RESUMO
Bacterial poly(hydroxyalkanoates) (PHAs) vary in the composition of their monomeric units. Besides saturated side-chains, unsaturated ones can also be found. The latter leads to unwanted by-products (THF ester, secondary alcohols) during acidic cleavage of the polymer backbone in the conventional analytical assays. To prevent these problems, we developed a new method for the reductive depolymerization of medium chain-length PHAs, leading to monomeric diols that can be separated and quantified by HPLC/MS. Reduction is performed at room temperature with lithium aluminum hydride within 5-15 min. The new method is faster and simpler than the previous ones and is quantitative. The results are consistent with the ones obtained by quantitative (1)H NMR.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas/métodos , Poli-Hidroxialcanoatos/análise , Álcoois/química , Compostos de Alumínio/análise , Biopolímeros/química , Cromatografia/métodos , Ésteres/química , Fermentação , Compostos de Lítio/análise , Espectroscopia de Ressonância Magnética/métodos , Modelos Químicos , Poli-Hidroxialcanoatos/química , Polímeros/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Temperatura , Fatores de TempoRESUMO
Bacterial polyhydroxyalkanoate (PHA) is an attractive biopolyester for medical applications due to its biocompatibility. However, inappropriate extraction of PHA from bacterial biomass results in contamination by pyrogenic compounds (e.g. lipopolysaccharides) and thus influences medical testing. This problem was solved by a temperature-controlled method for the recovery of poly(3-hydroxyoctanoate-co-3-hydroxyhexanaote) (PHO) from Pseudomonas putida GPo1. In contrast to other methods, precipitation of PHO was triggered by cooling the hot solution to a particular temperature. N-hexane and 2-propanol were found to be optimal solvents for such procedure. Quantitative extraction with n-hexane took place at 50 degrees C and optimal precipitation occurred between 0 and 5 degrees C. The purity was >97% (w/w) and the endotoxicity between 10 and 15 EU/g PHO. Additional re-dissolution in 2-propanol at 45 degrees C and precipitation at 10 degrees C resulted in a purity of close to 100% (w/w) and the minimal endotoxicity of 2 EU/g PHO. The polydispersity (M(w)/M(n)) of PHO was decreased from 2.0 to 1.5 for this optimized procedure.
Assuntos
Materiais Biocompatíveis/isolamento & purificação , Poliésteres/isolamento & purificação , Pseudomonas putida/metabolismo , 2-Propanol/química , Biomassa , Precipitação Química , Cromatografia Gasosa , Endotoxinas/isolamento & purificação , Peso Molecular , Oxirredução , Solubilidade , TemperaturaRESUMO
Standard chromatographic methods for the quantification of bacterial poly(3-hydroxyalkanoate) (PHA) proved to be inappropriate for the analysis of medium-chain-length PHA (mcl-PHA). Transesterification catalyzed by protic acids is not quantitative for mcl-PHA under common conditions due to slow reaction kinetics and formation of side-products in case of functionalized side-chains. To circumvent these limitations, an improved method for the quantification of mcl-PHA by GC-FID was developed. Boron trifluoride in methanol was successfully applied to quantitatively methanolyse different mcl-PHA (recovery >94%). This novel method is well-suited for the analysis of purified mcl-PHA as well as for mcl-PHA in biomass.
