Comprehensive two-dimensional field-flow fractionation-liquid chromatography in the analysis of large molecules

A novel, comprehensive two-dimensional asymmetric field-flow fractionation-liquid chromatographic system is described (AsFlFFF-RPLC). The interface is based on a switching valve, and the whole sample is analyzed in both dimensions. The system proved to be repeatable and quantitative in the characterization of egg white proteins. Four peaks at 4, 5.5-6.0, 7.5-8.0, and 10.0-11.0 nm, and corresponding to lysozyme, ovalbumin, transferrin, and a dimer of transferrin, were obtained in the AsFlFFF first-dimension system. Lysozyme also produced an additional peak, which overlapped with ovalbumin. Twelve compounds were separated in the LC second-dimension system. Identifications were made with the help of standards (ovalbumin, ovotransferrin, lysozyme) and by comparison of the peak areas, particle sizes, and retention data with values given in the literature. The effect of heat on egg white denaturation was studied, and the unfolding of peptide bonds of the protein was found to be pronounced when the sample was heated in phosphate solution.