High-performance liquid chromatography chiral stationary phases based on low-molecular-mass selectors

A review of HPLC chiral stationary phases (CSPs) based on low molecular mass selectors is given. The review is focused on brush- and monomeric-type CSPs obtained by covalent linkage of chiral selectors, with emphasis on those obtained by total synthesis. Emphasis is given to new, emerging aspects like enantioseparations on receptor-like chiral stationary phases and dynamic enantioselective chromatography of stereolabile compounds.     

Stereoselective interactions of chiral dipeptides on amylose based chiral stationary phases

Dipeptides are stereo-specifically involved in several biological functions that are challenging to separate enantiomerically. Elution order of enantiomers is an important issue in chiral chromatography. Amylose tris-(3,5-dimethylphenylcarbamate) chiral stationary phase(CSP) is the best and most-widely-used CSP in chiral separations, but experimental data of enantiomeric separation of dipeptides on this CSP is lacking. Simulation studies were conducted to determine the order of elution and the chiral recognition mechanism of didpetides on this CSP. Results indicated that the docking energy of SR-enantiomers were higher than SS-antipodes. The range of docking energies for SR-enantiomers was -7.44 to -5.92 kcal/mol with CSP, but -7.15 to -5.87 kcal/mol for SS-stereoisomers. Therefore it is predicted that SS-enantiomer will elute first, followed by SR-antipode. Furthermore, hydrogen bondings, van der Waal’s interactions and electrostatic interactions were observed among SR- and SSenantiomers and chiral grooves of CSP. The number of hydrogen bonds was one in each enantiomer binding except S-Ala-R-Tyr, which contained two hydrogen bonds. No hydrogen bond was found in S-Ala-R-Trp, S-Leu-S-Trp, and S-Leu-S-Tyr dipeptides bindings. The chiral recognition mechanisms dictate different strengths of stereoselective bindings of the enantiomers on CSP.     

Evaluation of novel dendrimer chiral stationary phases using HPLC

Summary The reversed phase chromatographic properties of the [G1]-L-glutamic and ethyl ester-AC-silica (1), [G2]-L-glutamic acid ethyl ester-AC-silica (2) and the [G1]-L-glutamic acidt-butyl ester-AC-silica (3) dendrimer stationary phases were evaluated. Initial studies involved the comparison between these phases with a classic reversed phase (i.e. ODS1) by the separation of a standard reversed phase test mixture composed of dimethylphthalate, nitrobenzene, anisole, diphenylamine and fluorene. Separations were achieved with comparable performance to those obtained with the conventional reversed phase (ODS1). However, it was apparent that the chromatographic selectivity exhibited by the dendrimer stationary phases was different from that of the ODS1 phase. On a per mole basis, the dendrimers exhibited similar (and sometimes greater) affinity for these analytes compared with the ODS1 ligand. Subsequent chromatographic experiments were conducted upon the dendrimer chiral stationary phases using chiral analytes under reversed phase and normal phase conditions. Chiral resolution was not observed.     

Preparation and evaluation of novel chiral stationary phases based on quinine derivatives comprising crown ether moieties

The C9‐position of quinine was modified by meta‐ or para‐substituted benzo‐18‐crown‐6, and immobilized on 3‐mercaptopropyl‐modified silica gel through the radical thiol‐ene addition reaction. These two chiral stationary phases were evaluated by chiral acids, amino acids, and chiral primary amines. The crown ether moiety on the quinine anion exchanger provided a ligand‐exchange site for primary amino groups, which played an important role in the retention and enantioselectivity for chiral compounds containing primary amine groups. These two stationary phases showed good selectivity for some amino acids. The complex interaction between crown ether and protonated primary amino group was investigated by the addition of inorganic salts such as LiCl, NH₄Cl, NaCl, and KCl to the mobile phase. The resolution results showed that the simultaneous interactions between two function moieties (quinine and crown ether) and amino acids were important for the chiral separation.     

Dynamic HPLC on chiral stationary phases: a powerful tool for the investigation of stereomutation processes

Dynamic HPLC on enantioselective stationary phases has become a well-established technique to investigate chiral molecules with internal motions that result in stereoinversion and occur on the time scale of the separation process. Kinetic parameters for the on-column interconversion phenomena can be extracted from experimental peak profiles by computer simulation or by direct calculation methods. The technique has been used in a wide range of temperatures and is complementary in scope to dynamic NMR spectroscopy.     

Investigation of brush-type chiral stationary phases based on O,O'-diaroyl tartardiamide and O,O'-bis-(arylcarbamoyl) tartardiamide

Four chiral organosilanes based on O,O'-dibenzoyl tartardiamide, O,O'-bis-(3,5-dimethylbenzoyl) tartardiamide, O,O'-bis-(phenylcarbamoyl) tartardiamide and O,O'-bis-[(3,5-dimethylphenyl)carbamoyl] tartardiamide were synthesized and immobilized on silica to afford corresponding brush-type chiral stationary phases (CSPs) with well-defined structures. Using 54 compounds containing a wide variety of structures as analytes, the enantioselectivities of the four CSPs were evaluated under normal-phase modes. 3,5-Dimethyl substituent in the aryl group was found to significantly affect the enantioselectivity of CSPs containing aryl ester moieties. Aryl carbamate moieties in CSPs were observed more beneficial for enantioseparation than aryl ester moieties. The additional hydrogen-bond donors (NH) present in the carbamate groups contributed greatly to the enantioselectivity of CSPs, which is contrary to the results that have been found in network-polymeric CSPs.     

Synthesis and chromatographic properties of HPLC chiral stationary phases based upon β-cyclodextrins

Chromatographia - β-cyclodextrin chiral stationary phases (CD-CSP) having different spacer lengths have been prepared and evaluated (retention, selectivity, resolution) for the liquid...     

Chiral recognition by enantioselective liquid chromatography: Mechanisms and modern chiral stationary phases

An overview of the state-of-the-art in LC enantiomer separation is presented. This tutorial review is mainly focused on mechanisms of chiral recognition and enantiomer distinction of popular chiral selectors and corresponding chiral stationary phases including discussions of thermodynamics, additivity principle of binding increments, site-selective thermodynamics, extrathermodynamic approaches, methods employed for the investigation of dominating intermolecular interactions and complex structures such as spectroscopic methods (IR, NMR), X-ray diffraction and computational methods. Modern chiral stationary phases are discussed with particular focus on those that are commercially available and broadly used. It is attempted to provide the reader with vivid images of molecular recognition mechanisms of selected chiral selector–selectand pairs on basis of solid-state X-ray crystal structures and simulated computer models, respectively. Such snapshot images illustrated in this communication unfortunately cannot account for the molecular dynamics of the real world, but are supposed to be helpful for the understanding. The exploding number of papers about applications of various chiral stationary phases in numerous fields of enantiomer separations is not covered systematically.     

Improved preparation of chiral stationary phases via immobilization of polysaccharide derivative‐based selectors using diisocyanates

The classical method for the preparation of immobilized polysaccharide‐based chiral stationary phases (CSPs) with a diisocyanate was improved. Cellulose or amylose was directly coated onto 3‐aminopropyl silica gel after it was dissolved in a mixture of N,N‐dimethylacetamide, LiCl, and pyridine, then immobilized onto silica gel with a diisocyanate, and finally allowed to react with an excess of corresponding isocyanate. Four polysaccharide derivatives, 3,5‐dimethylphenylcarbamate and 3,5‐dichlorophenylcarbamate of cellulose, and 3,5‐dimethylphenylcarbamate and 5‐chloro‐2‐methylphenylcarbamate of amylose, were immobilized onto silica gel utilizing this method. Compared with the classical diisocyanate method, the improved procedure avoided the derivatization and regeneration of 6‐hydroxyl groups of cellulose and amylose, and thus showed an advantage for simple and economical preparation. The relationships among the amount of diisocyanate used, immobilization efficiency, and enantioseparation on the cellulose‐based CSPs were investigated. Also, the solvent durability of the obtained CSPs was examined with eluents containing chloroform or THF. By utilizing these eluents, the chiral recognition abilities of the obtained CSPs for some of the tested racemates were improved.     

Chromatographic properties in normal-mode HPLC of chiral stationary phases based on substituted β-cyclodextrins

Summary Substituted β-cyclodextrin chiral stationary phases having different types of phenyl carbamate substituents have been prepared and evaluated (retention, selectivity, resolution) for the liquid chromatographic separation of several series of enantiomers. The influence on separations of the degree of substitution and the structure of the substituent are discussed. Different mechanisms are suggested to explain separations in normal mode conditions.     

The dynamic chromatographic behavior of tri-o-thymotide on HPLC chiral stationary phases

The interconverting stereoisomers of tri-o-thymotide have been separated by HPLC on chiral stationary phases and the temperature dependence of the dynamic chromatographic patterns has been interpreted in terms of exchange between enantiomeric propeller and helical conformations. Computed low energy structures and CD spectra were used in absolute configuration assignment.     

Synthesis and evaluation of a new biselector s-triazine based chiral stationary phase for enantioselective HPLC: potentiality of the approach and perspectives

A s-triazine scaffold bearing a free and a protected amino group was used for connecting two different chiral auxiliaries, whose enantiodiscriminating capabilities in enantioselective chromatography are well documented. A biselector system was synthesized and, after linkage to silica gel, used for the chromatographic resolution of different racemic analytes, chosen among the compounds resolved by the two different isolated chiral auxiliaries. The obtained chromatographic results were compared with literature data related to the use of the two chiral stationary phases (CSPs) whose chiral moieties constitute the biselector CSP, allowing us to gain useful information about potentialities and limitations of this approach for obtaining independent CSPs having broader applicability with respect to the classical independent monoselector CSPs.     

Preparation and evaluation of novel chiral stationary phases covalently bound with chiral pseudo-18-crown-6 ethers

Novel chiral stationary phases consisting of silica gel covalently bound with chiral pseudo-18-crown-6 type hosts, which possess either an OH or OMe group as a binding functionality, were prepared for enantiomer-separation of lipophilic amines.     

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.     

A combinatorial approach to recognition of chirality: preparation of highly enantioselective aryl-dihydropyrimidine selectors for chiral HPLC

A parallel library of 108 4-aryl-1,4-dihydropyrimidine (DHPM) enantiomers, which are potential selectors for chiral HPLC separations, was synthesized using the single-step Biginelli multicomponent condensation. The individual compounds were screened by observing the enantioselectivity for resolution on a "brush-type" L-(3,5-dinitrobenzoyl)leucine-based chiral stationary phase, and separation factors alpha up to 12 were achieved. The best candidates from the library contained an ortho-substituted aromatic group at C4 carbon atom of the pyrimidine ring and an alkyl substituent at N1 nitrogen atom. Resolution of the enantiomers of the lead compound, 4-(9-phenanthryl)-DHPM 8, using semipreparative chiral HPLC followed by attachment to monodisperse macroporous aminomethacrylate beads, provided the novel polymer based chiral stationary phase with good enantioselectivities in the resolution of several pi-acidic aryl-dihydropyrimidines and derivatized profens. In addition, 3,5-dinitrobenzamido derivatives of alpha-amino acids could be resolved under normal phase HPLC conditions with separation factors up to 8.     

Direct HPLC separation of carnosine enantiomers with two chiral stationary phases based on penicillamine and teicoplanin derivatives

Carnosine is present in high concentrations in specific human tissues such as the skeletal muscle, and among its biological functions, the remarkable scavenging activity toward reactive carbonyl species is noteworthy. Although the two enantiomers show almost identical scavenging reactivity toward reactive carbonyl species, only d ‐carnosine is poorly adsorbed at the gastrointestinal level and is stable in human plasma. Direct methods for the enantioselective analysis of carnosine are still missing even though they could find more effective applications in the analysis of complex matrices. In the present study, the use of two different chiral stationary phases is presented. A chiral ligand‐exchange chromatography stationary phase based on N,S‐dioctyl‐d‐ penicillamine resulted in the direct enantioseparation of carnosine. Indeed, running the analysis at 25°C and 1.0 mL/min with a 1.5 mM copper(II) sulfate concentration allowed us to obtain separation and resolution factors of 3.37 and 12.34, respectively. However, the use of a copper(II)‐containing eluent renders it hardly compatible with mass spectrometry detectors. With the teicoplanin‐based stationary phase, a mass spectrometry compatible method was successfully developed. Indeed, a water/methanol 60:40 v/v pH 3.1 eluent flowed at 1.0 mL/min and with a 25°C column temperature produced separation and resolution factors of 2.60 and 4.16, respectively.     

Novel enantioselective strong cation exchangers based on sulfodipeptide selectors: evaluation for enantiomer separation of chiral bases by nonaqueous capillary electrochromatography

Strong cation exchange (SCX)-type chiral stationary phases (CSPs) based on beta-amino sulfonic acid-terminated dipeptide derivatives as chiral selectors, immobilized on thiol-modified silica particles (3.5 microm), were synthesized and applied to enantiomer separations of chiral bases by nonaqueous capillary electrochromatography (CEC). The effect of structural variations of the sulfodipeptide selectors on the separation factors alpha was investigated. These studies included variation of the acid-terminal amino sulfonic acid residue, variation of the configurations, i.e., comparison of the diastereomeric (S,S)- and (R,S)-configurations of the sulfodipeptides, and finally comparison of sulfodipeptide selectors with corresponding beta-amino sulfonic acid analogs. In general, the capillary columns (100 microm ID) packed with the new SCX-type CSPs showed enantioselectivity for an elaborated set of chiral basic drugs in CEC acting by an enantioselective cation-exchange retention mechanism. N-[N-(4-Allyloxy-3,5-dichlorobenzoyl)-leucyl]-2-amino-3,3-dimethylbutane sulfonic acid, in particular with (R,S)-configuration, turned out to be a more effective SCX-type selector than a more rigid analog based on N-[N-(4-Allyloxy-3,5-dichlorobenzoyl)-leucyl]-2-pyrrolidinemethane sulfonic acid. Both of the former diastereomers were capable to baseline-resolve the enantiomers of ca. 40% of the tested basic chiral solutes including sympathomimetics and beta-blockers, while for the latter SCX-type CSPs only 10-20% of the selected solutes afforded resolutions > 1.5.     

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.     

Liquid chromatographic enantiomer separations of novel quinazolone derivatives on quinine carbamate based chiral stationary phases using hydro-organic mobile phases

Quinine carbamate-type weak chiral anion-exchange selectors (SOs) and the respective chiral stationary phases (CSPs) have been used for the direct liquid chromatographic enantiomer separation of a wide range of chiral acids. In the present work, we demonstrate that these CSPs can also be extended to chiral discrimination of a set of neutral polar potential NMDA (N-methyl-D-aspartic acid) and/or AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) antagonist imidazo-quinazoline-dione derivatives (selectands, SAs) using acetonitrile and methanol containing hydro-organic and buffered mobile phases. The influence of mobile phase composition, column temperature and structure variation of the SAs and SOs on retention and enantioselectivity was systematically investigated to gain insight into the overall chiral recognition mechanism. As was expected for the reversed-phase mode, acetonitrile has a stronger eluotropic effect compared to methanol. Except for two analytes, the acetonitrile containing mobile phases provided baseline resolution (R(S)) of the enantiomers with R(S) values ranging between 1.68 and 2.76. Using methanol as the organic modifier enhanced the enantioselectivity. The enthalpic and entropic terms for the SO-SA association were calculated from the linear van't Hoff plots. Data reveal that the enantiomer separations are predominantly enthalpically driven.