Retention in reversed-phase liquid chromatography as a function of mobile-phase composition

The pH calibration procedures that lead to the different pH scales in acetonitrile−water mixtures used as mobile phases in reversed-phase liquid chromatography are discussed. Appropriate buffers of known pH value in acetonitrile−water mixtures are selected and used to establish the relationship (δ values) between the two rigorous acetonitrile−water pH scales:  and (pH measured in acetonitrile−water mixtures and referred to acetonitrile−water or water, respectively, as standard state). These δ values allow one to convert pH values measured in acetonitrile−water with electrode systems calibrated with aqueous buffers ( scale) to values, which are directly related to the thermodynamic acid−base constants. This offers an easy way to measure the pH of acetonitrile−water mobile phases and to relate this pH to the chromatographic retention of acids and bases through the thermodynamic acid−base constants. The relationships are tested for the variation of the retention of acids and bases with the pH of the mobile p...

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Retention of ionizable compounds on HPLC. 5. pH scales and the retention of acids and bases with acetonitrile-water mobile phases

Computerized design of separation strategies by reversed-phase liquid chromatography: development of DryLab software

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.

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

Influence of mobile phase acid-base equilibria on the chromatographic behaviour of protolytic compounds.

Computer simulation for the prediction of separation as a function of pH for reversed-phase high-performance liquid chromatography. II. Resolution as a function of simultaneous change in pH and solvent strength.

L. R. Snyder

Papers

Computer simulation for the prediction of separation as a function of pH for reversed-phase high-performance liquid chromatography. II. Resolution as a function of simultaneous change in pH and solvent strength.