Adsorption behavior of the three species of the biprotic peptide Phe-Ala onto an end-capped C18-bonded organic/inorganic hybrid stationary phase.

We recorded the overloaded elution band profiles of the dipeptide phenylalanine-alanine (Phe-Ala) on a column packed with C18-bonded organic (ethyl)/inorganic (silica) hybrid porous particles (BEH), eluted with a series of buffered methanol-water mobile phases (20/80, v/v). The (W)(S)pHs of the mobile phases were successively adjusted with addition of suitable buffers to values of 1.67, 2.44, 3.83, 4.94, 7.41, and 10.71 (where the notation (W)(S)pH means that the pH of the solution is directly measured in the solution (S) after the electrode was calibrated in pure water (W)). The ionic strength of the eluent was kept constant at 20 mM. The injected samples had different sizes and concentrations. The retention of the low-concentration samples was minimum at an intermediate (W)(S)pH (k' approximately 0.5) and maximum for the lowest (k' approximately 1.3) and highest (k' approximately 3.5) (W)(S)pHs showing that the zwitterion (+Phe-Ala-) is less strongly adsorbed than the positively (+Phe-Ala) and negatively (Phe-Ala-) charged species of the dipeptide onto BEH-C18. The elution profiles of the concentrated samples demonstrated that the adsorption isotherm of the zwitterionic species is an anti-Langmuirian Moreau isotherm due to significant adsorbate-adsorbate interactions, whereas the isotherms of the charged species are more conventional Langmuir isotherms. A simple ternary isotherm for the coadsorption of the three dipeptide species is proposed. It accounts well for the complex band profiles observed when large concentrated samples of the peptide are injected under controlled pH conditions. The slight departure between the calculated and the experimental band profiles obtained under uncontrolled pH conditions suggests the possible adsorption of the buffer components onto the packing material and the possible influence of microenvironment effects near the stationary phase surface, which would affect the local pH along the column. Preparative chromatography of peptides should preferentially be operated at intermediate (W)(S)pHs or with high ionic strength buffers under acidic or basic (W)(S)pHs.