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

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Abstract

The gradient retention behaviour in reversed-phase high-performance liquid chromatography of a series of polypeptides related to human β-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 μm and 6 μm. Decreasing the pore size from 30 nm to 7.3 nm resulted in decreased S and log k0 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 bandbroadening was observed for very steep and very shallow gradients, i.e. b > 0.7 or b < 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 peptides.

Original languageEnglish
Pages (from-to)47-70
Number of pages24
JournalJournal of Chromatography A
Volume397
Issue numberC
DOIs
Publication statusPublished - 26 Jun 1987

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