High-performance Liquid Chromatographic Enantioseparation Research Articles

High-performance liquid chromatographic enantioseparation of unusual amino acid derivatives with axial chirality on polysaccharide-based chiral stationary phases

The successful enantioseparation of axially chiral amino acid derivatives containing a cyclohexylidene moiety on an analytical and semipreparative scale was achieved for the first time by HPLC using polysaccharide-based chiral stationary phases. Racemic methyl N-benzoylamino esters, easily obtained by methanolysis of the corresponding 5(4H)-oxazolones, were subjected to chiral HPLC resolution using chiral stationary phases based on immobilized 3,5-dimethylphenylcarbamate derivatives of amylose (Chiralpak® IA column) or cellulose (Chiralpak® IB column). The behaviour of both selectors under different elution conditions was evaluated and compared. The amylose column showed better performance than the cellulose column for all enantiomers tested. The semipreparative resolution of axially chiral amino acid derivatives with different side chains has been achieved on a 250mm×20mm ID Chiralpak® IA column using the appropriate mixture of n-hexane/chlorofom/ethanol as eluent by successive injections of a solution of the sample in chloroform. Using this protocol up to 120mg of each enantiomer of the corresponding axially chiral amino acid derivative were obtained from 300mg of racemate. [(Sa)-2a, 105mg; (Ra)-2a, 60mg, [(Sa)-2b, 105mg; (Ra)-2b, 90mg, [(Sa)-2c, 120mg; (Ra)-2c, 100mg].

Influence of the nature of alkyl substituents on the high-performance liquid chromatography enantioseparation and retention of new atropisomeric 1,1′-bibenzimidazole derivatives on amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phase

Six new atropisomeric heteroarenes were synthesized by connecting two 2-alkylbenzimidazole fragments via N–N junction. They differ by the substituent nature (methyl, ethyl, propyl, butyl, pentyl and hexyl) of the aliphatic function. The novel atropisomeric compounds were used as chiral probes to study the chromatographic behavior of the amylose tris(3,5-dimethylphenyl carbamate) (Chiralpak AD-3) chiral stationary phase (CSP) under normal phase mode. The pivotal role of the length and flexibility of the 2,2′-alkyl groups on retention, enantioselectivity and enantiomer elution order was demonstrated by enantioselective HPLC analysis. Additional information on the chiral recognition mechanism was obtained from the evaluation of the correlated thermodynamic data.

High‐performance liquid chromatography enantioseparation of polyhalogenated 4,4′‐bipyridines on polysaccharide‐based chiral stationary phases under multimodal elution

An investigation on the high-performance liquid chromatography enantioseparation of 12 polyhalogenated 4,4'-bipyridines on polysaccharide-based chiral stationary phases is described. The overall study was directed toward the generation of efficient separations in order to obtain pure atropisomers that will serve as ligands for building homochiral metal organic frameworks. Four coated columns--namely, Lux Cellulose-1, Lux Cellulose-2, Lux Cellulose-4, and Lux Amylose-2--and two immobilized columns--namely, Chiralpak IC and IA--were used under normal, polar organic, and reversed-phase elution modes. Moreover, Chiralcel OJ was considered under normal-phase and polar organic conditions. The effect of the chiral selector and mobile phase composition on the enantioseparation, the enantiomer elution order and the beneficial effect of nonstandard solvents were studied. The effect of water in the mobile phase on the enantioselectivity and retention was investigated and retention profiles typical of hydrophilic interaction liquid chromatography were observed. Interesting phenomena of solvent-induced enantiomer elution order reversal occurred under normal-phase mode. All the considered 4,4'-bipyridines were enantioseparated at the multimilligram level.

Direct high-performance liquid chromatographic enantioseparation of secondary amino acids on Cinchona alkaloid-based chiral zwitterionic stationary phases. Unusual temperature behavior

Two chiral stationary phases containing a quinine- or a quinidine-based zwitterionic ion-exchanger as chiral selector were applied for the enantioseparation of 27 unusual cyclic secondary α-amino acids. The effects of the nature and concentration of the bulk solvent, the acid and base additives, the structures of the analytes and temperature on the enantioresolution were investigated. To study the effects of temperature and to obtain thermodynamic parameters, experiments were carried out at constant mobile phase compositions in the temperature range −5 to 55°C. The thermodynamic parameters indicated that in most cases the separations were enthalpy-driven, but some entropy-driven separations were also observed. The sequence of elution of the enantiomers was determined in most cases.

High-performance liquid chromatographic enantioseparation of 3,5-disubstituted hydantoins analogs and temperature-induced reversals of elution orders on a polysaccharide-based chiral stationary phase

Enantioseparations were achieved for eleven 3,5-disubstituted hydantoins in HPLC under the normal phase mode using Chiralpak IA. The effects of polar alcoholic modifier and column temperature on retention and enantioseparation were determined. Importantly, we found two kinds of enantiomer elution order (EEO) reversals, which include solvent-induced EEO reversal for compound 9 and temperature-induced EEO reversals for compound 3 and compound 6. The phenomena of these EEO reversals were described for the first time in present work, which is helpful to elucidate the chiral separation mechanism of these hydantoins.

Insights into the impact of shape and electronic properties on the enantioseparation of polyhalogenated 4,4′-bipyridines on polysaccharide-type selectors. Evidence of stereoselective halogen bonding interactions

Starting from the high-performance liquid chromatography (HPLC) enantioseparation data collected by using twelve polyhalogenated 2,2′-dichloro-3-substituted-5,5′-dihalo-4,4′-bipyridines as test probes on seven polysaccharide-based chiral stationary phases (CSPs) under multimodal elution, the impact of substitution pattern, shape and electronic properties of the molecules on the separation behaviour was investigated through the evaluation of the chromatographic parameters (k, α, Rs) and molecular properties determined by means of quantum chemistry calculations. The computational/chromatographic screening furnished relevant structure-chromatographic behaviour relationships and some molecular interactions involved in the chiral discrimination process could be identified. In particular, a halogen bonding interaction (I.O) could reasonably explain the high enantioseparation (α=1.80, Rs=8.2) observed for the 2,2′-dichloro-3,5′-diiodo-5-bromo-4,4′-bipyridine on Lux Cellulose-1. To the best of our knowledge, this is the first report supporting the involvement of a stereoselective halogen bonding interaction in polysaccharide-based CSPs. Moreover, having at disposal a sufficient set of data, the unknown absolute configurations of the eluted enantiomers of 3-methyl-, 3-thiomethyl- and 3-diphenylphosphinoyl-2,2′-dichloro-5,5′-dibromo-4,4′-bipyridines could be deduced by chromatographic correlation with the enantiomer elution order (EEO) of the related compounds of known absolute configuration.

High-Performance Liquid Chromatographic Enantioseparation of Racemic Drugs Based on Homochiral Metal–Organic Framework

Homochiral metal-organic frameworks with fine-tuned pore sizes/walls and large surface areas are promising porous materials for enantioseparation considering the traditional zeolite molecular sieves have no chirality. Using enantiopure pyridyl-functionalized salen [(N-(4-Pyridylmethyl)-L-leucine·HBr)] as a starting material, we have prepared a noninterpenetrated three-dimensional homochiral metal organic framework {[ZnLBr]·H2O}n, which was further used as a chiral stationary phase for high-performance liquid chromatography to enantioseparate racemic drugs, showing excellent performances in enantioseparation of drugs. The metal-organic framework can be regarded as a novel molecular sieve-like material with a chiral separation function based on the relative sizes of the chiral channels and the resolved molecules.

High performance liquid chromatographic enantioseparation of atrolactic acids using chiral mobile phase additive

A method was established for the enantioseparation of atrolactic acids using high performance liquid chromatography with sulfobutyl ether-beta-cyclodextrin (SBE-beta-CD) as the chiral mobile phase additive, and a C18 reversed-phase column. The influences of the different types of cyclodextrin derivatives, the concentration of chiral mobile phase additive, pH value of the mobile phase, the flow rate and column temperature on the peak resolution were investigated. The separation mechanism for atrolactic acids by high performance liquid chromatography using chiral mobile phase additive, as well as the formation of inclusion complex between SBE-beta-CD and atrolactic acid was studied. In addition, the formation constant was calculated. The enantioseparation was carried out on a reversed-phase column YMC-Pack ODS-A C18 (250 mm x 4.6 mm, 5 microm) with chiral mobile phase consisted of 0.1 mol/L phosphate buffer at pH 2.68 containing 25 mmol/L SBE-beta-CD-acetonitrile (90:10, v/v). The detection wavelength was 220 nm. The flow rate was 0.6 mL/min and the column temperature was 30 degrees C. The retention times of atrolactic acids were 26.65 and 28.28 min, and the peak resolution of 1.68 was achieved. The above established method proves to be a simple and economical way for analytical enantioseparation of atrolactic acid with high peak resolution.

Chiral Recognition Mechanisms of four β-Blockers by HPLC with Amylose Chiral Stationary Phase.

The high performance liquid chromatography (HPLC) enantioseparation of four β-blocking agents metoprolol, bisoprolol, propranolol and atenolol was performed on amylose tris-(3,5-dimethylphenylcarbamate) chiral stationary phase using n-hexane-ethanol-diethylamine (DEA) as the mobile phase and related chiral recognition mechanisms were discussed. Enantiomeric separation of the four β-blockers was a result of more than one type of interaction between solutes and CSP. Besides hydrogen bonding, there was another type interaction that was independent of solvent polarity and responsible for enantiomeric selectivity, such as - interactions. Both the groups close to the chiral centers and the substituent groups on the phenyl rings, which were far away from the chiral centers, could contribute to the good separation. The separations of the four β-blocker enantiomers were all enthalpy driven process. In the range of 293-308K (20-35 ℃), as the temperature increased, the retention as well as the resolution decreased. The molecular size rather than concentration of the alcohol modifiers affected the resolution and retention.

Comparative HPLC Enantioseparation of Thirty‐Six Aromatic Compounds on Four Columns of the Lux® Series: Impact of Substituents, Shapes and Electronic Properties

With the aim to define a combined computational/chromatographic empirical approach useful for the high-performance liquid chromatography (HPLC) method development of new chiral compounds, 36 racemic aromatic compounds with different chemical structures were used as test probes on four polysaccharide-based chiral stationary phases (CSPs) of the Lux series, namely Lux Cellulose-1, Lux Cellulose-2, Lux Cellulose-4, and Lux Amylose-2, using classical n-hexane/2-propanol mixtures as mobile phase. Electrostatic potential surfaces (EPSs) determined using Density Functional Theory (DFT) calculations were used to derive size, shape, and electronic properties of each analyte. Then a comparative HPLC screening was carried out in order to evaluate the impact of substituents, shapes, and electronic properties of the analytes on the chromatographic behavior as the column changes. The four CSPs showed good complementary recognition ability. The elution sequence was determined in 30 cases out of 36. The success rate to afford baseline separations (R(s) ≥ 1.5) was estimated: 29 compounds out of 36 showed baseline enantioseparation on at least one of the four selected CSPs. The combined computational-chromatographic screening furnished useful collective structure-chromatographic behavior relationships and a map of the chiral discrimination abilities of the considered CSPs towards the analytes. On this basis, the chromatographic behavior of new analytes on a set of polysaccharide-based CSPs can be mapped through the qualitative correlation of chromatographic parameters (k, α, R(s)) to computed molecular properties of the analytes.

Direct high-performance liquid chromatographic enantioseparation of free α-, β- and γ-aminophosphonic acids employing cinchona-based chiral zwitterionic ion exchangers

We report a chiral high-performance liquid chromatographic enantioseparation method for free α-aminophosphonic, β-aminophosphonic, and γ-aminophosphonic acids, aminohydroxyphosphonic acids, and aromatic aminophosphinic acids with different substitution patterns. Enantioseparation of these synthons was achieved by means of high-performance liquid chromatography on CHIRALPAK ZWIX(+) and ZWIX(-) (cinchona-based chiral zwitterionic ion exchangers) under polar organic chromatographic elution conditions. Mobile phase characteristics such as acid-to-base ratio, type of counterion, and solvent composition were systematically varied in order to investigate their effect on the separation performance and to achieve optimal separation conditions for the set of analytes. Under the optimized conditions, 32 of 37 racemic aminophosphonic acids studied reached baseline separation when we employed a single generic mass-spectrometry-compatible mobile phase, with reversal of the elution order when we used (+) and (-) versions of the chiral stationary phase.

High‐performance liquid chromatographic enantioseparation of isoxazoline‐fused 2‐aminocyclopentanecarboxylic acids on a chiral ligand‐exchange stationary phase

The application of a chiral ligand-exchange column for the direct high-performance liquid chromatographic enantioseparation of unusual β-amino acids with a sodium N-((R)-2-hydroxy-1-phenylethyl)-N-undecylaminoacetate-Cu(II) complex as chiral selector is reported. The investigated amino acids were isoxazoline-fused 2-aminocyclopentanecarboxylic acid analogs. The chromatographic conditions were varied to achieve optimal separation. The effects of temperature were studied at constant mobile phase compositions in the temperature range 5-45°C, and thermodynamic parameters were calculated from plots of lnk or lnα versus 1/T. Δ(ΔH°) ranged from -2.3 to 2.2 kJ/mol, Δ(ΔS°) from -3.0 to 7.8 J mol(-1) K(-1) and -Δ(ΔG°) from 0.1 to 1.7 kJ/mol, and both enthalpy- and entropy-controlled enantioseparations were observed. The latter was advantageous with regard to the shorter retention and greater selectivity at high temperature. Some mechanistic aspects of the chiral recognition process are discussed with respect to the structures of the analytes. The sequence of elution of the enantiomers was determined in all cases.

Synthesis and HPLC Enantioseparation of Derivatives of the 3-hydroxyphenylethanone

AbstractWithin the framework of the study of the synthesis and high-performance liquid chromatography (HPLC) enantioseparation the series of 9 derivatives of 3-hydroxyphenylethanone was prepared by a well-tried method. The structure of the prepared compounds was confirmed on the basis of interpretation of the IR, UV, 1H NMR and 13C NMR spectra. An enantioseparation of prepared compounds was performed using HPLC on a native teicoplanin (Chirobiotic T) and the amylose tris (3,5-dimethylphenylcarbamate) (Chiralpak AD) chiral stationary phases, which is more suitable for the enantioseparation of all prepared compounds especially with heterocycles in the basic part of a molecule.

Access to Enantiomerically Pure cis‐ and trans‐β‐Phenylproline by High‐Performance Liquid Chromatography Resolution

The preparation of all four stereoisomers of the proline analog that bears a phenyl group attached to the β carbon either cis or trans to the carboxylic acid (cis- and trans-β-phenylproline, respectively) has been addressed. The methodology developed allows access to multigram quantities of the target amino acids in enantiomerically pure form and suitably protected for use in peptide synthesis. Racemic precursors of cis-β-phenylproline and trans-β-phenylproline were prepared from easily available starting materials and subjected to high-performance liquid chromatography enantioseparation. Semipreparative columns (250 × 20 mm) containing chiral stationary phases based on amylose (Chiralpak IA) (Daicel-Chiral Technologies Europe, Illkirch, France) or cellulose (Chiralpak IC) were used respectively for the resolution of the cis- and trans-β-phenylproline precursors.

Semi-Preparative Enantiomeric Separation of Ofloxacin by HPLC

A direct semi-preparative high performance liquid chromatography (HPLC) enantioseparation of ofloxacin was performed on chemically immobilized cyclodextrin derivative-mono (6A-azido-6A-deoxy)-per(p-chlorophenyl carbamoylated) β-CD chiral stationary phase. Conditions for semi-preparative separations were established using a 250 × 4.6 mm i.d. column and subsequently extended to a 250 × 10.0 mm i.d. column that enabled separations on a milligram scale. Optimization of the chromatographic conditions (mobile phase and column load) with respect to better efficiency, resolution and peak retention resulted in a system capable of separating up to 304 mg of (-)-(S)-ofloxacin and 56 mg of (+)-(R)-ofloxacin of the racemate over 6 h. The purities of the separated enantiomers were determined by HPLC. Moreover, both separated enantiomers were characterized by mass spectrometry; then, the absolute configuration of the products was clearly confirmed by polarimetry.

A Validated LC Method for the Determination of the Enantiomeric Purity of Aliskiren Hemifumarate in Bulk Drug Samples

High-performance liquid chromatography enantioseparation of aliskiren hemifumarate was accomplished on an immobilized-type Chiralpak IC chiral stationary phase under both polar organic and reversed-phase modes. A simple analytical method was developed and validated using a mixture of acetonitrile-n-butylamine 100:0.1 (v/v/) as a mobile phase with a flow rate maintained at 1.0 mL/min. Ultraviolet detection was carried out at 228 nm. Resolution between the two enantiomers was greater than 3.0. This method was capable of detecting the R-isomer to a level of 0.2 μg/mL. The method was validated as per International Conference on Harmonization guidelines and found to be robust. The method is very useful for routine evaluation of the quality of aliskiren hemifumarate in bulk drug manufacturing units.

High-performance liquid chromatography enantioseparation of atropisomeric 4,4′-bipyridines on polysaccharide-type chiral stationary phases: Impact of substituents and electronic properties

The high performance liquid chromatography (HPLC) enantioseparation of eleven atropisomeric 4,4′-bipyridines was performed in the normal and polar organic phase mode using three cellulose-based chiral stationary phases (CSPs), namely Lux® Cellulose-1, Lux® Cellulose-2, Lux® Cellulose-4, and two amylose-based CSPs, Chiralpak® AD-H and Lux® Amylose-2. n-Hexane/2-propanol mixtures and pure ethanol were employed as mobile phases. The combined use of Chiralpak® AD-H and Lux® Cellulose-2 allowed to enantioseparate all the considered bipyridines. Ten bipyridines were enantioseparated at the multimilligram level allowing the elution sequence determination of the enantiomers as well as their future use for the preparation of homochiral metal organic frameworks (MOFs). Moreover, the performance of the CSPs regarding the same bipyridine was different and dependent on the backbone as well as on the side chain of the polymer. The impact of substitution pattern, shape and electronic properties of the molecules on the separation behavior was investigated through the evaluation of retention factors (k), separation factors (α), resolution (Rs) and molecular properties determined using density functional theory (DFT) calculations. In this regard, the substituents at the 3,3′,5,5′ positions of the 4,4′-bipyridyl rings exhibited a pivotal role on the enantioseparation.

Comparison of separation performances of novel β-cyclodextrin-based chiral stationary phases in high-performance liquid chromatographic enantioseparation

Three β-cyclodextrin-based chiral stationary phases were developed applying novel bonding chemistry. The separation performances of β-cyclodextrin, (R,S)-2-hydroxypropyl-β-cyclodextrin, and permethyl-β-cyclodextrin-based CSPs were compared in the resolution of structurally divergent analytes, such as coumarins, dansyl amino acids, and propionic acid derivatives. Separations were carried out in reversed phase mode applying 0.1% triethylammonium phosphate (pH 3.5)/MeOH mobile phase systems in different compositions. Of the three novel CSPs the permethyl-β-cyclodextrin bonded phase proved to be the most effective one for the enantioseparation of investigated analytes.

(S)‐Naproxen as a platform to develop chiral derivatizing reagent for reversed‐phase high‐performance liquid chromatographic enantioseparation of analytes having a carbonyl functional group

(S)-Naproxen was used to synthesize a chiral reagent, (S)-2-(6-methoxynaphthalen-2-yl)propanehydrazide, by itsreaction with hydrazine hydrate in the presence of dicyclohexylcarbodiimide as coupling agent. The reagent was characterized and its chiral purity was established. It was used as a chiral derivatizing reagent for the synthesis of hydrazone diastereomers, under microwave irradiation, of certain chiral aldehydes and ketones. The respective diastereomers were separated by reversed-phase high-performance liquid chromatography using a binary solvent combination containing trifluoroacetic acid. The diastereomers were detected at 231 nm. The method was validated for accuracy, precision, and limit of detection (LOD). For a series of hydrazones the LOD was found to be in the range 1.62-1.65 pmol/mL.

Comparative high-performance liquid chromatography enantioseparations on polysaccharide based chiral stationary phases prepared by coating totally porous and core–shell silica particles

This article reports comparative high-performance liquid chromatographic separations of enantiomers with chiral stationary phases (CSPs) prepared by coating cellulose tris(4-chloro-3-methylphenylcarbamate) on totally porous and on core–shell type silica of comparable particle diameter. Several interesting observations were made: (1) the selectivity of separation was higher on core–shell type CSP compared to totally porous CSP at comparable content of chiral selector (polysaccharide derivative); (2) much flatter dependence of plate height on the mobile phase flow rate was observed for columns packet with CSP prepared with core–shell silica compared to the ones packed with CSPs prepared with totally porous particles; (3) at low mobile phase flow rates core–shell CSP provided lower resolving ability compared to a commercially available CSP having four times higher content of chiral selector along with higher retention of chiral analytes. However, at high flow rates core–shell type CSP performed similarly or better than the commercial column in regards of plate count (N) and peak resolution (Rs) per column length and within a given total analysis time. The advantage of CSP prepared with core–shell silica is obvious from the viewpoint of plate numbers and resolution calculated per unit time (i.e. speed of analysis).