Affinity capillary electrophoresis coupling with partial filling technique and field‐amplified sample injection for enantioseparation and determination of DL‐tetrahydropalmatine

A novel, simple and sensitive method for the enantioseparation and determination of DL ‐tetrahydropalmatine (DL ‐THP) was developed using ACE in combination with partial filling technique and field‐amplified sample injection. A chiral selector, i.e. BSA, was used for the enantioseparation of DL ‐THP in ACE. Effects of BSA concentration, pH and separation voltage on the effectiveness of the enantiomer separation were evaluated. In an optimal condition, D ‐ and L ‐THP were completely enantio‐separated in less than 9 min by partially filling an electrophoretic capillary with 50 μmol/L BSA (50 mbar, 100 s) and carrying out an electrophoresis with 20 mmol/L phosphate buffer (pH 7.4) at 15 kV. The sensitivity was further improved by making use of field‐amplified sample injection to lower the LOD (defined as S/N=3) down to 6 ng/mL. Real samples were also tested and promising results for the determination of DL ‐THP enantiomers were obtained.     

Enantioseparation of aromatic amino acids using CEC monolith with novel chiral selector,N‐methacryloyl‐l‐histidine methyl ester

A new type of polymethacrylate‐based monolithic column with chiral stationary phase was prepared for the enantioseparation of aromatic amino acids, namely d ,l ‐phenylalanine, d ,l ‐tyrosine, and d ,l ‐tryptophan by CEC. The monolithic column was prepared by in situ polymerization of butyl methacrylate (BMA), N‐methacryloyl‐l ‐histidine methyl ester (MAH), and ethylene dimethacrylate (EDMA) in the presence of porogens. The porogen mixture included DMF and phosphate buffer. MAH was used as a chiral selector. FTIR spectrum of the polymethacrylate‐based monolith showed that MAH was incorporated into the polymeric structure via in situ polymerization. Some experimental parameters including pH, concentration of the mobile phase, and MAH concentration with regard to the chiral CEC separation were investigated. Single enantiomers and enantiomer mixtures of the amino acids were separately injected into the monolithic column. It was observed that l ‐enantiomers of aromatic amino acids migrated before d ‐enantiomers. The reversal enantiomer migration order for tryptophan was observed upon changing of pH. Using the chiral monolithic column (100 μm id and 375 μm od), the best chiral separation was performed in 35:65% ACN/phosphate buffer (pH 8.0, 10 mM) with an applied voltage of 12 kV in CEC. SEM images showed that the chiral monolithic column has a continuous polymeric skeleton and large through‐pore structure.     

Chiral separation of phenylalanine and tryptophan by capillary electrophoresis using a mixture of β‐CD and chiral ionic liquid ([TBA] [l‐ASP]) as selectors

In this paper, a simple, effective and green capillary electrophoresis separation and detection method was developed for the quantification of underivatized amino acids (dl ‐phenylalanine; dl ‐tryptophan) using β‐Cyclodextrin and chiral ionic liquid ([TBA] [l ‐ASP]) as selectors. Separation parameters such as buffer concentrations, pH, β‐CD and chiral ionic liquid concentrations and separation voltage were investigated for the enantioseparation in order to achieve the maximum possible resolution. A good separation was achieved in a background electrolyte composed of 15 mm sodium tetraborate, 5 mm β‐CD and 4 mm chiral ionic liquid at pH 9.5, and an applied voltage of 10 kV. Under optimum conditions, linearity was achieved within concentration ranges from 0.08 to 10 µg/mL for the analytes with correlation coefficients from 0.9956 to 0.9998, and the analytes were separated in less than 6 min with efficiencies up to 970,000 plates/m. The proposed method was successfully applied to the determination of amino acid enantiomers in compound amino acids injections, such as 18AA‐I, 18AA‐II and 3AA. Copyright © 2013 John Wiley & Sons, Ltd.     

Preparative enantioseparation of propafenone by counter‐current chromatography using di‐n‐butyl l‐tartrate combined with boric acid as the chiral selector

This paper extends the research of the utilization of borate coordination complexes in chiral separation by counter‐current chromatography (CCC). Racemic propafenone was successfully enantioseparated by CCC with di‐n‐butyl l ‐tartrate combined with boric acid as the chiral selector. The two‐phase solvent system was composed of chloroform/ 0.05 mol/L acetate buffer pH 3.4 containing 0.10 mol/L boric acid (1:1, v/v), in which 0.10 mol/L di‐n‐butyl l ‐tartrate was added in the organic phase. The influence of factors in the enantioseparation of propafenone were investigated and optimized. A total of 92 mg of racemic propafenone was completely enantioseparated using high‐speed CCC in a single run, yielding 40–42 mg of (R)‐ and (S)‐propafenone enantiomers with an HPLC purity over 90–95%. The recovery for propafenone enantiomers from fractions of CCC was in the range of 85–90%.     

Use of cyclodextrin‐modified gold nanoparticles for enantioseparations of drugs and amino acids based on pseudostationary phase‐capillary electrochromatography

The application of chemical‐modified gold nanoparticles (GNPs) as chiral selector for the enantioseparation based on pseudostationary phase‐CEC (PSP‐CEC) is presented. GNPs modified by thiolated β‐CD were characterized by NMR and FT‐IR. The nanoparticle size was determined to be of 9.5 nm (+2.5 nm) by Transmission Electron Microscopy (TEM) and UV spectra. Four pairs of dinitrophenyl‐labeled amino acid enantiomers (DL‐Val, Leu, Glu and Asp) and three pairs of drug enantiomers (RS‐chlorpheniramine, zopiclone and carvedilol) were analyzed by using modified GNPs as the chiral selector in PSP‐CEC. Good theoretical plate number (up to 2.4×10⁵ per meter) and separation resolution (up to 4.7) were obtained even with low concentration of modified GNPs (0.8–1.4 mg/mL). The corresponding concentration of β‐CD in the buffer was only 0.30?0.53 mM, which was much lower than the optimum concentration of 15 mM if pure β‐CD was used as chiral selector. Our results showed that thiolated β‐CD modified GNPs have more sufficient interaction with the analytes, resulting in significant enhancement of enantioseparation. The study shed light on potential usage of chemical modified GNPs as chiral selector for enantioseparation based on PSP‐CEC.     

A novel C2 symmetric chiral stationary phase with N‐[(4‐Methylphenyl)sulfonyl]‐l‐leucine as chiral side chains

In this study, a series of chiral stationary phases based on N‐[(4‐methylphenyl)sulfonyl]‐l ‐leucine amide, whose enantiorecognition property has never been studied, were synthesized. Their enantioseparation abilities were chromatographically evaluated by 67 enantiomers. The chiral stationary phase derived from N‐[(4‐methylphenyl)sulfonyl]‐l ‐leucine showed much better enantioselectivities than that based on N‐(4‐methylbenzoyl)‐l ‐leucine amide. The construction of C₂ symmetric chiral structure greatly improved the enantiorecognition performance of the stationary phase. The C₂ symmetric chiral stationary phase exhibited superior enantioresolutions to other chiral stationary phases for most of the chiral analytes, especially for the chiral analytes with C₂ symmetric structures. By comparing the enantioseparations of the enantiomers with similar structures, the importance of hydrogen bond interaction, π–π interaction, and steric hindrance on enantiorecognition was elucidated. The enantiorecognition mechanism of trans‐N,N′‐(1,2‐diphenyl‐1,2‐ethanediyl)bis‐acetamide, which had an excellent separation factor on the C₂ symmetric chiral stationary phase, was investigated by ¹H‐NMR spectroscopy and 2D ¹H‐¹H nuclear overhauser enhancement spectroscopy.     

Predefine resolution of enantiomers in partial filling capillary electrophoresis and two discontinuous function plugs coupling in‐capillary

In common partial filling CE (PF‐CE), the capillary contains the selectors plug between the injection and detector end to avoid the selector going into the detector zone. To expand this method, we propose a mode of two discontinuous function plugs coupling in‐capillary, named as plug–plug PF‐CE (ppPF‐CE). Initially, we present the method to predefine the effective length of chiral selector to meet the requirement of enantiomers' resolution, which could avoid some experimental procedures. With α‐CD as a chiral selector, a satisfactory resolution of enantiomers d,l ‐tryptophan and d,l ‐tyrosine was obtained with a partial filling α‐CD plug of optimal length and concentration. Subsequently, a second plug containing hydroxypropyl methylcellulose, organic solvents (acetonitrile and methanol), anionic and cationic surfactants (SDS and CTAB), and different concentrations of sodium phosphate buffer was inserted after the selector plug. Effects of plug–plug filling on enantiomers' migration and resolution are discussed. The ppPF‐CE might be a new flexible mode for CE application.     

Amino Acid Ionic Liquids as Chiral Ligands in Ligand‐Exchange Chiral Separations

Recently, amino acid ionic liquids (AAILs) have attracted much research interest. In this paper, we present the first application of AAILs in chiral separation based on the chiral ligand exchange principle. By using 1‐alkyl‐3‐methylimidazolium L ‐proline (L ‐Pro) as a chiral ligand coordinated with copper(II), four pairs of underivatized amino acid enantiomers—dl ‐phenylalanine (dl ‐Phe), dl ‐histidine (dl ‐His), dl ‐tryptophane (dl ‐Trp), and dl ‐tyrosine (dl ‐Tyr)—were successfully separated in two major chiral separation techniques, HPLC and capillary electrophoresis (CE), with higher enantioselectivity than conventionally used amino acid ligands (resolution (R_s)=3.26–10.81 for HPLC; R_s=1.34–4.27 for CE). Interestingly, increasing the alkyl chain length of the AAIL cation remarkably enhanced the enantioselectivity. It was inferred that the alkylmethylimidazolium cations and L ‐Pro form ion pairs on the surface of the stationary phase or on the inner surface of the capillary. The ternary copper complexes with L ‐Pro are consequently attached to the support surface, thus inducing an ion‐exchange type of retention for the dl ‐enantiomers. Therefore, the AAIL cation plays an essential role in the separation. This work demonstrates that AAILs are good alternatives to conventional amino acid ligands for ligand‐exchange‐based chiral separation. It also reveals the tremendous application potential of this new type of task‐specific ILs.     

l‐Lysine‐derived ionic liquids as chiral ligands of Zn(II) complexes used in ligand‐exchange CE

Amino acid ionic liquids (AAILs) with l ‐lysine (l ‐Lys) as anion were synthesized and applied as new chiral ligands in Zn(II) complexes for chiral ligand‐exchange CE. After effective optimization, baseline enantioseparation of seven pairs of dansylated amino acids was achieved with a buffer of 100.0 mM boric acid, 5.0 mM ammonium acetate, 3.0 mM ZnSO₄, and 6.0 mM [C₆mim][l ‐Lys] at pH 8.2. To validate the unique behavior of AAILs, a comparative study between the performance of Zn(II)‐l ‐Lys and Zn(II)‐[C₆mim][l ‐Lys] systems was conducted. In Zn(II)‐[C₆mim][l ‐Lys] system, it has been found that the improved chiral resolution could be obtained and the migration times of the three test samples were markedly prolonged. Then the separation mechanism was further discussed. The role of [C₆mim][l ‐Lys] indicated clearly that the synthesized AAILs could be used as chiral ligands and would have potential utilization in separation science in future.     

Combined use of l‐alanine tert butyl ester lactate and trimethyl‐β‐cyclodextrin for the enantiomeric separations of 2‐arylpropionic acids nonsteroidal anti‐inflammatory drugs

In this study, a new CE method, employing a binary system of trimethyl‐β‐CD (TM‐β‐CD) and a chiral amino acid ester‐based ionic liquid (AAIL), was developed for the chiral separation of seven 2‐arylpropionic acid nonsteroidal anti‐inflammatory drugs (NSAIDs). In particular, the enantioseparation of ibuprofen, ketoprofen, carprofen, indoprofen, flurbiprofen, naproxen, and fenoprofen was improved significantly by supporting the BGE with the chiral AAIL l ‐alanine tert butyl ester lactate (l ‐AlaC₄Lac). Parameters, such as concentrations of TM‐β‐CD and l ‐AlaC₄Lac, and buffer pH, were systematically examined in order to optimize the chiral separation of each NSAID. It was observed that the addition of the AAIL into the BGE improved both resolution and efficiency significantly. After optimization of separation conditions, baseline separation (R_s>1.5) of five of the analytes was achieved in less than 11 min, while the resolution of ibuprofen and flurbiprofen was approximately 1.2. The optimized enantioseparation conditions for all analytes involve a BGE of 5 mM sodium acetate/acetic acid (pH 5.0), an applied voltage of 30 kV, and a temperature of 20°C. In addition, the results obtained by computing the %‐RSD values of the EOF and the two enantiomer peaks, demonstrated excellent run‐to‐run, batch‐to‐batch, and day‐to‐day reproducibilities.     

Enantioseparation of dansyl amino acids by ligand‐exchange capillary electrophoresis with zinc(II)‐L‐phenylalaninamide complex

A novel method of chiral ligand‐exchange CE was developed with L ‐amino acylamides as a chiral ligand and zinc(II) as a central ion. It has been demonstrated that these chiral complexes, such as Zn(II)‐L ‐alaninamide, Zn(II)‐L ‐prolinamide, and Zn(II)‐L ‐phenylalaninamide, are suitable for use as chiral selectors for the enantioseparation of either individual pair of or mixed dansyl amino acids. The optimal separation running buffer consisted of 5 mM ammonium acetate, 100 mM boric acid, 4 mM ZnSO₄·7 H₂O, and 8 mM L ‐amino acylamides at pH 8.2. The experiments showed that apart from the effect of the concentration of the complexes on the resolution and the migration time, the buffer pH also had a sharp influence on resolution. The employed chiral ligands exhibited different enantioselectivities and enantiomer migration orders. D ‐Amino acids migrate faster than L ‐amino acids when Zn(II)‐L ‐alaninamide and Zn(II)‐L ‐phenylalaninamide are used as chiral selectors, but it was observed that the migration order is reversed when Zn(II)‐L ‐prolinamide is used as the chiral selector. Furthermore, the amount of dansylated amino acids is found to be highly dependent on the labeling temperature.     

Indirect reversed‐phase high‐performance liquid chromatographic and direct thin‐layer chromatographic enantioresolution of (R,S)‐Cinacalcet

Enantioresolution of the calcimimetic drug (R,S)‐Cinacalcet was achieved using both indirect and direct approaches. Six chiral variants of Marfey's reagent having l ‐Ala‐NH₂, l ‐Phe‐NH₂, l ‐Val‐NH₂, l ‐Leu‐NH₂, l ‐Met‐NH₂ and d ‐Phg‐NH₂ as chiral auxiliaries were used as derivatizing reagents under microwave irradiation. Derivatization conditions were optimized. Reversed‐phase high‐performance liquid chromatography was successful using binary mixtures of aqueous trifluoroacetic acid and acetonitrile for separation of diastereomeric pairs with detection at 340 nm. Thin silica gel layers impregnated with optically pure l ‐histidine and l ‐arginine were used for direct resolution of enantiomers. The limit of detection was found to be 60 pmol in HPLC while in TLC it was found to be in the range of 0.26–0.28 µg for each enantiomers. Copyright © 2010 John Wiley & Sons, Ltd.     

Preparation and evaluation of a deoxycholic‐calix[4]arene hybrid‐type receptor as a chiral stationary phase for HPLC

We report the synthesis and enantioseparation characteristics of two novel covalently immobilized deoxycholic acid derivatives as chiral stationary phases for high‐performance liquid chromatography. In the structure of the first stationary phase, the 3‐position of deoxycholic acid is substituted with a 3,5‐dinitrophenylcarbamoyl group and the second one has an additional calix[4]arene attached to the carboxylic group of the deoxycholic acid. The chromatographic performance of the stationary phases was evaluated with enantioseparation of N‐(3,5‐dinitrobenzoyl)‐dl ‐leucine, N‐(3,5‐dinitrobenzoyl)‐dl ‐valine, omeprazole, diclofop‐methyl, dl ‐mandelic acid and (RS)‐pregabalin. Comparison of the performance characteristics of the prepared chiral stationary phases provided evidence for the active involvement of the calix[4]arene unit in the chiral recognition process. Both stationary phases are chemically bonded to the silica and can be used in both normal‐phase and reversed‐phase modes.     

Enantioselective separation of chiral vicinal diols in capillary electrophoresis using a mono‐6A‐aminoethylamino‐β‐cyclodextrin as a chiral selector

This paper describes an improved access to mono‐6^A‐aminoethylamino‐β‐CD (β‐CDen), a very efficient cationic chiral selector for CZE in the separation of eight chiral aromatic vicinal diols. The β‐CDen concentration has a strong influence on the efficiency of enantioseparation. The effects of the pH and concentration of the BGE, the capillary temperature, and the applied voltage on the resolution and separation selectivity have been studied. Excellent chiral resolution was achieved under the optimal conditions of β‐CDen 10 mM, pH 10, 200 mM borate buffer at 15 kV and 20°C within 20 min. Moreover, the developed method was successfully applied to the determination of the enantiomeric purity of the catalytic asymmetric dihydroxylation (AD) reaction products.     

Chiral separation of brompheniramine enantiomers by recycling high‐speed countercurrent chromatography using carboxymethyl‐β‐cyclodextrin as a chiral selector

A recycling high‐speed countercurrent chromatography protocol was proposed for the enantioseparation of brompheniramine by employing β‐cyclodextrin derivatives as a chiral selector. The two‐phase solvent system of n‐hexane/isobutyl acetate/0.10 mol/L phosphate buffer solution with a volume ratio of 2:4:6 was selected by a series of extraction experiments. Factors that affected the distribution of the enantiomers over the two‐phase system (e.g., the type and concentration of β‐cyclodextrin derivatives = pH value of the aqueous solution, and the separation temperature) were also investigated. In addition, the theory of thermodynamics is applied to verify the feasibility of the enantioseparation process and the corresponding results demonstrate that this separation process is feasible. The optimized conditions include carboxymethyl‐β‐cyclodextrin concentration of 0.010 mol/L, pH of 7.5, and temperature of 5°C. Under the optimal conditions, the purities of both monomer molecules were over 99%, and the recovery yields were 88% for (+)‐brompheniramine and 85% for (–)‐brompheniramine, respectively.     

Self‐Reporting Inhibitors: A Single Crystallization Process To Obtain Two Optically Pure Enantiomers

Collection of two optically pure enantiomers in a single crystallization process can significantly increase the chiral separation efficiency but this is difficult to realize. Now a self‐reporting strategy is presented for visualizing the crystallization process by a dyed self‐assembled inhibitor made from the copolymers with tri(ethylene glycol)‐grafting polymethylsiloxane as the main chain and poly(N⁶‐methacryloyl‐l ‐lysine) as side chains. When applied with seeds together for the fractional crystallization of conglomerates, the inhibitors can label the formation of the secondary crystals and guide the complete separation process of two enantiomers with colorless crystals as the first product and red crystals as the second. This method leads to high optical purity of d /l ‐Asn?H₂O (99.9 % ee for d ‐crystals and 99.5 % for l ‐crystals) in a single crystallization process. It requires a small amount of additives and shows excellent recyclability.     

Polymer‐based monolithic column with incorporated chiral metal–organic framework for enantioseparation of methyl phenyl sulfoxide using nano‐liquid chromatography

A new approach to the preparation of enantioselective porous polymer monolithic columns with incorporated chiral metal–organic framework for nano‐liquid chromatography has been developed. While no enantioseparation was achieved with monolithic poly(4‐vinylpyridine‐co‐ethylene dimethacrylate) column, excellent separations of both enantiomers of (±)‐methyl phenyl sulfoxide were achieved with its counterpart prepared after admixing metal–organic framework [Zn₂(benzene dicarboxylate)(l‐lactic acid)(dmf)], which is synthesized from zinc nitrate, l ‐lactic acid, and benzene dicarboxylic acid in the polymerization mixture. These novel monolithic columns combined selectivity of the chiral framework with the excellent hydrodynamic properties of polymer monoliths, may provide a great impact on future studies in the field of chiral analysis by liquid chromatography.     

HPLC enantioseparation of β2‐homoamino acids using crown ether‐based chiral stationary phase

RP high‐performance liquid chromatographic methods were developed for the enantioseparation of eleven unusual β²‐homoamino acids. The underivatized analytes were separated on a chiral stationary phase containing (+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid as chiral selector. The effects of organic (alcoholic) and acidic modifiers, the mobile phase composition and temperature on the separation were investigated. The structures of the substituents in the α‐position of the analytes substantially influenced the retention and resolution. The elution sequence was determined in some cases: the S enantiomers eluted before the R enantiomers.     

Preparative enantioseparation of loxoprofen precursor by recycling countercurrent chromatography with hydroxypropyl‐β‐cyclodextrin as a chiral selector

Recycling countercurrent chromatography was successfully applied to the resolution of 2‐(4‐bromomethylphenyl)propionic acid, a key synthetic intermediate for synthesis of nonsteroidal anti‐inflammatory drug loxoprofen, using hydroxypropyl‐β‐cyclodextrin as chiral selector. The two‐phase solvent system composed of n‐hexane/n‐butyl acetate/0.1 mol/L citrate buffer solution with pH 2.4 (8:2:10, v/v/v) was selected. Influence factors for the enantioseparation were optimized, including type of substituted β‐cyclodextrin, concentration of hydroxypropyl‐β‐cyclodextrin, separation temperature, and pH of aqueous phase. Under optimized separation conditions, 50 mg of 2‐(4‐bromomethylphenyl)propionic acid was enantioseparated using preparative recycling countercurrent chromatography. Technical details for recycling elution mode were discussed. The purities of both the S and R enantiomers were over 99.0% as determined by high‐performance liquid chromatography. The enantiomeric excess of the S and R enantiomers reached 98.0%. The recovery of the enantiomers from eluted fractions was 40.8–65.6%, yielding 16.4 mg of the S enantiomer and 10.2 mg of the R enantiomer. At the same time, we attempted to enantioseparate the anti‐inflammatory drug loxoprofen by countercurrent chromatography and high‐performance liquid chromatography using a chiral mobile phase additive. However, no successful enantioseparation was achieved so far.     

Chirality‐Driven Transportation and Oxidation Prevention by Chiral Selenium Nanoparticles

The chirality of nanoparticles directly influences their transport and biological effects under physiological conditions, but the details of this phenomenon have rarely been explored. Herein, chiral GSH‐anchored selenium nanoparticles (G@SeNPs) are fabricated to investigate the effect of their chirality on their transport and antioxidant activity. G@SeNPs modified with different enantiomers show opposite handedness with a tunable circular dichroism signal. Noninvasive positron emission tomography imaging clearly reveals that ⁶⁴Cu‐labeled l ‐G@SeNPs experience distinctly different transport among the major organs from that of their d ‐and dl ‐counterparts, demonstrating that the chirality of the G@SeNPs influences the biodistribution and kinetics. Taking advantage of the strong homologous cell adhesion and uptake, l ‐G@SeNPs have been shown here to effectively prevent oxidation damage caused by palmitic acid in insulinoma cells.