Kater ˇina Vc ˇ eláková 1 Iva Zusková 1 Ernst Kenndler 2 Bohuslav Gas ˇ 1 1 Faculty of Science, Charles University, Prague, Czech Republic 2 Institute for Analytical Chemistry, University of Vienna, Vienna, Austria Determination of cationic mobilities and pK a values of 22 amino acids by capillary zone electrophoresis The effective mobilities of the cationic forms of common amino acids – mostly protein- ogenic – were determined by capillary zone electrophoresis in acidic background elec- trolytes at pH between 2.0 and 3.2. The underivatized amino acids were detected by the double contactless conductivity detector. Experimentally measured effective mo- bilities were fitted with the suitable regression functions in dependence on pH of the background electrolyte. The parameters of the given regression function corresponded to the values of the actual mobilities and the mixed dissociation constants (combining activities and concentrations) of the compound related to the actual ionic strength. McInnes approximation and Onsager theory were used to obtain thermodynamic dis- sociation constants (pK a ) and limiting (absolute) ionic mobilities. Keywords: Amino acids / Capillary electrophoresis / Conductivity detection / Mobility / pK a value DOI 10.1002/elps.200305751 1 Introduction 1.1 General aspects The separation principle of capillary zone electrophoresis (CZE) in free solution is based on the different migration velocity of electrically charged analytes in the electric field. As the commercial instruments for CZE are readily available, this method is now applied for many analytical problems routinely, especially in the biological and bio- chemical fields. In additions to these analytical tasks, CZE can be used for the determination of physical and chemical properties of solutes such as acid dissociation constants [1] and/or ionic mobilities. Various methods have been used for the determination of pK a values and ionic mobilities. One of the most useful is isotachophoresis (ITP); here, a great deal of work was made by Hirokawa and his co-workers [2–6]. The advan- tage of ITP is its separation ability, which enables to deter- mine the mobilities of substances that are not pure, or of several analytes present together in mixtures, without sample pretreatment. An even more important advantage of the ITP method is its accuracy, as it mostly utilizes in a hydrodynamically closed system; the electrosmotic flow (EOF), an important possible source of bias, is eliminated here. Nevertheless, there is also a substantial drawback of ITP when used for the determination of pK a values and mobilities: it is limited by the pH value of the leading elec- trolyte. When the pH is too acidic (pH , 3) or too alkaline (pH . 11), steady-state isotachophoretic zones cannot be formed due to the high concentration of hydroxonium or hydroxide ions. For this reason, the cationic limiting mo- bilities of amino acids and peptides, which possess pK a values less than 3, cannot be determined isotachophoret- ically. As CZE is a nonsteady-state method, it is thus not subject to such restrictions. Therefore, dissociation con- stants [7–9] and limiting mobilities [10–12] of some spe- cies, mainly inorganic and organic ions and pharmaceuti- cally interesting substances have been determined by CZE even under more extreme pH conditions. The importance of correct pK a values and ionic mobilities of compounds lays, among other reasons, in their need in software for optimization of background electrolytes (BGEs) for capillary electrophoresis (CE). Our laboratory developed the program PeakMaster [13], which enables to vary separation parameters, mainly the composition of the BGE, and to find favorable configurations for a particu- lar separation problem. The real behavior of the systems can be proved in practice then. Instead of the usual experi- mental trial-and-error approach a faster computer simula- tion can be utilized, which, by the way, might save hazar- dous and toxic chemicals. Importantly, PeakMaster soft- ware also forecasts some disturbing phenomena often encountered in practice, like system eigenpeaks, unusual electromigration dispersion of some peaks due to reso- nance, etc. The correct functioning of the optimization Correspondence: Dr. Iva Zusková, Department of Physical Chemistry, Faculty of Science, Charles University, Albertov 2030, CZ-128 40 Prague 2, Czech Republic E-mail: zuskova@natur.cuni.cz Fax: 1420-2-2491-9752 Abbreviations: CCD, contactless conductivity detector; DAD, diode array detector; OH-Pro, hydroxyprolin Electrophoresis 2004, 25, 309–317 309 CE and CEC  2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim