IONIZABLE COMPOUNDS ON HPLC

Ionizable Compounds on HPLC

Abstract

HPLC retention of ionic and ionizable analytes is known to be dependent on pH of the mobile phase, type of salt added to the mobile phase and ionic strength. The influence of the type of salt and its concentration on the analyte's retention has been attributed to a so-called “chaotropic” mechanism. Methanol and acetonitrile demonstrate principal difference in the influence of the counter ions on the retention of ionic analytes. The ability of acetonitrile to form thick adsorbed layer on the adsorbent surface and its high pi-electron density create a favorable environment for the retention of inorganic ions with significant charge delocalization. This favorable environment results in significant retention increase of analyte possessing opposite charge. Similar effect is completely eliminated when acetonitrile is substituted with methanol.

Our studies have demonstrated that different inorganic ions do not influence analyte retention in the similar sequence as the position of these ions in Hofmeister series. In acetonitrile/water mobile phases all ions exhibit “chaotropic” effect, even the ions known to have “kosmotropic” properties (favorable for the formation of solvation shell) show the increase of the analyte retention with the increase of their concentration.

CHAPTER 1

Retention Of Ionic Analytes

Understanding the chemistry of the retention process will greatly simplify method development in liquid chromatography (LC) which is today often a trial and error process. The total number of experiments needed to establish a separation methodology can be minimized by using accurate quantitative relationships able to predict the elution of substances under different separation conditions.

Once a stationary phase is chosen, and due to the specific acid-base characteristics of ionogenic solutes, the two most useful optimization parameters are the pH and the organic modifier concentration. In comparison with optimizing the organic modifier concentration alone (a problem addressed with considerable success in previous works [1, 2, 3]), the inclusion of pH as one of the optimization parameters raises additional problems. The theoretical interpretation of the effects of pH on retention of ionizable analytes is hampered by the lack of a rigorous treatment of protolytic equilibria in hydroorganic mixtures. Thermodynamically valid pH and pKa values must be used to interpret the ionic equilibria in the aqueous-organic media used as mobile phases in LC. Moreover, activity coefficients can be neglected in water but must be considered in these hydroorganic mixtures.

The measurement of pH in a mixed solvent is based upon the operational definition of pH [4, 5] in which pH is estimated by(1)pHX=pHS+(ES-EX)/kg,where the unknown pH of solution X, pHX, is related to the pH of a standard reference solution, pHS, and the emf values of the potentiometric cell contain the standard, ES, and the unknown solution, EX. kg must be used for practical measurements, usually carried out in cells with glass electrodes, and corresponds to the practical slope of the E vs. pH function [6, 7]. Thus, the availability of standard buffer solutions of known pH, in the desired solvent mixture, is the key to pH measurements in these media [4, ...