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ABSTRACT: Immobilized amyloglucosidase was used as a chiral stationary phase (CSP). First, the retention and enantioselectivity of several model chiral amines and acids were investigated. We found that this CSP was unable to separate the enantiomers of acids, though all selected amines could be resolved. The adsorption of (R)- and (S)-propranolol and its influence on column temperature and 2-propanol content in the eluent were then studied in detail, using a three-step methodology. The adsorption was first evaluated using Scatchard plots; thereafter, the adsorption was characterized in detail by calculating the adsorption energy distribution. With this model-independent information, a better judgment could be made of the possible adsorption models selected in the last step, the model fitting to the data. In the case examined, the bi-Langmuir model (containing nonselective and enantioselective sites) describes the system well. The retention of (R)- and (S)-propranolol at low temperatures increases with the content of 2-propanol in the eluent, due to the increased saturation capacity of the enantioselective sites. The retention is an enthalpy-driven process at both types of sites, whereas the enantioseparation is due to differences between the entropy changes of the two enantiomers at the enantioselective sites. The enthalpy of adsorption at the nonselective sites is almost identical at the two concentrations of 2-propanol in the eluent. Enantioselective adsorption, on the other hand, is more exothermic at higher modifier content (20%). Thus, at high temperatures the retention decreases with increasing modifier content, whereas the opposite (unusual) trend is the case at low temperatures.
Journal of Chromatography 08/2007; 1156(1-2):3-13. · 4.53 Impact Factor
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ABSTRACT: The enantioseparation of 2,2,2-trifluoro-1-(9-anthryl)ethanol on silica-bonded quinidine carbamate was examined under linear chromatographic conditions. The significant impact of nonselective adsorption on the retention was demonstrated. The influences of a polar additive in the mobile phase on the retention, the selectivity and the thermodynamic quantities of the retention were measured. A small effect of the pressure on the selectivity and on the accuracy of the thermodynamic measurements was observed.
Journal of Chromatography 11/2005; 1091(1-2):183-6. · 4.53 Impact Factor
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ABSTRACT: In many studies of nonlinear or preparative chromatography, chromatographic signals must be recorded for relatively concentrated solutions and the detectors, that are designed for analytical applications and are highly sensitive, must be used under such experimental conditions that their responses are often nonlinear. Then, a calibration curve is needed to derive the actual concentration profiles of the eluates from the measured detector response. It becomes necessary to derive a relationship between the concentration of the eluent and the detector signal at any given time. The simplest approach consists in preparing a series of solutions of known concentrations and in flushing them successively through the detector cell, recording the height of the plateau response obtained. However, this method requires relatively large amounts of the pure solutes being studied and these are not always available or they may be most costly, although these solutions can be recovered. We describe and validate an alternative procedure providing this calibration from a series of peaks recorded upon the injection of increasingly large pulses of the studied compound.
Journal of Chromatography 06/2005; 1076(1-2):141-7. · 4.53 Impact Factor
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ABSTRACT: The adsorption isotherms of the enantiomers of 2,2,2-trifluoro-1-(9-anthryl)-ethanol from a toluene-acetonitrile solution onto a Chiris Chiral AX:QD1 column were measured using the pulse method. The isotherm data were modeled with a bi-Langmuir isotherm model, indicating the presence of two different types of adsorption sites on this stationary phase, nonselective and enantioselective sites. The latter are homogeneous but interact with both enantiomers, albeit with different energies. The thermodynamic characteristics of these two types of sites were characterized by their adsorption constants and saturation capacities and by the influence of the temperature on these different parameters.
Journal of Chromatography 01/2005; 1059(1-2):43-52. · 4.53 Impact Factor
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ABSTRACT: The influence of the pH on the complexation equilibria between (S)- or (R)-alprenolol and the cellulase Cel7A was investigated by isothermal titration calorimetry. The results obtained agree with those of previous, similar studies of the same equilibria in which the protein was immobilized on silica particles, packed in a chromatographic column. The association constant and the complexation enthalpy and entropy of the (S)-enantiomer increase with increasing pH. For (R)-alprenolol, the binding is endothermic at all pH values. Thus, for both enantiomers in the pH range 5.5-6.8, the binding is an entropically driven process.
Journal of Chromatography 09/2004; 1046(1-2):49-53. · 4.53 Impact Factor
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ABSTRACT: The difference between the average energies of the high-energy modes of the adsorption energy distributions of (S)-alprenolol and (R)-alprenolol on a chiral stationary phase calculated by the expectation maximization method agree well with the difference between the adsorption energies of these two compounds measured by isothermal titration calorimetry.
Langmuir 09/2004; 20(16):6521-3. · 4.19 Impact Factor
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ABSTRACT: The distributions of the adsorption energies (AED) of two enantiomers, (R)-1- indanol and (S)-1-indanol, on a chiral stationary phase were measured and the results are discussed. The chiral phase used is made of cellulose tribenzoate coated on porous silica. The AEDs were determined using the expectation maximization method, a numerical method that uses directly the raw experimental isotherm data, inverts this set of data into an AED, and introduces no arbitrary information in the calculation. However, it uses the Langmuir equation as the local isotherm. The experimental data fit very well to the bi-Langmuir isotherm model for the more retained enantiomer. Our results show that the AEDs of these two enantiomers have no energy modes that would be identical (same mean energy, mode profile, and mode area), in contrast to numerous cases previously studied, e.g., that of the beta-blockers on a Cel7A column. This indicates a significantly different retention mechanism.
Analytical Chemistry 02/2004; 76(1):197-202. · 5.86 Impact Factor
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ABSTRACT: The distributions of the adsorption energies (AED) of two enantiomers on a chiral stationary phase were measured, and the results are discussed. The enantiomers studied are two β-blockers, alprenolol and propranolol. The chiral phase is made of the protein Cel7A immobilized on porous silica. The determination of these AEDs was carried out using the expectation-maximization method, a numerical method that uses directly the raw experimental isotherm data and inverts this set of data into an AED, using in the process the Langmuir equation as the local isotherm. The distributions obtained were found to be bimodal and consistent with observations previously made on the isotherms and reported. The AEDs of two enantiomers have the same low-energy mode (same mean energy, mode profile, and mode area). Their high-energy modes are different. Depending on the eluent pH, the mode for the (S)-enantiomer has a larger mean energy or a larger area than the one for the (R)-enantiomer. The experimental data fit very well to the bi-Langmuir isotherm model. At low mobile phase pH, the difference between the retention of the two enantiomers is due to the difference of their saturation capacities at the enantioselective (or high-energy) sites. By contrast, at high pH, their separation is governed by the difference in their adsorption energies. This work illustrates how the consideration of the AEDs of two enantiomers may be informative on the validity of the method of determination of these distributions and on the properties of the surface.
07/2003;
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ABSTRACT: A complete census was made of the interactions between enantiomeric solutes and the chiral protein column CHIRAL-AGP with the theory of nonlinear LC as tool. The surface is heterogeneous, having a small number of strong enantioselective adsorption sites and a large number of weak nonselective ones. When the eluent pH was increased, the "linear" retention of (i) the amines increased strongly as a result of a strong increase in the enantioselective binding strength, whereas (ii) the retention of the aprot increased slightly as a result of an increase in both the enantioselective binding strength and its capacity. The retention of (iii) the acid has a maximum originating solely from the enantioselective binding energy, whereas the nonselective equilibria decreased steadily. For all compounds, the enantioselective equilibrium constants increase relatively more than the nonselective ones with increasing pH.
Analytical Chemistry 08/2002; 74(13):2950-9. · 5.86 Impact Factor
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ABSTRACT: 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, αapp, and the true, αtrue, separation factors. The existence of nonselective sites is responsible for αapp being less than αtrue. 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 β-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. Chirality 12:558–564, 2000. © 2000 Wiley-Liss, Inc.
Chirality 06/2000; 12(7):558 - 564. · 2.35 Impact Factor
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ABSTRACT: We reported previously on the unusual thermodynamic characteristics of the enantioselective interactions between the enantiomers of the β-blocker propranolol and the protein cellobiohydrolase I immobilized on silica. The adsorption of the more retained enantiomer, (S)-propranolol, is endothermic while that of the (R)-propranolol is exothermic. This causes a rapid increase of the selectivity factor with increasing temperature. In this work, we study the complex dependence of the selectivity factor on the pH of the solvent. We determined the equilibrium isotherms of (R)- and (S)-propranolol in a wide concentration range (0.25 μM to 1.1 mM) at six different mobile-phase pHs (4.7, 5.0, 5.2, 5.5, 5.7, and 6.0) and fitted the data obtained to the bi-Langmuir model. This gave the saturation capacity and the binding constant of the nonselective contribution for the two enantiomers. It also gave these parameters for the enantioselective contributions of each of them. The dependence of these parameters on the pH is discussed and interpreted in terms of the retention mechanism. Our conclusions are in excellent agreement with recent, independent results on the structure of the protein obtained by X-ray crystallography.
01/1999;
