RESUMO
A new kinetic method is proposed for the simultaneous determination of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) based on the different rate of the 3-hydroxybutyrate dehydrogenase-catalysed reactions of these compounds with coenzyme NAD(+). A flow injection system with two reactors of immobilised 3-hydroxybutyrate dehydrogenase and dual detection is used. The concentrations of NADH produced after two different reaction times are measured by fluorometry or spectrophotometry and multivariate linear calibration is applied for quantification. Concentrations of 3HB and 3HV between 1x10(-6) and 1x10(-4)M can be determined at an average sampling frequency of 20h(-1). In contrast to usual methods, the proposed here makes possible the discrimination of 3HB and 3HV without previous separation so that usual extraction with chlorinated solvents and/or chromatographic separation is not required. The method is of interest in a wide variety of fields concerning PHAs, as it can provide information on the degradation rate and mechanism, composition and structure of these polymers. Its applicability has been proved through the determination of 3HB and 3HV in the digests of some chemically degraded commercial PHAs.
Assuntos
Ácido 3-Hidroxibutírico/análise , Ácido 3-Hidroxibutírico/metabolismo , Ácidos Pentanoicos/análise , Ácidos Pentanoicos/metabolismo , Poli-Hidroxialcanoatos/metabolismo , Calibragem , Enzimas Imobilizadas/metabolismo , Análise de Injeção de Fluxo , Hidroxibutirato Desidrogenase/metabolismo , Cinética , Modelos Lineares , Análise Multivariada , NAD/metabolismo , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
Acetone and diazotized anthranilic acid react in alkaline solution, giving a fluorescent intermediate that can be measured at excitation and emission wavelengths of 305 and 395 nm, respectively. Based on this, a fluorimetric flow-injection method is proposed for the determination of acetone in aqueous solution. Under the proposed conditions, acetone can be detected at concentrations higher than 8 x 10(-7)M, with a linear application range from 1 x 10(-6) to 2 x 10(-4)M and an R.S.D. of 2.7% (1.0 x 10(-5)M, n=10). A sampling frequency of 24h(-1) is achieved. Some potentially interfering species are investigated.
Assuntos
Acetona/análise , Acetona/química , Compostos Azo/química , Análise de Injeção de Fluxo/métodos , Espectrometria de Fluorescência/métodos , ortoaminobenzoatos/química , Álcalis , Análise de Injeção de Fluxo/instrumentação , Concentração de Íons de Hidrogênio , Hidróxido de Sódio , Soluções , Espectrometria de Fluorescência/instrumentação , Temperatura , Fatores de TempoRESUMO
A flow injection enzymatic method for the spectrophotometric determination of l-phenylalanine has been developed. l-phenylalanine is deaminated in the presence of l-amino acid oxidase and the keto acid formed is made to react with borate to give a coloured enol-borate complex that can be detected at 282 nm. Catalase is added to the catalyzed reaction to prevent the keto acid being destroyed by the hydrogen peroxide generated. Kinetic determinations are performed by measuring the change in absorbance between 2 and 4 min. The proposed procedure, involving both merging-zones and stopped-flow techniques, can be applied to the quantitation of l-phenylalanine between 10 and 260 mg l(-1). Detection limit and R.S.D. are 1.1 mg l(-1) and 3.0%, respectively.
