High-performance liquid chromatography of amino acids, peptides and proteins. LXV. Studies on the optimisation of the reversed-phase gradient elution of polypeptides: evaluation of retention relationships with β-endorphin-related polypeptides

The gradient retention behaviour of 29 polypeptides related to β-endorphin has been investigated using a 15-nm pore diameter octadecylsilica stationary phase and 0.1% trifluoroacetic acid in water-acetonitrile as mobile phases. In particular, this study has examined the influence of changes in gradient time and flow-rate on relative retention, resolution, and bandwidth. The utility of quantitative expressions derived from linear solvent strength gradient theory has been further assessed. Evaluation of capacity factor (k′) dependencies on the mole fraction of organic solvent (φ{symbol}) has been used to determine the various solute parameters, including the S and log k′_o values from gradient data. Methods to adjust band-spacing via changes in gradient steepness parameters are documented. Peak capacities for various polypeptides have also been determined under different gradient conditions. The results demonstrate that solute-specific variation in peak capacity can arise, presumably due to anomalous band-broadening changes, even with small peptides under low pH gradient elution conditions with n-alkylsilicas. The determination of solute selectivity parameters (τ) as an adjunct to the characterisation of structural group effects with peptide analogues is examined. This information appears particularly useful for the evaluation in physicochemical terms of solute-solvent and solute-stationary phase interactions as well as other distribution processes associated with polypeptide retention to n-alkylsilicas.