information in compound characterization. Poly($\beta$-hydroxyalkanoates) investigated were poly($\beta$-hydroxybutyrate) (PHB), copolymer of $\beta$-hydroxybutyrate and $\beta$-hydroxyvalerate, P(HB-co-HV), and PHA containing functionality in the pendant group. The major pyrolyzates are unsaturated carboxylic compounds related to the monomer, dimer, trimer and tetramer units of the polymer. The pyrolyzate from the HB monomer unit is crotonic acid and for the HV unit is 2-pentenoic acid. The pyrolyzates can be chromatographed on normal non-polar column stationary phases with poor chromatographic peak shape or better peak shape can be obtained using acid deactivated stationary phases. Investigations were made into using Py-GC on whole cells as a method to monitor the polymer content without prior isolation of the polymer from the cell. The pyrolyzates were also derivatized on-line using trimethylanilinium hydroxide (TMAH) as the methylation reagent to obtain compounds which exhibit better chromatographic behavior. This reagent allows the methyl esters to be formed in the presence of heat, either from the pyrolysis or from the injection port of the gas chromatograph. This procedure allows up to tetramer units of PHB to be eluted from a non-polar column. Two bacteria strains, Alcaligenes eutrophus and Rhodobacter sphaeroides, were grown in the presence of acetate-$d\sb3$ and D$\sb2$O producing PHB containing deuterium. The PHB samples were analyzed by Py-GC/MS and FTIR to determine the level of deuteration, showing a significant level of deuterium incorporation. Synthesized 1,3-dioxaphospholane and 1,3-oxathiaphospholane compounds were separated into their stereoisomers by GC with a mid-polarity stationary phase (cyanopropylphenyl-methyl silicone). Mass spectrometry was used as the GC detector to assist in structure confirmation