Simultaneous measurement of pHWS and overloaded band profiles of small peptides when insufficiently buffered mobile phases are used in preparative liquid chromatography

We recorded simultaneously the pHWS and the elution band profiles of phenylalanine-alanine (Phe-Ala, pKa,1WS=3.6, pKa,2WS=7.5) 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). Using different buffers, the pHsWS of the mobile phases was set to values between 2.5 and 9.8. The ionic strength of the mobile phases was kept constant at 20 mM. The injected solutions were prepared with Phe-Ala as its zwitterionic species. Analytical to overloaded sample sizes were used, under strongly or insufficiently buffered conditions.With insufficiently buffered mobile phases, the experimental results show significant variations of pHWS during band elution, which proves a significant change of the concentration ratio of the different sample species during elution. This result confirms and validates our earlier general adsorption isotherm model of acido-basic compounds in RPLC. The pHWS record shows also the system peak of the buffer component, consecutive to sample injection. The phosphate II and carbonate II ions are adsorbed and may compete with the peptide for adsorption onto the active sites of BEH-C18. This suggests refinements of the general adsorption isotherm model, depending on the nature of the buffer components, including competitive adsorption of the sample and the buffer species.