Adsorption behavior of the (+/-)-Tröger's base enantiomers in the phase system of a silica-based packing coated with amylose tri(3,5-dimethyl carbamate) and 2-propanol and molecular modeling interpretation

The binary adsorption isotherms of the enantiomers of Tr?ger's base in the phase system made of Chiral Technologies ChiralPak AD [a silica-based packing coated with amylose tri(3,5-dimethyl carbamate)] as the chiral stationary phase (CSP) and 2-propanol as the mobile phase were measured by the perturbation method. The more retained enantiomer exhibits a S-shaped adsorption isotherm with a clear inflection point, the concentration of the less retained enantiomer having practically no competitive influence on this isotherm: In the entire range of concentrations studied, dq2/dC1 approximately 0. By contrast, the less retained enantiomer has a Langmuir adsorption isotherm when pure. At constant mobile phase concentrations, however, its equilibrium concentration in the adsorbed phase increases with increasing concentration of the more retained enantiomer and dq1/dC2 > 0. This cooperative adsorption behavior, opposed to the classical competitive behavior, is exceedingly rare but was clearly demonstrated in this case. Two adsorption isotherm equations that account for these physical observations were derived. They are based on the formation of an adsorbed multi-layer, as suggested by the isotherm data. The excellent agreement between the experimental overloaded elution profiles of binary mixtures and the profiles calculated with the equilibrium-dispersive model validates this binary isotherm model. The adsorption energies calculated by molecular mechanics (MM) and by molecular dynamics (MD) indicate that the chiral recognition arising from the different interactions between the functional groups of the CSP and the molecules of the Tr?ger's base enantiomers are mainly driven by their Van der Waals interactions. The MD data suggest that the interactions of the (-)-Tr?ger's base with the CSP are more favored by 8+/-(5) kJ/mol than those of (+)-Tr?ger's base. This difference seems to be a contributing factor to the increased retention of the - enantiomer on this chromatographic system. The modeling of the data also indicates that both enantiomers can form high stoichiometry complexes while binding onto the stationary phase, in agreement with the results of the equilibrium isotherm studies.     

High performance liquid chromatographic separation of dipeptide and tripeptide enantiomers using a chiral crown ether stationary phase

A chiral stationary phase (CSP) based on (-)-(18-crown-6)-2,3,11,12-tetracarboxylic acid was evaluated for the direct resolution of the enantiomers of dipeptides and tripeptides. The type and concentration of the acid and the methanol content were optimized with regard to retention time and resolution using Ala-Phe as model peptide. A mobile phase consisting of 10 mM sulfuric acid in 70% aqueous methanol was applied to the separation of a set of 16 structurally diverse dipeptides and tripeptides. Generally, the configuration of the amino acid at the N-terminus determined the enantiomer elution order. With a few exceptions the LL- and LD-enantiomers interacted stronger with the CSP compared to the corresponding DD- or DL-enantiomers. The experimental conditions also allowed the simultaneous separation of all four stereoisomers of Ala-Phe. Addition of ammonium sulfate generally reduced retention times and enantiomer resolution. Addition of triethylamine as modifier led to an overall increase of the retention times while the resolution did not show a general trend, increasing in the case of Ala-Ala but decreasing in the case of Ala-Phe.     

Determination of chromatographic separation parameters of tryptophan enantiomers on a Chirosil-SCA chiral stationary phase by using the inverse method based on the initial guesses estimated from elution by characteristic point method

An effective chiral stationary phase (CSP) for enantioseparation of amino acids was established previously by bonding (18-crown-6)-2, 3, 11, 12-tetracarboxylic acid to silica gel. This CSP has recently been commercialized under the name of Chirosil-SCA. As a first step for developing a Chirosil-SCA simulated moving bed chromatographic process for separation of tryptophan enantiomers, the adsorption isotherm and mass-transfer parameters of each tryptophan enantiomer on the Chirosil-SCA CSP were determined in this study while using only water as a mobile phase. For this task, inverse method (IM) was applied on the basis of the initial guesses estimated from elution by characteristic point (ECP) method, which was found to be more advantageous in the aspects of both accuracy and computational efficiency than the case of utilizing individually only IM or ECP method. The results revealed that the adsorption behavior of each tryptophan enantiomer on the Chirosil-SCA could be well described by the Langmuir-Freundlich isotherm. The model predictions based on the determined parameter values were in close agreement with the experimental chromatograms from a series of single-component or mixture pulse tests that were performed under various feed concentrations and flow rates. It was also found that the Langmuir-Freundlich isotherm parameters of each enantiomer were largely affected by temperature. Such a marked dependence of the parameters on temperature was investigated quantitatively. The results of such an investigation indicated that as the temperature decreases, the adsorption affinities of both enantiomers become higher and the heterogeneity of the Chirosil-SCA becomes more pronounced.     

Comparison of vancomycin-based stationary phases with different chiral selector coverage for enantioselective separation of selected drugs in high-performance liquid chromatography

Two vancomycin-based chiral stationary phases (CSPs) with different coverage of the chiral selector vancomycin (Chirobiotic V and Chirobiotic V2) were compared. beta-Blockers and profens, as structurally diverse groups of drugs, were chosen as analytes. Retention and enantioseparation of beta-blockers were studied in reversed-phase (RP) and polar-organic (PO) separation modes. Higher retention and better enantioresolution were obtained on the CSP with higher coverage of vancomycin in the both separation modes. Baseline separation of four beta-blockers (eight enantiomers) in the PO mode was achieved on the Chirobiotic V2 column within 15 min. The enantioseparation of profens did not bring so excellent and easy to interpret results. Higher retention of profens on the Chirobiotic V2 column was not always accompanied by an improvement of their chiral separation in the RP mode. The polar-organic mode was not suitable for these derivatives at all. The most interesting result was obtained with flobufen; its chiral center is further away from the rigid part of the molecule, which mostly causes difficulties in enantioselective recognition. Nevertheless, the enantiomers of flobufen were shown to be much better (baseline) resolved on the CSP with lower coverage of the chiral selector (Chirobiotic V).     

Capillary electrochromatographic separation of enantiomers under aqueous mobile phases on a covalently bonded cellulose derivative chiral stationary phase

A chemically bonded cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase (CSP) was prepared by a radical polymerization reaction. The prepared CSP was packed into fused-silica capillaries with inner diameter of 75 microm to perform enantiomer separations in CEC. The electrochromatographic behavior of the CSP was investigated. On the prepared CSP, high EOF could be generated under acidic mobile phases, which represented an advantage for the separation of acidic enantiomers. Several neutral, acidic, and basic enantiomers were resolved on the prepared CSP under aqueous mobile phases. The column efficiencies were between 20,000 and 100,000 plates/m, which were much higher than those of HPLC. In addition, it was observed that the separation of some enantiomers benefited from the adoption of THF as mobile phase modifier.     

Chiral HPLC separation and absolute configuration assignment of a series of new triazole compounds

A series of novel chiral triazole compounds were synthesized. They were separated into enantiomers by liquid chromatography on an amylose tris(3,5-dimethylphenylcarbamate) (ADMPC) chiral stationary phase (CSP). The absolute configuration of each enantiomer of the investigated compounds was established by combined use of chemical correlation, chiral HPLC and circular dichroism (CD) spectra analysis methods. The influence of the mobile-phase modifiers and the structure of chiral triazole compounds on the chiral separation and retention were investigated. Reversal of the elution order of some enantiomeric pairs upon using different mobile-phase modifier was observed. The temperature effect on the chiral separation and the thermodynamic properties including enthalpy and entropy change of binding to the ADMPC-CSP were also investigated.     

Contribution of ionically immobilized bovine serum albumin to the retention of enantiomers.

The retention of the enantiomers of mandelic acid and N-benzoylalanine was studied on columns prepared by immobilizing bovine serum albumin (BSA) on an anion exchanger. The amount of BSA fixed on the column is easy to adjust and measure. The adsorption isotherms were determined. For each enantiomer, the isotherm is well accounted for by a bi-Langmuir equation. One term of the isotherm (which is the same for both enantiomers) corresponds to non-selective interactions and the other term to the chiral selective interactions. The column saturation capacity of this second term is 8% larger for the less strongly retained enantiomer. This saturation capacity corresponds approximately to one enantiomer molecule adsorbed for five BSA molecules immobilized. This result is in agreement with the assumption of the hydrophobic cavity of BSA being the chiral selective site.     

Measurement and modeling of the equilibrium behavior of the Tröger's base enantiomers on an amylose-based chiral stationary phase

The binary isotherms of the two enantiomers of Tr?ger's base were measured on a system made of pure 2-propanol as the mobile phase and of Chiralpak AD, a chiral stationary phase (CSP) based on amylose tri-(3,5-dimethylphenyl carbamate). The experimental data were acquired using both frontal analysis and the perturbation method. The results obtained are most unusual. The adsorption of the more-retained (-)-enantiomer is not competitive: the amount adsorbed onto the CSP at equilibrium with a constant concentration of the (-)-enantiomer is independent of the concentration of the (+) enantiomer. On the other hand, the adsorption of the less-retained enantiomer is cooperative: the amount of this (+)-enantiomer adsorbed by the CSP at equilibrium with a constant concentration of this enantiomer increases with increasing concentration of the (-)-enantiomer. Such a phenomenon has hardly ever been reported. A model equation is proposed that accounts well for all these isotherm data.     

A new application of stopped-flow chiral HPLC: inversion of enantiomer elution order

A newly developed procedure to reverse the enantiomer elution order of compounds resolved on chiral stationary phases (CSPs) for HPLC is presented. The optimized analytical protocol is based on the effect of temperature on enantioselectivity and does not involve any changing in mobile phase composition or type of CSP. In essence, the approach entails variable temperature chromatography at two temperatures. The enantiomer separation is performed at a low column temperature, with stopping the flow prior to elution of the less retained enantiomer. Then, the column temperature is changed with the peaks trapped inside the column, followed by elution with the same mobile phase in reverse direction. Under these conditions, the more pronounced loss in free energy of binding for the more strongly bound enantiomer results in an inversion of the elution order. This procedure may be applied to each enantiomer pair that is separated by chiral HPLC under an appreciable enthalpy-control.     

Analysis of the band profiles of the enantiomers of phenylglycine in liquid chromatography on bonded teicoplanin columns using the stochastic theory of chromatography.

The retention behaviour of the enantiomers of underivatized phenylglycine was studied on a Chirobiotic T column packed with amphoteric glycopeptide teicoplanin covalently bonded to the surface of silica gel. The retention and the selectivity of separation of the enantiomers increase with rising concentration of ethanol or of methanol in aqueous-organic mobile phases. The band profiles of the less retained L-phenylglycine are symmetrical, but the band profiles of the more strongly retained D-phenylglycine are tailing in all mobile phases tested. The band broadening does not diminish even at very low concentrations of phenylglycine, so that it cannot be attributed to possible column overload. The analysis of the band profile using the stochastic theory of chromatography suggests that the broadening can be attributed to at least two additional chiral centres of adsorption in the stationary phase contributing to the retention of the more strongly retained enantiomer in addition to the adsorption of the less retained one. This behaviour can be explained by the complex structure of the teicoplanin chiral stationary phase.     

Preparation of a positively charged cellulose derivative chiral stationary phase with copolymerization reaction for capillary electrochromatographic separation of enantiomers

A positively charged chiral stationary phase (CSP) was prepared by chemically immobilizing cellulose 3,5-dimethylphenylcarbamate onto methacryloyldiethylenetriaminopropylated silica (MCDEAPS) via a radical copolymerization reaction. The prepared CSP was evaluated for enantiomer separation in nonaqueous capillary electrochromatography (CEC). Electroosmotic flow (EOF) generated on the prepared CSP could be significantly improved with introduction of positive charges into the CSP, and separation of enantiomers in CEC has been achieved with mobile phases of ethanol and hexane-ethanol, respectively. In addition, we investigated the solvent versatility of the immobilized CSP on enantioseparations in CEC and capillary liquid chromatography (CLC) due to the elimination of dissolution of chiral selector in a number of solvents. Chiral resolution of some enantiomers was improved by adopting tetrahydrofuran (THF) and chloroform as mobile-phase modifiers, respectively.     

Separation of chiral sulfoxides by liquid chromatography using macrocyclic glycopeptide chiral stationary phases

A set of 42 chiral compounds containing stereogenic sulfur was prepared. There were 31 chiral sulfoxide compounds, three tosylated sulfilimines and eight sulfinate esters. The separations were done using five different macrocyclic glycopeptide chiral stationary phases (CSPs), namely ristocetin A, teicoplanin, teicoplanin aglycone (TAG), vancomycin and vancomycin aglycone (VAG) and seven eluents, three normal-phase mobile phases, two reversed phases and two polar organic mobile phases. Altogether the macrocyclic glycopeptide CSPs were able to separate the whole set of the 34 sulfoxide enantiomers and tosylated derivatives. Five of the eight sulfinate esters were also separated. The teicoplanin and TAG CSPs were the most effective CSPs able to resolve 35 and 33 of the 42 compounds. The three other CSPs each were able to resolve more than 27 compounds. The normal-phase mode was the most effective followed by the reversed-phase mode with methanol-water mobile phases. Few of these compounds could be separated in the polar organic mode with 100% methanol mobile phases. Acetonitrile was also not a good solvent for the resolution of enantiomers of sulfur-containing compounds, neither in the reversed-phase nor in the polar organic mode. The structure of the chiral molecules was compared to the enantioselectivity factors obtained with the teicoplanin and TAG CSP. It is shown that the polarity, volume and shape of the sulfoxide substituents influence the solute enantioselectivity factor. Changing the oxidation state of the sulfur atom from sulfoxides to sulfinate esters is detrimental to the compound's enantioselectivity. The enantiomeric retention order on the teicoplanin and TAG CSPs was very consistent: the (S)-(+)-sulfoxide enantiomer was always the less retained enantiomer. In contrast, the (R)-(-)-enantiomer was less retained by the ristocetin A, vancomycin and vancomycin aglycone columns, showing the complementarity of these CSPs. The macrocyclic glycopeptide CSPs provided broad selectivity and effective separations of chiral sulfoxides.     

用半制备高效液相色谱法制备少量的阿苯达唑亚砜单一对映体

涂敷直链淀粉-三(3,5-二甲基苯基氨基甲酸酯)(ADMPC)于自制的球形氨丙基硅胶上,制备了手性固定相。用高效液相色谱法(HPLC)在正相条件下,用该固定相在分析柱上直接拆分了广谱驱虫药物阿苯达唑亚砜外消旋体(Albendazole Sulfoxide,ABZSO)。然后,将分析色谱方法扩展到了半制备色谱,进行该药物的半制备分离,考察了不同进样量对半制备色谱的参数的影响。在最大进样量下,83h制备了各约1g的阿苯达唑亚砜的两种单一对映体。( )ABZSO的产率大于98．0％,纯度大于99．9％;(-)ABZSO的产率大于94．0％,纯度大于99．0％。     

Intermolecular (1)H-(1)H Two-Dimensional Nuclear Overhauser Enhancements in the Characterization of a Rationally Designed Chiral Recognition System

Chiral stationary phases (CSPs) for liquid chromatography derived from N-(acyl)proline-3,5-dimethylanilides separate the enantiomers of N-(3,5-dinitrobenzoyl)-alpha-amino esters and amides with high levels of selectivity. These CSPs have been used to assemble a large body of chromatographic data which indirectly supports the validity of the mechanistic rationale originally used in the design of these CSPs. We herein report (1)H and (13)C chemical shift data obtained when the (S)-enantiomer of chiral solvating agent (CSA) 3, a soluble analogue of the selector used in CSP (S)-1, acts on each of the enantiomers of the dimethylamide of N-(3,5-dinitrobenzoyl)leucine, 2. The changes in chemical shift in the mixture of (S)-2 and (S)-3 support the existence of those interactions thought to be essential to chiral recognition in this system. In addition, significant intermolecular NOESY enhancements are observed in this mixture. These NOE data are consistent with the structure expected for the more stable diastereomeric adsorbate formed between (S)-2 and the (S)-proline-derived CSP 1. No intermolecular NOEs are observed for corresponding mixtures of the chiral solvating agent (S)-3 and (R)-2, the enantiomer least retained on (S)-CSP 1.     

New chiral crown ether stationary phase for the liquid chromatographic resolution of alpha-amino acid enantiomers

A new chiral stationary phase (CSP) for the liquid chromatographic separation of enantiomers was prepared by bonding a novel enantiopure (diphenyl-substituted 1,1'-binaphthyl) crown ether to 5 microm silica gel. The resulting CSP was applied to the separation of the enantiomers of various natural and unnatural alpha-amino acids. All alpha-amino acids tested were resolved very well on the new CSP, with the exception of proline, which does not contain a primary amino group. The resolution of alpha-amino acid enantiomers on this new CSP was found to be dependent on the type and amounts of organic and acidic modifiers, and on column temperature.     

Characterization of an unusual adsorption behavior of racemic methyl-mandelate on a tris-(3,5-dimethylphenyl) carbamoyl cellulose chiral stationary phase

An interesting adsorption behavior of racemic methyl mandelate on a tris-(3,5-dimethylphenyl)carbamoyl cellulose chiral stationary phase was theoretically and experimentally investigated. The overloaded band of the more retained enantiomer had a peculiar shape indicating a type V adsorption isotherm whereas the overloaded band of the less retained enantiomer had a normal shape indicating a type I adsorption behavior. For a closer characterization of this separation, adsorption isotherms were determined and analyzed using an approach were Scatchard plots and adsorption energy distribution (AED) calculations are combined for a deeper analysis. It was found that the less retained enantiomer was best described by a Tóth adsorption isotherm while the second one was best described with a bi-Moreau adsorption isotherm. The latter model comprises non-ideal adsorbate-adsorbate interactions, providing an explanation to the non-ideal adsorption of the more retained enantiomer. Furthermore, the possibility of using the Moreau model as a local model for adsorption in AED calculations was evaluated using synthetically generated raw adsorption slope data. It was found that the AED accurately could predict the number of adsorption sites for the generated data. The adsorption behavior of both enantiomers was also studied at several different temperatures and found to be exothermic; i.e. the adsorbate-adsorbate interaction strength decreases with increasing temperature. Stochastic analysis of the adsorption process revealed that the average amount of adsorption/desorption events increases and the sojourn time decreases with increasing temperature.     

Enantiomeric separation using temperature-responsive chiral polymers composed of L-valine diamide derivatives in aqueous liquid chromatography

This paper describes enantiomer separation by aqueous liquid chromatography using chiral stationary phases (CSPs) in which temperature-responsive polymers derived from acryloyl-L-valine N-methylamide (1) and its N,N-dimethylamide analogue (2) were bound on silica gel supports. The linear polymers composed of monomer 1 and monomer 2 are temperature-responsive in solution and their aggregation and extension states related to water solubility are reversible at particular critical temperatures. During chromatography, enantioselectivity and retentivity for solute enantiomers were controlled by column temperature, which changes the aggregation and extension states of the chiral polymers depending upon their interior hydrophobic nature. Two different types of CSPs were made: a temperature-responsive linear polymer derived from 3-mercaptopropyl silica gel, and another polymer cross-linked with ethylene dimethacrylate from 3-methacryloyloxypropyl silica gel. The former CSP could separate racemic N-(3,5-dinitrobenzoyl(DNB))amino acid isopropyl esters. Retention of the amino acid derivatives was prolonged with an increase in column temperature. Enantioselectivity was also enhanced with temperature increase until the particular critical temperature. The latter, cross-linked CSP could not provide enantioselectivity for the amino acid derivatives in aqueous media, although the chiral valine diamide moieties were effective for enantiomer separation in non-aqueous media. The degree of hydrophobicity and volume of the bonded phase formed by the polymers on the support surface was determined by measuring the fluorescence of pyrene.     

Enantiomeric separation of chiral pesticides by high-performance liquid chromatography on an amylose tris-(S)-1-phenylethylcarbamate chiral stationary phase

Amylose tris-(S)-1-phenylethylcarbamate chiral stationary phase (CSP) was prepared. The direct enantiomeric separation of chiral pesticides on this CSP had been studied by HPLC. The mobile phase was n-hexane-isopropanol at a flow rate of 1.0 mL/min. The effects of isopropanol content and column temperature on retention and enantioselectivity were investigated. Thirty-two samples were tested, of which ten interacted enantioselectively with the CSP. Five samples were completely resolved and another five underwent near-baseline or partial resolution. The enantiomers were identified by a circular dichroism detector. Linear van't Hoff plots were established and the thermodynamic parameters were thus calculated.