Change of mobile phase pH during gradient reversed-phase chromatography with 2,2,2-trifluoroethanol–water as mobile phase and its effect on the chromatographic hydrophobicity index determination

We have shown previously that using a trifluoroethanol containing mobile phase provides a unique chromatographic selectivity. This is essential to derive molecular descriptors by HPLC which requires retention data from several systems. It also requires that the ionisation is suppressed so that retention times reflect the properties of the neutral molecules. Therefore the pH change of the mobile phase during gradient elution and its effect on the solute ionisation have been studied. During gradient elution of mixtures of ammonium acetate and butylammonium formate with trifluoroethanol as an organic modifier it was found that the pH was almost constant when the gradient started with a low pH. However, when the starting mobile phase pH was above 8 the pH dropped very quickly as the trifluoroethanol concentration increased in the mobile phase. The CHI descriptor (a retention index derived directly from gradient retention times) of several basic compounds as a function of starting mobile phase pH has been measured using trifluoroethanol gradient. The effect of the trifluoroethanol on the pKa change of the compounds has been investigated. The experimental data fit closely to a previously derived equation that describes gradient retention times as a function of mobile phase pH and analyte ionisation constant (pKa). This equation makes it possible to predict the CHI descriptor for ionisable compounds at various pH values. We have used butylamine for high pH mobile phase preparation as is more basic than ammonia and for many basic drugs the retention of the neutral form could be obtained directly (without extrapolation).