Characterization of wide-pore reversed-phase columns for biopolymer separations. I. Single-parametric evaluation

Nine prepacked wide-pore columns recommended for protein separation were studied. The stationary phases investigated were widely different with respect to the type of ligands. In order to characterize the columns, measurements were made with six different test mixtures containing two different homologous series, substituted benzenes and amino acids, peptides and proteins. The isopotential eluent compositions obtained for the homologous series revealed the polar-apolar nature of the stationary phases. The relative hydrophobicity calculated for the individual test compounds provided more thorough but still partial characterization. The involvement of substituted benzenes resulted in the most complete description. The characterization with amino acids, peptides and proteins furnished results similar to those obtained for smaller test compounds having low or intermediate polarity. The examination of the behaviour of proteins revealed that the retention is determined by the hydrophobicity of the stationary phases and the other characteristics affect the selectivity