High-performance liquid chromatography of amino acids, peptides and proteins. LXXIII. Investigations on the relationships between molecular structure, retention and band-broadening properties of polypeptides separated by reversed-phase high-performance liquid chromatography.

The gradient retention behaviour in reversed-phase high-performance liquid chromatography of a series of polypeptides related to human beta-endorphin has been investigated using different n-alkylsilica stationary phases and 0.1% trifluoroacetic acid in water-acetonitrile as mobile phase. In particular, the influence of changes in gradient time and flow-rate on retention parameters has been assessed with five different porous octadecylsilica phases with average particle diameters of 4 microns and 6 microns. Decreasing the pore size from 30 nm to 7.3 nm resulted in decreased S and log k'o values for most solutes. The effect of changes in the gradient steepness parameter, b, on the bandwidth behaviour of these polypeptides has also been investigated. Anomalous band-broadening was observed for very steep and very shallow gradients, i.e. b greater than 0.7 or b less than 0.2. The significance of these results is discussed in relation to the presence of hydrophobic domains in the linear sequence, the probability of the formation of highly stabilised secondary structures and the possible involvement of multiple folded forms of a single solute in the chromatographic performance of these polypeptides.