Retention of ionizable compounds on HPLC. 4. Mobile-phase pH measurement in methanol/water

The different procedures used in HPLC to measure the pH of a mobile phase a re evaluated in terms of the rigorous IUPAC definition of pH. The three pro cedures evaluated are as follows: measurement of the pH of the aqueous HPLC buffer before mixing it with the organic modifier, measurement of the pH o f the HPLC buffer after mixing it with the organic modifier using a pH elec trode system calibrated with aqueous buffers, and measurement of the pH of the HPLC buffer after mixing it with the organic modifier but calibrating t he electrode system with reference buffers prepared in the same mixed solve nt used as mobile phase. Following IUPAC definitions and recommendations, t he three pH values can be related with the pH scales: (w)(w)pH, (s)(w)pH, a nd (s)(s)pH, respectively. The relationships between these three pH scales are also presented. The retention of several compounds with acid/ base beha vior in a C-18 and a polymeric column with buffered methanol/water as mobil e phase is related to the mobile phase pH value measured in the three pH sc ales. It is demonstrated that the (s)(w)pH and (sPH)-P-s scales give better relationships than the (w)(w)pH scale (pH measured in the aqueous buffer b efore mixing it with the organic modifier), commonly used on HPLC. The (s)( w)pH scale is specially recommended because of its simplicity of measuremen t: the pH is measured after mixing the aqueous buffer with the organic modi fier, but the pH calibration is performed with the common aqueous reference buffers.