Extraordinary chiral discrimination in inclusion gas chromatography. thermodynamics of enantioselectivity between a racemic perfluorodiether and a modified gamma-cyclodextrin

The enantiomers of the perfluorodiether "compound B" [2-(fluoromethoxy)-3-methoxy-1,1,1,3,3-pentafluoropropane], a decomposition product of the inhalational anesthetic sevoflurane [2-(fluoromethoxy)-1,1,1,3,3,3-hexafluoropropane], were separated by gas chromatography on octakis(3-O-butanoyl-2,6-di-O-n-pentyl)-gamma-cyclodextrin (Lipodex E), dissolved in polysiloxane PS 255 (30% w/w), with an unexpectedly high separation factor of alpha = 10.6 at 26 degrees C. Using the concept of the retention increment R', non-enantioselective and enantioselective contributions to retention were separated and thus reliable thermodynamic parameters of enantioselectivity, i.e. - deltaS,R(deltaG) = 5.7 (0.05) kJ/mol at 303 K, - deltaS,R(deltaH) = 20.1 (0.64) kJ/mol, deltaS,R(deltaS) = -47.4 (2.0) J/K mol and T(isoenant) = 424 (30) K or approximately 150 degrees C, were determined by temperature-dependent measurements. The enantiomeric bias represents the largest values ever measured in enantioselective gas chromatography. An equation is presented which allows calculation of the non-enantioselective contributions to retention from measurements at two arbitrary concentrations of Lipodex E in polysiloxane. Surprisingly, the enantioselectivity is greatly reduced when employing the beta-cyclodextrin analogue and breaks down completely with the alpha-cyclodextrin analogue of Lipodex E.     

Chiral recognition of peptide enantiomers by cinchona alkaloid derived chiral selectors: mechanistic investigations by liquid chromatography, NMR spectroscopy, and molecular modeling

The chiral recognition mechanism of a cinchona alkaloid based chiral selector for N-protected peptide enantiomers was investigated. A chiral stationary phase derived from this selector was employed for liquid chromatographic enantiomer separations. It showed exceptionally high enantiomer discrimination for the (all-R)- and (all-S)-enantiomers of dialanine (alpha = 20), while a pronounced loss of chiral recognition occurred upon the insertion of an additional alanine residue into the peptide backbone. This reduction of enantioselectivity was investigated in great detail by NMR spectroscopy of complexes of the chiral selector and the analyte enantiomers accompanied by molecular modeling studies. Investigation of intramolecular NOEs provided the conformational states of the free and complexed forms of the selector. The analysis of complexation-induced shifts yielded information on intermolecular interactions and allowed us to propose binding models, which were further supported by the observation of intermolecular NOEs, indicating the relative arrangements of selector and analytes. Stochastic molecular dynamics simulations were able to reproduce the chromatographic retention orders and energy differences, as well as the intermolecular NOEs. The computational data were used to evaluate the intermolecular forces responsible for analyte binding. In addition, the relative contributions of the fragments of the chiral selector to the enantioselective binding event were assessed. A spatial arrangement of the chiral selector and the analyte allowing the primary ionic interaction as well as hydrogen bonding and pi-pi-stacking to take place simultaneously was found to be essential to obtain very high enantioselectivities.     

Apparent and true enantioselectivity of single- and binary-selector chiral stationary phases in gas chromatography

Almost all gas-chromatographic chiral stationary phases (CSPs) are complex systems containing one or more chiral selector(s) dissolved in, or bonded to, an achiral solvent such as squalane or poly(dimethylsiloxane). The presence of different components in the total CSP, interacting independently with the analyte enantiomers, impairs the elucidation of enantiorecognition mechanisms and complicates the optimization of enantioseparations. In the present work a quantitative analysis of the influence of different factors on the observed enantioselectivity is performed. The parameters varied in this study were the composition of the CSP, the concentration and the enantiomeric excess of the chiral selector(s) and the presence of achiral selectors (including racemic compositions). Special attention is given to the determination of distribution and association constants, as well as apparent and true enantioseparation factors.     

Use of immobilized amyloglucosidase as chiral selector in chromatography. Control of enantioselective retention and resolution in liquid chromatography

Summary Several mobile phase parameters were investigated for controlling enantioselective retention and resolution on a chiral stationary phase made in-house. The chiral selector was the enzyme amyloglucosidase, which was immobilized onto a silica support via reductive amination. The influences of the mobile phase pH, concentration and type of uncharged organic modifier, ionic strength and column temperature on enantios-electivity were studied. The analysis time for resolving enantiomers could be adjusted with only a minor decrease in enantioselectivity by using a high ionic strength mobile phase buffer. This indicated a retention mechanism involving ion-exchange interactions. It was further confirmed by the decreasing enantioselectivity of amines when using a mobile phase pH below the isoelectric point of the native protein. Interesting effects were observed when the organic modifier concentration was increased and also when the column temperature was raised. Both retention and enantioselectivity increased with increasing concentration of 2-propanol in the mobile phase. Examples are given where both enantioselectivity and retention increased with increasing column temperature. Thermodynamic studies were performed to calculate the entropy and enthalpy constants. The results showed that, depending on mobile phase composition, the enantioselective retention may be caused by differences in entropy or enthalpy.     

Study of tryptophan enantiomer binding to a teicoplanin-based stationary phase using the perturbation technique. Investigation of the role of sodium perchlorate in solute retention and enantioselectivity

The retention of D,L-tryptophan enantiomers on an immobilized teicoplanin column was investigated in relation to the mobile phase sodium perchlorate concentration using the perturbation method to determine the solute distribution isotherms. From the experimental data, it appeared that the bi-Langmuir model was able to describe D- and L-enantiomer retention on the immobilized selector over the salt concentration range. An increase in the apparent enantioselectivity with an increase in sodium perchlorate concentration was observed. The chiral recognition enhancement was governed by (i) an increase in the difference of the adsorption constants for binding to the high-affinity site (aglycone pocket) between the two enantiomers and (ii) enhancement of the number of aglycone chiral regions interacting with D-tryptophan. It is suggested that an ion-pair formation mechanism between perchlorate and solute and/or selector is responsible for this behavior. In addition, this work shows that additional secondary sites on the teicoplanin surface are involved in the apparent enantioselectivity at low sodium perchlorate concentrations.     

Nonlinear effects in enantioselective chromatography: prediction of unusual elution profiles of enantiomers in non-racemic mixtures on an achiral stationary phase doped with small amounts of a chiral selector

Nonlinear effects caused by molecular association of enantiomers in non-racemic mixtures can cause unexpected effects in chiroptics, NMR spectroscopy, homogeneous catalysis, and chromatography. Herein we present a theoretical model to simulate and verify unusual elution orders of enantiomers on an achiral stationary phase doped with a small amount of a chiral selector or achiral columns coupled with columns doped with a chiral selector. Scenarios with strong, medium, and weak associations of enantiomers, different separation efficiencies typical for flash chromatography and liquid chromatography, and the influence of the enantioselectivity of the chiral selector on the complex equilibria have been investigated. The findings presented here are of importance for the validation of the determination of enantiomeric ratios in not fully separated elution zones as well as for the preparative separation of non-racemic enantiomeric mixtures on chiral stationary phases bonded to achiral matrices.     

An instance of temperature-dependent elution order of enantiomers from a chiral brush-type HPLC column

Typically, reduction of column temperature increases the enantioselectivity of a chiral stationary phase. An instance in which progressive reduction of temperature initially reduces enantioselectivity, then inverts the elution order of the enantiomers, and finally causes enantioselectivity to increase has been observed. This behavior is related to the nature and concentration of the polar modifier in the mobile phase, and requires particular chiral phase-analyte-mobile phase combinations. A rationalization is presented as to the possible origin of this behavior. This rationale may aid in finding other examples of this temperature-dependent elution order of enantiomers and ultimately increase our understanding of chiral recognition processes.     

Recent innovations in enantiomer separation by electrochromatography utilizing modified cyclodextrins as stationary phases.

Enantiomer separation by electrochromatography employing modified cyclodextrins as stationary phases is performed in two ways. (i) Polysiloxane-linked permethylated beta-cyclodextrin (Chirasil-Dex 1) or related selectors are coated and immobilized onto the inner surface of a capillary column. Enantiomer separation is performed in the open tube and the method is referred to as open-tubular capillary electrochromatography (o-CEC). (ii) Silica-linked native beta-cyclodextrin, permethylated beta-cyclodextrin (Chira-Dex 2) or hydroxypropyl-beta-cyclodextrin are filled into a capillary column and the bed is secured by two frits. Enantiomer separation is performed in a packed column and the method is referred to as packed capillary electrochromatography (p-CEC). In a unified instrumental approach, method (i) as well as method (ii) can be operated both in the electro- and pressure-driven modes (o-CEC vs. open-tubular liquid chromatography (o-LC) and p-CEC vs. p-LC). It is demonstrated that the electro-driven variant affords higher efficiencies at comparable elution times. Employing a single open-tubular column coated with Chirasil-Dex 1, a unified enantioselective approach can be realized in which the same selectand is separated using all existing chromatographic modes for enantiomers, i.e., gas chromatography (GC), super-critical fluid chromatography (SFC), o-LC and o-CEC. As the chiral selector is utilized as a stationary phase, an additional chiral selector may be added to the mobile phase. In the resulting dual chiral recognition systems, enhancement of enantioselectivity (matched case) or compensation of enantioselectivity (mismatched case) may be observed. The overall enantioselectivity is dependent on the sense of enantioselectivity of the selectors chosen and their influence on the electrophoretic and electroosmotic migration of the enantiomers of a selectand.     

Chiral recognition: design and preparation of chiral stationary phases using selectors derived from ugi multicomponent condensation reactions and a combinatorial approach

Combinatorial approaches together with high-throughput screening have been used to develop highly selective stationary phases for chiral recognition. Libraries of potential chiral selectors have been prepared by the Ugi multicomponent condensation reactions and screened for their enantioselectivity using the reciprocal approach involving a chiral stationary phase with immobilized model target compound N-(3,5-dinitrobenzoyl)-alpha-l-leucine. The best candidates were identified from the library of phenyl amides of 2-oxo-azetidineacetic acid derivatives. This screening also enabled specification of the functionalities of the selector desired to achieve the highest level of chiral recognition. The substituents of the phenyl ring adjacent to the chiral center of the selector candidates exhibited the most profound effect on the chiral recognition. The best candidate was then synthesized on a larger scale, resolved into single enantiomers using preparative enantioselective HPLC, and attached to porous poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) beads via an ester linkage to afford the desired stationary phase. Selectivities alpha as high as 3.2 were found for the separation of a variety of amino acid derivatives.     

手性配位体交换流动相添加剂法拆分对映体

综述了手性配合基交换色谱法通常采用三种手性相系统中的流动相添加剂方法。主要内容有：（Ａ）手性配合基交换机制,给出了描述对映体对在色谱系统中的保留时间和分离选择性的公式,包括手性选择剂在固定相和流动相中的各种不同情况。公式表明整个色谱往系统的对映体选择性不同于溶液中所存在的选择剂与被选择物作用的情况;（Ｂ）影响配合交换的参数,讨论了金属离子、金属离子／配位体比率、金属离子络合物浓度、固定相、流动相ｐＨ、洗脱顺序、有机调节剂、离子对试剂、流动相离子强度、温度、立体选择性和手性交互识别;（Ｃ）应用。     

High-performance liquid chromatographic enantioseparation of beta-amino acid stereoisomers on a (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid-based chiral stationary phase

Direct reversed-phase high-performance liquid chromatographic methods were developed for the separation of enantiomers of 14 unnatural beta-amino acids, including several beta-3-homo-amino acids on a chiral stationary phase containing (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid bonded to 3-aminopropyl silica gel as chiral selector. The effects of the organic and the acidic modifiers and the mobile phase composition on the separation were investigated. The natures and positions of the substituents on the aromatic ring substantially influenced the retention and enantioseparation. The elution sequence in most cases was determined and the R enantiomers were eluteted before the S enantiomers.     

On the zopiclone enantioselective binding to human albumin and plasma proteins. An electrokinetic chromatography approach

In this work, a methodology for the chiral separation of zopiclone (ZPC) by electrokinetic chromatography (EKC) using carboxymethylated-β-cyclodextrin as chiral selector has been developed and applied to the evaluation of the enantioselective binding of ZPC enantiomers to HSA and total plasma proteins. Two mathematical approaches were used to estimate protein binding (PB), affinity constants (K(1)) and enantioselectivity (ES) for both enantiomers of ZPC. Contradictory results in the literature, mainly related to plasma protein binding reported data, suggest that this is an unresolved matter and that more information is needed. Discrepancies and coincidences with previous data are highlighted.     

Calculation of chromatographic band profiles with an implicit isotherm

The mechanisms of structure selective and enantioselective retentions of amines and acids on two chiral stationary phases based on wild type cellobiohydrolase I (CBH I) and its mutant D214N have been investigated. All the amino alcohols tested had an enantioselective site that overlaps with the catalytically active site of CBH I, whereas the enantioselectivity of prilocaine was not affected by the mutation. The hydroxyl group of the amino alcohols did not seem to be an important contributor to the total binding strength whereas a bromo substituent in the aromatic ring promotes a high enantioselectivity (alpha=7.05). Interestingly, the chiral recognition site of the acid warfarin overlaps with the binding site of the amino alcohols. Di-p-toluoyltartaric acid and dibenzoyltartaric acid were strongly retained probably due to electrostatic attraction, but no enantioselectivity was observed. The difference in retention characteristics for the amino alcohols on the two stationary phases was strongly pH-dependent. A change in elution order of different amino alcohols occurred when changing the pH from 5.0 to 7.0. The difference between the two phases was lower at low pH. The retention times could also be affected by ionic strength and by use of cellobiose as a mobile phase additive but no indication of ion-pair retention of the amines was observed, when adding hexanesulphonate as counter ion to the mobile phase. The temperature dependence of the retention of the enantiomers of propranolol at pH 7.0 on the mutant D214N was similar to what was earlier observed on the wild type CBH I at lower pH.     

Transient state of chiral recognition in a binary mixture of cyclodextrins in capillary electrophoresis

The transient state (as the defined point where no enantioseparation is obtained in a dual chiral selector system) of chiral recognition of aminoglutethimide in a binary mixture of neutral cyclodextrins (CDs) was studied by capillary electrophoresis (CE). The following three dual selector systems were used: alpha-cyclodextrin (alpha-CD) and beta-cyclodextrin (beta-CD); alpha-CD and heptakis(di-O-methyl-beta-cyclodextrin) (DM-beta-CD); alpha-CD and heptakis(tri-O-methyl-beta-cyclodextrin) (TM-beta-CD). The S-(-) enantiomer of the analyte was more strongly retained in the presence of either alpha-CD or TM-beta-CD at pH 2.5, 100 mM phosphate buffer, while the R-(+) enantiomer was more strongly retained in the presence of either beta-CD or DM-beta-CD. In the more simple case, the elution order is invariably kept if the enantiomers have the same elution order in either one of the two hosts of the binary mixture. In contrast, the elution order may be switched by varying the concentration ratio of two hosts that produce opposite elution order for this particular analyte. In such a dual selector system, the enantioselectivity will disappear at the transient state at a certain ratio of host1:host2. Moreover, the migration times of the two enantiomers with host, alone (diluted in buffer) is approximately equal to the migration times at the corresponding concentration of host2 alone (diluted in buffer), where the ratio of concentrations of host1:host2 is the same as in the binary mixture at the transient state. As found by nuclear magnetic resonance experiments, the analyte is forming a 1:1 complex with either one of the CDs applied. From this finding, a theoretical model based on the mobility difference of the two enantiomers was derived that was used to simulate the transient state.     

Gas chromatographic enantiomer separation of agrochemicals using modified cyclodextrins

The enantiomers of phenoxypropionic acid type herbicides have been resolved by capillary gas chromatography employing modified cyclodextrins as chiral stationary phases. Excellent separations were obtained with columns containing a 1:1 mixture of per-O-pentylated and per-O-methylated γ-cyclodextrin. The enantiomers of the methyl esters of mecoprop and dichlorprop were also resolved on octakis(3-O-butyryl-2,6-di-O-pentyl)-γ-cyclodextrin. On this phase the order of elution of the enantiomers was temperature-dependent, the elution order being reversed as the temperature passed through the isoenantioselective temperature. This is the first time such behavior has been observed with cyclodextrin derivatives. The enantiomers of the polychlorinated polycyclic pesticides cis- and trans-chlordane, oxychlordane, heptachlor, heptachlorepoxide, and three chiral organophosphorus pesticides could be resolved using selectively derivatized cyclodextrin derivatives.     

Selectivity tuning in chiral dual column gas chromatography

The enantioselective tuning of two columns coupled in series is investigated in chiral high-resolution gas chromatography. Two columns with opposite enantioselectivities (Chirasil-L-Val and Chirasil-D-Val) are coupled in series via a T connector, and the relative retention of enantiomers chromatographed on the system is changed by varying the individual carrier gas flow rates in the coupled columns. The flow-rate ratio necessary for the required selectivity is calculated on the basis of the measured retention factors on the individual columns. The performance of this method for adjusting selectivity is studied by the separation of enantiomers of the N-TFA-O-methyl esters of six amino acids. It is demonstrated that the change of the coupling point carrier gas pressure, at the constant inlet and outlet pressures, may change the enantioselectivity of the given column series to such an extent that the enantiomer elution order may be reversed.     

Temperature and Solute Molecular Size Effects on the Retention and Enantioselectivity of a Series of D, L Dansyl Amino Acids on a Vancomycin-Based Chiral Stationary Phase

Summary The influence of column temperature (0–28 °C) and solute molecular size on the retention and enantioselectivity of a series of D, L dansyl amino acids with a non-polar side chain (valine, leucine, phenylalanine and tryptophan) were investigated using a vancomycin-based chiral stationary phase (CSP). The enthalpic and entropic terms for the solute-CSP association were determined from the linear vant Hoff plots. Two solute groups were distinguished in relation to these thermodynamic quantities: the solute group I (dansyl valine, dansyl leucine, dansyl phenylalanine) for which large negative values of enthalpic terms were obtained and the solute group II (dansyl tryptophan) for which H value was much less negative. The enthalpy-entropy compensation study revealed that the interaction mechanism was identical for the group I solute enantiomers but changed for D, L dansyl tryptophan. This was further exemplified as the group I compound enantiomers were resolved over the temperature range while the enantiomers of dansyl tryptophan were not separated in the operating conditions. Relationships between both the solute retention factors and apparent enantioselectivity, and the accessible surface area of the amino acid side chain indicated that when the solute molecular size increased (i) the retention was enhanced by the hydrophobic effect and (ii) the chiral discrimination decreased dependent, at least in part, on a steric hindrance phenomenon at the vancomycin aglycone pocket.     

Direct chromatographic resolution of carnitine and O-acylcarnitine enantiomers on a teicoplanin-bonded chiral stationary phase.

R-(-)-Carnitine (vitamin B(T)) plays an important role in human energy metabolism, by facilitating the transport of long-chained fatty acids across the mitochondrial membranes. Its (S)-enantiomer acts as a competitive inhibitor of carnitine acetyltransferase, causing depletion of the body R-(-)-carnitine stock. Consequently, the separation of carnitine enantiomers is very important both to study their biological activities and to control the enantiomeric purity of pharmaceutical formulations. In the present paper we describe an easy, fast and convenient procedure for the separation of the enantiomers of carnitine and O-acylcarnitines by enantioselective HPLC on a laboratory-made chiral column containing covalently bonded teicoplanin as selector. High enantioselectivity factors (alpha values ranging from 1.31 to 3.02) and short-time analyses characterize the analytical procedure; in addition, analytes are easily detected by evaporative light scattering with no need for preliminary derivatization. The effects of pH and ionic strength of the mobile phase and of the nature of the organic modifier on the enantioselective separations were also investigated.     

Separation and control of the elution order of N-t-butyloxycarbonyl amino acids D/L isomers by reversed-phase HPLC using cyclodextrins as chiral selectors for the mobile phase.

The enantiomeric resolution of N-t-butyloxycarbonyl (N-t-Boc) amino acids D/L isomers by reversed-phase HPLC was investigated using cyclodextrins (CD's) as chiral selectors for the mobile phase. The use of a low pH (pH<4) for the mobile phase enabled the enantioseparation of N-t-Boc amino acids. The opposite elution order of D/L isomers was observed when hydroxypropyl-derivatized beta-CD was used instead of native beta-CD. A computer simulation of the enantioseparation showed that the ratio of the retention factors of the chiral selector and the sample determined the elution order and the resolution. When the retention factor of the chiral selector is smaller than that of the sample, an isomer having larger complex formation constant eluted faster. However, when the chiral selector had a larger retention factor than the sample, an opposite elution order of the isomers was obtained. The large difference in the retention factors between the chiral selector and the sample led to good enantiomeric separation.     

Determination of methylamphetamine and related compounds in human urine by high performance liquid chromatography/electrospray/mass spectrometry

The influence of tetrabutylammonium (TBA) as a hydrophobic charged additive of the mobile phase was investigated in a chromatographic system involving d,l-dansyl amino acids as the test solute enantiomers and immobilized human serum albumin as the chiral stationary phase. By varying the column temperature, van't Hoff plots for solute retention and enantioselectivity were performed and thermodynamic parameters were calculated. An enthalpy-entropy compensation study revealed that the type of interaction between the analyte and stationary phase was independent of TBA concentration in the eluent. The counterion dependence on retention indicated that the coulombic interactions between dansyl amino acid and the site II binding cavity were of crucial importance in this association process. Also, the increasing variations of chiral discrimination with a TBA addition were attributed, by an analysis of the thermodynamic parameter trends, to a great facilitation of enantioselective H-bonding between solute and polar residues at the cavity rim.