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

- Amorphous poly(3-hydroxybutyrate) films were degraded by enzyme molecules.
- A quantitative characterization of the metabolites was performed by applying a novel, vapor pressure analysis based detection method.
- The principle of the measurement is the different equilibrium vapor pressure of the eluent and the analyte.
- If an analyte is present in eluent flow, its equilibrium vapor pressure is reduced, which results in a decreasing evaporation rate.

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.