Enantiomer separation by countercurrent chromatography using cinchona alkaloid derivatives as chiral selectors

Cinchona-derived anion-exchange-type chiral selectors have been adapted and employed in countercurrent chromatography (CCC) for the separation of enantiomers of N-derivatized amino acids and 2-aryloxypropionic acids. The accurate optimization of the enantioseparation in terms of solvent system composition, pH values, ionic strength, and CCC operating conditions was performed. A wide range of solvent mixtures was evaluated. Successful resolutions were achieved in systems such as ammonium acetate buffer/tert-amyl alcohol/methanol/heptane and especially ammonium acetate buffer/methyl isobutyl ketone or diisopropyl ether. Up to 300 mg (0.92 mmol) of N-(3,5-dinitrobenzoyl)-(+/-)-leucine was totally resolved in a single run using a 10 mM concentration of chiral selector in 122 mL of stationary phase. This amount could be increased up to 900 mg (2.77 mmol) when pH-zone-refining mode was applied. The results here presented account for the high potential of CCC as a preparative enantiomer separation technique.     

Direct resolution of enantiomers in high-performance immunoaffinity chromatography under isocratic conditions

This paper describes the application of stereoselective antibodies as tailor-made chiral selectors for the separation of enantiomers in HPLC under isocratic conditions. Stereoselective monoclonal antibodies to D- and L-alpha-amino acids, raised against protein conjugates of p-amino-D- and L-phenylalanine, were immobilized on a synthetic high-flow-through support material and used for rapid enantiomer separation of a number of amino acids at flow rates between 0.1 and 10 mL/min. Since separations could be performed in a mild buffer, column lifetime considerably exceeded that of classical immunoaffinity systems. Using an anti-D-amino acid antibody as chiral selector, the L-enantiomers eluted with the void volume, while the D-enantiomers eluted second. Inverted elution orders were obtained on chiral stationary phases prepared from an anti-L-amino acid antibody. These results demonstrate, for the first time, that antibody-based chiral stationary phases are useful for routine enantiomer separation under true high-performance chromatographic conditions.     

Enantiomeric Separation and Detection of 2-Arylpropionic Acids Derivatized with [(N,N-Dimethylamino)sulfonyl]benzofurazan Reagents on a Modified Cellulose Stationary Phase by High-Performance Liquid Chromatography

(RS)-2-Arylpropionic acids (2-APAs) were derivatized with the fluorogenic reagents, 4-[(N,N-dimethylamino)sulfonyl]-7-piperazino-2,1,3-benzoxadiazole (DBD-PZ) and 4-[[(N-hydrazinoformyl)methyl]-N-methyl]amino-7-[N,N-(dimethylamino)sulfonyl]-2,1,3-benzoxadiazole (DBD-COHz), and their enantiomeric separation by a chiral stationary phase high-performance liquid chromatography was investigated in the reversed-phase mode with H(2)O/CH(3)CN or H(2)O/MeOH as the mobile phase on a column of cellulose tris(3,5-dimethylphenyl carbamate) coated on a silica gel support (Chiralcel OD-R). The derivatives with DBD-PZ were enantiomerically separated well under the elution condition of H(2)O/MeOH, based on the π-π interaction between the derivatives and the stationary phase. The rigid and bulky structure of DBD-PZ was demonstrated to be more effective as compared to the less rigid ones. The derivatives with DBD-COHz were more efficiently separated into each enantiomer with H(2)O/CH(3)CN as the eluent. The effective separation was based on hydrogen-bonding interaction between the acid hydrazide of the derivatives and the carbamoyl moiety of the stationary phase. There was a reversal in the elution order of the enantiomers between the two fluorescent derivatives. The detection limits obtained for each enantiomer were approximately 10-30 fmol on column. The derivatization with the reagent and the concomitant use of the reversed-phase and chiral stationary-phase HPLC were demonstrated to be useful for the enantiomeric quantification in rat plasma after intravenous administration of flurbiprofen racemate, a representative of 2-APAs.     

Tuneable peak deformations in chiral liquid chromatography

Modern chiral stationary phases are often combined with eluents comprising a mixture of organic solvents and polar additives. The latter may cause extreme deformations of the eluted enantiomer bands in both analytical and preparative separations. In this work, we give a theoretical background for these deformations. As an experimental verification, we separate the enantiomers of different beta-blockers on a teicoplanin stationary phase (Chirobiotic T) in the presence of triethylamine/acetic acid. We show that it is possible to tune the peak shapes of the two enantiomers by varying the organic solvent composition. An advantageous situation occurs when the first eluted peak is transformed to an anti-Langmuirian shape while keeping the second enantiomer in a normal Langmuirian shape. In this situation, the two peaks tail in opposite directions with their sharp sides pointing closely to each other. It is then possible to obtain baseline resolution at higher load than when both enantiomer peaks tail in the same direction. Adsorption isotherm parameters were determined using the inverse method; no other method could be used due to the system complexity. Computer simulations, based on these parameters, agreed very well with the observed deformations, thus confirming our hypothesis of their origin.     

Prediction for the separation efficiency of a pair of enantiomers during chiral high-performance liquid chromatography using a quartz crystal microbalance

A simple and rapid screening method of the chiral stationary phase during high-performance liquid chromatography (HPLC) utilizing a quartz crystal microbalance (QCM) has been developed for the chiral separation of a pair of enantiomers. The outline of the method is as follows: a self-assembled monolayer (SAM) is constructed on the gold electrodes of the QCM sensor chips by utilizing the interaction between thiols and gold. The chiral selectors used as chiral stationary phases in the HPLC are then immobilized, and a pseudostationary phase is constructed on the electrodes. Subsequently, the sensors are equilibrated in the solutions, the targeted chiral samples are injected, and the frequency changes are observed. Four kinds of chiral molecules and three kinds of chiral stationary phases were examined in this study. When chiral separation is possible using the chiral stationary phase immobilized on the sensors, significant differences in the frequency changes are observed because the intensities based on interactions differ among the isomers. The developed method can predict not only the possibility for chiral separation but also the elution order from the chiral stationary phase column. Furthermore, the degree of the mutual separation of a pair of enantiomers seems to be roughly predictable from the difference in the frequency change (DeltaF) and first-order association rate constant (k(obs)). The method does not require several different kinds of chiral columns that are more expensive than achiral ones such as the octadecylsilica (ODS) column. The required amounts of the chiral stationary phases are extremely small, and the sensors with immobilized chiral selectors are reusable. In addition, the method requires only a few minutes to complete the analysis. Thus, considerable reductions in both cost and time are realized. By applying the developed method to many chiral molecules and chiral stationary phases, its superiority may be corroborated; thus, it is expected that the method can be effectively used for the selection of chiral stationary phases.     

Direct high-performance liquid chromatographic separation of peptide enantiomers: study on chiral recognition by systematic evaluation of the influence of structural features of the chiral selectors on enantioselectivity

All-R/all-S enantiomers of oligoalanines (Ala(n), n = 1-10) with N-terminal protection group have been separated by HPLC on chiral stationary phases based on various cinchona alkaloid selectors. Structure-enantioselectivity relationships derived by extensive selector structure optimization provided insights into binding mechanisms and chiral recognition. Their interpretation was supported by X-ray crystal structures of amino acid and dipeptide, respectively, in complex with chiral selector. Optimized selectors have bulky elements representing steric barriers and deep binding pockets that afforded very high enantioselectivities; e.g., for the all-R and all-S enantiomers of N-(3,5-dinitrobenzoyl)alanylalanine, an alpha-value of 20.0 (corresponding to deltadeltaG of -7.43 kJ/mol) was obtained with a chiral stationary phase based on 6'-(neopentoxy)-9-O-tert-butylcarbamoylcinchonidine. Further, a chiral stationary phase based on 1,4-bis(9-O-quinidinyl)phthalazine was able to distinguish between the all-R and all-S enantiomers of hepta- to decaalanine peptides with enantioselectivity values between 1.8 and 1.9, corresponding to deltadeltaG of -1.46 and -1.59 kJ/mol, respectively.     

Theory and use of the pseudophase model in gas-liquid chromatographic enantiomeric separations

The theory and use of the "three-phase" model in enantioselective gas-liquid chromatography utilizing a methylated cyclodextrin/polysiloxane stationary phase is presented for the first time. Equations are derived that account for all three partition equilibria in the system, including partitioning between the gas mobile phase and both stationary-phase components and the analyte equilibrium between the polysiloxane and cyclodextrin pseudophase. The separation of the retention contributions from the achiral and chiral parts of the stationary phase can be easily accomplished. Also, it allows the direct examination of the two contributions to enantioselctivity, i.e., that which occurs completely in the liquid stationary phase versus the direct transfer of the chiral analyte in the gas phase to the dissolved chiral selector. Six compounds were studied to verify the model: 1-phenylethanol, alpha-ionone, 3-methyl-1-indanone, o-(chloromethyl)phenyl sulfoxide, o-(bromomethyl)phenyl sulfoxide, and ethyl p-tolylsulfonate. Generally, the cyclodextrin component of the stationary phase contributes to retention more than the bulk liquid polysiloxane. This may be an important requirement for effective GC chiral stationary phases. In addition, the roles of enthalpy and entropy toward enantiorecognition by this stationary phase were examined. While enantiomeric differences in both enthalpy and entropy provide chiral discrimination, the contribution of entropy appears to be more significant in this regard. The three-phase model may be applied to any gas-liquid chromatography stationary phase involving a pseudophase.     

Enantioselective interactions at the solid-liquid interface of an HPLC column under working conditions

A technique is presented which allows studying the enantioselective interactions occurring at the solid-liquid interface of a chiral stationary phase (CSP) and a racemate relevant to high performance liquid chromatography (HPLC). A conventional chiral column (Chiralpak AS) was mounted on an attenuated total reflection-infrared (ATR-IR) cell mimicking an HPLC setup equipped with an ATR-IR detector. Racemic pantolactone (PL) was used as the selectand. This setup in combination with modulation excitation spectroscopy (MES) allows for the identification of inter- and intramolecular hydrogen bonds being crucial for enantioseparation under HPLC operation conditions. The method is based on a two step strategy. In a first step, the enantiomers are separated by the chiral column similar to a standard HPLC experiment and upon adsorption on the identical CSP deposited on the internal reflection element (IRE), they are detected by ATR-IR spectroscopy. This experiment provides a retention time for each enantiomer. From the difference in retention, a suitable frequency is calculated which is used in a second experiment where the racemate concentration is varied alternately (modulation) in a way that the pulses of ( R)-PL and ( S)-PL exhibit a phase lag of 90 degrees after elution through the column. This procedure allows one to gain separate information of the enantioselective selectand-CSP interaction after performing a demodulation similar to a phase sensitive detection (PSD). A further benefit of this method is the strong enhancement of the signal-to-noise ratio. The effectiveness of the method is demonstrated by investigating the observed faster decrease in retention time of the later-eluted ( R)-PL, as compared to ( S)-PL, when separating at higher temperatures (from 12 to 36 degrees C). The origin is attributed to a weakening of a specific hydrogen bond between the C=O of ( R)-PL and the N-H of the CSP.     

Measurement of solution-phase chiral molecular recognition in the gas phase using electrospray ionization-mass spectrometry

Development of chiral selectors (SOs) is important both for understanding chiral molecular recognition processes and for their use in the separation of chiral species (selectands). Their evaluation by chromatographic procedures (e.g., as chiral stationary phase) can, however, be time-consuming. In this respect, electrospray ionization-MS (ESI-MS) is tested here as a possible alternative for screening enantioselective binding by SOs. The set of well-characterized cinchona alkaloid SOs are investigated with respect to their enantioselective binding to a set of model enantiomers, dinitrobenzoyl-(R)- and dinitrobenzoyl-(S)-leucine. MS-based enantioselectivity values from normalized gas-phase ion abundances for the diastereomeric complexes are compared empirically to chromatographic (HPLC) enantioselectivity results and shown to be consistent. Investigations into the fundamentals of measuring unbiased enantioselectivity values in the limit of dilute solution by correlation between experimental and modeled theoretical data are shown. Titration experiments are used to extract binding constants and are compared with published calorimetric (ITC) data. Results show that while the magnitude of binding affinities determined for various diastereomeric complexes is attenuated, the relative ranking and stereochemical preference in binding are consistently reproduced. This work represents a fundamental study of solution- versus gas-phase correlation for enantioselective systems by ESI-MS and indicates that, although not all questions and assumptions can be clearly engaged, for these enthalpically driven binding systems, the relative degree of binding affinity and selectivity is preserved.     

Separation and purification of methadone enantiomers by continuous- and interval-flow electrophoresis

Continuous- or free-flow electrophoresis is based upon a thin film of fluid flowing between two parallel plates. The electrolytes and the sample are continuously admitted at one end of the electrophoresis chamber and are fractionated by an array of outlet tubes at the other. Using the Octopus apparatus in a horizontal position, continuous preparative separation of methadone enantiomers in the presence of (2-hydroxypropyl)-β-cyclodextrin as a chiral selector was investigated under conditions of continuous-flow zone electrophoresis and continuous-flow isotachophoresis. The enantiomeric composition of methadone in the collected fractions was assessed by chiral capillary electrophoresis and circular-dichroism spectroscopy. In both electrophoretic modes, partial separation of the two enantiomers with an enrichment of about 80% and a throughput of 10-20 mg of racemic methadone per hour was obtained. Operating the Octopus apparatus with interrupted buffer flow during electrophoresis, a process termed interval-flow electrophoresis, resulted in complete separation of milligram quantities of the two methadone enantiomers. Furthermore, commencing with racemic methadone, continuous multistage isotachophoretic processing is shown to be suitable to purify (R)-(-)-methadone, the enantiomer with higher pharmacological activity, on a mg/h scale and at a mM concentration in the collected product stream.     

Homochiral Zeolitic Imidazolate Framework with Defined Chiral Microenvironment for Electrochemical Enantioselective Recognition

The recognition and separation of chiral molecules with similar structure are of great industrial and biological importance. Development of highly efficient chiral recognition systems is crucial for the precise application of these chiral molecules. Herein, a homochiral zeolitic imidazolate frameworks (c-ZIF) functionalized nanochannel device that exhibits an ideal platform for electrochemical enantioselective recognition is reported. Its distinct chiral binding cavity enables more sensitive discrimination of tryptophan (Trp) enantiomer pairs than other smaller chiral amino acids owing to its size matching to the target molecule. It is found that introducing neighboring aldehyde groups into the chiral cavity will result in an inferior chiral Trp recognition due to the decreased adsorption-energy difference of D- and L-Trp on the chiral sites. This study may provide an alternative strategy for designing efficient chiral recognition devices by utilizing the homochiral reticular materials and tailoring their chiral environments.     

Electroosmotic flow-based pump for liquid chromatography on a planar microchip

An electroosmotic flow (EOF)-based pump, integrated with a sol-gel stationary phase located in the electric field-free region of a microchip, enabled the separation of six nitroaromatic and nitramine explosives and their degradation products via liquid chromatography (LC). The integrated pump and LC system were fabricated within a single quartz substrate. The pump region consisted of a straight channel (3.0 cm x 230 microm x 100 microm) packed with 5-microm porous silica beads. The sol-gel stationary phase was derived from a precursor mixture of methyltrimethoxy- and phenethyltrimethoxysilanes and was synthesized in the downstream, field-free region of the microchip, resulting in a stationary-phase monolith with dimensions of 2.6 cm x 230 microm x 100 microm. Fluid dynamic design considerations are discussed, especially as they relate to integrating the EOF pump with the LC system. Pump and separation performance, as characterized by flow rate measurements, injection, elution, separation, and detection, point to a viable analytical chemistry platform that encompasses all of the benefits expected of portable, laboratory-on-chip systems, including reduced sample requirements and small packaging.     

Adsorption mechanisms in normal-phase chromatography. Mobile-phase modifier adsorption from dilute solutions and displacement effect

The adsorption equilibria of methanol from diluted solutions of the alcohol in dichloromethane (the weak solvent of the binary mixture) have been investigated in normal-phase conditions on an hybrid polymeric chiral stationary phase. This was done by analyzing the shapes of nonlinear perturbations of methanol, measured with a refractive index detector from a column in equilibrium with pure dichloromethane. Experiments have been set up to demonstrate the displacement effect, predictable from the adsorption data, by a nonlinear pulse of methanol on a racemic mixture of temazepam injected at trace level. A most used method in fundamental studies of band behavior in chromatography has been used for modeling the system. An extraordinary increase of the apparent column efficiency has been observed due to the displacement phenomenon, with the peak of the second eluted enantiomer noticeably higher and narrower than that of the first eluted component.     

Complete enantiomeric separation of phenylthiocarbamoylated amino acids on a tandem column of reversed and chiral stationary phases

The enantiomeric separation of phenylthiocarbamoyl derivatives of amino acids (PTC-AAs) was studied on a series of reversed phase HPLC columns coupled to the chiral phase HPLC columns. First, the five chiral phases (native, 0.2, 3.3, 7.5 and 16.9 phenylcarbamoylated/β-cyclodextrins, Ph/CD) were newly prepared by modification of β-cyclodextrin with phenyl isocyanate and were examined for the enantiomeric separation of PTC-AAs. Among them, the 3.3Ph/CD phase gave the best enantiomeric separation (α ≥ 1.04). However, the separation of the individual PTC-AAs was not sufficient. Next, these separations were investigated on various reversed phase HPLC columns, and octyl silica was selected in terms of the suitability of the mobile phase adopted for the enantiomeric separation mentioned above. The effects of the column temperature, the ion-pairing reagent, and the final content of methanol were also studied on the tandem column of octyl silica and the 3.3Ph/CD phase. Under the best conditions (100 mM ammonium acetate (pH 6.5) containing 1 mM butanesulfonate with 0-40% methanol as the mobile phase), all the individual PTC-AAs were well separated within 150 min. The applicability of the method was demonstrated by the sequence/configuration analysis of a peptide containing a d-amino acid ([d-Thr(2)]leucine enkephalin-Thr).     

An alternative procedure to screen mixture combinatorial libraries for selectors for chiral chromatography

An alternative process for the analysis of mixture library components for their potential as selectors for chiral chromatography is described. The procedure involves the immobilization of each enantiomer of the target racemic analyte to silica gel, followed by incubation of each resulting stationary phase with a mixture library. The adsorbed library components on the two stationary phases are then analyzed by reversed-phase liquid chromatography. A comparison of the resulting two chromatograms is made. Any peak of identical retention time but with a significant difference in intensity in the two chromatograms indicates that this component is most likely a chiral selector. Its chemical structure is then determined by LC-MS or LC-MS-MS. This new screening method significantly increases the efficiency of chiral selector determination by eliminating the need for multilibrary syntheses, as opposed to our previous method. This technique should also allow for the screening of much larger libraries as compared to our previous work.     

萘普生分子印迹拆分过程热力学研究

采用4-乙烯基吡啶为功能单体,三甲氧基丙烷三甲基丙烯酸酯为交联剂,制备出(S)-萘普生的分子印迹聚合物,并将其用作高效液相色谱固定相,对其进行了评价.研究了流动相流速、不同功能单体和柱温对拆分效果的影响,测定出分离过程的热力学参数,证明萘普生分子印迹拆分过程是焓控过程.     

Chiral separations performed by enhanced-fluidity liquid chromatography on a macrocyclic antibiotic chiral stationary phase

The use of enhanced-fluidity liquid chromatography (EFLC) for chiral separations was demonstrated on a macrocyclic antibiotic column, Chirobiotic-V. This technique was compared to high performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC) for the separation of chiral compounds in normal-phase mode. The highest resolution was always observed for EFLC condition. Higher efficiency and shorter retention time were also observed for most separations with portions of CO(2) in the range of 0-50 mol %. Larger amounts of CO(2) caused efficiency to decrease and retention time to be prolonged. For some separations, the temperature was elevated to bring the mobile phase to the supercritical condition. Improved efficiency was obtained in SFC, whereas resolution and selectivity were worse. The use of EFLC in reversed-phase chiral separations was also tested. Enantiomer resolution improved under the EFLC condition. For the tested methanol/H(2)O mixture, fluoroform provided more significant improvements in chromatographic performance than CO(2) when used as a fluidity enhancing liquid. The use of EFLC instead of HPLC also caused a markedly lower pressure drop across the column for commonly used flow rates. The low-pressure drop will allow the use of longer columns or multiple columns to increase the total efficiency of the separation. Since chiral columns are often inefficient, this attribute may be very important for chiral separations.     

Structure-function relationships in high-density octadecylsilane stationary phases by Raman spectroscopy. 2. Effect of common mobile-phase solvents

Raman spectroscopy is used to examine the effects of solvent, temperature, and surface grafting method (surface or solution polymerized) on alkyl chain rotational and conformational order in a series of high-density octadecylsilane stationary phases ranging in surface coverage from 3.09 to 6.45 micromol/m2. Rotational and conformational order is assessed using the intensity ratio of the antisymmetric to symmetric v(CH2) modes as well as the frequency at which these Raman bands are observed. Solvents studied include perdeuterated hexane, toluene, chloroform, tetrahydrofuran, benzene, methanol, acetone, acetonitrile, and water. Stationary-phase order was investigated at temperatures between 258 and 323 K. Alkyl chain rotational and conformational order, and hence, solvation of the stationary phase, is dependent on solvent parameters (polarity, size, etc.), temperature, and stationary-phase properties (polymerization method and surface coverage). Information on stationary-phase conformational order allows solvent-stationary-phase interactions to be described in terms of a combination of adsorption and partitioning models for reversed-phase liquid chromatography. Finally, a distinct interplay between solvent- and temperature-induced ordering of these stationary phases is documented that is also a function of solvent and stationary-phase properties.     

Tertiary amine appended derivatives of N-(3,5-dinitrobenzoyl)leucine as chiral selectors for enantiomer assays by electrospray ionization mass spectrometry

Derivatives of the chiral selector N-(3,5-dinitrobenzoyl)leucine were prepared and used as chiral selectors for enantiomer discrimination in single-stage electrospray ionization mass spectrometric experiments. The chiral selectors were designed to remove the ionization site from the sites required for effective chiral recognition. Addition of a chiral analyte to a solution of the two pseudoenantiomeric chiral selectors, which differ in absolute stereochemistry and the length of the tether connecting the tertiary amine site used for ionization via protonation and the rest of the chiral selector, affords selector-analyte complexes in the electrospray ionization mass spectrum where the ratio of these complexes is dependent on the enantiomeric composition of the analyte. The relationship between the ratio of the selector-analyte complexes in the electrospray ionization mass spectrum and the enantiomeric composition of the analyte can be used to relate the extent of enantioselectivity that is being observed and for quantitative enantiomeric composition determinations. Investigations into the scope and limitations of this method, plus a comparison to the enantioselectivities observed by chiral HPLC using a N-(3,5-dinitrobenzoyl)leucine-derived chiral stationary phase, is presented.