Separation of large denatured peptides by reverse phase high performance liquid chromatography. Trifluoroacetic acid as a peptide solvent.

A method employing reverse phase high performance liquid chromatography has been developed for the purification of large denatured peptides. The cyanogen bromide fragments of hemoglobin alpha, beta, and gamma chains (13 to 91 residues in length) could be separated and recovered in 71 to 95% yields using LiChrosorb C-8 columns and a binary solvent system consisting of 0.013 M trifluoroacetic acid in water and a limiting organic solvent. This procedure permits the nondestructive detection of peptides at low wavelength (210 nm) on as little as 50 ng of sample. The peptides are recovered directly by evaporation or lyophilization. An elutropic solvent series for the separation of large and small peptides on reverse phase supports is also described.