Super/subcritical fluid chromatography chiral separations with macrocyclic glycopeptide stationary phases

The chiral recognition capabilities of three macrocyclic glycopeptide chiral selectors, namely teicoplanin (Chirobiotic T), its aglycone (Chirobiotic TAG) and ristocetin (Chirobiotic R), were evaluated with supercritical and subcritical fluid mobile phases. A set of 111 chiral compounds including heterocycles, analgesics (nonsteroidal antiinflamatory compounds), beta-blockers, sulfoxides, N-protected amino acids and native amino acids was separated on the three chiral stationary phases (CSPs). All separations were done with an outlet pressure regulated at 100 bar, 31 degrees C and at 4 ml/min. Various amounts of methanol ranging from 7 to 67% (v/v) were added to the carbon dioxide along with small amounts (0.1 to 0.5%, v/v) of triethylamine and/or trifluoroacetic acid. The Chirobiotic TAG CSP was the most effective closely followed by the Chirobiotic T column. Both columns were able to separate, partially or fully, 92% of the enantiomers of the compound set. The ristocetin chiral selector could partially or baseline resolve only 60% of the enantiomers tested. All separations were done in less than 15 min and 70% were done in less than 4 min. The speed of the separations is the main advantage of the use of SFC compared to normal-phase HPLC. In addition, SFC is advantageous for preparative separations with easy solute recovery and solvent disposal.     

Separation of racemic sulfoxides and sulfinate esters on four derivatized cyclodextrin chiral stationary phases using capillary gas chromatography

The separation of 17 chiral sulfoxides and eight chiral sulfinate esters by gas chromatography (GC) on four derivatized cyclodextrin chiral stationary phases (CSPs) (Chiraldex G-TA, G-BP, G-PN, B-DM) is presented. Many of these compounds are structural isomers or part of a homologous series. Differences in enantioselectivity of the methyl phenyl sulfoxide isomers on the derivatized gamma cyclodextrin and the heptakis 2,6-di-O-methyl-beta-cyclodextrin (i.e. B-DM) CSPs are discussed. Under the conditions of this study, the molecular mass cut-off for the GC separation of these compounds was approximately 230. Compounds of higher molecular mass were not eluted from the CSPs at reasonable times and temperatures, but these higher molecular mass enantiomers can be separated by liquid chromatography and capillary electrophoresis. The enantiomeric separation and elution order of a sulfinate ester containing two stereogenic centers as well as 15 chiral sulfoxides is presented. The G-TA and B-DM CSPs generally gave opposite elution orders for most of the compounds studied.     

Comparison of enantioseparation of selected benzodiazepine and phenothiazine derivatives on chiral stationary phases based on β‐cyclodextrin and macrocyclic antibiotics

Enantioselective separation of some phenothiazine and benzodiazepine derivatives was studied on six different chiral stationary phases (CSPs) in HPLC. Selected CSPs, with respect to the structure of the separated compounds, were either based on β‐cyclodextrin chiral selectors – underivatized β‐cyclodextrin and hydroxypropyl ether β‐cyclodextrin, or on macrocyclic antibiotics – vancomycin, teicoplanin, teicoplanin aglycone, and ristocetin A. Measurements were carried out in a reversed‐phase separation mode. The influence of mobile phase composition on retention and enantioseparation was studied. Benzodiazepines could be enantioresolved with almost all the chiral stationary phases used, except for the vancomycin‐bonded CSP. Peak coalescence of oxazepam and lorazepam was observed if separation was carried out at laboratory temperature. Reduced temperature was required in some instances in order to avoid the on‐column racemization. Separation systems composed of teicoplanin‐bonded CSP and buffer‐methanolic or pure methanolic mobile phases were shown to be suitable even for preparative purposes due to high resolution values of the enantiomers. Enantioseparation of phenothiazine derivatives was more difficult to achieve but it was successful, at least partly, also with both types of the CSPs used (except for levomepromazine).     

Evaluation and comparison of a methylated teicoplanin aglycone to teicoplanin aglycone and natural teicoplanin chiral stationary phases

HPLC enantiomeric separations of a wide variety of racemic analytes was evaluated using chiral stationary phases (CSPs) based on the macrocyclic glycopeptides teicoplanin (T), teicoplanin aglycone (TAG), and methylated teicoplanin aglycone (Me-TAG) in two different mobile phase modes, i.e., the RP mode and the polar organic (PO) mode. Comparison of the enantiomeric separations using Chirobiotic T, Chirobiotic TAG, and the methylated form of TAG were conducted in order to gain a better understanding of the roles of the polar functional groups on the CSP. Substantial effects due to the cleavage of saccharides and/or methylation on chiral separations were observed in both separation modes. Improved separation efficiencies for many acidic analytes were obtained by methylating the H-bonding groups of TAG. These groups were believed to be a contributing factor to band broadening on TAG due to their negative effect on mass transfer between the stationary phase and mobile phase. Ionic/dipolar interactions between the carboxylate group of the analytes and the amine groups on T, TAG, or Me-TAG are important for chiral discrimination. Therefore, analytes possessing a carboxyl group are good candidates for successful separations on these CSPs. Hydrophobic interactions are important for enantiomeric separations in the RP mode where the H-bonding interactions between analytes and the chiral selectors are relatively weak. Me-TAG offers higher hydrophobicity, which can accentuate the interactions of analytes with hydrophobic moieties, but these interactions are not necessarily stereoselective. In the PO mobile phase, electrostatic/dipolar interactions between polar functional groups are the dominating interactions in chiral recognition. Another important factor is steric fit, which could be changed with every modification of the T structure. Therefore, substantial changes of enantioseparations were obtained within this studied group of CSPs. The PO mode was shown to be the most powerful mobile phase mode for enantiomeric separations on T-based stationary phases, mainly due to the improved efficiency. Methylation of the TAG proved to be a very useful tool for investigating the chiral recognition mechanism for this group of chiral selectors.     

Direct high-performance liquid chromatographic separation of unusual secondary amino acids and a comparison of the performances of Chirobiotic T and TAG columns

Two macrocyclic glycopeptide antibiotic-type chiral stationary phases (CSPs) based on native teicoplanin and teicoplanin aglycone, Chirobiotic T and TAG, respectively, were evaluated with regard to the high-performance liquid chromatographic separation of the enantiomers of 10 secondary alpha-amino acids (imino acids). The chromatographic results are given as the retention, separation and resolution factors, together with the enantioselective free energy difference corresponding to the separation of the enantiomers. By application of these two CSPs, excellent resolutions were achieved for the investigated compounds by using reversed-phase mobile mode systems. The separation conditions were optimized by variation of the mobile phase composition. The difference in enantioselective free energy between the aglycone CSP and the teicoplanin CSP for these particular amino acids ranged between 0.70 and -1.83 kJ mol(-1). It was established that better enantioseparations of the secondary alpha-amino acids were attained in most cases on the aglycone CSP.     

Derivatized vancomycin stationary phases for LC chiral separations

In this work, synthetic and natural chiral selectors were combined to form two different chiral stationary phases (CSPs). These were made by bonding R- or S-(1-naphthylethyl) carbamate (R-NEC or S-NEC)-derivatized vancomycin molecules to a silica gel support. The two CSPs were evaluated using a set of 60 enantiomeric pairs. The results were compared to the ones obtained with the commercial underivatized vancomycin CSP. Three Chromatographic modes were used: (i) the normal-phase mode using a nonpolar mobile phase with different ratios of hexane and ethanol; (ii) the reversed-phase mode with hydro-organic mobile phases; and (iii) the polar aprotic organic mode with nonaqueous acetonitrile plus small amounts of methanol and an acid and/or base to control retention and selectivity. It is shown that the polarity of the underivatized vancomycin phase is higher than that of the two R- and S-NEC-derivatized CSPs. In the pH range 4-7, there is no ionization change of the chiral selector for the three CSPs. 43% of the studied compounds were resolved by the NEC-derivatized phases when they could not be resolved by the vancomycin CSP. However, the enantiorecognition for 12% of the compounds on the native vancomycin CSP was lost upon NEC derivatization. 45% of the studied compounds were resolved by the NEC-derivatized and native CSPs. The NEC derivatization procedure may block some useful active sites on the vancomycin molecule. Also, the R- and S-NEC moieties are chiral themselves and can contribute additional interaction sites not available on the native vancomycin molecule.     

Temperature and enantioseparation by macrocyclic glycopeptide chiral stationary phases

Seventy-one chiral compounds were separated on four macrocyclic glycopeptide chiral selectors: teicoplanin, its aglycone, ristocetin A and vancomycin, using three possible separation modes: reversed phase with methanol/buffer mobile phases, normal phase with hexane/ethanol mobile phases and polar ionic mode (PIM) with 100% methanol mobile phase with trace amounts of acid and/or base. These 148 separations were studied in a 5-45 degrees C temperature range. Peak efficiencies always increased with temperature, but in only 17% of the separations studied a small increase of the enantioresolution factor was observed. In the majority (83%) of the cases, the enantioresolution decreased or even vanished when temperature increased. All 148 Van't Hoff plots were linear showing that the selector did not change in the temperature range studied. The calculated enthalpy and entropy variations showed that the interaction of the solute with the stationary phase was always enthalpy driven with normal and reversed mobile phases. It could be enthalpy as well as entropy driven with PIM mobile phases strongly dependent on the solute. The plots of delta(deltaH) versus delta(deltaS) were linear in most cases (enthalpy entropy compensation). This observation cannot be used to give clear information on chiral recognition mechanisms, but it allowed identifying specific stationary phase-solute interactions because the points corresponding to the respective thermodynamic parameters were clearly delineated from the general compensation lines.     

Comparison of the separation efficiencies of chirobiotic T and TAG columns in the separation of unusual amino acids

Two macrocyclic antibiotic type chiral stationary phases (CSPs), based on native teicoplanin and teicoplanin aglycone, Chirobiotic T and Chirobiotic TAG, respectively, were evaluated for the high-performance liquid chromatographic separation of enantiomers of 15 unnatural conformationally constrained alpha-amino acids, Phe and Tyr analogs, and 12 beta-amino acids having cycloalkane or cycloalkene skeletons. The chromatographic results are given as the retention, separation and resolution factors along with the enantioselective free energy difference corresponding to the separation of the enantiomers. It is clearly established that in most cases the aglycone is responsible for the enantioseparation of amino acids. The difference in enantioselective free energy between the aglycone CSP and the teicoplanin CSP was between 0.02 and 0.30 kcal mol(-1) for these particular amino acids. The resolution factors are higher with the aglycone CSP. Although the sugar units generally decrease the resolution of amino acid enantiomers, they can contribute significantly to the resolution of some unusual amino acid analogs. By application of these two CSPs excellent resolutions were achieved for most of the investigated compounds by using reversed phase or polar organic mobile mode systems. The separation conditions were optimized by variation of the mobile phase composition.     

High-performance liquid chromatographic enantioseparation of 2-aminomono- and dihydroxycyclopentanecarboxylic and 2-aminodihydroxycyclohexanecarboxylic acids on macrocyclic glycopeptide-based phases

The direct separation of the enantiomers of four 2-aminomono- or dihydroxycyclopentanecarboxylic acids and four 2-aminodihydroxycyclohexanecarboxylic acids was performed on chiral stationary phases containing macrocyclic glycopeptide antibiotics such as teicoplanin (Astec Chirobiotic T and T2), teicoplanin aglycone (Chirobiotic TAG) or ristocetin A (Chirobiotic R) as chiral selectors. The effects of the nature of organic modifiers, the pH, the mobile phase composition and the structures of the analytes on the separation were investigated. Chirobiotic TAG, and in some cases Chirobiotic T, proved to be the most useful of these columns. The elution sequence was determined in most cases.     

Thermodynamic origin of the chiral recognition of tryptophan on teicoplanin and teicoplanin aglycone stationary phases

The D-, L-tryptophan binding and the chiral recognition properties of the teicoplanin and teicoplanin aglycone (TAG) chiral stationary phase (CSPs) were compared at various column temperatures. The solute adsorption isotherms (bi-Langmuir model) were determined for both the two CSPs using the perturbation method. It was demonstrated that the sugar units were involved in the reduction of the apparent enantioselectivity through two phenomena: (i) the inhibition of some enantioselective contacts with low-affinity binding regions of the aglycone and (ii) a decrease in the stereoselective properties of the aglycone high-affinity binding pocket. The phenomenon (ii) was governed by both a decrease in the ratio of the enantiomer adsorption constant and a strong reduction of the site accessibility for D- and L-tryptophan. In addition, a temperature effect study was performed to investigate the chiral recognition mechanism at the aglycone high-affinity pocket. An enthalpy-entropy compensation analysis derived from the Grunwald model as well as the comparison with the literature data demonstrated that the enantioselective binding mode was dependent on an interface dehydration process. The change in the enantioselective process observed between the TAG and teicoplanin CSP was characterized by a difference of ca. 2-3 ordered water molecules released from the species interface.     

Separation of the enantiomers of substituted dihydrofurocoumarins by HPLC using macrocyclic glycopeptide chiral stationary phases

Enantiomer separations by HPLC using the macrocyclic glycopeptides teicoplanin (Chirobiotic T), teicoplanin aglycon (Chirobiotic TAG), and ristocetin A (Chirobiotic R) chiral stationary phases (CSP) have been achieved on a unique series of potentially biologically active racemic analogues of dihydrofurocoumarin. The macrocyclic glycopeptides have proven to be very selective for this class of compound. All of the 28 chiral analogues examined afforded baseline separation on at least one of the macrocyclic glycopeptide CSP. The teicoplanin CSP showed the broadest enantioselectivity with 24 of the compounds baseline separated. The TAG and the R CSP produced 23 and 14 baseline separations respectively. All three mobile phase modes, i.e. normal phase (NP), reversed phase (RP), and new polar organic modes (PO), have been evaluated. The NP mode proved to be most effective for the separation of chiral dihydrofurocoumarins on all CSP tested. In the reversed phase (RP) mode, all three CSP separated a similar number of compounds. It was observed that the structural characteristics of the analytes and steric effects are very important factors leading to chiral recognition. Hydrogen bonding was found to play a secondary role in chiral discrimination in the normal phase and polar organic modes. Hydrophobic interactions are important for chiral separation in the reversed-phase mode. Chromatographic retention data does not provide information on the absolute configuration of these chiral dihydrofurocoumarin derivatives. However, when coupled with circular dichroism using the exciton coupling chirality method, the enantiomer elution order and the absolute configuration of some chiral dihydrofurocoumarins were successfully determined.     

Comparison of Separation Efficiency of Macrocyclic Glycopeptide-Based Chiral Stationary Phases for the LC Enantioseparation of β-Amino Acids

Direct reversed-phase high-performance liquid chromatographic methods were developed for the separation of enantiomers of eighteen unnatural β-amino acids, including several β-3-homo-amino acids. The direct separations of the underivatized analytes were performed on chiral stationary phases containing macrocyclic glycopeptide antibiotics such as teicoplanin (Chirobiotic T and T2), teicoplanin aglycone (Chirobiotic TAG), vancomycin (Chirobiotic V and V2), and ristocetin A (Chirobiotic R) as chiral selectors. The effects of the organic modifier, mobile phase composition and pH on the separations were investigated. A comparison of the separation performances of the macrocyclic glycopeptide stationary phases revealed that the Chirobiotic T2 column exhibited better selectivity than the Chirobiotic T column for the separation of β-3-homo-amino acid enantiomers; vancomycin or ristocetin A exhibited lower selectivity. The elution sequence was determined in some cases: the S enantiomers eluted before the R enantiomers, with the exception of the Chirobiotic R column, where the elution sequence R < S was observed.     

Chiral separations using the macrocyclic antibiotics: a review

The macrocyclic antibiotics have recently gained popularity as chiral selectors in CE, HPLC and TLC. The macrocyclic antibiotics used for chiral separations include the ansamycins, the glycopeptides, and the polypeptide antibiotic thiostrepton. Although not strictly considered macrocyclic antibiotics, the aminoglycosides are antibiotics that have been used for chiral separations in CE. More chiral analytes have been resolved using the glycopeptides than with the other macrocyclic antibiotics combined. The glycopeptides vancomycin, ristocetin A and teicoplanin have been used extensively as chiral selectors in CE, with ristocetin A appearing to be the most useful chiral selector followed by vancomycin and teicoplanin, respectively. The macrocyclic antibiotics have also been used as chiral bonded phases in HPLC, and HPLC stationary phases based on vancomycin, ristocetin A and teicoplanin have been commercialized. Ristocetin A seems to be the most useful glycopeptide HPLC bonded phase, but its greater expense can be a drawback. The macrocyclic antibiotics have been used with micelles to improve efficiency, provide unique selectivity, and extend the range of separations to neutral solutes. Changing the macrocyclic antibiotic used in CE or HPLC can significantly alter the enantioselectivity of the separations. In fact, the glycopeptide antibiotics are complementary to one another, where if a partial enantioresolution is obtained with one glycopeptide, there is a high probability that a baseline or better separation can be obtained with another.     

Comparative study of three teicoplanin-based chiral stationary phases using the linear free energy relationship model

Teicoplanin (T) is a macrocyclic glycopeptide that is highly effective as a chiral selector for enantiomeric separations. In this study, we used three teicoplanin-based chiral stationary phases (CSPs) - native teicoplanin, teicoplanin aglycon (TAG) and recently synthesized methylated teicoplanin aglycon (MTAG). In order to examine the importance of various interaction types in the chiral recognition mechanism the three related CSPs were evaluated and compared using a linear free energy relationship (LFER). The capacity factors of 19 widely different solutes, with known solvation parameters, were determined on each of the columns under the same mobile phase conditions used for the chiral separations. The regression coefficients obtained revealed the magnitude of the contribution of individual interaction types to the retention on the compared columns under those specific experimental conditions. Statistically derived standardized regression coefficients were used to evaluate the contribution of individual molecular interactions within one stationary phase. It has been concluded that intermolecular interactions of the hydrophobic type significantly contribute to retention on all the CSPs studied here. Other retention increasing factors are n- and pi-electron interactions and dipole-dipole or dipole-induced dipole ones, while hydrogen donating or accepting interactions are more predominant with the mobile phase than with the stationary phases. However, these types of interactions are not equally significant for all the CSPs studied.     

Direct and indirect high-performance liquid chromatographic enantioseparation of beta-amino acids

Direct and indirect reversed-phase (RP) high-performance liquid chromatographic methods were developed for the separation of enantiomers of 18 unnatural beta-amino acids, including several beta-3-homo amino acids. The direct separations of the underivatized analytes were performed on chiral stationary phases (CSPs) containing macrocyclic glycopeptide antibiotic teicoplanin (Chirobiotic T column) and teicoplanin aglycone (Chirobiotic TAG column). The indirect method involved pre-column derivatization with a new chiral derivatizing agent (CDA), (S)-N-(4-nitrophenoxycarbonyl)phenylalanine methoxyethyl ester ((S)-NIFE), and subsequent separation of diastereomers on Discovery C18 and Hyperpep 300 C18 columns. The different methods were compared in systematic chromatographic examinations. The effects of organic modifier, mobile phase composition, pH and flow rate on the separation were investigated.     

Evaluation of ethoxynonafluorobutane as a safe and environmentally friendly solvent for chiral normal-phase LC-atmospheric pressure chemical ionization/electrospray ionization-mass spectrometry

Coupling normal-phase LC separation methods to atmospheric pressure ionization (API)-mass spectrometry (MS) for detection can be problematic because of the possible detonation hazard and because nonpolar solvents do not support ionization of the analyte. Unlike achiral separations, enantiomeric separations can be very sensitive to small changes in the separation environment. Thus, completely substituting the main mobile phase component of a normal-phase LC solvent for an environmentally friendly, nonflammable fluorocarbon-ether as a safe and effective solvent must be thoroughly evaluated before it can be recommended for enantioselective separations with API-MS detection. Ethoxynonafluorobutane (ENFB) was used as a normal-phase solvent for the enantioselective separation of 15 compounds on two macrocyclic glycopeptide chiral stationary phases (CSPs) and a new polymeric chiral stationary phase. The chromatographic figures of merit were compared between results obtained with the ENFB mobile phases and traditional heptane-based mobile phases. In addition, the limits of detection (LOD) using the API-MS compatible ENFB were examined, as well as flow rate sensitivities and compatibilities with common polar organic modifier. ENFB is a safe and effective solvent for enantioselective normal-phase/API-MS analyses.     

High-performance liquid chromatographic and capillary electrophoretic enantioseparation of plant growth regulators and related indole compounds using macrocyclic antibiotics as chiral selectors

Enantioseparation of plant growth regulators, such as 3-(3-indolyl)-butyric acid, abscisic acid and structurally related molecules including a variety of substituted tryptophan compounds, has been achieved by HPLC and/or CE. The covalently bonded macrocyclic antibiotics, teicoplanin, ristocetin A and vancomycin, were used as chiral stationary phases (CSPs) in HPLC. Most of the racemates were baseline resolved in the reversed-phase mode (EtOH-H2O) using the teicoplanin CSP. The chiral recognition mechanism is discussed in regard to the structure of the analytes. In CE, the three aforementioned macrocyclic antibiotics were used as chiral additives in a phosphate run buffer. The effect of pH and the concentration of the organic modifiers were considered. The results obtained by HPLC and CE were compared.     

Evaluation of the macrocyclic glycopeptide A-40,926 as a high-performance liquid chromatographic chiral selector and comparison with teicoplanin chiral stationary phase

A new macrocyclic antibiotic of the vancomycin family, referred to by its industrial designation as A-40,926, was bonded to 5 microm silica particles and utilised as a chiral stationary phase (CSP). Since A-40,926 is structurally related to teicoplanin, the A-40,926 CSP was compared to a commercially available teicoplanin CSP. A set of 28 chiral compounds, including amino-acids and related compounds, compounds with a ring containing the stereogenic centre, compounds bearing aromatic structures near their stereogenic centres and alcohols, was tested for enantioseparation on the two CSPs. The results are compared and discussed in terms of enantioselective Gibbs energy difference. The A-40,926 CSP was able to resolve one compound that was not resolved by the teicoplanin CSP. However, it could not separate four compounds that the teicoplanin CSP did separate. It is shown that the A-40,926 CSP is complementary to the teicoplanin CSP, thereby enlarging the number of enantiomers that can be separated by the macrocyclic glycopeptide based CSPs.     

HPLC separation of amino acid enantiomers and small peptides on macrocyclic antibiotic-based chiral stationary phases: a review

The search for new and effective chiral selectors capable of separating a wide variety of enantiomeric compounds is an ongoing process. In the past decade, macrocyclic antibiotics have proved to be an exceptionally useful class of chiral selectors for the separation of enantiomers of biological and pharmacological importance by means of HPLC, TLC and electrophoresis. More chiral analytes have been resolved through the use of glycopeptides than with all the other macrocyclic antibiotics combined (ansamycins, thiostrepton, aminoglycosides, etc.). The glycopeptides avoparcin, teicoplanin, ristocetin A and vancomycin have been extensively used as chiral selectors in the form of chiral bonded phases in HPLC, and HPLC stationary phases based on these glycopeptides have been commercialized. Teicoplanin, vancomycin, their analogs and ristocetin A seem to be the most useful glycopeptide HPLC bonded phases for the enantioseparation of proteins and unusal native and derivatized amino acids. In fact, the macrocyclic glycopeptides are to some extent complementary to one another: where partial enantioresolution is obtained with one glycopeptide, there is a high probability that baseline or better separation can be obtained with another. This review sets out to characterize the physicochemical properties of these antibiotics and their application in the enantioseparations of amino acids. The mechanism of separation, the sequence of elution of the stereoisomers and the relation to the absolute configuration are also discussed.     

Cyclodextrin-based liquid chromatographic enantiomeric separation of chiral dihydrofurocoumarins, an emerging class of medicinal compounds

A set of 28 racemic dihydrofurocoumarins in which the stereogenic center is located in the furan ring have been synthesized. Currently no effective asymmetric synthesis of this class of compounds exists, although their enantiomers are produced biologically by certain plants. Their diverse medicinal properties are being investigated in several laboratories. The enantioselective separation of these dihydrofurocoumarins by three native and six derivatized cyclodextrins has been evaluated in the reversed-phase mode, the polar organic mode, and normal-phase mode. The hydroxypropyl-beta-cyclodextrin is the most effective chiral stationary phase (CSP) at separating the dihydrofurocoumarins into enantiomers, showing some enantioselectivity for 22 dihydrofurocoumarins, and baseline resolving 16 of the 28 compounds in the reversed-phase mode. The acetyl-beta-cyclodextrin and 2,3-dimethyl-beta-cyclodextrin also showed enantioselectivity for a large number (18 and 17, respectively) of dihydrofurocoumarins in the reversed-phase mode. The native cyclodextrins are ineffective and the aromatic derivatized beta-cyclodextrins are only marginally effective at separating the furocoumarin enantiomers in the reversed-phase mode. The polar organic mode and the normal-phase mode have also been evaluated with these CSPs, but no enantioseparations were observed.