Compositional analysis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by pyrolysis-gas chromatography in the presence of organic alkali

Rapid and Direct Compositional Analysis of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in Whole Bacterial Cells by Thermally Assisted Hydrolysis and Methylation-Gas Chromatography

“…20 Among these products, the peak 8 (methyl 3-methoxybutanoate) and peak 9 (methyl 3-methoxypentanoate) were formed from 3HB and 3HV units, respectively, through the typical THM reaction while the other products with olefinic structures were considered to be generated by cis-elimination reaction of the ester linkages followed by the THM reaction at the adjacent ester bond. 17 Similarly to the case for the P(3HB-co-3HV) sample, the chromatogram of the C. necator sample (b) clearly showed the nine characteristic peaks derived from 3HB and 3HV units in the polymer chains. Although peaks 3 and 4 were slightly overlapped with those derived from the bacterial matrix components, such as lipids and proteins, the interference was not appriciable so that the sufficient reproducibility of the relative intensities for these nine peaks were observed (less than 10% of the relative standard deviation (RSD)).…”

“…20 mg were performed according to the procedure reported by Braunegg et al 10 The methyl esters of 3HB and 3HV, prepared through transmethylation with methanol containing 10% H2SO4 at 100 C for 3.5 h, were extracted with water, and then analyzed by an ordinary GC system under the same chromatographic conditions as for the THM-GC measurements. Figure 2 shows typical chromatograms of (a) P(3HB-co-3HV) and (b) intact C. necator cells cultured in the medium, in which the ratio of valeric acid to glucose (V/G) was set at 0.125, obtained by THM-GC in the presence of TMAH at 400 C. As was reported in our previous paper, 17 on the chromatogram of P(3HB-co-3HV) (a), a series of peaks attributed to the products from 3HB unit (peaks 1 -3, and 8) and 3HV unit (peaks 4 -7, and 9) were clearly observed after the elution of the reagentrelated products. Table 1 summarizes the assignment of these characteristic peaks together with their molecular structures, origins and effective carbon numbers (ECN) corresponding to the relative molar sensitivities for FID.…”

“…17 A vertical microfurnance pyrolyzer (PY-2020iD, Frontier Laboratories, Koriyama, Japan) was attached to a GC (GC2010 Plus, Shimadzu, Kyoto, Japan) equipped with a flame ionization detector (FID). A small platinum samples cup (2 mm i.d.…”

“…This phenomenon might be explained by the toxic behavior of the increased amount of valeric acid to C. necator, which leads to the inhibition of biosynthesis of 3HV units and/or the change in the metabolic pathway of the bacteria. As proposed in our previous paper, 17 the total molar yields for 3HB unit (Y3HB) and 3HV unit (Y3HV) in the chromatograms are calculated from the intensities of the nine characteristic products as follows: Table 1 for the peak assignment. Table 1 for the peak assignment.…”

“…0.1 mg or less) of isolated P(3HB-co-3HV). 17 It was revealed that P(3HB-co-3HV) subjected to pyrolysis in the presence of TMAH around 400 C underwent not only the typical THM reaction, but also cis-elimination of the ester linkages, followed by hydrolysis and methylation of the adjacent ester bond. Based on the peak intensities of the products formed from these reactions, the copolymer compositions were analyzed precisely and accurately without using any cumbersome pretreatment.…”