High-performance liquid chromatography of amino acids, peptides and proteins : XL. Further studies on the role of the organic modifier in the reversed-phase high-performance liquid chromatography of polypeptides. Implications for gradient optimisation

Abstract

The retention behaviour of a variety of polypeptide hormones on octadecyl-silica columns has been investigated as the volume fraction, ϕ s, of the organic solvent modifier, acetonitrile, was systematically varied over the range 0 < ϕ s < 0.8 under low pH, low ionic strength conditions. The results are compared with data obtained in related experiments using eluents of the same ph containing 15 m M triethylamine. Under both sets of conditions non-linear dependencies of the logarithmic capacity factor, log k′, values for these polypeptides, as well as for several phenylalanine oligomers, on ϕ s, were observed. In all cases examined the plots of log k′ versus ϕ s were bimodal with selectivity reversals from a dominant reversed-phase elution mode to a polar phase elution mode occurring at ϕ s values near to 0.5 for the acetonitrile-based eluents. Within the range of k′ values of interest in isocratic or gradient optimisation of reversed-phase separations of polypeptides, namely 1 < k′ < 10 with water-rich eluents of low pH, the dependency of log k′ on ϕ s has been evaluated in terms of a linear relationship approximation. Compared to small polar molecules, the slope, the values of the solvent strength parameters ( s), and the extrapolated k′ value intercepts at ϕ s  0, namely the k′ w values, derived from this linear relationship approximation, are considerably larger with peptides and polypeptides. Furthermore, the magnitudes of the extrapolated k′ w values and the estimated s values over this restricted range of ϕ s, values essentially follow the relative hydrophobicities of the polypeptides examined. The consequences of this retention behaviour on the choice of optimal gradient elution conditions for the separation of polypeptide mixtures on alkylsilicas are examined. In particular, the relationship between s value, gradient steepness and flow-rate, and the influence these parameters have on resolution and peak capacity under linear solvent strength conditions, have been further evaluated.