Fast, inexpensive, and reliable HPLC method to determine monomer fractions in poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

Abstract

The determination of the monomer fractions in polyhydroxyalkanoates is of great importance for research on microbial-produced plastic material. The development of new process designs, the validation of mathematical models, and intelligent control strategies for production depend enormously on the correctness of the analyzed monomer fractions. Most of the available detection methods focus on the determination of the monomer fractions of the homopolymer poly(3-hydroxybutyrate). Only a few can analyze the monomer content in copolymers such as poly(3-hydroxybutyrate-co-3-hydroxyvalerate), which usually require expensive measuring devices, a high preparation time or the use of environmentally harmful halogenated solvents such as chloroform or dichloromethane. This work presents a fast, simple, and inexpensive method for the analysis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with high-performance liquid chromatography. Samples from a bioreactor experiment for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with Cupriavidus necator H16 were examined regarding their monomer content using the new method and gas chromatography analysis, one of the most frequently used methods in literature. The results from our new method were validated using gas chromatography measurements and show excellent agreement.Key points∙ The presented HPLC method is an inexpensive, fast and environmentally friendly alternative to existing methods for quantification of monomeric composition of PHBV.∙ Validation with state of the art GC measurement exhibits excellent agreement over a broad range of PHBV monomer fractions.

Keywords: Bioplastic; High-pressure liquid chromatography; Polyhydroxyalkanoate; Quantitative measurement; UV/Vis spectrometry.

Fig. 1

Part of a poly(3-hydroxybuyrate-co-3-hydroxyvalerate) chain and the products after alkaline hydrolysis

Fig. 2

Chromatographic separation with different eluents. HPLC measurements were performed with an isocratic eluent: left panel, 100% H₂SO₄ (0.0025%); right panel, 92% H₂SO₄ (0.0025%) and 8% acetonitrile. Ten microliters standard mix consisting crotonic acid (0.05 g/L, early peak) and 2-pentenoic acid (0.01 g/L, later peak) were eluted for both cases

Fig. 3

Evaluation of HPLC with a standard GC method. The hydroxyalkanoate monomer (HA) amount of the total biomass (TBM) was measured from six replicates for each measurement method. A paired t-test was performed between the two groups. The p-values for 3HB and 3HV are 0.11 and 0.16, respectively

Fig. 4

HPLC/GC-correlation of 3HB (left) and 3HV measurements (right). Samples were taken between 0 and 35 h after inoculation and analyzed with HPLC and GC, respectively

Fig. 5

Measurements obtained by UV/Vis spectrometry (left) and HPLC/photodiode-array detector (right). By UV/Vis measurement crotonic (0.01 g/L, dashed lines) or 2-pentenoic acid (0.01 g/L, solid lines) can be detected with an absorbance maximum at 235 nm and 240 nm, respectively. By photodiode-array detection, both substances can be detected with an absorbance maximum at 210 nm