SIMULTANEOUS EFFECT OF TEMPERATURE AND SOLVENT COMPOSITION ON THE pH OF COMMON MOBILE PHASES USED IN RPLC.

LEONARDO G. GAGLIARDI; AGUSTÍN ACQUAVIVA; MARCOS TASCÓN; CECILIA B. CASTELLS

Florianópolis, Brasil

Congreso; Congreso Latinoamericano de Cromatografía y Técnicas Afines, (Co.La.Cro. XII); 2008

<!-- @page { margin: 2cm } P { margin-bottom: 0.21cm } --> The optimization of any separation requires to count on as many variables as possible. In liquid chromatography (LC) of ionizable compounds, the mobile phase pH is clearly the first optimization variable, then the solvent composition is carefully chosen to modulate retention times of the sample components. Nowadays, chromatographers are conscious that the addition of an organic solvent into an aqueous buffer solution produces important shifts on the pH measured due to the change in dissociation constants of buffer components. This shift depends on the amount of added solvent but mainly on the chemical nature of the buffer substance: e.g. an addition of 50% of acetonitrile can increase the aqueous pH in about 4 units when the buffer is prepared from a carboxylic acid, while the same addition can reduce the pH in 0.3 units when the buffer is prepared from a primary amine. At the time that last generations of stationary phases which are resistant to high temperatures and extreme pH values have been developed, the column temperature has became to be considered as another important parameter in LC optimization. The increase in the column temperature, which can be easily controlled from the software, leads to many effects not only on the fluid hydrodynamics but also on the thermodynamics of the chromatographic system. Thus, as the solvent composition has important influence on the pH, the temperature has also its own incidence. The effects can be additive or, even so, synergistic with the effect of a solvent addition. In order to understand and to predict the simultaneous effect of temperature and solvent composition on the pH and, therefore, on chromatographic retention of ionizable compounds, it is necessary to know the variation of the pKa of the substances with both variables. We developed a simple and fast method to determine pKa values at several solvent compositions and temperatures. Then, we have determined pKa of phosphate and tris buffer in acetonitrile/water mixtures from 0 to 90% and at temperatures from 20ºC to 60ºC. This data set is used to predict the retention of ionizable compounds at different temperatures and acetonitrile compositions when one or other buffer substance is used. The predicted values are compared with real chromatograms.