Isolation of denatured proteins and peptides by high-performance liquid chromatography. Effect of different perfluorinated acids, column length and large-pore supports.

The utilization of high-performance liquid chromatography for the isolation of peptides and proteins is extended by the demonstration that trifluoroacetic acid, pentafluoropropionic acid and heptafluorobutyric acid are effective peptide and protein solvents and as components of a mobile phase alter both the absolute and relative retention times of eluting peptides. In addition, studies related to stationary-phase performance indicate that: (1) column length (5-25 cm) does not significantly influence large peptide or protein retention times or resolution; (2) 300 A pore supports are ideal for the isolation of both large peptides and proteins; (3) peptide and protein conformation, even in the presence of denaturants, alters the steric interaction with the support; and (4) large peptides and proteins probably interact with the support by virtue of multi-site binding, not partitioning between the stationary and mobile phases.