Browsing by Subject "field-flow fractionation"

The aim of the literature review was to examine barley’s (Hordeum vulgare) alcohol-soluble proteins – hordeins and their technological attributes. Additionally, applicability of flow field flow fractionation (FFF) separation method as well as spectrophotometric and light scattering methods for protein characterization was under investigation. The objective of the experimental research was to determine a suitable extraction method for hordeins and subsequent analysis of their molecular weight distribution, size and conformation by the use of AF4 (asymmetric flow field flow fractionation) in combination with MALS-, UV- and RI-detectors. 40 % 1 propanol combined with mild sonication treatment proved to be the most efficient method to extract hordeins from barley flour. In order to prevent deterioration of the FFF channels the solvent had to be diluted to 20 %. Same dilution was used to measure hordeins’ extinction coefficient and to calculate ?n/?c theoretically. Berry plot was found to be the most suitable fit for the data analysis. Extracted hordeins were analysed with SDS PAGE. Extracts contained monomeric C, B and ? hordeins and polymeric B, D and ? hordeins. Also, small amounts of albumins, globulins and hydrolysed proteins were present. Extracts’ fractograms had five distinctive peaks. All of the peaks’ mass fractals and polydispersity indices were above 1, which means analysed aggregates were polydisperse and shaped as complex rods. This can be explained by 1 propanol influenced protein aggregation. Some inference in light scattering was identified in the MALS detector signal. This and the use of measured extinction coefficient and calculated index of refraction caused some errors in the data. The low sample yield (19–26 %) can be explained by the hordeins’ adhering to a syringe filter and adsorbing to the surface of AF4’s ultrafiltration membrane. Also, the use of over simplifying mathematical model to calculate the results and yield could cause some errors in the results. This study showed that it is possible to study Mw, size and conformation of polymeric hordeins with AF4 combined with MALS/UV-detectors and that hordeins form big aggregates in 20 % 1 propanol. For MALS proteins should be extracted in a solvent that will not interfere with subsequent analysis and proteins net charge, which creates a challenge to find proper solvent for hordeins. Nevertheless AF4 proved to be useful and delicate tool for characterizing cereal polymeric proteins.