Glycogen: A novel branched polysaccharide chiral selector in CE

Various chiral selectors have been employed in CE and among them linear polysaccharides exhibited powerful enantioselective properties. Different from linear polysaccharides, the use of branched polysaccharides as chiral selectors in CE has not been reported previously. In this study glycogen belonging to the class of branched polysaccharides was used as a novel chiral selector for the enantiomeric separations for the first time. Since glycogen is electrically neutral, the method is applicable to ionic compounds. Eighteen chiral compounds including 12 basic drugs and six acidic drugs have been tested to demonstrate the potential of this chiral selector. BGE and selector concentrations and buffer pH were systematically optimized in order to obtain successful chiral separations. Among the tested compounds, the enantiomers of ibuprofen, which is an acidic drug, were successfully recognized by 3.0% w/v glycogen with 90 mM Tris‐H₃PO₄ buffer (pH 7.0). The enantiomers of basic drugs such as citalopram, cetirizine and nefopam were also baseline‐resolved with 50 mM Tris‐H₃PO₄ buffer (pH 3.0) containing 3.0% glycogen. Amlodipine belonging to basic compound only gave partial enantioseparation under the above‐mentioned condition.     

High‐performance liquid chromatographic separations of stereoisomers of chiral basic agrochemicals with polysaccharide‐based chiral columns and polar organic mobile phases

The separation of the stereoisomers of 23 chiral basic agrochemicals was studied on six different polysaccharide‐based chiral columns in high‐performance liquid chromatography with various polar organic mobile phases. Along with the successful separation of analyte stereoisomers, emphasis was placed on the effect of the chiral selector and mobile phase composition on the elution order of stereoisomers. The interesting phenomenon of reversal of enantiomer/stereoisomer elution order function of the polysaccharide backbone (cellulose or amylose), type of derivative (carbamate or benzoate), nature, and position of the substituent(s) in the phenylcarbamate moiety (methyl or chloro) and the nature of the mobile phase was observed. For several of the analytes containing two chiral centers all four stereoisomers were resolved with at least one chiral selector/mobile phase combination.     

HPLC of fluoroquinolone antibacterials using chiral stationary phase based on enantiomeric (3,3′‐diphenyl‐1,1′‐binaphthyl)‐20‐crown‐6

A residual silanol group‐protecting chiral stationary phase (CSP) based on optically active (3,3′‐diphenyl‐1,1′‐binaphthyl)‐20‐crown‐6 was successfully applied to the resolution of fluoroquinolone compounds including gemifloxacin mesylate. The chiral recognition ability of the residual silanol group‐protecting CSP was generally greater than that of the residual silanol group‐containing CSP. From these results, it was concluded that the simple protection of the residual silanol groups of the latter CSP with lipophilic n‐octyl groups can improve its chiral recognition ability for the resolution of racemic fluoroquinolone compounds. The chromatographic resolution behaviors were investigated as a function of the content and type of organic and acidic modifiers and the ammonium acetate concentration in aqueous mobile phase and the column temperature. Especially, the addition of ammonium acetate to the mobile phase was found to be a quite effective means of reducing the enantiomer retentions without sacrificing the chiral recognition efficiency of the CSP.     

Enantioseparation of chiral aromatic acids by multiple dual mode counter‐current chromatography using hydroxypropyl‐β‐cyclodextrin as chiral selector

This work concentrates on extending the utilization of multiple dual mode (MDM) counter‐current chromatography in chiral separations. Two aromatic acids, 2‐(6‐methoxy‐2‐naphthyl)propionic acid (NAP) and 2‐phenylpropionic acid (2‐PPA), were enantioseparated by MDM counter‐current chromatography using hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) as chiral selector. The two‐phase solvent systems consisting of n‐hexane/ethyl acetate 0.1 mol/L phosphate buffer pH 2.67 containing 0.1 mol/L HP‐β‐CD (7.5:2.5:10 for NAP and 7:3:10 for 2‐PPA, v/v/v) were used. Conventional MDM and modified MDM were compared according to peak resolution under current separation mechanism. The influence of elution time after the first‐phase inversion and number of cycles for MDM were investigated. Peak resolution of NAP and 2‐PPA increased from 0.62 to 1.05 and 0.72 to 0.84, respectively, using optimized MDM conditions. Being an alternative elution method for counter‐current chromatography, MDM elution greatly improved peak resolution in chiral separations.     

Enantioseparation of selected chiral sulfoxides in high‐performance liquid chromatography with polysaccharide‐based chiral selectors in polar organic mobile phases with emphasis on enantiomer elution order

The separation of the enantiomers of 17 chiral sulfoxides was studied on polysaccharide‐based chiral columns in polar organic mobile phases. Enantiomer elution order (EEO) was the primary objective in this study. Two of the six chiral columns, especially those based on amylose tris(3,5‐dimethylphenylcarbamate) and cellulose tris(4‐chloro‐3‐methylphenylcarbamate) (Lux Cellulose‐4) proved to be most successful in the separation of the enantiomers of the studied sulfoxides. Interesting examples of EEO reversal were observed depending on the chiral selector or the composition of the mobile phase. For instance, the R‐(+) enantiomer of lansoprazole eluted before the S‐(?) enantiomer on Lux Cellulose‐1 in both methanol or ethanol as the mobile phase, while the elution order was opposite in the same eluents on amylose tris(3,5‐dimethylphenylcarbamate) with the S‐(?) enantiomer eluting before the R‐(+) enantiomer. The R‐(+) enantiomer of omeprazole eluted first on Lux Amylose‐2 in methanol but it was second when acetonitrile was used as the mobile phase with the same chiral selector. Several other examples of reversal in EEO were observed in this study. An interesting example of the separation of four stereoisomers of phenaminophos sulfoxide containing chiral sulfur and phosphor atoms is also reported here.     

Effect of the residual silanol group protection on the liquid chromatographic resolution of α‐amino acids and proton pump inhibitors on a ligand exchange chiral stationary phase

A new ligand exchange chiral stationary phase (new CSP) containing residual silanol group‐protecting n‐octyl groups on the silica surface was prepared by treating a ligand exchange CSP (original CSP) based on sodium N‐[(R)‐2‐hydroxy‐1‐phenylethyl]‐N‐undecylaminoacetate bonded to silica gel with excess n‐octyltriethoxysilane. The new and original CSPs containing an identical amount of chiral selector were applied to the resolution of α‐amino acids and proton pump inhibitors (PPIs) including omeprazole, pantoprazole, lansoprazole, and rabeprazole. The separation factors (α) and resolutions (R_S) were greater on the new CSP than on the original CSP except for the resolution of asparagine. The trends of the retention factors (k₁) for the resolution of α‐amino acids on the new and original CSPs with the variation of the organic modifier content in aqueous mobile phase were opposite to those for the resolution of PPIs. Removal of the nonenantioselective interactions between the residual silanol groups and the analytes and the improved lipophilicity of the new CSP were proposed to be responsible for the improved chiral recognition ability of the new CSP and the different retention behaviors of the enantiomers between the new and original CSPs.     

Investigation of the enantioselectivity of tetramethylammonium‐lactobionate chiral ionic liquid based dual selector systems toward basic drugs in capillary electrophoresis

Recently, chiral ionic liquids (CILs) have received increasing interest in chiral separation by CE. Nevertheless, the lack of deep perception of the specific mechanism about CILs in CE for enantioseparation still perplexes a legion of researchers, despite the strenuous efforts. In this paper, a lactobionic acid based ionic liquid, tetramethylammonium‐lactobionate (TMA‐LA) was applied for the first time in CE to establish dual selector system with clindamycin phosphate (CP) for enantiomeric separation. Compared to single CP system or single TMA‐LA system significantly improved separations of seven tested rameric drugs (propranolol, nefopam, citalopram, chlorphenamine, metoprolol, bisoprolol, and esmolol) were observed in the dual selector systems. Several crucial parameters such as type and proportion of organic modifier, buffer composition and pH, and concentration of TMA‐LA/CP were systematically investigated to achieve satisfied enantioseparation. Meanwhile, molecular modeling was applied to demonstrate the chiral recognition mechanism of the TMA‐LA/CP dual‐selector separation system using the molecular docking software Autodock, which well supported the experimental results. The existence of TMA‐LA/CP complex may give rise to a higher discriminatory ability against the enantiomers, indicating the reason of improved separation in TMA‐LA/CP system. All the influence factors evaluated by means of Statistical Product and Service Solutions (SPSS) to research the influences on the chiral separation system.     

Direct chiral resolution of underivatized amino acids on a stationary phase dynamically modified with the ion‐exchanger Nτ‐decyl‐l‐spinacine

Increasing attention has been devoted in the last decades to chiral chromatography, principally to high‐performance liquid chromatography techniques using a chiral stationary phase. Many chiral high‐performance liquid chromatography columns are commercially available, but, unfortunately, they are most often rather expensive. A cheap alternative to the commercial chiral columns is the dynamic‐coating procedure of a standard achiral stationary phase with a chiral selector containing both a chiral domain and a chain or a group able to tightly (but noncovalently) bind the achiral support. This is the case of N^τ‐decyl‐l ‐spinacine, already successfully employed to dynamically cover a reversed‐phase column to separate racemic mixtures of amino acids through the ligand‐exchange mechanism. In the present work, the same chiral selector is employed to separate racemic mixtures of amino acids and oligopeptides, in the absence of metal ions: no coordination complex is formed, but only electrostatic and weak nonbonding interactions between the chiral phase and the analytes are responsible for the observed enantioselectivity. The new method is simpler than the previous one, very effective in the case of aromatic amino acids and oligopeptides and also suitable for preparative purposes.     

Azithromycin as a new chiral selector in capillary electrophoresis

In capillary electrophoresis (CE), separation of enantiomers of a chiral compound can be achieved through the chiral interactions and/or complex formation between the chiral selector and the enantiomeric analytes on leaving their diastereomeric forms with different stability constants and hence different mobilities. A great number of chiral selectors have been employed in CE and among them macrocyclic antibiotics exhibited excellent enantioselective properties towards a wide number of racemic compounds. The use of azithromycin (AZM) as a chiral selector has not been reported previously. This work reports the use of AZM as a chiral selector for the enantiomeric separations of five chiral drugs and one amino acid (tryptophan) in CE. The enantioseparation is carried out using polar organic mixtures of acetonitrile (ACN), methanol (MeOH), acetic acid and triethylamine as run buffer. The influences of the chiral selector concentration, ACN/MeOH ratio, applied voltage and capillary temperature on enantioseparation are investigated. The results show that AZM is a viable chiral selector in CE for the enantioseparation of the type of chiral drugs investigated.     

In‐column preparation of a brush‐type chiral stationary phase using click chemistry and a silica monolith

Brush‐type chiral stationary phases (CSP) have been prepared both from a silica monolith and, separately, from 10 μm porous silica beads via a process of in‐column modification including attachment of the chiral selector via copper‐catalyzed azide–alkyne cycloaddition. Azide functionalities were first introduced on the pore surface of each type of support by reaction with 3‐(azidopropyl)trimethoxysilane, followed by immobilization of a proline‐derived chiral selector containing an alkyne moiety. This functionalization reaction was carried out in dimethylformamide (DMF) in the presence of catalytic amounts of copper (I) iodide. The separation performance of these triazole linked stationary phases was demonstrated in enantioseparations of four model analytes, which afforded separation factors as high as 11.4.     

Optimization of the LC enantioseparation of chiral pharmaceuticals using cellulose tris(4‐chloro‐3‐methylphenylcarbamate) as chiral selector and polar non‐aqueous mobile phases

The resolving power of a new commercial polysaccharide‐based chiral stationary phase, Sepapak‐4, with cellulose tris(4‐chloro‐3‐methylphenylcarbamate) coated on silica microparticles as chiral selector, was evaluated toward the enantioseparation of ten basic drugs with widely different structures and hydrophobic properties, using ACN as the main component of the mobile phase. A multivariate approach (experimental design) was used to screen the factors (temperature, n‐hexane content, acidic and basic additives) likely to influence enantioresolution. Then, the optimization was performed using a face‐centered central composite design. Complete enantioseparation could be obtained for almost all tested chiral compounds, demonstrating the high chiral discrimination ability of this chiral stationary phase using polar organic mobile phases made up of ACN and containing an acidic additive (TFA or formic acid), 0.1% diethylamine and n‐hexane. These results clearly illustrate the key role of the nature of the acidic additive in the mobile phase.     

Monolithic silica columns functionalized with substituted polyproline‐derived chiral selectors as chiral stationary phases for high‐performance liquid chromatography

In this study, two polyproline‐derived chiral selectors are bonded to monolithic silica gel columns. In spite of high chiral selector coverage, the derivatization was found to have only a slight effect on the hydrodynamics of the mobile phase through the column. The enantioseparation ability of the resulting chiral monolithic columns was evaluated with a series of structurally diverse racemic test compounds. When compared to analogous bead‐based chiral stationary phases, higher enantioseparation and broader application domain were observed for monolithic columns. Moreover, the increase in flow rate produces a minor reduction of resolution, which permits to shorten analysis time. Additionally, increased loadability defines chiral polyproline derived monoliths as adequate for preparative chromatography.     

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

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

Use of chiral amino acid ester‐based ionic liquids as chiral selectors in CE

In this study, the applicability of a chiral ionic liquid (CIL) as the sole chiral selector in CE was investigated for the first time. In particular, five amino acid ester‐based CILs were synthesized and used as additives in the BGE in order to evaluate their chiral recognition ability. The performance of these CILs as the sole chiral selectors was evaluated by using 1,1′‐binaphthyl‐2,2‐diylhydrogenphosphate (BNP) as the analyte and by comparing the resolution values. Different parameters were examined, such as the alkyl group bulkiness and the configuration of the cation, the anion type of the CIL and its concentration, and the pH of the BGE, in order to optimize the separation of the enantiomers and to demonstrate the effect that each parameter has on the chiral‐recognition ability of the CIL. Baseline separation of BNP within 13 min was achieved by using a BGE of 100 mM Tris/10 mM sodium tetraboratedecahydrate (pH 8) and a chiral selector of 60 mM l ‐alanine tert butyl ester lactate. The run‐to‐run and batch‐to‐batch reproducibilities were also evaluated by computing the %RSD values of the EOF and the two enantiomer peaks. In both cases, very good reproducibilities were observed, since all %RSD values were below 1%.     

Mono‐6A‐(4‐methoxybutylamino)‐6A‐β‐cyclodextrin as a chiral selector for enantiomeric separation

A new member of the family of methoxylalkylamino monosubstituted β‐cyclodextrins, mono‐6^A‐(4‐methoxybutylamino)‐6^A‐β‐cyclodextrin, has been developed as a chiral selector for enantioseparation in capillary electrophoresis. This amino cyclodextrin exhibited good enantioselectivities for 16 model acidic racemates including three dansyl amino acids at an optimum pH of 6.0. Excellent chiral resolutions over six were obtained for α‐hydroxy acids and 2‐phenoxypropionic acids with 3.0 mM chiral selector. The good chiral recognition for α‐hydroxyl acids was attributed to inclusion complexation, electrostatic interactions, and hydrogen bonding. The hydrogen‐bonding‐enhanced chiral recognition was revealed by NMR spectroscopy. The chiral separation of acidic racemates was further improved with the addition of methanol (≤10 vol%) as an organic additive.     

Molecular modelling experiments on chiral recognition in GC with specially derivatized cyclodextrins as selectors

The results of gas chromatographic enantiomeric separations of selected non-polar and polar test solutes (limonene and 1-phenylethanol) using two different derivatives of β-cyclodextrin, permethyl-β-CD and 2,3-dimethyl-tert-butyldimethylsilyl-β-CD (PMCD and TBCD) are interpreted in combination with the conclusions from molecular modelling calculations based on docking experiments. A comparison of the two selectors which were used as solution in a polysiloxane matrix (OV 1701) indicated that the less flexible TBCD which is blocked on one side by the bulky tert-butyldimethylsilyl groups seems to possess advantages for certain chiral separations, especially of the non-polar type. The special properties of TBCD as selector are also demonstrated by chiral separations of other compounds.     

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%.     

Preparation and HPLC application of chiral stationary phase from 4‐tert‐butylphenylcarbamates of cellulose and amylose immobilized onto silica gel

The 4‐tert‐butylphenylcarbamates of cellulose and amylose bearing a small amount of 3‐(triethoxysilyl)propyl residues were synthesized by a one‐pot process and efficiently immobilized onto a silica gel through intermolecular polycondensation of the triethoxysilyl groups. The obtained chiral packing materials (CPMs) were evaluated by HPLC. The polysaccharide derivatives containing about 1–2% of the 3‐(triethoxysilyl)propyl residue were efficiently immobilized with a high chiral recognition ability. The immobilized CPMs could be used with the eluents containing chloroform and tetrahydrofuran (THF), which cannot be used with the conventional coated‐type CPMs. By using these eluents, the chiral recognition for many racemates was improved.     

Immobilization of 3,5‐dimethylphenylcarbamates of cellulose and amylose onto silica gel using (3‐glycidoxypropyl)triethoxysilane as linker

The 3,5‐dimethylphenylcarbamates of cellulose and amylose were effectively immobilized onto plain silica gels as chiral packing materials (CPMs) for HPLC by means of intermolecular polycondensation of triethoxysilyl groups introduced with (3‐glycidoxypropyl)triethoxysilane. The immobilization and chiral recognition abilities of the obtained CPMs prepared with different amounts of (3‐glycidoxypropyl)triethoxysilane were investigated. In addition, the solvent compatibilities of the immobilized‐type CPMs were examined with eluents containing chloroform and THF. When these eluents were used, for most of the tested racemates, the chiral resolving abilities of the obtained CPMs were improved.     

Advances in enantiomeric resolution on monolithic chiral stationary phases in liquid chromatography and electrochromatography

During the last decade, chiral monolithic stationary phases have been prepared and used for rapid enantioseparations in CEC and HPLC. Various chiral selectors are used to prepare these CSPs. The preparation, properties, and applications of these CSPs are discussed in this paper. Attempts have been made to describe optimization strategies and the chiral recognition mechanisms. A comparison of chiral separations in CEC and HPLC is described. Efforts have also been made to predict the future perspectives and challenges of chiral monolithic stationary phases. The most effective chiral selectors include polysaccharides, cyclodextrins, and macrocyclic glycopeptide antibiotics. These chiral phases produced acceptable analytical enantiomeric separation of a variety of racemates. However, the development of these CSPs for preparative‐scale separations is needed.