Separation of peptides from myoglobin enzymatic digests by RPLC. Influence of the mobile-phase composition and the pressure on the retention and separation.

The influence of the mobile-phase composition and the pressure on the chromatographic separation of the peptides from the enzymatic digest of myoglobin was studied under linear conditions. The retention behavior of these tryptic peptides was measured under isocratic conditions with different mobile-phase compositions, ranging from 9 to 28% (v/v) acetonitrile in 0.1% (v/v) aqueous trifluoroacetic acid. The effect of the pressure was studied by analyzing the separation of the tryptic peptides under different average column pressures between 14 and 220 bar, at 13, 20, and 26% (v/v) acetonitrile. The differences between the partial molar volumes of these peptides in the stationary and mobile phases were derived from these results. All the measurements were performed on a 10-cm-long C18-bonded, end-capped monolithic column. The results obtained illustrate the highly complicated behavior of the complex peptide mixtures afforded by tryptic digestion. The capacity factors of the analyzed peptides do not depend linearly on the acetonitrile concentration but follow exactly a quadratic relationship. The adsorption changes of partial molar volumes are in good agreement with other literature data. The consequences of the influence of the average column pressure (hence of the flow rate) on the column phase ratio and on the retention factors of the peptides are discussed. The retention pattern of the complex mixture is affected by both the mobile-phase composition and the pressure, and the resolution of certain peptide pairs is so much affected by the pressure that inversions in the elution order of some pairs are observed.