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.     

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.     

Retention mechanism of high-performance liquid chromatographic enantioseparation on macrocyclic glycopeptide-based chiral stationary phases

The development of methods for the separation of enantiomers has attracted great interest in the past 20 years, since it became evident that the potential biological or pharmacological applications are mostly restricted to one of the enantiomers. 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 high-performance liquid chromatography (HPLC), thin-layer chromatography and electrophoresis. 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. 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 macrocyclic glycopeptide antibiotics and, through their application, endeavors to demonstrate the mechanism of separation on macrocyclic glycopeptides. The sequence of elution of the stereoisomers and the relation to the absolute configuration are also discussed.     

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.     

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.     

Chromatographic enantioseparation of amino acids using a new chiral stationary phase based on a macrocyclic glycopeptide antibiotic

The separation of the enantiomers of several a-amino acids was studied on a new chiral stationary phase (CSP) which is based on the macrocyclic glycopeptide antibiotic eremomycin attached to silica particles. Retention and separation factors were determined under analytical conditions at ambient temperature for different mobile phase compositions. In order to evaluate the potential with respect to preparative separations the adsorption isotherms of D- and L-methionine were determined for one mobile phase composition applying the elution by characteristic point method. The isotherms were validated by comparing experimentally determined elution profiles with predictions based on the equilibrium dispersive model. Finally, the performance of the eremomycin CSP was compared with a commercially available CSP based on the macrocyclic antibiotic teicoplanin. After determining the isotherms of D- and L-methionine also for the teicoplanin phase, the equilibrium dispersive model was used for both CSP to identify optimal operating conditions. For the separation and conditions considered the new eremomycin CSP revealed a better performance compared to the teicoplanin CSP.     

Polysaccharide-based chiral stationary phases for high-performance liquid chromatographic enantioseparation

Recent developments of polysaccharide-based chiral stationary phases (CSPs) for the direct separation of enantiomers in high-performance liquid chromatography (HPLC) are mainly reviewed together with the results on mechanistic studies by means of chromatography, NMR and mass spectroscopies, and computational methods. Miscellaneous applications of polysaccharide derivatives to the newly developed, chiral dynamic high-performance liquid chromatography (DHPLC) for obtaining a nonracemic compound are also described.     

双选择体手性固定相中选择体的构型对分离性能的影响

为研究选择体的构型对双选择体固定相手性识别的影响,以(1S,2S)-(~)-二苯基乙二胺及L-(~)-二苯甲酰酒石酸为手性源,合成了一种新的双选择体固定相,并用不同结构的手性样品测试了其手性分离能力。结果表明,这种固定相与以(1R,2R)-(+)-二苯基乙二胺及L(~)-二苯甲酰酒石酸为手性源制备的双选择体固定相有相当的手性分离能力,但这两种固定相所能分离的化合物不尽相同。对双选择体固定相中两个选择体的构型对固定相手性识别的影响进行了探讨。在手性识别中,以不同手性源制备的两个选择体的立体构型不能同时与一个手性样品的立体构型相匹配,从而导致相应的双选择体固定相手性分离能力的下降。     

Monolithic chiral stationary phases for liquid‐phase enantioseparation techniques

This short overview summarizes the development in the field of enantioselective monolithic chromatographic media and their application for pressure‐driven and electrokinetic separations. The major emphasis is put on the currently existing problems and the author's vision for their solution is provided. Due to the author's personal experience silica‐based monoliths are discussed in more detail although the key developments in the field of organic monolithic materials for separation of enantiomers are also discussed.     

Advanced dress-up chiral columns: New removable chiral stationary phases for enantioseparation of chiral carboxylic acids

This paper describes the preparation of new dress-up columns featuring reproducibly removable and replaceable chiral stationary phases. After synthesizing perfluroalkylated quinine and quinidine derivatives as chiral stationary phase compounds (F-CSPs), we adsorbed them reversibly onto a fluorous LC column through pumping of their solutions. Using this dress-up chiral column and fluorophobic elution of aqueous ammonium formate/MeOH mixtures, we could enantioseparate four racemic N-acetyl amino acids, dichlorprop, and sixteen fluorescent 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC)-derivatized amino acids. Dressing and undressing of the coated F-CSPs could be controlled by varying the fluorophilicity and fluorophobicity of the eluent. The relative standard deviations of the retention times, the retention factors, the number of theoretical plates, the enantioseparation factors, and the resolutions of each of four preparations of such dress-up columns were all less than or equal to 5.26% (from 20 repeated analyses); the reproducibilities from four different preparations were all less than or equal to 10.6%. These columns also facilitated highly sensitive and selective analyses of AQC-amino acids when detected using LC–MS/MS.     

Synthesis of novel chiral imidazolium stationary phases and their enantioseparation evaluation by high-performance liquid chromatography

Two novel chiral stationary phases (CSPs) were prepared by bonding chiral imidazoliums on the surface of silica gel. The chiral imidazoles were derivatized from chiral amines, 1-phenylethylamine and 1-(1-naphthyl)ethylamine. The obtained CSPs were characterized by Fourier Transform Infrared (FT-IR) spectroscopy and elemental analysis (EA), demonstrating the bonding densities of CSP 1 and CSP 2 were 0.43 mmol g⁻¹ and 0.40 mmol g⁻¹, respectively. These two CSPs could be used to availably separate 8 pharmaceuticals, 7 mandelic acid/its derivatives, 2 1-phenylethylamine derivatives, 1 1,1′-bi-2-naphthol, and 1 camphorsulfonic acid in high-performance liquid chromatography (HPLC). It is found that CSP 1 could effectively enantioseparate most chiral analytes, especially the acidic components, while CSP 2 could enantiorecognize all chiral analytes, although a number of components did not achieve baseline separation. Additionally, the effects of mobile phase composition, mobile phase pH and salt content, chiral selector structures, and analyte structures on the enantiorecognitions of the two CSPs were investigated. It is found that high acetonitrile content in mobile phases was conducive to enantiorecognition. Mobile phase pH and salt content could alter the retention behaviors of different enantiomers of the same chiral compound, resulting in better enantioresolution. Moreover, both chiral selector structures and substituted groups of analytes played a significant role in the separation of chiral solutes.     

High-performance liquid chromatographic enantioseparation of aminonaphthol analogs on polysaccharide-based chiral stationary phases

High-performance liquid chromatographic methods were developed for the separation of the enantiomers of five new aminonaphthol analogs possessing two chiral centers. The direct separations were performed on chiral stationary phases containing either amylose-tris-3,5-dimethylphenyl carbamate (Kromasil^® AmyCoat™ column) or cellulose-tris-3,5-dimethylphenyl carbamate (Kromasil^® CelluCoat™ column) as chiral selector. The experimental data are utilized to discuss the effects of the mobile phase composition, the nature of the alcoholic modifier and the specific structural features of the analytes on the retention and separation. The elution sequence was determined in all cases; no general regularities could be established.     

High-performance liquid chromatographic enantioseparation of drugs containing multiple chiral centers on polysaccharide-type chiral stationary phases

Enantioseparation of drugs with multiple chiral centers is challenging. This article describes resolution of some drugs with multiple chiral centers using polysaccharide-type chiral stationary phases. Also, the use of the column-switching technique is demonstrated to achieve the resolution of this type of compounds.     

New chiral stationary phase with macrocyclic glycopeptide antibiotic eremomycin chemically bonded to silica

A new chiral stationary phase (CSP) was prepared by attachment of macrocyclic glycopeptide antibiotic eremomycin to the epoxy-activated silica under mild conditions. In contrast to CSP with immobilized vancomycin, which is a close structural analogue of eremomycin, the prepared CSP reveals high enantioselectivity for separation of amino acids enantiomers. It was demonstrated by the example of ristocetin A CSP that method of the immobilization of macrocyclic glycopeptide antibiotics affects remarkably the resulting enantioselectivity.     

Low-molecular-weight chiral cation exchangers: novel chiral stationary phases and their application for enantioseparation of chiral bases by nonaqueous capillary electrochromatography

Cation exchange type chiral stationary phases (CSPs) based on 3,5-dichlorobenzoyl amino acid and amino phosphonic acid derivatives as chiral selectors (SOs) and silica as chromatographic support were developed and applied to enantiomer separations of chiral bases by nonaqueous capillary electrochromatography (NA-CEC). As a rationale for efficient CSP development we adopted the combined use of the "reciprocity principle of chiral recognition" and nonaqueous ion-pair CE as screening assay. Thus, (S)-atenolol was employed as chiral counter-ion added to the BGE in CE and a series of N-derivatized amino acids and amino phosphonic acids were screened to derive reciprocally information on their chiral recognition abilities for atenolol enantiomers. Two SO candidates, namely N-(3,5-dichlorobenzoyl)-O-allyl-tyrosine and N-(4-allyloxy-3,5-dichlorobenzoyl)-1-amino-3-methylbutane phosphonic acid that have been identified as potential SOs in the CE screening were, after immobilization on thiol-modified silica, evaluated in cation-exchange NA-CEC. The strong chiral cation exchanger with the free phosphonic acid group exhibited enhanced enantioselectivity compared to the weak chiral cation exchanger with the carboxylic acid group. A wide variety of chiral bases could be successfully resolved on the strong chiral cation exchanger with alpha-values up to 2.2 and efficiencies up to 375000 m-1 including beta-blockers and other amino alcohols, local anesthetics like etidocaine, antimalarial agents like mefloquine, Tr?ger's base, phenothiazines like promethazine, and antihistaminics. The influence of several experimental parameters (electrolyte concentration, acid-base ratio and acetonitrile-methanol ratio) was evaluated.     

High-performance liquid chromatographic enantioseparation of amino compounds on newly developed cyclofructan-based chiral stationary phases

Direct separation of the enantiomers of amino acid amines, amino alcohols, and diamines was performed on recently developed chiral stationary phases containing isopropyl carbamate-cyclofructan 6 (IP-CF6), (R)-naphthylethylcarbamate cyclofructan 6 (RN-CF6), or dimethylphenylcarbamate cyclofructan 7 (DMP-CF7) as chiral selectors, using n-hexane/alcohol/TFA as mobile phase. The effects of the mobile phase composition, the nature and concentrations of the alcoholic and acidic modifiers, and the structures of the analytes on the retention and resolution were investigated. In some cases, separations were carried out at constant mobile phase composition in the temperature range 5-40 °C. Thermodynamic parameters and T(iso) values were calculated from plots of lnk versus 1/T. It was found that the enantioseparations were enthalpy driven. The sequences of elution of the stereoisomers were determined but no general rule could be established.     

Mechanism of enantioseparation on zeolite-supported chiral stationary phases in the presence of lipophilic buffer

Summary Chiral stationary phases have been prepared on a zeolite A support. In addition to polar hydrophilic chiral selector molecules derived from DNB-L-Leu and DNB-L-Phe amides, dynamic modification of the underivatized free surface hydroxyl (silanol) groups of the zeolite can be used to increase the overall hydrophobic character. On these non-capped chiral stationary phases, in the presence of lipophilic buffer salts in the aqueous organic phase, the enantioseparation of ion-paired D,L-mandelic acid was facilitated and was studied in more detail. The enantioseparation might be based on hydrogen bonding and π-π interaction supported by simultaneous hydrophobic interaction. Presented at: Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 3–5, 1997.     

新型直链淀粉类手性固定相的制备与手性拆分性能

通过对糖单元2-位进行选择性酯化以及6-位保护与去保护,运用区域选择性方法合成了5种新型直链淀粉类衍生物,分别为直链淀粉-2-苯甲酸酯-3-(4-氯苯基氨基甲酸酯)-6-(3,5-二甲基苯基氨基甲酸酯)、直链淀粉-2-苯甲酸酯-3-(4-氯苯基氨基甲酸酯)-6-(3,5-二氯苯基氨基甲酸酯)、直链淀粉-2-苯甲酸酯-3,6-二(4-氯苯基氨基甲酸酯)、直链淀粉-2-(4-氯苯甲酸酯)-3,6-二(3,5-二氯苯基氨基甲酸酯)和直链淀粉-2-(4-氯苯甲酸酯)-3,6-二(环己基氨基甲酸酯),并将其涂覆在氨丙基硅胶表面制备了HPLC手性固定相。利用核磁共振-氢谱(¹H-NMR)和傅里叶变换红外光谱(FT-IR)技术对所合成衍生物的结构进行了表征和分析,并用HPLC法评价所合成衍生物的手性识别能力。与具有单一取代基直链淀粉类手性固定相的对比分析表明,所合成的新型直链淀粉类手性固定相对于某些对映体具有更为优异的拆分结果。进一步分析表明,2-、3-和6-位取代基的性能和引入位置对直链淀粉衍生物的手性识别能力均有较大的影响。     

Enantioselective high-performance liquid chromatographic separation of N-methyloxycarbonyl unsaturated amino acids on macrocyclic glycopeptide stationary phases

This paper describes the enantiomeric resolution of a series of unsaturated N-methyloxycarbonyl-alpha-H-alpha-amino acids (N-MOC-alpha-amino acids) on macrocyclic glycopeptide stationary phases by means of high-performance liquid chromatography (HPLC). Three types of glycopeptide phases, i.e. Chirobiotic T, V and R, were evaluated in both reversed-phase (RP) and polar ionic mode (PIM). The best results in terms of enantioselectivity and resolution were obtained on Chirobiotic R phase, with the PIM mobile phase giving the highest resolution per min. Investigation of the pH of the reversed-phase mobile phase in the pH range 4.1-5.9 showed little effect on enantioselectivity. The method was applied for monitoring the conversion and product enantiomeric excess of an enzymatic hydrolysis reaction using N-MOC-alpha-H-alpha-amino acid esters as substrate.     

Synthesis and enantioseparation behaviors of novel immobilized 3,5‐dimethylphenylcarbamoylated polysaccharide chiral stationary phases

Two new polysaccharide‐derived chiral selectors, namely, 6‐azido‐6‐deoxy‐3,5‐dimethylphenylcarbamoylated amylose and 6‐azido‐6‐deoxy‐3,5‐dimethylphenyl carbamoylated cellulose, were synthesized under homogeneous conditions and immobilized onto aminized silica gel by the Staudinger reaction, resulting in two new immobilized polysaccharide chiral stationary phases (CSPs). Their enantioseparation performances were investigated under normal‐phase mode by HPLC. Among 17 analytes, baseline separations of 12 pairs of enantiomers are achieved on the immobilized cellulose CSP, which demonstrates that this new cellulose material exhibits almost the same enantioseparation performance as the coated cellulose CSP. In addition, the amylose‐derived CSP presents limited enantiorecognition ability but certain complementarity with the immobilized and coated cellulose‐based materials. Neither metolachlor nor paclitaxel side chain acids are separated on two cellulose‐derived CSPs, but effective separations are obtained on the immobilized amylose column.