Effects of a strongly adsorbed additive on process performance in chiral preparative chromatography.

The shapes of elution profiles are often significantly influenced by the presence of strongly adsorbed additives in the mobile phase. This aspect needs to be considered in quantitative optimization of preparative chromatography. The theoretical study carried out here is based on available thermodynamic information for the enantiomers of three beta-blockers, alprenolol, propranolol, and atenolol, on a teicoplanin chiral stationary phase (Chirobiotic T) using methanol/acetonitrile as the mobile phase and acetic acid/triethylamine as the additive. The properties of this strong additive made it possible to tune the binary elution profiles in any combination of the following apparent band shapes: anti-Langmuir/anti-Langmuir, anti-Langmuir/Langmuir and Langmuir/Langmuir. Optimization of the productivity and yield, when performing repetitive batch injections, was investigated using the equilibrium dispersive model. We show that it is important to consider the invisible additive perturbation peak when defining the cycle time and therefore a model-based optimization needs to take this into account. Furthermore, both productivity and yield could be improved for the two unusual shape combinations in comparison to the traditional Langmuir/Langmuir case.

Influence of the solute hydrophobicity on the enantioselective adsorption of beta-blockers on a cellulase protein used as the chiral selector.

Adsorption isotherm data were acquired at different eluent pH values for the enantiomers of several beta-blockers on cellobiohydrolase I on silica gel. They fit well to the biLangmuir model, allowing the determination of the equilibrium constants and the monolayer capacities for chiral and nonselective adsorption. The adsorption of the S-enantiomers (eluted second) is exothermic at low pH, endothermic at high pH, and athermal in a narrow pH range depending on the beta-blocker. This transition pH range is lower for S-alprenolol than for the more hydrophobic S-propranolol, although their endothermic adsorption originates from hydrophobic interactions. This surprising observation is explained by the relative values of the isotherm coefficients. S-Alprenolol seems to have a more pronounced endothermic behavior than S-propranolol because the nonselective interactions of both compounds with the stationary phase are exothermic but their contribution to retention, relative to that of the endothermic chiral interactions, is less important for alprenolol. The order of increasing energy of the chiral interactions is the same as that of hydrophobicity, propranolol>alprenolol>metoprolol.

Theoretical study of the accuracy of the elution by characteristic points method for bi-langmuir isotherms.

The bi-Langmuir equation has recently been proven essential to describe chiral chromatographic surfaces and we therefore investigated the accuracy of the elution by characteristic points method (ECP) for estimation of bi-Langmuir isotherm parameters. The ECP calculations was done on elution profiles generated by the equilibrium-dispersive model of chromatography for five different sets of bi-Langmuir parameters. The ECP method generates two different errors; (i) the error of the ECP calculated isotherm and (ii) the model error of the fitting to the ECP isotherm. Both errors decreased with increasing column efficiency. Moreover, the model error was strongly affected by the weight of the bi-Langmuir function fitted. For some bi-Langmuir compositions the error of the ECP calculated isotherm is too large even at high column efficiencies. Guidelines will be given on surface types to be avoided and on column efficiencies and loading factors required for adequate parameter estimations with ECP.

Retention mechanism of beta-blockers on an immobilized cellulase. Relative importance of the hydrophobic and ionic contributions to their enantioselective and nonselective interactions.

The adsorption isotherms of the enantiomers of three beta-blockers, metoprolol, alprenolol, and propranolol, were measured on cellobiohydrolase I (CBH I) immobilized on silicagel, in the concentration range between 0.25 microM and 1.7 mM, at pH = 5.0, 5.5, and 6.0. In agreement with previous results, these data are accounted for by a two-sites physical model and fit closely to a Bilangmuir equation. The saturation capacities and the binding constants were determined for each enantiomer on the chiral and the nonchiral sites. The chiral sites are shown to be strongly ionic, in contrast to the nonchiral ones, which are mainly hydrophobic. However, the chiral binding of (S)-propranolol is endothermic, with a high adsorption entropy, in contrast to the chiral interactions of (R)-propranolol and to the nonchiral interactions, which are all exothermic. This indicates that hydrophobic interactions also play a role in the chiral binding. The dependence of the adsorption parameters on the hydrophobicity of the solute is discussed and interpreted in terms of the retention mechanism. The results are compared with the structure of the protein, recently elucidated by X-ray crystallography.

Apparent and true enantioselectivity in enantioseparations.

The separation factor of two compounds in chromatography is the ratio of their equilibrium constants or retention factors. This parameter is universally employed to investigate their resolution and to optimize the experimental conditions of their analysis. In enantioseparations, the situation is more complex because there is a mixed retention mechanism. The retention factor is the sum of two contributions, one enantioselective, the other nonselective. Although both contribute to retention, the latter being identical for the two enantiomers and does not contribute to their separation. We show how these two contributions can be measured and how it becomes necessary to distinguish between the apparent, alpha(app), and the true, alpha(true), separation factors. The existence of nonselective sites is responsible for alpha(app) being less than alpha(true). Depending on the difference between these two factors, the more effective approach to improve a separation is either to increase the enantioselectivity or to reduce the nonselective interactions. Practical applications to separations of different beta-blockers on cellobiohydrolase are discussed. The apparent enantioselectivity of alprenolol is larger and increases faster with increasing pH than that of the more hydrophobic propranolol, in spite of the importance of hydrophobic interactions in the enantioselective mechanism. These two unexpected properties are discussed and explained.

Chiral assay of atenolol present in microdialysis and plasma samples of rats using chiral CBH as stationary phase.

Two different enantioselective chiral chromatographic methods were developed and validated to investigate the disposition of the beta 1-receptor antagonist atenolol in blood and in brain extracellular fluid of rats (tissue dialysates). System A for the plasma samples was a one-column chromatographic system with a chiral CBH column with an aqueous buffer as mobile phase into which cellobiose was added for selective regulation of the retention of the internal standard, (S)-metoprolol. The plasma samples were analysed after a simple extraction procedure. The limit of quantitation was 0.2 micrograms/ml for the atenolol enantiomers. The repeatability of the medium concentration quality control plasma sample (6.0 micrograms rac-atenolol/ml) was 11-18% for the enantiomers. The dynamic linear range of the plasma samples was 0.5-20 micrograms/ml. For system B, since atenolol is an extremely hydrophilic drug, the tissue dialysate sample required a much more sensitive system as compared to the plasma samples. A coupled column system was used for peak compression of the enantiomers in the eluate after the separation on the Chiral CBH column, hence increasing the detection sensitivity. The limit of quantification was 0.045 micrograms/ml for the atenolol enantiomers in artificial CSF. The repeatability of the medium concentration quality control samples (0.1 and 4.0 micrograms rac-atenolol/ml in artificial CSF and Hepes Ringer, respectively) was 2.8-9.3% for the two enantiomers. The dynamic linear range of the brain samples was 0.05-1.0 and 0.5-20 micrograms/ml in artificial CSF and Hepes Ringer, respectively.

Experimental and theoretical study of the adsorption behavior and mass transfer kinetics of propranolol enantiomers on cellulase protein as the selector.

The thermodynamics and mass transfer kinetics of the retention of the R and S enantiomers of propranolol were investigated on a system comprising an acetic acid buffer solution as the mobile phase and the protein cellobiohydrolase I immobilized on silica as the stationary phase. The bi-Langmuir isotherm model fitted best to each set of single-component isotherm data. The monolayer capacity of the nonchiral type of adsorption sites was 22.9 mM. For the chiral type of sites, it was 0.24 mM for the R enantiomer and 0.64 mM for the S enantiomer. Peak tailing was observed, even at very low concentrations allowing operation of the low-capacity chiral sites under linear conditions. This tailing can be explained on the basis of heterogeneous mass transfer kinetics. At higher concentrations, which are often used in analytical applications, the isotherms on the chiral sites no longer have a linear behavior, and peak tailing is consequently more pronounced. Under those conditions, peak tailing originates from the combined effect of heterogeneous thermodynamics and heterogeneous mass transfer kinetics. These complex phenomena are explained and modeled using the transport-dispersive model with a solid film linear driving force model modified to account for heterogeneous mass transfer kinetics. The rate coefficient of the mass transfer kinetics was found to be concentration dependent.

Peak distortion effects of suramin due to large system peaks in bioanalysis using ion-pair adsorption chromatography.

Sample injections in ion-pair adsorption chromatography lead to changes in the established equilibria of the eluent components at the top of the column, which in turn lead to migrating concentration changes in the column (system peaks). Large system peaks contain big concentration deviations of the mobile phase components compared with the bulk composition in the eluent. Analyte peaks are distorted upon combined elution with large system peaks. In bioanalysis, samples that deviate considerably from the eluent are often injected, resulting in large system peaks. An illustrative example of such analyte peak distortions in bioanalysis is given for the case of suramin, and guidelines are given for avoiding the effects.