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Anal Biochem. 2017 Mar 15;521:20-27. doi: 10.1016/j.ab.2017.01.004. Epub 2017 Jan 6.

The novel technique of vapor pressure analysis to monitor the enzymatic degradation of PHB by HPLC chromatography.

Author information

1
Laboratory of Plastics and Rubber Technology, Department of Physical Chemistry and Materials Science, Budapest University of Technology and Economics, H-1521, Budapest, P.O. Box 91, Hungary; Institute of Materials and Environmental Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences, H-1519, Budapest, P.O. Box 286, Hungary.
2
Department of Applied Biotechnology and Food Science, Budapest University of Technology and Economics, H-1521, Budapest, P.O. Box 91, Hungary; Laboratory of Genome Metabolism and Repair, Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, H-1519, Budapest, P.O. Box 286, Hungary.
3
Laboratory of Plastics and Rubber Technology, Department of Physical Chemistry and Materials Science, Budapest University of Technology and Economics, H-1521, Budapest, P.O. Box 91, Hungary; Institute of Materials and Environmental Chemistry, Research Centre for Natural Sciences, Hungarian Academy of Sciences, H-1519, Budapest, P.O. Box 286, Hungary. Electronic address: bpukanszky@mail.bme.hu.

Abstract

A novel method was introduced for the quantitative determination of substances in aqueous solutions by using the evaporative light scattering (ELS) detector of a high performance liquid chromatograph (HPLC). The principle of the measurement is the different equilibrium vapor pressure of the solvent and the analyte resulting in decreasing evaporation rate, larger droplets and stronger signal with increasing concentration. The new technique based on vapor pressure analysis was validated with traditional UV-Vis detection carried out with a diode array detector (DAD). The new technique was used for monitoring the concentration of solutions obtained during the enzymatic degradation of poly(3-hydroxybutyrate) yielding the 3-hydroxybutyrate monomer as the product. The accuracy of the measurement allowed the determination of degradation kinetics as well. The results obtained with the two techniques showed excellent agreement at small concentrations. Deviations at larger concentrations were explained with the non-linear correlation between analyte concentration and detector signal and the linear regression used for calibration. Mathematical analysis of the method made possible the determination of the evaporation enthalpy of the analyte as well. The new approach is especially suitable for the quantitative analysis of compounds, which do not absorb in the detection range of the DAD detector or if their characteristic absorbance is close to the lower end of its wavelength range.

KEYWORDS:

Degradation kinetics; Enzymatic degradation; Liquid chromatography; Poly(3-hydroxybutyrate); Vapor pressure analysis

PMID:
28069452
DOI:
10.1016/j.ab.2017.01.004
[Indexed for MEDLINE]

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