High-performance liquid chromatography of amino acids, peptides and proteins. LXVII. Evaluation of bandwidth relationships of peptides related to human beta-endorphin, separated by gradient-elution reversed-phase high-performance liquid chromatography

Abstract

The bandwidths of several polypeptides related to human beta-endorphin have been investigated with different n-alkylsilica stationary phases and different elution gradients of 0.1% trifluoroacetic acid-water-acetonitrile mobile phases. In particular, we have examined the influence of changes in the gradient steepness parameter, b, on peakwidth with five different octadecylphases, chemically bonded to porous spherical silica particles of nominally 4 micron and 6 micron average particle diameter, respectively. The effect on the zone dispersion of these polypeptide solutes as the average pore diameter of the silica matrix was increased from 7.3 nm to 30 nm with stationary phases of similar ligand densities packed into columns of identical configuration has been further documented. The experimental data on solute bandwidths and peak capacities are comparable with the corresponding bandwidth and peak capacity values calculated from analytical equations, derived from the general plate height theory and from gradient elution theory. These comparisons clearly demonstrate that anomalous bandbroadening phenomena may occur when polypeptides are eluted with steep gradients, i.e. with gradients of large b values. Moreover, as the relative chromatographic residence times of beta-endorphin peptides capable of forming a C-terminal amphiphilic secondary structures is increased, i.e. as the dwell times and median capacity factors, k, for such peptides are increased, significant divergencies arise between the observed peakwidth behaviour and the behaviour predicted by analytical relationships which describe either the dependency of peak bandwidth (as 4 sigma v) on the gradient steepness parameter, b, or the dependency of peak capacity on gradient time, tG, median capacity factor, k, and the Knox parameter, C, respectively. The importance of these divergences from the predicted bandwidth and peak capacity behaviour for polypeptides separated on reversed phases, and for resolution optimisation in particular, is evaluated. These investigations thus enable further assessment of the quantitative relevance of current models that describe polypeptide zone migration under gradient elution reversed-phase chromatographic conditions in which solute-dependent slow equilibria, mediated by conformational or solvation effects, may still occur.