Retention of Ionizable Compounds on HPLC. 12. The Properties of Liquid Chromatography Buffers in Acetonitrile -Water Mobile Phases That Influence HPLC Retention Sonia Espinosa, Elisabeth Bosch, and Martı´ Rose ´s* Departament de Quı ´ mica Analı ´ tica, Universitat de Barcelona, Diagonal 647, E-08028 Barcelona, Spain The addition of acetonitrile to aqueous buffers to prepare RP HPLC mobile phases changes the buffer properties (pH and buffer capacity). This variation is studied for ace- tate, phosphate, phthalate, citrate, and ammonia buffers in acetonitrile-water mixtures up to 6 0 % in acetonitrile (v/ v). Equations are proposed to relate pH and buffer capacity change of these buffers to the initial aqueous pH value and to the volume fraction of acetonitrile added. It is demonstrated that the pH change of the buffer depends not only on the initial aqueous pH of the buffer and on the percentage of acetonitrile added but also on the particular buffer used. The proposed equations allow an accurate prediction of this ionization for the studied buffers. Since the retention of acid/ base compounds shows a strong dependence of their degree of ionization, the equations are used to predict the change in this ionization with addition of acetonitrile when the RP HPLC mobile phase is prepared. This prediction allows estima- tion of the retention of an acid/ base compound in a particular acetonitrile-water buffered mobile phase. Buffers are widely used for pH control of chemical processes. 1 In reversed-phase high-performance liquid chromatography (RP HPLC) separations of compounds with acid/ base properties, appropriate buffered solutions are needed for calibration of the electrode system used to measure pH and for control of the mobile-phase pH, since the latter influences analyte ionization and thus retention. pH standardization of electrode systems in water and in the most used RP HPLC mobile phases has been substantially achieved. The IUPAC has endorsed rules for pH standardization in water and in water-organic solvent mixtures of moderate to high permittivities and proposed some buffered solutions for pH standardization in these solvents. 2-4 Some additional buffers in acetonitrile-water have been studied by Barbosa et al. 5-7 On the basis of IUPAC rules and recommendations, we have studied the different pH scales that are employed in pH measurement of RP HPLC mobile phases. Three different pH scales have been used. The most common is the aqueous pH scale ( w w pH), which is obtained when the electrode system is calibrated with aqueous buffers and the pH measured in the RP HPLC aqueous buffer before mixing it with the organic modifier. If the electrode system is calibrated with aqueous buffers, but the pH is measured in the mobile phase obtained after mixing the aqueous buffers with the organic modifier, the pH scale in the mobile-phase solvent (s) relative to water (w) as standard-state solvent is obtained ( w s pH). Finally, if the electrode system is calibrated with buffers prepared in the same mixed solvent used as mobile phase (s) and the pH is measured in the mobile phase (s), the pH scale in the mobile phase referred to the same mobile phase as standard-state solvent is obtained ( s s pH). Whenever possible, we recommend s s pH and w s pH scales because they give much better general relationships between pH and retention. 8-10 The w s pH scale is specially recom- mended because of its simplicity of measurement, since it does not require pH standards for each different mobile-phase composi- tion. However, the main acid/ base properties of the buffers used to control the pH of RP HPLC mobile phases has not been systematically studied. In a pioneer work, Bates discussed the properties of acid/ base aqueous buffers and its applicability to chemical processes. 1 Bates remarked that in choosing a suitable buffer system, one should not only consider the pH required but should also take into account the nature of the reaction to be regulated. A high buffer capacity is essential for the control of an acid/ base reaction, and a low dilution value is desirable if dilution of the medium is likely to occur. The dilution value is defined as the increase of pH suffered by a solution when it is diluted with an equal volume of pure solvent. Salt effects and temperature changes may be important in some instances. The chemical nature * Corresponding author. Fax: 34 93 402 12 33. E-mail: marti@ apolo. qui.ub.es. (1) Bates, R. G. Determination of pH: Theory and Practice, 2nd ed.; Wiley: New York, 1964. (2) IUPAC Compendium of Analytical Nomenclature. Definitive Rules 1997, 3rd ed.; Blackwell: Oxford, U.K., 1998. (3) Mussini, T.; Covington, A. K.; Longhi, P.; Rondinini, S. Pure Appl. Chem. 1985 , 57, 865-876. (4) Mussini, P. R.; Mussini, T.; Rondinini, S. Pure Appl. Chem. 1997 , 69, 1007- 1014. (5) Barbosa, J.; Sanz-Nebot, V. Anal. Chim. Acta 1993 , 283, 320-325. (6) Barbosa, J.; Butı ´, S.; Sanz-Nebot, V. Talanta 1994 , 41, 825-831. (7) Barbosa, J.; Sanz-Nebot, V. Mikrochim. Acta 1994 , 116, 131-141. (8) Canals, I.; Portal, J. A.; Bosch, E.; Rose ´s, M. Anal. Chem. 2000 , 72, 1802- 1809. (9) Espinosa, S.; Bosch, E.; Rose ´s, M. Anal. Chem. 2000 , 72, 5193-5200. (10) Canals, I.; Oumada, F. Z.; Rose ´s, M.; Bosch, E. J. Chromatogr., A 2001 , 911, 191-202. Anal. Chem. 2002, 74, 3809-3818 10.1021/ac020012y CCC: $22.00 © 2002 American Chemical Society Analytical Chemistry, Vol. 74, No. 15, August 1, 2002 3809 Published on Web 07/03/2002