Cecilia B. Castells

Fast enantioseparations of basic analytes by high-performance liquid chromatography using cellulose tris(3,5-dimethylphenylcarbamate)-coated zirconia stationary phases.

In this work, we study the influence of the mobile phase and column temperature on the enantioresolution of basic compounds on microparticulate porous zirconia coated with cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC). The chiral analytes are amino compounds, including a number of beta-blockers. Analytes are eluted with hexane-alcohol mobile phases. We investigated the effect of alcohol (type and concentration), basic eluent additives, and column temperature on the parameters that control resolution (column efficiency, retention and selectivity). Conditions for achieving an adequate separation in the least time have been determined for numerous racemic mixtures. For most solutes, baseline resolution of the enantiomeric pair was achieved in less than 1 min; 12 of 13 pairs were separated in less than 2 min.

A Study of the Thermodynamics and Influence of Temperature on Chiral High-Performance Liquid Chromatographic Separations Using Cellulose tris(3,5-dimethylphenylcarbamate) Coated Zirconia Stationary Phases

SummaryIn chiral HPLC, the separation is based on the differential interaction of a pair of enantiomeric molecules with a chiral selector. Temperature will affect such interactions. Most studies indicate that a decrease in temperature increases chromatographic selectivity. This is consistent with an enthalpy-controlled separation, but a more complete characterization of the physicochemical interactions is required to understand the driving forces for chiral recognition.In this work, we studied the separation of a number of enantiomers on cellulosetris(3,5-dimethylphenylcarbamate) supported on porous zirconia, over the temperature range of 0 to 55°C usingn-hexane/2-propanol mixtures as the eluent. The differences in the enthalpy (Δ(ΔH°)) and entropy (Δ(ΔS°)) of transfer of the enantiomers from the mobile to the chiral stationary phase were estimated from van’t Hoff plots. These relationships allow the study of the origin of the differences in interaction energies. The most interesting finding is that while most solutes show a negative Δ(ΔH°) difference, the two most easily resolved enantiomeric pairs were separated by an entropy dominated process. Studies of the relationship between the thermodynamics of transfer of these two entropically controlled separations and the eluent composition showed a substantial change in the interaction energies of these two solutes with the chiral polymer when the alcohol was reduced to 2% (ν/gn). Finally, we show that there is virtually no correlation between Δ(ΔG°) and overall retention, between Δ(ΔH°) and ΔH°, and little or no enthalpy-entropy compensation. These findings indicate the extreme difficulty in predicting or even correlating chiral selectivity with overall intermolecular interactions.

Effect of temperature on the chromatographic retention of ionizable compounds. III. Modeling retention of pharmaceuticals as a function of eluent pH and column temperature in RPLC

We propose a general simple equation for accurately predicting the retention factors of ionizable compounds upon simultaneous changes in mobile phase pH and column temperature at a given hydroorganic solvent composition. Only four independent experiments provide the input data: retention factors measured in two pH buffered mobile phases at extreme acidic and basic pH values (e. g., at least +/- 2 pH units far from the analyte pK(a)) and at two column temperatures. The equations, derived from the basic thermodynamics of the acid-base equilibria, additionally require the knowledge of the solute pK(a )and enthalpies of acid-base dissociation of both the solute and the buffer components in the hydroorganic solvent mixture. The performance of the predictive model is corroborated with the comparison between theoretical and experimental retention factors of several weak acids and bases of important pharmacological activity, in mobile phases containing different buffer solutions prepared in 25% w/w ACN in water and at several temperatures.

Determination of thermodynamic binding constants by affinity capillary electrophoresis

A strategy to study thermodynamic binding constants by affinity capillary electrophoresis (ACE) is presented. In order to simplify mathematical treatment, analogy with acid-base dissociation equilibrium is proposed: instead of ligand concentration [X], negative logarithm of ligand concentration (or activity), pXu202f=u202f-log[X], is used. On this base, and taking into account ionic activities, a general procedure for obtaining thermodynamic binding constants is proposed. In addition, the method provides electrophoretic mobilities of the free analyte and analyte-ligand complex, even when binding constants are low and thus, the complexed analyte fraction is also low. This is useful as a base to rationally analyze a diversity of situations, i.e., different mathematical dependencies are obtained when analytes and ligands with different charges are combined. Practical considerations are given for carrying out a full experimental design. Enantiomeric ACE separation based on the use of chiral selectors is addressed. 2-hydroxypropyl-β-cyclodextrin was chosen as a model ligand, and both enantiomeric forms of four pharmaceutical drugs (propranolol, pindolol, oxprenolol and homatropine methylbromide) were considered as model analytes. Practical aspects are detailed and thermodynamic binding constants as well as free and complexed analytes mobilities are determined.