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 pho sphoric acid, on the energetics of peptide retention in reversed-phase chromatography was investigated using Hy-Tach C-18 micropellicular an d Vydac C-4 and C-18 totally porous stationary phases. The effect of t he relatively low phase ratio of columns packed with micropellicular s orbents was also examined. The logarithmic retention factors, of two m odel peptides, Ac-RGGGGLGLGK-amide and Ac-RGAGGLGLGK-amide, were evalu ated with different columns and additives in a practical range of elue nt strength. The dependence of the logarithmic retention factor on the concentration of acetonitrile in the mobile phase was linear in all c ases. 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 l ogarithmic retention factors were linear. The plots of data obtained w ith the two additives has unit slopes and thus reveal homoenergetic re tention behavior. On the other hand data obtained on two different col umns manifest homeoenergetic retention, the slopes of plots are differ ent from unity. The analysis has yielded consistent results and valida ted the assumption that the retention free energy can be divided into two components arising from mobile phase and stationary phase contribu tions, The approach also allowed an estimation of the relative phase r atios 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.