The different procedures used in HPLC to measure the pH of a mobile phase are evaluated in terms of the rigorous IUPAC definition of pH. The three procedures evaluated are as follows: measurement of the pH of the aqueous HPLC buffer before mixing it with the organic modifier, measurement of the pH of the HPLC buffer after mixing it with the organic modifier using a pH electrode system calibrated with aqueous buffers, and measurement of the pH of the HPLC buffer after mixing it with the organic modifier but calibrating the electrode system with reference buffers prepared in the same mixed solvent used as mobile phase. Following IUPAC definitions and recommendations, the three pH values can be related with the pH scales: w(w)pH, s(w)pH, and s(s)pH, respectively. The relationships between these three pH scales are also presented. The retention of several compounds with acid/base behavior in a C-18 and a polymeric column with buffered methanol/water as mobile phase is related to the mobile phase pH value measured in the three pH scales. It is demonstrated that the s(w)pH and s(s)pH scales give better relationships than the w(w)pH scale (pH measured in the aqueous buffer before mixing it with the organic modifier), commonly used on HPLC. The s(w)pH scale is specially recommended because of its simplicity of measurement: the pH is measured after mixing the aqueous buffer with the organic modifier, but the pH calibration is performed with the common aqueous reference buffers.
SummaryThe retention of ionizable compounds in reversed-phase liquid chromatography with methanol-water mobile phases has been related to the pH, composition and polarity of the mobile phase. The ionization of the compound is calculated from the pH measured in the mobile phase (WpH) and the p~ of the compound in the same mobile phase (SpKa) which can be estimated from the aqueous p~ (WpKa) by means of general equations previously established. The retention factors are calculated from the degree of ionization and the retention factors of the neutral and ionic forms of the compounds. Two different linear models have been tested to relate the retention factor of the acid-base forms of the studied compounds to methanol-water composition or polarity
SummaryA global LSER model that relates HPLC retention to mobile phase composition and pH is tested for a varied group of solutes, both neutral and ionizable, in a polymeric column and methanol-water mobile phases. It is compared to the local LSER model developed only for a given mobile phase, i.e., a fixed organic modifier content, and to the global LSER model set only for neutral solutes.The global LSER model for neutral and ionizable solutes requires a few su pplementary parameters over the other models tested, but it accounts for retention under any experimental conditions for a given column and methanol-water mobile phases, describing properly the interactions established in the HPLC system (hyclrophobicity, hydrogen-bond acidity and basicity, clipolarity/polarizability...).
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.