Effect of mobile phase additives on peptide retention in reversed-phase chromatography with pellicular and totally porous sorbents

The effect of two mobile phase additives, trifluoroacetic acid and phosphoric acid, on the energetics of peptide retention in reversed-phase chromatography was investigated using Hy-Tach C-18 micropellicular and Vydac C-4 and C-18 totally porous stationary phases. The effect of the relatively low phase ratio of columns packed with micropellicular sorbents was also examined. The logarithmic retention factors, of two model peptides, Ac-RGGGGLGLGK-amide and Ac-RGAGGLGLGK-amide, were evaluated with different columns and additives in a practical range of eluent strength. The dependence of the logarithmic retention factor on the concentration of acetonitrile in the mobile phase was linear in all cases. The higher sensitivity of the retention to the organic modifier concentration in the case of the Hy-Tach C-18 column is attributed to the relatively low phase ratio of this column. Pairwise plots of the logarithmic retention factors were linear. The plots of data obtained with the two additives has unit slopes and thus reveal homoenergetic retention behavior. On the other hand data obtained on two different columns manifest homeoenergetic retention, the slopes of plots are different from unity. The analysis has yielded consistent results and validated the assumption that the retention free energy can be divided into two components arising from mobile phase and stationary phase contributions, The approach also allowed an estimation of the relative phase ratios of the columns and the Vydac C-18 column was found to have an 3 and 8 times higher phase ratio than the Vydac C-4 and the Hy-Tach C-18 column, respectively.