Rapid chiral separation methods development by cyclodextrin-mediated capillary electrophoresis for acidic and basic compounds

Chiral separation methods development using conventional techniques such as GC or HPLC requires a lot of experience, effort, and expense, due to the wide diversity of the optically active solutes and their possible chiral selectors. Capillary electrophoresis has received increased attention as an alternative technique for chiral separation due to its inherent high efficiencies and ease of methods development. However, due to the wide variety of chiral selectors available in CE, the benefits of this technique might be diminished without an appropriate methods development scheme. In this paper detailed examples are shown for fast, efficient, and predictable chiral capillary electrophoresis separation methods development based on a new and systematic theory. Optimized separations and their parameters are presented for several enantiomeric acids and bases. All the three possible cases, such as the use of low and high pH, as well as pH = pK buffer systems are thoroughly discussed. © 1995 Wiley-Liss, Inc.     

The stereochemical resolution of enantiomeric free and derivatized amino acids using an HPLC chiral stationary phase based on immobilized alpha-chymotrypsin: chiral separation due to solute structure or enzyme activity

The stereochemical separation of free and derivatized amino acids on active alpha-chymotrypsin bonded to silica is governed by two mechanisms based on the structure of the solutes or on the enzymatic activity of the enzyme. The deactivation of the hydrolytically active site of the enzyme demonstrated that a significant portion of the retention on this support is due to hydrophobic interactions at other sites. These sites appear to be stereoselective for the ester derivatives of amino acids but not for the other studied solutes.     

Use of Sulfated Cyclofructan 6 and Sulfated Cyclodextrins for the Chiral Separation of Four Basic Pharmaceuticals by Capillary Electrophoresis

Sulfated cyclofructan 6 (S‐CF6) and sulfated cyclodextrins (S‐α‐, β‐, γ‐CDs) are highly selective chiral selectors for the enantioseparation of basic solutes. In this study, S‐CF6 was introduced for the enantiomeric separation of four basic pharmaceuticals (including tamsulosin, tiropramide, bupivacaine, and norephedrine) by capillary electrophoresis (CE), and the enantiomeric separation performance was compared with S‐α‐, β‐, γ‐CDs. The effects of the chiral selector type, chiral selector concentration, operating voltage, and column temperature were examined and optimized. Excellent resolutions were obtained for all solutes on these chiral selectors. Chirality 25:735–742, 2013. © 2013 Wiley Periodicals, Inc.     

Enantioselective separation of chiral arylcarboxylic acids on an immobilized human serum albumin chiral stationary phase

A series of 12 chiral arylcarboxylic acids were chromatographed on an immobilized human serum albumin chiral stationary phase (HSA-CSP). The effects of solute structure on chromatographic retentions and enantioselective separations were examined by linear regression analysis and the construction of quantitative structure-enantioselective retention relationships. Competitive displacement studies were also conducted using R-ibuprofen as the displacing agent. The results indicate that the enantioselective retention of the solutes takes place at the indole-benzodiazepine site (site II) on the HSA molecule and that chiral recognition is affected by the hydrophobicity and steric volume of the solutes. The displacement studies also identified a cooperative allosteric interaction induced by the binding of R-ibuprofen to site II. Chirality 9:178–183, 1997. © 1997 Wiley-Liss, Inc.     

Cellulose type chiral stationary phase based on reduced graphene oxide@silica gel for the enantiomer separation of chiral compounds

The graphene oxide (GO) was covalently coupled to the surfaces of silica gel (SiO₂) microspheres by amide bond to get the graphene oxide@silica gel (GO@SiO₂). Then, the GO@SiO₂ was reduced with hydrazine to the reduced graphene oxide@silica gel (rGO@SiO₂), and the cellulose derivatives were physically coated on the surfaces of rGO@SiO₂ to prepare a chiral stationary phase (CSP) for high performance liquid chromatography. Under the optimum experimental conditions, eight benzene‐enriched enantiomers were separated completely, and the resolution of trans‐stilbene oxide perfectly reached 4.83. Compared with the blank column of non‐bonded rGO, the separation performance is better on the new CSP, which is due to the existence of rGO to produce special retention interaction with analytes, such as π‐π stacking, hydrophobic effect, π‐π electron‐donor–acceptor interaction, and hydrogen bonding. Therefore, the obtained CSP shows special selectivity for benzene‐enriched enantiomers, improves separation selectivity and efficiency, and rGO plays a synergistic effect with cellulose derivatives on enantioseparation.     

Chiral separation principles in chromatographic and electromigration techniques

Almost half of the drugs in use today are chiral. It is well established that the pharmacological activity is mostly restricted to one of the enantiomers (eutomer). There can be qualitative and quantitative differences in the activity of the enantiomers. In many cases, the inactive enantiomer (distomer) shows unwanted side effects or even toxic effects. Even if the side effects are not that drastic, the distomer has to be metabolized and this represents an unnecessary burden for the organism. Therefore, the development of methods for the separation of enantiomers, both on analytical and preparative scale, has become increasingly important. Chromatographic techniques such as thin layer chromatography (TLC), gas chromatography (GC), supercritical fluid chromatography (SFC), and above all high-performance liquid chromatography (HPLC) have been used for enantiomer separation for about two decades. More recently, electromigration techniques, such as capillary electrophoresis and capillary electrochromatography, have been shown to be powerful alternatives to chromatographic methods. This review gives a short overview of different chiral separation principles and their application. Several new developments are discussed.     

The development of biphasic chiral recognition in chiral separation

In chiral separation, enantioseparation factor is an important parameter which influences the resolution of enantiomers. In this current overview, a biphasic chiral recognition method is introduced to the readers. This method can significantly improve the enantioseparation factor in two‐phase solvent through adding lipophilic and hydrophilic chiral selectors which have opposite chiral recognition ability to organic and aqueous phases, respectively. This overview presents the development and applications of biphasic chiral recognition in liquid‐liquid extraction and counter current chromatography. It mainly focuses on the topics of mechanism, advantages and limitations, applications, and key factors of biphasic chiral recognition. In addition, the future outlook on development of biphasic chiral recognition also has been discussed in this overview.     

Capillary electrophoresis of herbicides. III. Evaluation of octylmaltopyranoside chiral surfactant in the enantiomeric separation of phenoxy acid herbicides

A chiral alkylglucoside surfactant, namely n-octyl-β-D-maltopyranoside (OM), was evaluated in the enantiomeric separation of phenoxy acid herbicides. The enantiomeric resolution of the phenoxy acid herbicides could be manipulated readily by adjusting the surfactant concentration, ionic strength, pH, the percent organic modifier and separation temperature. The optimum surfactant concentration needed for maximum enantiomeric resolution varied among the different analytes, and was an inverse function of the hydrophobicity of the phenoxy acid herbicides with the most hydrophobic solute requiring less surfactant concentration for attaining a baseline enantiomeric resolution. Due to the ionic nature of the phenoxy acid herbicides, increasing the pH of the running electrolyte increased the degree of ionization of the acidic herbicides thus decreasing their association with the chiral micelles and in turn their enantiomeric resolution. Increasing the ionic strength of the running electrolyte seems to enhance both the solubilization of the solute in the micelle and the chiral interaction of the solute with the micelle with a net increase in enantiomeric resolution. The percent of added methanol had a varying effect on the resolution of the various enantiomers in the sense that it enhanced the enantiomeric resolution for the most hydrophobic solutes while it decreased the enantiomeric resolution for the weakly hydrophobic ones. Thermostating the capillary column at subambient temperature improved enantiomeric resolution. © 1996 Wiley-Liss, Inc.     

Central composite design for the rapid optimisation of ruggedness and chiral separation of amlodipine in capillary electrophoresis

Systematic optimisation with central composite design offers an efficient route for rapid optimisation of resolution with multiple interacting parameters in chiral CE. This is illustrated by separations of amlodipine with α-CD as chiral selector in the running buffer, for which the predicted performance of central composite design is assessed. The utility of response surface methodology for locating optimum ruggedness in CE is also described. © 1995 Wiley-Liss, Inc.     

Evaluation of the macrocyclic antibiotic vancomycin as a chiral selector for capillary electrophoresis

Vancomycin is one of a family of related macrocyclic glycopeptide antibiotics that were discovered by scientists at the Eli Lilly Company in the 1950s. It has been used to treat severe staphylococcal infections, particularly when bacterial resistance to other antibiotics has developed. Vancomycin is a naturally occurring chiral compound and has a number of stereogenic centers. Furthermore, it contains a variety of functionalities that are known to be useful for enantioselective interactions (e.g., hydrogen bonding groups, hydrophobic pockets, aromatic groups, amide linkages, etc.). The physicochemical properties of vancomycin, including its stability in solution, are discussed as they pertain to capillary electrophoresis. Over 100 racemates were resolved including many nonsteroidal antiinflammatory drugs, antineoplastic compounds and N-derivatized amino acids. Many of these compounds had very high resolution factors. Optimization and the effect of different experimental parameters on the enantioselective separations are discussed. © 1994 Wiley-Liss, Inc.     

Preparation and enantioseparation characteristics of two chiral stationary phases based on mono(6(A)-azido-6(A)-deoxy)-perphenylcarbamoylated alpha- and gamma-cyclodextrin

Two chiral stationary phases, ph-alpha-CD and ph-gamma-CD, were prepared from mono(6(A)-azido-6(A)-deoxy)perphenylcarbamoylated alpha- and gamma-cyclodextrin immobilized onto silica gel via the Staudinger reaction. The chromatographic characteristics of these two chiral stationary phases were evaluated. The influence of different cyclodextrins (CDs) on the enantioselectivities was also investigated in this study. Compared to ph-gamma-CD, ph-alpha-CD exhibited quite good enantioselectivity toward the analytes with bulky molecular structures. It was found that the formation of inclusion complex might play a quite important role in the chiral recognition not only under reverse phases but also under normal phases.     

Chiral separation of vinpocetine using cyclodextrin-modified micellar electrokinetic chromatography

A cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) technique has been developed for enantioseparation of vinpocetine using an inexpensive 2-hydroxypropyl-β-CD (HP-β-CD) as the chiral selector (CS). The best chiral separation was achieved using 40 mM HP-β-CD as the CS in 50 mM phosphate buffer (pH 7.0) consisting of 40 mM sodium dodecyl sulfate (SDS) at a separation temperature and separation voltage of 25°C and 25 kV, respectively. To the author's best knowledge, this is the first CD-MEKC study able to successfully separate the four stereoisomer of vinpocetine in separation time of 9.5 min and resolution of 1.04-3.87.     

Use of cyclodextrins in capillary zone electrophoresis for the separation of optical isomers: Resolution of racemic tryptophan derivatives

In this study capillary zone electrophoresis has been used for the separation of racemic tryptophan derivatives in their enantiomers. The effect of cyclodextrins with different shape, added to the background electrolyte, on the migration time of 10 compounds, including methyl tryptophan, hydroxy tryptophan, and tryptophan ester derivatives, has been studied. Furthermore, the effect of cyclodextrins with different shape and that of the composition of the background electrolyte on the enantiomer resolution are discussed. Among different cyclodextrins used α-cyclodextrin and heptakis(2,6-di-O-methyl)-β-cyclodextrin were found to possess the best complexing capacity and thus the resolution power toward analysed compounds.     

Comparison between cellulose and amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phases for enantiomeric separation of 17 amidotetralins

Direct enantiomeric separations of 17 chiral amidotetralins by means of high performance liquid chromatography were performed on stationary phases composed of tris(3,5-dimethylphenylcarbamate) derivatives of cellulose and amylose, coated on silica gel. The enantiomers of 15 out of 17 amidotetralins were resolved with a resolution of more than 1.5 by at least one of the chiral stationary phases. The stationary phases showed complementary results with regard to the separation of the amidotetralins, that is, pairs that did not separate on the cellulose-type column were well separated on the amylose-type column, and vice versa. There was no significant correlation between the chromatographic properties of the chiral stationary phases. © 1993 Wiley-Liss, Inc.     

The Enantiomeric Separation of Tetrahydrobenzimidazoles Cyclodextrins‐ and Cyclofructans

High performance liquid chromatography (HPLC) and capillary electrophoresis (CE) were used to examine the enantiomeric separation of a series of 17 racemic tetrahydrobenzimidazole analytes. These compounds were prepared as part of a synthetic program directed towards a select group of pyrrole‐imidazole alkaloids. This group of natural products has a unique framework of pyrrole‐ and guanidine‐containing fused rings which can be constructed through the intermediacy of a tetrahydrobenzimidazole scaffold. Several bonded cyclodextrin‐ (both native and derivatized) and derivatized cyclofructan‐based chiral stationary phases were evaluated for their ability to separate these racemates via HPLC. Similarly, several cyclodextrin derivatives and derivatized cyclofructan were evaluated for their ability to separate this set of chiral compounds via CE. Enantiomeric selectivity was observed for the entire set of racemic compounds using HPLC with resolution values up to 3.0. Among the 12 different CSPs, enantiomeric recognition was most frequently observed with the Cyclobond RN and LARIHC CF6‐P, while the Cyclobond DMP yielded the greatest number of baseline separations. Fifteen of the analytes showed enantiomeric recognition in CE with resolution values as high as 5.0 and hydroxypropyl‐γ‐cyclodextrin was the most effective chiral additive. Chirality 25:133–140, 2013. © 2012 Wiley Periodicals, Inc.     

The stereochemical resolution of the enantiomers of aspartame on an immobilized alpha-chymotrypsin HPLC chiral stationary phase: the effect of mobile-phase composition and enzyme activity

The enantioselective and diastereoselective resolutions of the stereoisomers of N alpha-aspartyl-phenylalanine 1-methyl ester (APME) have been accomplished on an HPLC chiral stationary phase based upon alpha-chymotrypsin (the ACHT-CSP) with observed enantioselectivities (alpha 1) for the DL-/LD-enantiomer of as high as 29.17 and for the DD-/LL-enantiomers of as high as 28.97. In addition, the effect on the chromatographic retention of the APME stereoisomers of the activity of the ACHT and the composition of the mobile phase--structure of the anionic component, molarity, and pH--have been studied. The results of this study suggest that the aspartyl moiety and/or the aspartyl-phenylalanine amide linkage play key roles in the observed enantioselectivity; the APME stereoisomers containing L-phenylalanine, i.e., DL- and LL-APME, bind at a different site in the ACHT molecule (the L-Phe site) than the APME stereoisomers containing D-phenylalanine (the D-Phe site); and the observed enantioselectivity is a measure of the difference in the binding affinities at the two sites rather than the consequence of differential affinities at a single site.     

Optimizing the sample size and the retention parameters to achieve maximum production rates for enantiomers in chiral chromatography

The optimum experimental conditions (sample size and mobile phase composition) are calculated for maximum production rate of either one of two enantiomers contained in feeds of different compositions (1/1, 1/10, and 10/1). The products are obtained at 99% purity. The calculations use the equilibrium-dispersive model of chromatography and the equilibrium isotherms determined experimentally from the rear, diffuse boundary of overloaded elution profiles. The production rate measured experimentally under the optimum conditions calculated agree with 4% of the calculated values. There is an optimum value for the retention factor which is higher than predicted by a model assuming constant separation factor, because both separation factor and retention decrease with increasing organic solvent concentration in the mobile phase. (c) 1992 John Wiley & Sons, Inc.     

Enantioselective separation of enantiomeric amides on three amylose-based chiral stationary phases: Effects of backbone and carbamate side chain chiralities

A series of enantiomeric amides have been chromatographed on three amylose-based chiral stationary phases (CSPs): amylose tris(3,5-dimethylphenylcarbamate) (AD-CSP), amylose tris (S-phenylethylcarbamate) (AS-CSP), and amylose tris(R-phenylethyl-carbamate) (AR-CSP). The relative retentions and enantioselectives of the solutes on the three CSPs were compared and basic structure-retention relationships developed to describe the chromatographic results. The data indicate that for these solutes the observed elution order was a function of the chirality of the amylose backbone, while the magnitude of the enantioselective separations was affected by the chirality of the carbamate side chain. Chirality 9:173–177, 1997. © 1997 Wiley-Liss, Inc.     

Possible uses of micellar electrokinetic capillary chromatography and high-performance liquid chromatography for the chiral discrimination of some pyrethroids

Micellar electrokinetic capillary chromatography (MECC) and high-performance liquid chromatography (HPLC) were used for the separation of stereoisomers of the lipophilic uncharged pyrethroids cypermethrin, alphamethrin, permethrin, and fenpropathrin. Different kinds of cyclodextrin (β-cyclodextrin, hydroxypropyl-β-cyclodextrin, dimethyl-β-cyclodextrin, and γ-cyclodextrin), surfactants (sodium dodecyl sulphate [SDS] and cetyltrimethylammonium bromide [CTAB]), and cations of background electrolyte (sodium, ammonium, TRIS, and Ammediol) were tested. Optimized conditions (background electrolyte: 50 mmol/l sodium phosphate, pH ≈ 2.5, 150 mmol/l SDS, 150 mg/ml γ-cyclodextrin) allowed the separation of alphamethrin, the eight cypermethrin stereoisomers being eluted in seven peaks and the separation of two enantiomers of fenpropathrin with resolution R_s = 10 and with n ≃ 500,000 theoretical plates. Different experimental conditions, e.g., mobile phase composition, temperature, injected amount, and flow rate, were also optimized in HPLC experiments. The optimal conditions (stationary phase: ChiraDex, 5 μm; mobile phase: 150 mmol triethylamine/l with H₂SO₄ in water (pH = 3.5) with methanol or acetonitrile; flow rate: 0.8 or 0.6 ml/min; temperature: ambient or 30°, 20°, or 10°C; experimental conditions were modified according to the type of analysis) allow chiral discrimination of alphamethrin enantiomers and analysis of permethrin stereoisomers. MECC offers higher efficiency and shorter analysis time than HPLC, but under tested conditions it was shown that the methods complement each other. Chirality 9:162–166, 1997. © 1997 Wiley-Liss, Inc.     

HPLC on chiral support with polarimetric detection: application to conglomerate discovery

In conglomerates, each single crystal contains only one of the two possible enantiomeric forms--either dextrorotatory or levorotatory. The analysis of a single crystal by liquid chromatography on chiral support associated with chiroptical detection is a very efficient tool to reveal the occurrence of a conglomerate. In terms of rapidity and easiness, this method compares favorably with the classical methods used to show this occurrence. Two examples are provided.