Asymmetrical Flow Field-Flow Fractionation in the Application to Biological Macromolecules

Abstract: Asymmetrical flow field-flow fractionation (AsFlFFF) is especially useful in the application to biological macromolecules due to the absence of a stationary phase in the separation channel. As a result no upper exclusion limit occurs and low shear forces affect the sample components. In addition, native condition, most suitable for the samples analysed, can be used since no consideration has to be taken to a stationary phase. Due to the last decade’s increased interest in biological science and biological macromolecules new separation systems, well suited for fast and cost-efficient quantitative and qualitative characterisations, are in high demand. In this thesis the suitability of AsFlFFF in the separation and characterisation of biological macromolecules was demonstrated by two biological macromolecules in need of new separation methods – the ultra large wheat protein glutenin and the ribosomal particles, involved in the protein synthesis. AsFlFFF was proven to be a fast alternative to ultracentrifugation in the separation of ribosomal particles. A correlation between cell growth and ribosomal composition was established. Using a time-minimised ribosome preparation high frequency, at-line determinations, of ribosome and tRNA levels in cell cultures during cultivation, were made possible. This resulted in the complete ribosome and tRNA profiles, as well as evidence for the 100S ribosomal particle in the exponential growth phase. The AsFlFFF method displayed its usefulness for at-line monitoring during recombinant protein production. AsFlFFF connected to multiangle light scattering (MALS) detection, in combination with refractive index (RI) detection, enabled the complete molecular weight distribution as well as the weight average molecular weight of glutenin to be determined. Molecular weights above 108 were observed and the average weight average molecular weight was (2 – 3) x 107. The AsFlFFF-MALS-RI combination was also used to illustrate the impact on the physical breakdown of the glutenin molecules when using sonication to dissolve glutenin, compared to when using gentle stirring. Finally the nonideal behaviour of ultra-large macromolecules in the AsFlFFF channel, exemplified by the glutenin, was carefully investigated and minimised.