An Insight to Chiral Monolith for Enantioselective Nano and Micro HPLC: Preparation and Applications

This review gives an overview of chiral separation principles and their application in enantioselective nano/micro high performance liquid chromatography (n/μ‐HPLC) using chiral monolith. In particular, developments in silica and polymer chiral monolithic stationary phases are presented. The preparation and applications of chiral monoliths, the basic chiral separation principles and the mechanisms are discussed. Chirality 25:314–323, 2013. © 2013 Wiley Periodicals, Inc.     

Separation of Tryptophan Enantiomers by Ligand‐Exchange Chromatography With Novel Chiral Ionic Liquids Ligand

Chiral ionic liquids (CILs) with amino acids as cations have been applied as novel chiral ligands coordinated with Cu²⁺ to separate tryptophan enantiomers in ligand exchange chromatography. Four kinds of amino acid ionic liquids, including [L‐Pro][CF₃COO], [L‐Pro][NO₃], [L‐Pro]₂[SO₄], and [L‐Phe][CF₃COO] were successfully synthesized and used for separation of tryptophan enantiomers. To optimize the separation conditions, [L‐Pro][CF₃COO] was selected as the model ligand. Some factors influencing the efficiency of chiral separation, such as copper ion concentration, CILs concentration, methanol ratio (methanol/H₂O, v/v), and pH, were investigated. The obtained optimal separation conditions were as follows: 8.0 mmol/L Cu(OAc)₂, 4.0 mmol/L [L‐Pro][CF₃COO] ,and 20% (v/v) methanol at pH 3.6. Under the optimum conditions, acceptable enantioseparation of tryptophan enantiomers could be observed with a resolution of 1.89. The results demonstrate the good applicability of CILs with amino acids as cations for chiral separation. Furthermore, a comparative study was also conducted for exploring the mechanism of the CILs as new ligands in ligand exchange chromatography. Chirality 26:160–165, 2014. © 2014 Wiley Periodicals, Inc.     

Effect of Basic and Acidic Additives on the Separation of Some Basic Drug Enantiomers on Polysaccharide‐Based Chiral Columns With Acetonitrile as Mobile Phase

The separation of enantiomers of 16 basic drugs was studied using polysaccharide‐based chiral selectors and acetonitrile as mobile phase with emphasis on the role of basic and acidic additives on the separation and elution order of enantiomers. Out of the studied chiral selectors, amylose phenylcarbamate‐based ones more often showed a chiral recognition ability compared to cellulose phenylcarbamate derivatives. An interesting effect was observed with formic acid as additive on enantiomer resolution and enantiomer elution order for some basic drugs. Thus, for instance, the enantioseparation of several β‐blockers (atenolol, sotalol, toliprolol) improved not only by the addition of a more conventional basic additive to the mobile phase, but also by the addition of an acidic additive. Moreover, an opposite elution order of enantiomers was observed depending on the nature of the additive (basic or acidic) in the mobile phase. Chirality 27:228–234, 2015. © 2015 Wiley Periodicals, Inc.     

Enantioseparation of Racemic Flurbiprofen by Aqueous Two‐Phase Extraction With Binary Chiral Selectors of L‐dioctyl Tartrate and L‐tryptophan

A novel method for chiral separation of flurbiprofen enantiomers was developed using aqueous two‐phase extraction (ATPE) coupled with biphasic recognition chiral extraction (BRCE). An aqueous two‐phase system (ATPS) was used as an extracting solvent which was composed of ethanol (35.0% w/w) and ammonium sulfate (18.0% w/w). The chiral selectors in ATPS for BRCE consideration were L‐dioctyl tartrate and L‐tryptophan, which were screened from amino acids, β‐cyclodextrin derivatives, and L‐tartrate esters. Factors such as the amounts of L‐dioctyl tartrate and L‐tryptophan, pH, flurbiprofen concentration, and the operation temperature were investigated in terms of chiral separation of flurbiprofen enantiomers. The optimum conditions were as follows: L‐dioctyl tartrate, 80 mg; L‐tryptophan, 40 mg; pH, 4.0; flurbiprofen concentration, 0.10 mmol/L; and temperature, 25 °C. The maximum separation factor α for flurbiprofen enantiomers could reach 2.34. The mechanism of chiral separation of flurbiprofen enantiomers is discussed and studied. The results showed that synergistic extraction has been established by L‐dioctyl tartrate and L‐tryptophan, which enantioselectively recognized R‐ and S‐enantiomers in top and bottom phases, respectively. Compared to conventional liquid–liquid extraction, ATPE coupled with BRCE possessed higher separation efficiency and enantioselectivity without the use of any other organic solvents. The proposed method is a potential and powerful alternative to conventional extraction for separation of various enantiomers. Chirality 27:650–657, 2015. © 2015 Wiley Periodicals, Inc.     

Chiral Separation of Cathinone and Amphetamine Derivatives by HPLC/UV Using Sulfated ß‐Cyclodextrin as Chiral Mobile Phase Additive

In the last years the identification of new legal and illegal highs has become a huge challenge for the police and prosecution authorities. In an analytical context, only a few analytical methods are available to identify these new substances. Moreover, many of these recreational drugs are chiral and it is supposed that the enantiomers differ in their pharmacological potency. Since nonenantioselective synthesis is easier and cheaper, they are mainly sold as racemic mixtures. The goal of this research work was to develop an inexpensive method for the chiral separation of cathinones and amphetamines. This should help to discover if the substances are sold as racemic mixtures and give further information about their quality as well as their origin. Chiral separation of a set of 6 amphetamine and 25 cathinone derivatives, mainly purchased from various Internet shops, is presented. A LiChrospher 100 RP‐18e, 250 x 4 mm, 5 µm served as the stationary phase. The chiral mobile phase consisted of methanol, water, and sulfated ß‐cyclodextrin. Measurements were performed under isocratic conditions in reversed phase mode using UV detection. Four model compounds of the two substance classes were used to optimize the mobile phase. Under final conditions (methanol:water 2.5:97.5 + 2% sulfated ß‐cyclodextrin) enantiomers of amphetamine and five derivatives were baseline separated within 23 min. In all, 17 cathinones were completely or partially chirally separated. However, as only 3 of 25 cathinones were baseline resolved, the application of this method is limited for cathinone analogs. Additionally, the results were compared with an RP‐8e column. Chirality 26:411–418, 2014. © 2014 Wiley Periodicals, Inc.     

Chiral Ligand‐Exchange Resolution of Underivatized Amino Acids on a Dynamically Modified Stationary Phase for RP‐HPTLC

The synthesis of Spi(τ‐dec), derived from the selective alkylation of L‐spinacine (4,5,6,7‐tetrahydro‐1H‐imidazo[4,5‐c]pyridine‐6‐carboxylic acid) at the τ‐nitrogen of its heteroaromatic ring, with a linear hydrocarbon chain of 10 carbon atoms, is described here for the first time. Spi(τ‐dec) was successfully employed in the past to prepare home‐made chiral columns for chiral ligand‐exchange high‐performance liquid chromatography. In the present article a new method is described, using Spi(τ‐dec) as a chiral selector in high‐performance thin‐layer chromatography (HPTLC): commercial hydrophobic plates were first coated with Spi(τ‐dec) and then treated with copper sulfate. The performance of this new chiral stationary phase was tested against racemic mixtures of aromatic amino acids, after appropriate optimization of both the conditions of preparation of the plates and the mobile phase composition. The enantioselectivity values obtained for the studied compounds were higher than those reported in the literature for similar systems. The method employed here for the preparation of chiral HPTLC plates proved practical, efficient, and inexpensive. Chirality 26:313–318, 2014. © 2014 Wiley Periodicals, Inc.     

Preparation and Studies of Chiral Stationary Phases Containing Enantiopure Acridino‐18‐Crown‐6 Ether Selectors

The enantiomeric separation ability of the newly prepared chiral stationary phases containing acridino‐18‐crown‐6 ether selectors was studied by high‐performance liquid chromatography (HPLC). The chiral stationary phases separated the enantiomers of selected protonated primary aralkylamines efficiently. The best results were found for the separation of the mixtures of enantiomers of NO₂‐PEA. Chirality 26:651–654, 2014. © 2014 Wiley Periodicals, Inc.     

Identification of N‐octylnortadalafil and its Stereoisomers in Dietary Supplements with Chiral Liquid Chromatography–Circular Dichroism

A direct chiral liquid chromatography–circular dichroism (LC‐CD) method was developed for the simple and rapid identification of N‐octylnortadalafil [(6R, 12aR)‐6‐(1,3‐benzodioxol‐5‐yl)‐2‐octyl‐2,3,6,7,12,12a‐hexahydropyrazino[1’,2’:1,6]pyrido[3,4‐b]indole‐1,4‐dione; RR‐OTDF] and its stereoisomers in dietary supplements. Samples were extracted with methanol. Compounds were then separated by chiral LC‐CD using Chiralcel OD‐RH (4.6 × 1 50 mm, 5 µm) with 5 mM ammonium formate (pH 3)/0.1% formic acid in acetonitrile (95:5, v/v) mixture solution (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). The isocratic elution used was mobile phase A / mobile phase B (3:7, v/v) at a flow rate of 1 ml/min. The column temperature was held at 30°C. RR‐OTDF and its stereoisomers were separated within 20 min with the resolution factors being over 2.0. Using this method, RR‐OTDF and (6R, 12aS)‐6‐(1,3‐benzodioxol‐5‐yl)‐2‐octyl‐2,3,6,7,12,12a‐hexahydropyrazino[1’,2’:1,6]pyrido[3,4‐b]indole‐1,4‐dione were detected in a dietary supplement. Chirality 28:204–208, 2016. © 2016 Wiley Periodicals, Inc.     

Substituent effects on chiral resolutions of derivatized 1‐phenylalkylamines by heptakis(2,3‐di‐O‐methyl‐6‐O‐tert‐butyldimethylsilyl)‐β‐cyclodextrin GC stationary phase

Chiral resolutions of trifluoroacetyl‐derivatized 1‐phenylalkylamines with different type and position of substituent were investigated by capillary gas chromatography by using heptakis(2,3‐di‐O‐methyl‐6‐O‐tert‐butyldimethylsilyl)‐β‐cyclodextrin diluted in OV‐1701 as a chiral stationary phase. The influence of column temperature on retention and enantioselectivity was examined. All enantiomers of meta‐substituted analytes as well as fluoro‐substituted analytes could be resolved. Temperature had a favorable influence on enantioselectivity for small amines with substituents at the ortho‐position. The type of substituent at the stereogenic center of amines also had a crucial effect as the ethyl group led to poor enantioseparation. Among all analytes studied, trifluoroacetyl‐derivatized 1‐(2′‐fluorophenyl)ethylamine exhibited baseline resolution with the shortest analysis time.     

Enantiomeric Separation and Simulation Studies of Pheniramine,Oxybutynin, Cetirizine,and Brinzolamide Chiral Drugs on Amylose‐Based Columns

Solid phase extraction ( SPE)‐chiral separation of the important drugs pheniramine, oxybutynin, cetirizine, and brinzolamide was achieved on the C₁₈ cartridge and AmyCoat (150 x 46 mm) and Chiralpak AD (25 cm x 0.46 cm id) chiral columns in human plasma. Pheniramine, oxybutynin, cetirizine, and brinzolamide were resolved using n‐hexane‐2‐PrOH‐DEA (85:15:0.1, v/v), n‐hexane‐2‐PrOH‐DEA (80:20:0.1, v/v), n‐hexane‐2‐PrOH‐DEA (70:30:0.2, v/v), and n‐hexane‐2‐propanol (90:10, v/v) as mobile phases. The separation was carried out at 25 ± 1 ºC temperature with detection at 225 nm for cetirizine and oxybutynin and 220 nm for pheniramine and brinzolamide. The flow rates of the mobile phases were 0.5 mLmin^‐1. The retention factors of pheniramine, oxybutynin, cetirizine and brinzolamide were 3.25 and 4.34, 4.76 and 5.64, 6.10 and 6.60, and 1.64 and 2.01, respectively. The separation factors of these drugs were 1.33, 1.18, 1.09 and 1.20 while their resolutions factors were 1.09, 1.45, 1.63 and 1.25, and 1.15, respectively. The absolute configurations of the eluted enantiomers of the reported drugs were determined by simulation studies. It was observed that the order of enantiomers elution of the reported drugs was S‐pheniramine > R‐pheniramine; R‐oxybutynin > S‐oxybutynin; S‐cetirizine > R‐cetirizine; and S‐brinzolamide > R‐brinzolamide. The mechanism of separation was also determined at the supramolecular level by considering interactions and modeling results. The reported SPE‐chiral high‐performance liquid chromatography ( HPLC) methods are suitable for the enantiomeric analyses of these drugs in any biological sample. In addition, simulation studies may be used to determine the absolute configuration of the first and second eluted enantiomers. Chirality 26:136–143, 2014. © 2014 Wiley Periodicals, Inc.     

Chiral separation of flurbiprofen enantiomers by preparative and simulated moving bed chromatography

This study presents the chiral resolution of flurbiprofen enantiomers by preparative liquid chromatography using the simulated moving bed (SMB) technology. Flurbiprofen enantiomers are widely used as nonsteroidal anti‐inflammatory drugs, and although demonstrate different therapeutic actions, they are still marketed as a racemic mixture. The results presented here clearly show the importance of the selection of the proper solvent composition for the preparative separation of flurbiprofen enantiomers. Chiral SMB separation is carried out using a laboratory‐scale unit (the FlexSMB‐LSRE®) with six columns, packed with the Chiralpak AD® stationary phase (20 μm). Results presented include the experimental measurement of equilibrium and kinetic data for two very different solvent compositions, a traditional high hydrocarbon content [10%ethanol/90%n‐hexane/0.01% trifluoroacetic acid (TFA)] and a strong polar organic composition (100%ethanol/0.01%TFA). Experimental data, obtained using the two mobile phase compositions, are used to predict and optimize the SMB operation. After selecting 10%ethanol/90%n‐hexane/0.01%TFA as the most appropriate solvent composition, three feed concentrations of racemic flurbiprofen were considered. Using 40 g/l of racemic flurbiprofen feed solution, the purities for both outlet streams were above 99.4%, the productivity was 13.1 g_feed/(L_bed h), and a solvent consumption of 0.41 L_solvent/g_feed was achieved. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

Geometric enantiomerism in cyclic compounds: Chiral dibrominated 1,3‐dioxanes

The first geometric enantiomers in the cyclic compounds series are reported. The investigated compounds are 2,2‐disubstituted‐5‐methyl‐1,3‐dioxane derivatives in which the substituents at position 2 bear chiral centers with identical substituents but with opposite configurations. The structure of the unlike isomers was determined from the solid state molecular structure of the compounds obtained by single crystal X‐ray diffractometry and the enantiomers of these diastereoisomers were observed by chiral column HPLC base‐line separation. The investigated compounds were obtained by a diastereoselective bromination reaction of the corresponding 2,2‐dialkyl and 2,2‐dibenzyl‐5‐methyl‐1,3‐dioxanes. Chirality, 2011. © 2010 Wiley‐Liss, Inc.     

Enantiomeric resolution of some nonsteroidal antiinflammatory and anticoagulant drugs using β‐cyclodextrins by capillary electrophoresis

β‐cyclodextrin (CD) and its derivatives HP‐β‐CD, DM‐β‐CD, and TM‐β‐CD have been employed as chiral selectors for the separation of three nonsteroidal antiinflammatory drugs (NSAIDs) and anticoagulant at relatively low concentration (8–15 mM) by capillary zone electrophoresis (CZE). In this study, baseline separation was achieved for ibuprofen, ketoprofen, naproxen, and warfarin. It was found that the addition of 0.1% hydroxypropyl methyl cellulose (HPMC) was effective for separation. Under these conditions, the S‐(+) enantiomer eluted before R‐(−) in terms of ibuprofen; the calculated energy values obtained from the molecular modeling correlated well with the elution order. An equation for calculating the pK_a values by capillary electrophoresis was introduced, and the pK_a values of the four chiral drugs at 25°C were obtained based on the equation. The value pK_a + 0.5 is proposed to be the suitable pH of the background electrolyte for the separation of chiral compounds containing a carboxylic group. Chirality 11:56–62, 1999. © 1999 Wiley‐Liss, Inc.     

Dynamic Behavior of Clobazam on High‐Performance Liquid Chromatography Chiral Stationary Phases

Clobazam, a 1,5‐benzodiazepin‐2,4‐dione, is a chiral molecule because its ground state conformation features a nonplanar seven‐membered ring lacking reflection symmetry elements. The two conformational enantiomers of clobazam interconvert at room temperature by a simple ring‐flipping process. Variable temperature HPLC on the Pirkle type (R)‐N‐(3,5‐dinitronenzoyl)phenylglycine and (R,R)‐Whelk‐O1 chiral stationary phases (CSPs) allowed us to separate for the first time the conformational enantiomers of clobazam and to observe peak coalescence‐decoalescence phenomena due to concomitant separation and interconversion processes occurring on the same time scale. Clobazam showed temperature dependent dynamic high‐performance liquid chromatography (HPLC) profiles with interconversion plateaus on the two CSPs indicative of on‐column enantiomer interconversion. (enantiomerization) in the column temperature range between T_col = 10°C and T_col = 30°C, whereas on‐column interconversion was absent at temperature close to or lower than T_col = 5°C. Computer simulation of exchange‐deformed HPLC profiles using a program based on the stochastic model yielded the apparent rate constants for the on‐column enantiomerization and the corresponding free energy activation barriers. At T_col = 20°C the averaged enantiomerization barriers, ΔG^?, for clobazam were found in the range 21.08–21.53 kcal mol^?1 on the two CSPs. The experimental dynamic chromatograms and the corresponding interconversion barriers reported in this article are consistent with the literature data measured by DNMR at higher temperatures and in different solvents. Chirality 28:17–21, 2016. © 2015 Wiley Periodicals, Inc.     

Preparation and characterization of a new open‐tubular capillary column for enantioseparation by capillary electrochromatography

In order to use the enantioseparation capability of cationic cyclodextrin and to combine the advantages of capillary electrochromatography (CEC) with open‐tubular (OT) column, in this study, a new OT‐CEC, coated with cationic cyclodextrin (1‐allylimidazolium‐β‐cyclodextrin [AI‐β‐CD]) as chiral stationary phase (CSP), was prepared and applied for enantioseparation. Synthesized AI‐β‐CD was characterized by infrared (IR) spectrometry and mass spectrometry (MS). The preparation conditions for the AI‐β‐CD‐coated column were optimized with the orthogonal experiment design L₉(3⁴). The column prepared was characterized by scanning electron microscopy (SEM) and elemental analysis (EA). The results showed that the thickness of stationary phase in the inner surface of the AI‐β‐CD‐coated columns was about 0.2 to 0.5 μm. The AI‐β‐CD content in stationary phase based on the EA was approximately 2.77 mmol·m^?2. The AI‐β‐CD‐coated columns could separate all 14 chiral compounds (histidine, lysine, arginine, glutamate, aspartic acid, cysteine, serine, valine, isoleucine, phenylalanine, salbutamol, atenolol, ibuprofen, and napropamide) successfully in the study and exhibit excellent reproducibility and stability. We propose that the column, coated with AI‐β‐CD, has a great potential for enantioseparation in OT‐CEC.     

Chiral discrimination by HPLC and CE and antifungal activity of racemic fenticonazole and its enantiomers

Fenticonazole is a chiral antifungal agent, used in therapy as the racemic mixture. The investigation on the chirality of fenticonazole is reported in this study. rac-Fenticonazole was resolved by HPLC and by capillary electrophoresis (CE). The chiral stationary phase (CSP), used in HPLC, was Daicel OD-H, a commercial phase, which allowed the separate collection of the two enantiomers. The chiral selectors used for CE were some cyclodextrin derivatives. The analysis time required from CE was about the half the HPLC enantioseparation time. The biological activity of the rac-mixture and each individual enantiomer was tested against Cryptococcus neoformans and two Aspergillus nidulans strains. The minimum inhibitory concentration (MIC) evaluation showed that the eutomer was the enantiomer chromatographically more retained and had a longer migration time in the electrophoretic enantioseparation. The CD spectrum of the eutomer showed a positive Cotton effect.     

Enantiomeric Separation of 13 New Amphetamine‐Like Designer Drugs by Capillary Electrophoresis,Using Modified‐‐Cyclodextrins

An easy‐to‐prepare chiral CE method for the enantiomeric separation of 13 new amphetamine‐like designer drugs, using CDs as chiral selectors, was developed. Sulfated‐β‐CD was found to be the best chiral selector among the three used (sulfated‐β‐CD, caroboxymethyl‐β‐CD, dimethyl‐β‐CD). The separation of the analytes was achieved in a fused‐silica gel capillary at 20 °C using an applied voltage of +25 kV. The optimized background electrolyte consisted of 63.5 mM H₃PO₄ and 46.9 mM NaOH in water. Several electrophoretic parameters such as CD type, CD concentration (1 ? 40 mg/mL), buffer pH (2.6, 3.6, 5.0, 6.0), length of the capillary (70 ? 40 cm total length), amount of the organic solvent (methanol and acetonitrile) were investigated and optimized. Chirality 25:617–621, 2013. © 2013 Wiley Periodicals, Inc.     

Screening Approach for Chiral Separation of β‐Aminoketones by HPLC on Various Polysaccharide‐Based Chiral Stationary Phases

Nine β‐aminoketones were synthesized via Mannich reaction when benzaldehyde was condensed with some primary amines and acetophenone. The purified compounds were identified by using spectroscopic methods. The enantiomeric separation of these derivatives was carried out by high‐performance liquid chromatography (HPLC) using several coated and immobilized polysaccharide stationary phases, namely, Chiralcel^® OD‐H, Chiralcel^® OD, Chiralcel^® OJ, Chiralpak^® AD, Chiralpak^® IA, and Chiralpak^® IB using different mobile phases composed of n‐hexane and alcohol mixed in various ratios or pure ethanol or isopropanol. The retention behavior and selectivity of these chiral stationary phases were examined in isocratic normal phase mode. The results indicate that cellulose derivatives have higher enantioselectivity than amylose derivatives for the separation of racemic β‐amino ketones. Chirality 27:332–338, 2015. © 2015 Wiley Periodicals, Inc.     

Chiral Separation of Four Stereoisomers of Ketoconazole Drugs Using Capillary Electrophoresis

This work aimed to develop a chiral separation method of ketoconazole enantiomers using electrokinetic chromatography. The separation was achieved using heptakis (2, 3, 6‐tri‐O‐methyl)‐β‐cyclodextrin (TMβCD), a commonly used chiral selector (CS), as it is relatively inexpensive and has a low UV absorbance in addition to an anionic surfactant, sodium dodecyl sulfate (SDS). The influence of TMβCD concentration, phosphate buffer concentration, SDS concentration, buffer pH, and applied voltage were investigated. The optimum conditions for chiral separation of ketoconazole was achieved using 10 mM phosphate buffer at pH 2.5 containing 20 mM TMβCD, 5 mM SDS, and 1.0% (v/v) methanol with an applied voltage of 25 kV at 25 °C with a 5‐s injection time (hydrodynamic injection). The four ketoconazole stereoisomers were successfully resolved for the first time within 17 min (total analysis time was 28 min including capillary conditioning). The migration time precision of this method was examined to give repeatability and reproducibility with RSDs ≤5.80% (n =3) and RSDs ≤8.88% (n =9), respectively. Chirality 27:223–227, 2015. © 2014 Wiley Periodicals, Inc.