Chirobiotic TAG Research Articles

Reversed-phase HPLC enantioseparation of pantoprazole using a teicoplanin aglycone stationary phase-Determination of the enantiomer elution order using HPLC-CD analyses.

A direct HPLC method was developed for the enantioseparation of pantoprazole using macrocyclic glycopeptide-based chiral stationary phases, along with various methods to determine the elution order without isolation of the individual enantiomers. In the preliminary screening, four macrocyclic glycopeptide-based chiral stationary phases containing vancomycin (Chirobiotic V), ristocetin A (Chirobiotic R), teicoplanin (Chirobiotic T), and teicoplanin-aglycone (Chirobiotic TAG) were screened in polar organic and reversed-phase mode. Best results were achieved by using Chirobiotic TAG column and a methanol-water mixture as mobile phase. Further method optimization was performed using a face-centered central composite design to achieve the highest chiral resolution. Optimized parameters, offering baseline separation (resolution = 1.91 ± 0.03) were as follows: Chirobiotic TAG stationary phase, thermostated at 10°C, mobile phase consisting of methanol/20mM ammonium acetate 60:40 v/v, and 0.6 mL/min flow rate. Enantiomer elution order was determined using HPLC hyphenated with circular dichroism (CD) spectroscopy detection. The online CD signals of the separated pantoprazole enantiomers at selected wavelengths were compared with the structurally analogous esomeprazole enantiomer. For further verification, the inline rapid, multiscan CD signals were compared with the quantum chemically calculated CD spectra. Furthermore, docking calculations were used to investigate the enantiorecognition at molecular level. The molecular docking shows that the R-enantiomer binds stronger to the chiral selector than its antipode, which is in accordance with the determined elution order on the column-S- followed by the R-isomer. Thus, combined methods, HPLC-CD and theoretical calculations, are highly efficient in predicting the elution order of enantiomers.

Effects of high concentrations of mobile phase additives on retention and separation mechanisms on a teicoplanin aglycone stationary phase in supercritical fluid chromatography

The objective of this study is to understand the behavior of a peptide in a medium containing supercritical carbon dioxide mixed with an alcohol (methanol) and acidic or basic additives in uncommonly high concentrations. Chirobiotic TAG is a chromatographic column made of silica bonded with a macrocyclic peptide, teicoplanin aglycone. With this stationary phase, two additives (trifluoroacetic acid and isopropylamine) were tested under extreme concentration conditions to observe the behavior of this peptide. Indeed, concentrations exceeding 1 M in the methanol co-solvent (>0.1 M overall concentration in the CO2-methanol mixture) were used whereas usual additive concentrations employed in supercritical fluid chromatography (SFC) rarely exceed 50 mM in the co-solvent. One purpose was to modify the apparent pH of the fluid, which is normally slightly acidic (around 5) and consequently possibly changing the ionization state of the peptide. Firstly, the effect of acidic and basic additives on the polarity and the apparent pH were evaluated with the help of color indicators. This served to assess the ionization state of the peptide under the selected operating conditions. Secondly, 54 achiral and 24 chiral molecules were injected in the chromatographic column at different levels of additives. The achiral species served at establishing retention models based on linear solvation energy relationships (LSER), while the chiral species were examined for their enantioresolution. From the LSER equations and observation of chromatograms, it appeared that specific interactions between the peptide-based stationary phase and the analytes evolved when increasing the concentration of additives, particularly hydrogen bonds and ionic interactions. A bare silica stationary phase (Acquity BEH) served as reference to deconvolute the contributions of silica support and bonded peptide. This study, with these extreme conditions of mobile phase, could be useful to understand the behavior of such peptides in SFC mobile phases and also improve the knowledge of the effects of additives in SFC, which should be helpful in the future prospect of analyzing large biomolecules in SFC.

Characterization of three macrocyclic glycopeptide stationary phases in supercritical fluid chromatography

Macrocyclic glycopeptides have been used as chromatographic stationary phases for over twenty years, particularly for their ability to separate enantiomers. While they are mostly used with buffered aqueous liquid mobile phases, they can also be used in supercritical fluid chromatography (SFC) with mobile phases comprising pressurized carbon dioxide and a co-solvent (like methanol), possibly comprising acidic or basic additives.In the present study, we compared three macrocyclic glycopeptide stationary phases (Chirobiotic V2, Chirobiotic T and Chirobiotic TAG) in SFC with carbon dioxide – methanol (90:10) containing no additives. First, the interactions contributing to retention are evaluated with a modified version of the solvation parameter model, comprising five Abraham descriptors (E, S, A, B, V) and two additional descriptors to take account of interactions with ionizable species (D− and D+). Linear solvation energy relationships (LSER) are established based on the retention of 145 achiral analytes.Secondly, the contributions of interactions to enantioseparations are discussed, based on the analysis of 67 racemates. The individual success rate on each phase was observed to be moderate, especially as these phases are known to be more efficient when acidic or basic additives are employed. Chirobiotic TAG proved more successful than the other two phases. Discriminant analyses were computed to gain some insight on retention mechanisms, but only Chirobiotic TAG provided interpretable results.Finally, the effects of a small proportion of acidic or basic additive on enantioseparation with Chirobiotic T stationary phase are briefly discussed.

Simultaneous stereoselective determination of seven β-agonists in pork meat samples by ultra-performance liquid chromatography-tandem mass spectrometry

This paper presents the stereoselective multi-residue analysis of seven β-agonists including salbutamol, clorprenaline, terbutaline, tulobuterol, metaproterenol, cimaterol and formoterol in pork muscles by chiral UPLC-MS/MS. The simultaneous enantioseparation of seven analytes was accomplished on a teicoplanin aglycone-based Astec Chirobiotic TAG column under isocratic condition at a flow rate of 0.6 mL min−1. The effects of the mobile phase composition, the type and content of the organic modifier, and the ratio and content of the additive on the retention and resolution of the enantiomers were investigated and optimized. To obtain the best extraction yields, the analytes were extracted from meat muscles by enzymatic hydrolysis, and then cleaned up using Cleanert PCX solid phase extraction cartridge. Then, the proposed method was validated in terms of selectivity, sensitivity, linearity, accuracy and precision, matrix effect, stability. Quantification of the analytes was achieved using matrix-matched standard calibration curve, providing a liner correlation over the concentration range of 0.1–50 ng g−1 for each enantiomer. The limits of detection (LODs) and quantification (LOQs) were in the range of 0.014–0.033 ng g−1 and 0.035–0.070 ng g−1, respectively. The method recoveries were within 84.9–106.9%, while the matrix effects were in the range of 84.3–108.3%. The intra- and inter-precision (expressed as RSD%) were found to be <11.2%, which confirmed the precision of this method. Taking into consideration obtained values of validation parameters, the method seems to be suitable for routine analysis of the target seven β-agonists in pork meats by UPLC-MS/MS.

Enantiomeric quality control of R-Tofisopam by HPLC using polysaccharide-type chiral stationary phases in polar organic mode.

A novel, fast and economic chiral HPLC method was developed and validated for the resolution of the four isomers of tofisopam. The separation capacity of eleven different chiral columns: six polysaccharide-type including three amylose-based (Chiralpak AD, Chiralpak AD-RH and Chiralpak AS) and three cellulose-based (Chiralcel OD, Chiralcel OJ and Lux Cellulose-4); three cyclodextrin- (Quest-BC, Quest-C2 and Quest-CM) and two macrocyclic glycopeptide antibiotic-type (Chirobiotic T and Chirobiotic TAG) were screened using polar organic or reversed-phase mode. Chiralpak AD, based on amylose tris(3,5-dimethylphenylcarbamate) as chiral selector with neat methanol was identified as the most promising system. In order to improve resolution, an orthogonal experimental design was employed, altering the concentration of 2-propanol, column temperature, and flow rate in a multivariate manner. Using the optimized method (85/15 v/v methanol/2-propanol, 40°C, flow rate: 0.7mL/min) we were not only able to separate the four isomers but also detect 0.1% S-enantiomer as chiral impurity in R-tofisopam. This is important since the latter is under development as a single enantiomeric agent. Thermodynamic investigation revealed an unusual entropy and enthalpy-entropy co-driven controlled enantioseparation on Chiralcel OJ and on Chiralpak AD column, respectively. Our newly developed HPLC method was validated according to the ICH guidelines and its application was tested on a pharmaceutical formulation containing the racemic mixture of the drug. As a further novelty, a separate circular dichroism method was applied for the investigation of the interconversion kinetics of tofisopam conformers, which proved to be crucial for sample preparation and method validation.

Simultaneous Preconcentration, Identification, and Quantitation of Selenoamino Acids in Oils by Enantioselective High Performance Liquid Chromatography and Mass Spectrometry.

Selenium is an essential micronutrient for humans. In food, selenium can be present in both inorganic and organic forms, the latter mainly being selenomethionine, Se-methyl-selenocysteine, and selenocystine. Selenoamino acid speciation rarely involves the chirality of selenoamino acids. In this work, a 5 cm long CHIROBIOTIC TAG chromatographic column was used for enantioresolution of selenoamino acids (d- and l-selenomethionine, Se-methyl-l-selenocysteine, d-, l- and meso-selenocystine); in the optimized conditions, the complete resolution of the analytes was achieved within 15 min by using a very polar aqueous mobile phase (gradient elution by methanol/acetonitrile/H2O, 45:45:10 ( v/ v/ v) with 10 mmol L-1 of ammonium formate and 0.5% formic acid as the mobile phase A and acetonitrile/H2O, 20:80 ( v/ v) with 20 mmol L-1 of ammonium formate at apparent pH 4 as the mobile phase B). The affinity of the teicoplanin aglycone was further exploited to devise a preconcentration method for selenoamino acids in oils. In particular, the CHIROBIOTIC TAG precolumn was used to directly concentrate the selenoamino acids after simple dilution of oil samples with dichloromethane. An optimized procedure for selenoamino acid trapping and preconcentration under normal phase conditions was developed. The enrichment procedure also ensured band focusing during the subsequent separation. The target analytes were finally identified and quantified by triple quadrupole selected reaction monitoring. The method allowed obtainment of recovery values up to 73%, with limits of detection between 280 and 750 ng and limits of quantification between 375 and 960 ng for the different selenoamino acids. The method was applied to commercial oil samples, and only l-selenomethionine was detected.

Liquid chromatographic enantioseparation of carbocyclic β-amino acids possessing limonene skeleton on macrocyclic glycopeptide-based chiral stationary phases

Polar-ionic and reversed-phase high-performance liquid chromatographic separations of limonene-based cyclic β-amino acid enantiomers were carried out by using macrocyclic glycopeptide-based chiral selectors applying Chirobiotic T, TAG and R columns. The effects of additives, concentration of the co- and counter-ions and the temperature in polar-ionic mobile phase systems were studied. The influence of pH, MeOH content and alcohol additives were investigated in the reversed-phase mode. The difference in the change in standard enthalpy Δ(ΔH°), entropy Δ(ΔS°), and free energy Δ(ΔG°) was calculated from the linear van't Hoff plots derived from the ln α vs 1/T curves in the temperature range 5–40°C. Unusual temperature behavior was observed on Chirobiotic TAG for most of the analytes: decreased retention times were accompanied with increased separation factors with increasing temperature, and separation was entropically-driven. For two of the studied analytes enthalpically-driven enantioseparations were observed. The elution sequence was determined in all cases, but no general rule could be established.

Integration of simulated moving bed chromatography and enzymatic racemization for the production of single enantiomers

Integration of enantioseparation by simulated moving bed (SMB) and mild enzymatic racemization enables the production of single enantiomers from a racemic mixture in theoretically 100% yield and hence overcomes the 50% yield limitation of conventional SMB processes. We implemented such a process consisting of a Chirobiotic TAG column-SMB, an amino acid racemase-containing enzyme membrane reactor, and a nanofiltration unit for concentration of the distomer-enriched SMB raffinate prior to racemization on lab-scale for the production of enantiopure D-methionine. The integrated process scheme was operated continuously for over 30h without significant variations in product concentration and purity and with a yield of 93.5%, demonstrating the feasibility of this integrated process concept. Furthermore, a rational analysis of the integrated process on the basis of a short-cut model was conducted. The process model consists of a true moving bed equilibrium stage model to represent the SMB, a continuous stirred tank reactor model with reversible Michaelis–Menten kinetics to represent the enzyme membrane reactor, a nanofiltration model and feed node mass balances, and enabled the identification of optimal operating points (flow rate ratios, enzyme concentration) at a variety of process specifications and objectives. Optimal operating points were calculated for different cost distributions between the applied materials such as stationary phase, enzyme, solvent, and nanofiltration membrane. By assigning plausible pricing data and lifetimes to the respective materials, variable costs for the specific process considered in this work were estimated.

In Vitro Investigation of D- and Lenantiomer Synergistic Efects of Some Amino Acids

Abstract D-amino acids can arise from endogenous microbial flora, from ingestion with the diet or from spontaneous racemization of L-amino acids during ageing. In this work, the behavior of methionine, homocysteine and cysteine enantiomers was investigated in human serum in vitro during 0-72 h at incubation temperature 37°C. The separation of enantiomers was realized in two dimensional on-line system (the connection of an achiral column Purospher RP-18 endcapped and a chiral column Chirobiotic TAG). This system allowed simultaneous monitoring all tested amino acids and their enantiomers. The possible effect D-enantiomer on the behavior of its L-enantiomer (the synergistic effect) was evaluated during incubation time. The first results have showed that no synergistic effect of D-enantiomer on its Lisomer has been observed in our experimental conditions in vitro.

Two-dimensional high performance liquid chromatography for determination of homocysteine, methionine and cysteine enantiomers in human serum

A two-dimensional HPLC system with electrochemical detection was used for determination of homocysteine, methionine and cysteine enantiomers in biological samples. The amino acid separations were not possible only by using a chiral column. The compounds were separated from each other on an achiral column (Purospher RP-18 endcapped 250–4mm, 5μm) and their enantiomers were separated on Chirobiotic TAG (250–4.6mm, 5μm) column in an on-line system. The mobile phase composition and a choice of electrode potentials for detection were investigated. The l-enantiomers always eluted before the d-enantiomers. The proposed method was applied to the analysis of human serum of healthy volunteers and patients with multiple sclerosis. The limit of detection (LOD) and quantitation (LOQ) were defined as the concentration that produced a signal-to-noise ratio (S/N) of 3 and 10. The method LOD values were found to be between 0.05 and 0.50μgmL−1. The range of LOQ values were between 0.17 and 1.67μgmL−1, respectively.

D- and L-amino acids in Antarctic lakes: assessment of a very sensitive HPLC-MS method.

Amino acids represent a fraction of organic matter in marine and freshwater ecosystems, and a source of carbon, nitrogen and energy. L-Amino acids are the most common enantiomers in nature because these chiral forms are used during the biosynthesis of proteins and peptide. To the contrary, the occurrence of D-amino acids is usually linked to the presence of bacteria. We investigated the distribution of L- and D-amino acids in the lacustrine environment of Terra Nova Bay, Antarctica, in order to define their natural composition in this area and to individuate a possible relationship with primary production. A simultaneous chromatographic separation of 40 L- and D-amino acids was performed using a chiral stationary phase based on teicoplainin aglycone (CHIROBIOTIC TAG). The chromatographic separation was coupled to two different mass spectrometers--an LTQ-Orbitrap XL (Thermo Fisher Scientific) and an API 4000 (ABSciex)--in order to investigate their quantitative performance. High-performance liquid chromatography coupled with mass spectrometry methods were evaluated through the estimation of their linear ranges, repeatability, accuracy and detection and quantification limits. The high-resolution mass spectrometer LTQ-Orbitrap XL presented detection limits between 0.4 and 7 μg L (-1), while the triple quadrupole mass spectrometer API 4000 achieved the best detection limits reported in the literature for the quantification of amino acids (between 4 and 200 ng L (-1)). The most sensitive method, HPLC-API 4000, was applied to lake water samples.

Enantioresolution of Chiral Derivatives of Xanthones on Different Types of Liquid Chromatography Stationary Phases: A Comparative Study

Since the development of chiral stationary phases (CSPs), liquid chromatography has emerged as one of the most useful methods for analyses and preparation of enantiomerically pure bioactive compounds. However, the key for success of enantioseparation lies in the first place in the choice of a suitable CSP having enantioselectivity for the target enantiomers. Thus, this work describes a comparative study of resolution of seven chiral derivatives of xanthones (CDXs) on nine CSPs of different nature (four macrocyclic antibiotics, two Pirkle-type and three polysaccharide-based), under multimodal elution conditions. The polysaccharide-based CSPs have proved to be most suitable to separate this group of compounds, since all the CDXs were enantioseparated with excellent enantioselectivity and resolution. Six of the seven CDXs were separated on macrocyclic antibiotic-based CSPs, however with low enantioresolution. The Pirkle-type CSPs exhibited specificity on chiral discrimination behavior, presenting very high enantioselectivity for CDXs with aromatic group linked to the chiral moiety. This comparative study provides valuable information in the field of enantioresolution, and constitutes a useful background for subsequent studies of this important class of compounds. Keywords: Amylose tris-3, 5-dimethylphenylcarbamate, amylose tris-3, 5-dimethoxyphenylcarbamate, cellulose tris-3, 5- dimethylphenylcarbamate, chiral derivatives of xanthones, chiral stationary phases, chirality, chirobiotic R, chirobiotic T, chirobiotic TAG, chirobiotic V, enantiomerically pure, enantioresolution, enantioselectivity, liquid chromatography, Lphenylglycine, macrocyclic antibiotic, multimodal elution, Pirkle-type stationary phases, polysaccharide-based stationary phases, (S, S)-Whelk-O1.

Simultaneous, sequential quantitative achiral–chiral analysis by two‐dimensional liquid chromatography

In general, chromatographic analysis of chiral compounds involves a minimum of two methods; a primary achiral method for assay and impurity analysis and a secondary chiral method for assessing chiral purity. Achiral method resolves main enantiomeric pairs of component from potential impurities and degradation products and chiral method resolves enantiomeric pairs of the main component and diastereomer pairs. Reversed-phase chromatographic methods are preferred for assay and impurity analysis (high efficiency and selectivity) whereas chiral separation is performed by reverse phase, normal phase, or polar organic mode. In this work, we have demonstrated the use of heart-cutting (LC-LC) and comprehensive two-dimensional liquid chromatography (LC × LC) in simultaneous, sequential achiral and chiral analysis and quantitation of minor, undesired enantiomer in the presence of major, desired enantiomer using phenylalanine as an example. The results were comparable between LC-LC and LC × LC with former offering better sensitivity and accuracy. The quantitation range was over three orders of magnitude with undesired D-phenylalanine detected at approximately 0.3% in the presence of predominant, desired L-phenylalanine (99.7%). The limit of quantitation was comparable to conventional high-performance liquid chromatography. A reversed-phase C18 achiral column in the primary and reversed-phase Chirobiotic Tag chiral column in the secondary dimension were used with a compatible mobile phase.

Enantioseparation and chiral recognition mechanism of new chiral derivatives of xanthones on macrocyclic antibiotic stationary phases

A chiral HPLC method using four macrocyclic antibiotic chiral stationary phases (CSPs) has been investigated for determination of the enantiomeric purity of fourteen new chiral derivatives of xanthones (CDXs). The separations were performed with the CSPs Chirobiotic T, Chirobiotic TAG, Chirobiotic V and Chirobiotic R under multimodal elution conditions (normal-phase, reversed-phase and polar ionic mode). The analyses were performed at room temperature in isocratic mode and UV and CD detection at a wavelength of 254nm. The best enantioselectivity and resolution were achieved on Chirobiotic R and Chirobiotic T CSPs, under normal elution conditions, with RS ranging from 1.25 to 2.50 and from 0.78 to 2.06, respectively. The optimized chromatographic conditions allowed the determination of the enantiomeric ratio of eight CDXs, always higher than 99%. In order to better understand the chromatographic behavior at a molecular level, and the structural features associated with the chiral recognition mechanism, computational studies by molecular docking were carried out using VDock. These studies shed light on the mechanisms involved in the enantioseparation for this important class of chiral compounds.

Simulated moving bed enantioseparation of amino acids employing memory effect-constrained chromatography columns

Teicoplanin aglycone-based chromatography columns (Chirobiotic TAG) enable amino acid enantioseparation with aqueous mobile phases, which perfectly accommodates the distinct hydrophilicity of most amino acids. Therefore, this stationary phase constitutes a promising option in particular for preparative-scale separations that require high feed concentrations for economic operation. However, detailed studies revealed a solute-related memory effect when this column is subjected to high loadings of amino acids, conditions that prevail in SMB operation. High loadings yield an activation of the column as indicated by increased retention times when comparing finite injection chromatograms obtained before and after the column had been subjected to a concentrated amino acid feed. This effect can be slowly reversed by flushing the column with solvent devoid of amino acid. Obviously, the activation of the stationary phase needs to be accounted for in the determination of adsorption isotherms that are used for SMB design. In this work we introduce a perturbation method adapted specifically to capture the stationary phase behaviour at SMB-like conditions. The adsorption isotherms obtained from this method indeed allowed for accurate SMB design of a methionine enantioseparation as judged by the very good agreement of experimentally obtained and model-predicted purities. Furthermore, SMB operation over 3 days with constant purities (besides deviations originating from a dip in temperature) was accomplished indicating that the adsorption behaviour in the activated state is indeed time invariant and stable long-term SMB operation with these columns is principally feasible.

High-performance liquid chromatographic enantioseparation of unusual isoxazoline-fused 2-aminocyclopentanecarboxylic acids on macrocyclic glycopeptide-based chiral stationary phases

The enantiomers of four unusual isoxazoline-fused 2-aminocyclopentanecarboxylic acids were directly separated on chiral stationary phases containing macrocyclic glycopeptide antibiotics teicoplanin (Astec Chirobiotic T and T2), teicoplanin aglycone (Chirobiotic TAG), vancomycin (Chirobiotic V) and vancomycin aglycone (Chirobiotic VAG) as chiral selectors. The effects of the mobile phase composition, the structure of the analytes and temperature on the separations were investigated. Experiments were performed at constant mobile phase compositions in the temperature range 5–45°C to study the effects of temperature, and thermodynamic parameters were calculated from plots of lnk or lnα versus 1/T. Some mechanistic aspects of the chiral recognition process are discussed with respect to the structures of the analytes. It was found that the enantiomeric separations were in most cases enthalpy-driven. The sequence of elution of the enantiomers was determined in all cases.

High-performance liquid chromatographic enantioseparation of monoterpene-based 2-amino carboxylic acids on macrocyclic glycopeptide-based phases

The enantiomers of five monoterpene-based 2-amino carboxylic acids were directly separated on chiral stationary phases containing macrocyclic glycopeptide antibiotics such as teicoplanin (Astec Chirobiotic T and T2) and teicoplanin aglycone (Chirobiotic TAG) as chiral selectors. The effects of pH, the mobile phase composition, the structure of the analyte and temperature on the separations were investigated. Experiments were performed at constant mobile phase compositions in the temperature range 10–40 °C to study the effects of temperature and thermodynamic parameters on separations. Apparent thermodynamic parameters and T iso values were calculated from plots of ln k or ln α versus 1/ T. Some mechanistic aspects of the chiral recognition process are discussed with respect to the structures of the analytes. It was found that the enantioseparations were in most cases enthalpy driven. The sequence of elution of the enantiomers was determined in all cases.

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.

Comparison of performance of Chirobiotic T, T2 and TAG columns in the separation of β2‐ and β3‐homoamino acids

The enantiomers of eight unusual beta(2)- and beta(3)-homoamino acids were separated on chiral stationary phases containing the macrocyclic glycopeptide antibiotic teicoplanin (Chirobiotic T or T2) or teicoplanin aglycone (Chirobiotic TAG) as chiral selectors. The effects of the organic modifier, the mobile phase composition, and temperature on the separations were investigated. Linear van't Hoff plots were observed in the studied temperature range, 280-318 K, and the changes in enthalpy, Delta(DeltaH(o)), entropy, Delta(DeltaS(o)), and free energy, Delta(DeltaG(o)) were calculated. The values of the thermodynamic parameters depended on the nature of the selectors, the structures of the analytes, and especially the positions of the substituents on the analytes. A comparison of the separation performances of the macrocyclic glycopeptide stationary phases revealed that the Chirobiotic TAG column exhibited much better selectivity for beta(2)-homoamino acids, while the separation of beta(3)-homoamino acid enantiomers was better on Chirobiotic T or T2. The elution sequence was determined in some cases and was observed to be R < S.

High‐performance liquid chromatographic chiral separation of β2‐homoamino acids

Reversed-phase high-performance liquid chromatographic methods were developed for the separation of enantiomers of eleven unnatural beta(2)-homoamino acids on chiral stationary phases containing macrocyclic glycopeptides (teicoplanin-containing Chirobiotic T and T2) or the macrocyclic peptide teicoplanin aglycone (Chirobiotic TAG) as chiral selectors. The effects of the organic modifier, the mobile phase composition, temperature, and the structures of the analytes on the separations were investigated. Separations were carried out at constant mobile phase compositions in temperature range 7-45 degrees C and the changes in enthalpy, Delta(DeltaH(o)), entropy, Delta(DeltaS(o)), and free energy, Delta(DeltaG(o)), were calculated. The -Delta(DeltaG(o)) values obtained on the three columns indicated that Chirobiotic TAG, without sugar units, may promote the interactions of the enantiomers of beta(2)-homoamino acids with branched alkyl or aryl side-chains, whereas for beta(2)-homoamino acids with alkyl side-chains Chirobiotic T and T2 seem to be more favorable. The elution sequence was determined in some cases and was observed to be R < S.