Chiral zwitterionic stationary phases based on Cinchona alkaloids and dipeptides: Application in chiral separation of dipeptides under reversed phase conditions.

Chromatographic behavior of novel chiral stationary phases with bonded selectors based on Cinchona alkaloids modified with dipeptides was studied using dipeptides as probe molecules. Buffer-free and salt containing hydro-organic solutions were used as the mobile phases. The selectors exhibit pseudoenantiomeric behavior with respect to the L/D or LL/DD enantiomers and do not behave so with respect to the LD/DL enantiomers. The alkaloid part of the selectors is the driver of enantioselectivity, while the dipeptide substituent plays a modulating role. The quinidine-based selectors demonstrate stronger adsorption affinity and higher enantioselectivity as compared to the quinine-based selectors. The dipeptide analytes containing a glycyl fragment are weaker retained and their enantiomers are worse separated comparing to dipeptides with both units being larger amino acids. Moreover, a phenyl group in the structure of a dipeptide analyte facilitates enantioseparation. The effect of the mobile phase composition on retention depends on the hydrophobicity of an analyte. Hydrophobic dipeptides are better eluted by methanol-rich solvents, hydrophilic dipeptides are better eluted with water-rich solvents, and dipeptides with an intermediate hydrophobicity demonstrate a U-shaped or more complicated dependence of the retention factor on the percentage of methanol. Even a small buffer addition to the mobile phase decreases retention, but the ion-exchange mechanism was not confirmed. The effect of an electrolyte is rather due to the shielding of the charged groups of the selector reducing thereby electrostatic interaction between the selector and analyte. Efficiency of the novel columns is comparable to that of other brush-type chiral columns, the highest achieved number of the theoretical plates per 1 m varying between 30,000 and 40,000.

Elucidation of retention mechanism of dipeptides on a ristocetin A-based chiral stationary phase using a combination of chromatographic and molecular simulation techniques.

Two chiral stationary phases virtually reproducing the Nautilus-R column were modeled in silico to study the enantiorecognition mechanism of some selected dipeptides, taking into consideration the two different anchoring alternatives to the silica layer involving the two ristocetin A amino groups. A mobile phase composed of water-methanol (40:60, v/v) was included in the system. The analyses of the trajectories supported the experimental L(LL)

Enantioselective adsorption dynamics of leucyl-leucine in a Chirobiotic R column.

The dynamics of adsorption of the Leu-Leu stereoisomers in a chromatographic column packed with the Chirobiotic R chiral stationary phase bearing grafted antibiotic ristocetin A was studied by means of measurement and analysis of van Deemter plots. Similar measurements were carried out with weakly retained Gly-Gly for the sake of comparison. The bulk diffusion coefficients of the investigated dipeptides were also determined. It is found that the van Deemter plots of both the Leu-Leu stereoisomers and Gly-Gly have an uncommon convex-upward shape. Besides, the van Deemter B coefficients for the Leu-Leu stereoisomers, but not for Gly-Gly, have unusually high values. It is suggested that a high transcolumn contribution to eddy dispersion, which turned out to be enantioselective, accounts for these findings. Adsorption kinetics of all the dipeptides considered is relatively slow, the adsorption rate constant (kads) being of order of magnitude 20-60 s-1. kads does not depend on the configuration of Leu-Leu stereoisomers, although their affinity toward the chiral selector depends on this factor. This supports the above hypothesis that eddy dispersion is mainly responsible for the observed peculiarities in the dynamic behavior of dipeptides, and adsorption kinetics has secondary importance in this phenomenon.

Enantioselective retention mechanisms of dipeptides on antibiotic-based chiral stationary phases. II. Effect of the methanol content in the mobile phase.

Effect of mobile phase (water-methanol) composition on the enantioseparation of dipeptides on the chiral stationary phase Chirobiotic R was investigated using Ala-Ala, Leu-Leu, Gly-Leu, and Leu-Gly as case studies. The lipophilicity of dipeptides was found to be an essential factor in the dependence of their retention on the methanol percentage, the retention factor of lipophobic dipeptides increasing monotonously and that of lipophilic dipeptides changing according to an asymmetric U-shaped trajectory as methanol concentration increases. The behavior of enantioselectivity as a function of the methanol content also depends on the lipophilicity of dipeptide. For lipophilic Leu-Leu, the dependence has a dome-like shape, and for more lipophobic dipeptides, Ala-Ala and Gly-Leu, it is an increasing function of the methanol concentration. The importance of solvation equilibria in the bulk liquid and on the surface of the stationary phase for the total retention is discussed from the thermodynamic point of view. Special consideration is given to the adsorption of the water-methanol mixture on the surface of the Chirobiotic R stationary phase.

Enantioselective retention mechanisms of dipeptides on antibiotic-based chiral stationary phases: Leucyl-leucine, glycyl-leucine, and leucyl-glycine as case studies.

Chromatographic behaviors of dipeptides consisting of leucine and glycine were studied on two antibiotic-based chiral stationary phases (CSPs) with teicoplanin (Chirobiotic T) or ristocetin A (Chirobiotic R) as chiral selectors under reversed-phase conditions. The effect of mobile phase pH on the retention of stereoisomers of dipeptides was investigated and thermodynamic characteristic of adsorption were measured at different pH values. It was shown that the retention of dipeptides depends on the ionization of their molecules in the mobile phase, as different ionic forms have different affinity towards antibiotic selectors. Enantioselectivity of the bound antibiotics with respect to Leu-Leu stereoisomers was achieved via steric modulation of ion-ion interactions between the solute and the selector, while in the case of Gly-Leu enantiomers non-ionic interactions such as hydrogen bonding might play the key role. In both cases, the dipeptides terminating in D-Leu were retained stronger than their optical antipodes, whereas the enantiomers of Leu-Gly were hardly separated. The regression analysis of the retention data applying the Horvath-Melander-Molnar model revealed that different types of enantioselectivity resides in particular ionic forms of the compounds: cations are responsible for the separation of diastereomeric pairs and the anionic and zwitterionic forms have a universal enantioselectivity on the Chirobiotic T CSP, and the anions and zwitterions are the enantioselective forms for the Chirobiotic R CSP.

Unusual van Deemter plots of optical isomers on a chiral brush-type liquid chromatography column.

Unusual dynamic behavior of the enantiomers of 9-bromo-11b-(tert-butyl)-2,3,6,11b-tetrahydrooxazolo[3',2':1,5]pyrrolo[3,4-b]quinoline-5,11-dione (I) was observed on a Nautilus-R column packed with silica grafted with antibiotic ristocetin. It consisted in (i) antibatic behavior of the van Deemter plots of the enantiomers and (ii) high and strongly enantiomer dependent values of the A- and B-terms of the van Deemter equation. Although rare, such a pattern has been found earlier in chiral chromatography, with all reported cases limited to brush-type chiral stationary phases. Adsorption dynamics in this system was studied by means of the moment method and the peak parking technique; hydrodynamic properties of the column were explored by using unretained tracers. It was shown that the peculiar shape of the van Deemter curves for the enantiomers of I is conditioned by imperfect packing of the stationary phase, which result in high transcolumn eddy dispersion, and by slow adsorption/desorption kinetics. It was proven that the whole void volume of the column available to an eluent is not accessible to the studied analyte because it cannot penetrate the space between neighboring grafted ligands. Its mass transfer in pores is also affected by the fact that the stagnant layer of the binary water-acetonitrile mobile phase differs in composition from the bulk liquid due to preferential adsorption of water that influences the apparent molecular diffusivity of solutes. An effect of the structures of analyte and chiral selector on the adsorption kinetics is also briefly discussed.

Van't Hoff analysis in chiral chromatography.

The present review discusses the theory and application of van't Hoff analysis in chiral chromatography, with main focus on liquid chromatography. The topics considered include the physical meaning of van't Hoff equation's parameters, interpretation of thermodynamic data in terms of retention and enantioseparation mechanisms, abnormal behavior of van't Hoff plots, and best practices to avoid biased results.

Adsorption of aqueous organic mixtures on a chiral stationary phase with bound antibiotic eremomycin.

The adsorption of two typical hydro-organic mobile phases, with methanol and acetonitrile as the organic component, on an antibiotic based chiral stationary phase (CSP) Nautilus-E was studied by the minor perturbation method. In both cases, the excess adsorption of water was positive over a wide range of concentrations from 0 to ∼75 or 90 mol% for MeOH or MeCN containing mobile phases, respectively. Such hydrophilic properties of the CSP were attributed to multiple polar functional groups of the chiral ligand and to the residual silanol groups of the silica support. The adsorbed phase was found to be thinner for H2O-MeOH (∼1.1Å) and thicker for H2O-MeCN (∼9.4Å). The measurements of the column hold-up volume by different methods allowed us to suggest a model of the adsorbed phase consisting of the volume between bound chiral selectors inaccessible to large size molecules and of the stagnant layer of the mobile phase adsorbed on the external surface of the chiral selectors.

Effect of competing binding modes on retention in chromatography and capillary electrophoresis. A theoretical consideration.

A mathematical formalism has been developed to describe equilibrium in a system involving a single selector and a selectand molecule capable of binding to this selector by different modes. The generalization of this model to a multiselector system has also been considered. Applications of the developed equations in chromatography and CE are discussed.

The adsorption of Naproxen enantiomers on the chiral stationary phase (R,R)-Whelk-O1 under reversed-phase conditions: the effect of mobile phase composition.

The adsorption of the Naproxen enantiomers on the Whelk-O1 chiral stationary phase from acidic (0.01 M CH3COOH) methanol-water solutions was studied using the frontal analysis method and band profile calculations. The adsorption model that best accounted for these data is one assuming the coexistence of three types of adsorption sites, two of them being enantioselective, associated with the chiral selectors, the third type being the nonselective type of sites associated with residual surface silanol groups. The adsorption equilibrium is influenced by secondary processes that result in variations of the model parameters with the mobile phase composition. Several qualitative correlations between the model parameters and the methanol-water ratio were observed and are discussed.

Retention of Naproxen enantiomers on the chiral stationary phase Whelk-O1 under reversed-phase conditions. A reconsideration of the adsorption mechanism in the light of new experimental data.

A model of adsorption of the Naproxen enantiomers on the (S,S)-Whelk-O1 chiral stationary phase that we recently proposed was found to be inconsistent with new experimental data. A new model taking into account the variation of the dissociation coefficient of the analyte during elution of the band is discussed.