SEC-MALS Characterization of Microbial Polyhydroxyalkanoates

Abstract

Characterization of poly-3-hydroxybutyric acid (PHB) and poly-3-hydroxybutyric-co-valeric acid (PHBV, 13% valerate) in chloroform was performed using size exclusion chromatography coupled to a multi-angle light scattering detector (SEC-MALS). Absolute molar mass averages, molar mass distribution, and the radius of gyration were determined. Three sample preparation methods were examined: dissolution in chloroform (1) at room temperature, (2) at 60 degrees C, and (3) after thermal pretreatment of samples (annealing at 180 degrees C with subsequent quenching in liquid nitrogen). Dissolution at 60 degrees C and dissolution of thermally pretreated samples gave molecularly dissolved PHB and PHBV. At 60 degrees C using acid free chloroform, there was no indication of degradation for up to 120 min dissolution time, whereas thermal degradation of polymers did take place during annealing at 180 degrees C. The degradation rate constants for number and weight average degree of polymerization at 180 degrees C were slightly higher for PHB (5.19 x 10(-5) min(-1), 4.95 x 10(-5) min(-1)) than for PHBV (4.99 x 10(-5) min(-1), 4.54 x 10(-5) min(-1)). The dependence of the radii of gyration on molar mass showed that both polymers form random coils in chloroform. The relationship between the absolute molar masses and relative SEC results was determined. DSC and NMR characterization also gave evidence of the progress of degradation.

Influence of the thermomechanical degradation on the formation of the crystalline structure of PHBV evaluated by temperature‐resolved SAXS experiments

“…The olefinic protons in PHBV are observed in the 1 H‐NMR spectra when a higher degree of chain scission is present. [ 31 ]…”

“…The olefinic protons in PHBV are observed in the 1 H-NMR spectra when a higher degree of chain scission is present. [31] T A B L E 1 Steady-state torque (T), mechanical energy (E m ), shear rate (_ γ), viscosity (η), and viscosimetric molar mass (M v ) for PHBV at different processing conditions [2] Additionally, other structural transition was reported and can be described by two models of lamellar crystals for HV contents below and above 10 mol%, respectively: from sandwich lamella, in which the core is composed entirely of the major unit with the counit located only at the edge of the lamella, to a uniform lamella where the counits are uniformly distributed within the lamella. [32] The diffractograms in Figure 3 show that the variation of the rotation speed did not alter the crystalline structure of the polymer since no new reflections were observed in the range of 2θ from 1.5 to 40 .…”

Secondary crystallisation and degradation in P(3HB-co-3HV): an assessment of long-term stability

“…This can be ascribed to the onset of degradation which has been reported previously. 16,35 Although the temperatures adopted in these papers are greater than the 150 °C used here (190 and 180 °C respectively) it is probable that the far greater exposure times adopted in this study (up to 672 hours compared to 80 and 300 minutes) produced similar effects. The glass transition temperature could not be detected using DSC due to the low levels of amorphous material present.…”

Techniques for tracing PHA‐producing organisms and for qualitative and quantitative analysis of intra‐ and extracellular PHA

“…To overcome the restrictions of GPC in obtaining reliable data for absolute molar masses, characterization of chloroform solutions of PHB and PHBV using SEC‐MALS was performed and reported by Žagar and Kržan. Using this method, it was possible to determine absolute molar mass averages, molar mass distribution, and even the radius of gyration of the PHA helix, hence, even conformational information is provided by SEC‐MALS .…”