Influence of mobile phase gradients on the retention and separation of peptides from a cytochrome-c digest by reversed-phase liquid chromatography

The retention data of the components of the peptide mixture obtained by the enzymatic digestion of cytochrome-c were measured under different gradient conditions, using different organic modifiers (acetonitrile, methanol, and tetrahydrofuran) in a 0.1% (v/v) aqueous solution of trifluoroacetic acid. The influences of the nature of the organic modifier, of the gradient steepness, and of the column length on the retention and the resolution of these peptides were studied. Peak capacity and retention factor calculations for gradient separations were helpful in determining a desired gradient for this separation. All three organic modifiers are shown to cause reversal orders of some peaks in the cytochrome-c digest separation.