Separation of Enantiomers and Control of Elution Order of β-Lactams by GC Using Cyclodextrin-Based Chiral Stationary Phases

Enantiomers of 19 racemic β-lactams, with 3 and 4-position substitutions, were separated using gas chromatography. Excellent results were achieved on derivatized cyclodextrin-based GC chiral stationary phases (CSPs). All 19 compounds were baseline separated, most with high resolution factors. The Chiraldex G-TA was found to be the most powerful CSP with the broadest enantioselectivity, while Chiraldex B-DM produced the fastest separations for most of the compounds assayed. Results obtained in this work suggest that GC can serve as a potential method for the enantiomeric separation of sufficiently volatile solid β-lactams.     

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.     

Chromatographic peak deconvolution of constitutional isomers by multiple-reaction-monitoring mass spectrometry

Highly efficient and sophisticated separation techniques are available to analyze complex compound mixtures with superior sensitivities and selectivities often enhanced by a 2nd dimension, e.g. a separation technique or spectroscopic and spectrometric techniques. For enantioselective separations numerous chiral stationary phases (CSPs) exist to cover a broad range of chiral compounds. Despite these advances enantioselective separations can become very challenging for mixtures of stereolabile constitutional isomers, because the on-column interconversion can lead to completely overlapping peak profiles. Typically, multidimensional separation techniques, e.g. multidimensional GC (MDGC), using an achiral 1st separation dimension and transferring selected analytes to a chiral 2nd separation are the method of choice to approach such problems. However, this procedure is very time consuming and only predefined sections of peaks can be transferred by column switching to the second dimension. Here we demonstrate for stereolabile 1,2-dialkylated diaziridines a technique to experimentally deconvolute overlapping gas chromatographic elution profiles of constitutional isomers based on multiple-reaction-monitoring MS (MRM-MS). The here presented technique takes advantage of different fragmentation probabilities and pathways to isolate the elution profile of configurational isomers.     

Separation of the enantiomers of 2-phenylcyclopropanecarboxylate esters by capillary gas chromatography on derivatized cyclodextrin stationary phases

Summary Separation of the enantiomers of 2-phenylcyclopropanecarboxylate esters has been investigated on derivatized cyclodextrin chiral stationary phases (CD CSPs) to enable direct determination of the enantiomeric purity of the products of enantioselective cyclopropanation. Four stereoisomers of these chiral compounds could be resolved to baseline on permethylated β-cyclodextrin CSP. Some unusual phenomena, iso-enthalpy retention behavior and entropically driven chiral separation, were observed for the enantioseparation of 2-phenylcyclo-propanecarboxylates on the CD CSPs. Thermodynamic parameters were evaluated and an enthalpy-entropy compensation effect was observed forn-alkyl esters of 2-phenylcyclopropanecarboxylate separated on CD CSPs.     

Urea bonded cyclodextrin derivatives onto silica for chiral HPLC

Several structurally well-defined perfunctionalised cyclodextrin chiral stationary phases (CD CSPs) for high performance liquid chromatography have been successfully prepared by immobilisation of perfunctionalised cyclodextrins on silica through urea linkage(s) using the Staudinger reaction. These CSPs show high chiral recognition efficiency and are utilised in the resolution of various types of racemic compounds. This paper reviews the development of sixteen perfunctionalised cyclodextrin-based CSPs, their preparation, and their application to enantioseparation of seventy-seven racemic compounds under a range of separation conditions.     

4,6-Di-O-pentyl-3-O-trifluoroacetyl/propionyl cyclofructan stationary phases for gas chromatographic enantiomeric separations

4,6-Di-O-pentyl-3-O-trifluoroacetyl cycloinulohexaose (DP-TA-CF6) and 4,6-di-O-pentyl-3-O-propionyl cycloinulohexaose (DP-PN-CF6) were synthesized and used as chiral stationary phases (CSPs) in gas chromatography (GC). The chiral recognition ability of the two CSPs was investigated. A total of 47 racemic compounds were separated on the two new CSPs, including derivatized amino acids, amino alcohols, amines, alcohols, tartrates and lactones. Interestingly, several analytes were only separated on either the DP-TA-CF6 or the DP-PN-CF6 phase. The chiral recognition mechanism was evaluated through thermodynamic analysis. The result indicated there was no inclusion complex formation involved in the chiral recognition process.     

Separation of chiral furan derivatives by liquid chromatography using cyclodextrin-based chiral stationary phases

The enantiomeric separation of a set of 30 new chiral furan derivatives has been achieved on native and derivatized beta-cyclodextrin stationary phases using high performance liquid chromatography (HPLC). The hydroxypropyl-beta-cyclodextrin (Cyclobond RSP), the 2,3-dimethyl-beta-cyclodextrin (Cyclobond DM), and the acetyl-beta-cyclodextrin (Cyclobond AC) stationary phases are the most effective chiral stationary phases (CSPs) for the separation of these racemates in the reverse phase mode. No enantioseparations have been observed on the native beta-cyclodextrin chiral stationary phase (Cyclobond I 2000) and only a few separations have been attained on the S-naphthylethyl carbamate beta-cyclodextrin (Cyclobond SN) and 3,5-dimethylphenyl carbamate beta-cyclodextrin (Cyclobond DMP) chiral stationary phases in the reverse phase mode. The polar organic and the normal phase mode on these CSPs are not effective for separation of these compounds. The characteristics of the analytes, including steric bulk, hydrogen bonding ability, and geometry, play an important role in the chiral recognition process. The pH affects the enantioseparation of compounds with ionizable groups and the addition of 0.5% methyl tert-butyl ether to the mobile phase significantly enhances the separation efficiency for some highly retained compounds.     

Enantiomer separation of mandelates and their analogs on cyclodextrin derivative chiral stationary phases by capillary GC.

Enantiomer separation of mandelates and their analogs, which are important intermediates in asymmetric synthetic and pharmaceutical chemistry, was investigated by capillary gas chromatography using different cyclodextrin derivative chiral stationary phases (CD CSPs). The used cyclodextrin derivatives included permethylated beta-CD (PMBCD), permethylated gamma-CD, heptakis(2,6-di-O-butyl-3-O-butyryl)-beta-CD, heptakis(2,6-di-O-pentyl-3-O-acetyl)-beta-CD and heptakis(2,6-di-O-nonyl-3-O-trifluoroacetyl)-beta-CD (DNTBCD), respectively. Among all the CSPs used, PMBCD and DNTBCD exhibited the broadest and best enantioselectivity for all the racemates investigated. Some thermodynamic parameters were evaluated and an enthalpy-entropy compensation effect was observed in enantiomer separation processes of mandelates and their analogs. Based on thermodynamic data and molecular mechanics calculations, the chiral recognition mechanism of mandelate derivatives on CD CSPs is discussed.     

Use of native and derivatized cyclodextrin chiral stationary phases for the enantioseparation of aromatic and aliphatic sulfoxides by high performance liquid chromatography

Summary The enantioselectivity of native and derivatized cyclodextrin stationary phases for aromatic and aliphatic chiral sulfoxides was evaluated using high performance liquid chromatography (HPLC). Many sulfoxide enantiomers could be baseline resolved using the derivatized cyclodextrin stationary phases in the reverse phase mode. The most important factor influencing enantioselectivity is the presence of steric bulk alpha to the chiral center. However, substituents on an aromatic ring bonded to the sulfoxide have less pronounced effects on enantioselectivity. The 2,3-dimethyl β-cyclodextrin exhibits the broadest anantioselectivity for neutral chiral sulfoxides. Native cyclodextrins and hydroxypropyl-β-cyclodextrins were much less effective in separating this class of molecules. The hydrogen bonding ability of the organic modifier does not significantly affect enantioselectivity.     

Separation of positional isomers by the use of coupled shape-selective stationary phase columns

The successful separation of 2- and 3-methyl-substituted positional isomers of butanol, butyl acetate, and butanoic acid and its ethyl ester, is reported. These compounds are of interest in the study of wine flavour, however the separation of the 2- and 3-methyl isomers may present problems, and more so in the presence of the wine matrix components, when single capillary column gas chromatography (GC) is used. The strategy to achieve separation was based on the use of shape-selective cyclodextrin derivative (CDD) capillary columns (commonly referred to as chiral columns). These columns provide simultaneous resolution of the enantiomeric pairs of the 2-methyl isomers, and at the same time the ability to separate the 3-methyl isomer from the 2-methyl is achieved in all but the case of the ( S)-2- and 3-methylbutanol. The advantages of using shape-selective columns to perform this study is demonstrated, with coupling of two CDD columns giving improved separations of these compounds. Although these compounds are relatively volatile, cryogenic modulated comprehensive two-dimensional GC was shown to provide good pulsed peak profiles with chiral separation in the first dimension when a thicker film trapping column segment was employed. The components of interest were well separated from other wine matrix components.     

Enantiomeric separation of citalopram analogues by HPLC using macrocyclic glycopeptide and cyclodextrin based chiral stationary phases

The enantiomeric separation of a novel series of twenty-eight racemic mixtures of citalopram analogues was performed by high performance liquid chromatography (HPLC). Due to the effectiveness of citalopram as an antidepressant drug, the development of new compounds based on its chemical structure is interesting, and their enantiomeric separation is needed to allow further pharmacokinetic studies. Several bonded cyclodextrin (both native and derivatized) and macrocyclic glycopeptide based chiral stationary phases (CSPs) were evaluated for their ability to separate this set of compounds via HPLC. Polar ionic, polar organic, and reversed phase modes were tested. Twenty-five of the racemic mixtures were separated with resolutions and enantiomeric selectivities up to 2.9 and 1.33, respectively. A total of eighteen baseline separations were achieved, while seven compounds were partially separated. Vancomycin based columns operated in the polar ionic mode resulted in the greatest number of separations. Lastly, the chromatographic behaviors of similar compounds were compared based on their chemical structure and also on the chiral selectors used.     

General access to asymmetric γ-cyclodextrins for gas chromatographic applications by insertion of a selectively modified sugar unit

Gas chromatography (GC) using chiral stationary phases (CSPs) based on modified cyclodextrins is the most commonly used technique to quantify enantiomeric purity of synthesized volatile racemic drugs. The fully methylated cyclodextrin derivatives are the most used because of their easy synthesis, their thermal stability and their ability to recognize a wide range of compounds. To complete the background on molecular recognition, we describe a route to access fully characterized new asymmetric cyclodextrins derived from fully methylated ones, thereby directly useful in chiral gas chromatography. The synthesis of these compounds involves a three-step procedure: ring opening of fully methylated cyclodextrins, elongation of the chain with correctly modified monosaccharide derivatives and, finally, macrocyclization to obtain the desired compounds. This strategy is applied to achieve the synthesis of a series of asymmetric γ-cyclodextrins, by insertion of glucopyranosic derivatives. Appropriate selection of the glycosylating reagents during chain elongation and macrocyclization steps allows satisfactory anomeric selectivities to be reached.     

Enantiomeric Resolution of a Chiral Sulfoxide Series by LC on Synthetic Polymeric Columns with Multimodal Elution

The liquid chromatography enantiomeric separation of a series of 17 chiral sulfoxides was systematically investigated using multimodal elution with the new synthetic polymeric stationary phases P-CAP, P-CAP DP and DEAVB. The sulfoxide series was composed of aryl alkyl sulfoxides, benzoimidazole sulfoxides and the drugs modafinil, albendazole sulfoxide, omeprazole, lansoprazole, pantoprazole and rabeprazole. This work examines the effectiveness of the polymeric chiral stationary phases for the separation of chiral sulfoxides and describes the superiority of DEABV for these separations in three different elution modes. The first ever reversed phase enantiomeric separations on these columns is demonstrated.     

Synthesis of cationic beta-cyclodextrin derivatives and their applications as chiral stationary phases for high-performance liquid chromatography and supercritical fluid chromatography

Four cationic beta-cyclodextrin derivatives, namely mono-6-(3-methylimidazolium)-6-deoxy-perphenylcarbamoyl-beta-cyclodextrin chloride (MPCCD), mono-6-(3-methylimidazolium)-6-deoxyper(3,5-dimethylphenylcarbamoyl)-beta-cyclodextrin chloride (MDPCCD), mono-6-(3-octylimidazolium)-6-deoxyperphenylcarbamoyl-beta-cyclodextrin chloride (OPCCD) and mono-6-(3-octylimidazolium)-6-deoxyper(3,5-dimethylphenylcarbamoyl)-beta-cyclodextrin chloride (ODPCCD), have been synthesized and physically coated onto porous spherical silica gel to obtain novel chiral stationary phases (CSPs). The performances of these CSPs are studied on high-performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC) using 18 racemic aryl alcohols as test analytes. Among these four CSPs, OPCCD shows the best separation results for all analytes on both HPLC and SFC analyses. Chromatographic studies reveal that the CSPs consisting of an n-octyl group on the imidazolium moiety and phenylcarbamoyl groups on the cyclodextrin ring provide enhancement of analyte-chiral substrate interactions over CSPs bearing the methyl group on the imidazolium moiety and 3,5-dimethylphenylcarbamoyl groups on the cyclodextrin ring.     

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.     

Development of dinitrophenylated cyclodextrin derivatives for enhanced enantiomeric separations by high-performance liquid chromatography

The synthesis and evaluation of new dinitrophenyl (DNP) substituted beta-cyclodextrin (beta-CD) chiral stationary phases (CSPs) for the enantioseparation of various classes of chiral analytes by HPLC are presented. The dinitrophenyl substituted beta-CD derivatives are synthesized and covalently bonded to functionalized 5 microm spherical porous silica gel. These are the first reported derivatized cyclodextrin which contains pi-electron deficient substituents (i.e., pi-acidic moieties). The column performance in terms of their ability to separate enantiomers is evaluated. A variety of different dinitro-substituted aryl groups are investigated and compared. The pH of the mobile phase buffers, the buffer composition, the number and position of the dinitro groups on the phenyl ring substituent, the degree of substitution, and the bonding strategy all greatly affect the performance of the CSPs. A large variety of racemic compounds have been separated successfully on these CSPs. The bonded dinitrophenyl-derivatized cyclodextrins are stable in all three mobile phase modes, namely, the reversed-phase, polar organic, and normal phase modes. No degradation in column performance was observed in any mode of operation even after more than 1000 injections. The analytical applicability of these types of CSPs for enantiomeric separations is discussed in detail.     

Novel strong cation-exchange type chiral stationary phase for the enantiomer separation of chiral amines by high-performance liquid chromatography

The preparation of novel brush-type chiral cation-exchange materials based on de novo designed synthetic low molecular mass selectors (SOs) and their evaluation for enantioselective separation of chiral amines by HPLC are presented. The SO as the functional unit for enantioselectivity contains a beta-aminocyclohexanesulfonic acid moiety and is readily accessible via straightforward synthesis in both enantiomeric forms yielding chiral stationary phases (CSPs) with opposite configurations, CSPs 1 and 2, and reversed elution orders. For the evaluation of these novel CSPs by HPLC a sound set of chiral amines, mainly amino-alcohol type drug molecules, was selected. The chromatographic evaluations were carried out using polar organic mobile phase conditions. All of the analytes could be baseline separated, compared to common CSPs in parts with excellent peak efficiencies (up to 70000 theoretical plates per meter for the second eluted enantiomer). A number of experimental parameters have been varied to look at and prove the underlying ion-exchange process on CSPs 1 and 2, and to reveal suitable conditions for their operation. In this context, the influence of proton activity in the mobile phase and the effects of varying concentration and type of the counterion as well as type of co-ion and of bulk solvent components were thoroughly investigated.     

Ionic liquids as stationary phase solvents for methylated cyclodextrins in gas chromatography

Summary Room temperature ionic liquids (RTIL) are molten salts with melting points well below room temperature. 1-butyl-3-methylimidazolium chloride is a typical example of such RTIL. It was used as a solvent to dissolve permethylated-β-cyclodextrin (BPM) and dimethylated-?cyclodextrin (BDM) to prepare stationary phases for capillary columns in gas chromatography for chiral separation. The RTIL containing columns were compared to commercial columns containing the same chiral selectors. A set of 64 chiral compounds separated by the commercial BPM column was tested on the RTIL BPM column. Only 21 were enantioresolved. Similarly, a set of 80 compounds separated by the commercial BDM column was passed on the RTIL BDM column with only 16 positive separations. It is proposed that the imidazolium ion pair could make an inclusion complex with the cyclodextrin cavity, blocking it for chiral recognition. All the chiral compounds recognized by the RTIL columns had their asymmetric carbon that was part of a ring structure. The retention factors of the derivatized solutes were lower on the RTIL columns than those obtained on the commercial equivalent column. The peak efficiencies obtained with the RTIL capillary were significantly higher than that obtained with the commercial column. These observations may contribute to the knowledge of the mechanism of cyclodextrin-based GC enantioselective separations.     

New approach for chiral separation: from polysaccharide-based materials to chirality-responsive polymers

Chiral separation that is closely related to daily life is a meaningful research. Polysaccharide-(e.g., cellulose, amylose derivatives) based chiral packing materials afford powerful chiral stationary phases(CSPs) toward a broad range of racemic compounds. However, considering the explosive growth of specific chiral drugs, the separation efficiencies of these CSPs need further improvement, which calls for new approaches and strategies. Smart polymers can change their physical or chemical properties dynamically and reversibly according to the external stimuli(e.g., thermo-, pH, solvent, ion, light, critical parameters for chromatographic separation) exerted on them, subsequently resulting in tunable changes in the macroscopic properties of materials. In addition to their excellent controllability, the introduction of chiral characteristics into the backbones or side-chains of smart polymers provides a promising route to realize reversibly conformational transition in response to the chiral analytes. This dramatic transition may significantly improve the performance of materials in chiral separation through modulating the enantioselective interactions between materials and analytes. With the help of chirality-responsive polymers, intelligent and switchable CSPs could be developed and applied in column-liquid chromatography. In these systems, the elution order or enantioselectivity of chiral drugs can be precisely modulated, which will help to solve many challenging problems that involve complicated enantiomers. In this paper we introduce some typical examples of smart polymers that serve as the basis for a discussion of emerging developments of CPSs, and then briefly outline the recent CSPs based on natural and certain synthetic polymers.     

Chiral separation of labetalol stereoisomers in human plasma by capillary electrophoresis

A newly derivatized cyclodextrin [octakis-(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin] was investigated as a chiral selector in capillary zone electrophoresis in a study of the chiral separation of labetalol stereoisomers. Heptakis(2,3-diacetyl-6-sulfato)-beta-cyclodextrin (HDAS-beta-CD) and octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin (ODAS-gamma-CD) were shown to be effective in separating labetalol stereoisomers. Optimal separating conditions of the four stereoisomers of labetalol were achieved with 10 mM HDAS-beta-CD and 10 mM ODAS-gamma-CD in an acidic pH buffer of low molarity. Data illustrating the effects of capillary length and cyclodextrin concentration on the separation are presented. The longer capillary length and high voltage enabled the baseline separation of all isomers in less than 15 min. The optimized method was applied to the analysis of human control plasma containing labetalol utilizing solid-phase extraction (SPE) in the 96-well format.