Use of a naphthylethylcarbamoylated-β-cyclodextrin chiral stationary phase for the separation of drug enantiomers and related compounds by sub- and supercritical fluid chromatography

Enantiomeric separation of a variety of drugs and related compounds was achieved on an (S)-naphthylethylcarbamoylated-β-cyclodextrin (S-NEC-CD) chiral stationary phase (CSP) using sub- and supercritical fluid chromatography (SFC). Compounds previously resolved on native or derivatized cyclodextrin CSPs in liquid chromatography (LC) using reversed phase or polar organic mobile phase modes could be resolved in SFC using a simple carbon dioxide/methanol eluent. Resolution of cromakalim, which is not possible on the S-NEC-CD column in LC, was readily accomplished in SFC. The importance of modifier, temperature, and pressure was assessed in relation to retention, selectivity, and resolution. The nature of the modifier and the modifier concentration were found to be crucial parameters. © 1996 Wiley-Liss, Inc. Contribution of the National Institute of Standards and Technology. Not subject to copyright.     

New chiral stationary phases for liquid chromatography based on small molecules: Development,enantioresolution evaluation and chiral recognition mechanisms

Recently, we reported the development of new chiral stationary phases (CSPs) for liquid chromatography (LC) based on chiral derivatives of xanthones (CDXs). Based on the most promising CDX selectors, 12 new CSPs were successfully prepared starting from suitable functionalized small molecules including xanthone and benzophenone derivatives. The chiral selectors comprising one, two, three, or four chiral moieties were covalently bonded to a chromatographic support and further packed into LC stainless-steel columns (150 × 2.1 mm I.D.). The enantioselective performance of the new CSPs was evaluated by LC using different classes of chiral compounds. Specificity for enantioseparation of some CDXs was observed in the evaluation of the new CSPs. Besides, assessment of chiral recognition mechanisms was performed by computational studies using molecular docking approach, which are in accordance with the chromatographic parameters. X-Ray analysis was used to establish a chiral selector 3D structure.     

Enantioseparation of napropamide by supercritical fluid chromatography: Effects of the chromatographic conditions and separation mechanism

Supercritical fluid chromatography (SFC) is already used for enantioseparation in the pharmaceutical industry, but it is rarely used for the separation of chiral pesticides. Comparing with high performence liquid chromatography, SFC uses much more environmnetal friendly and economic mobile phase, supercritical CO₂. In our work, the enantioseparation of an amide herbicide, napropamide, using three different polysaccharide‐type chiral stationary phases (CSPs) in SFC was investigated. By studying the effect of different CSPs, organic modifiers, temperature, back‐pressure regulator pressures, and flow rates for the enantioseparation of napropamide, we established a rapid and green method for enantioseparation that takes less than 2 minutes: The column was CEL2, the mobile phase was CO₂ with 20% 2‐propanol, and the flow rate was 2.0 mL/min. We found that CEL2 demonstrated the strongest resolution capability. Acetonitrile was favored over alcoholic solvents when the CSP was amylose and 2‐propanol was the best choice when using cellulose. When the concentration of the modifiers or the flow rate was decreased, resolutions and analysis times increased concurrently. The temperature and back‐pressure regulator pressure exhibited only minor influences on the resolution and analysis time of the napropamide enantioseparations with these chiral columns. The molecular docking analysis provided a deeper insight into the interactions between the enantiomers and the CSPs at the atomic level and partly explained the reason for the different elution orders using the different chiral columns.     

Use of a new pirkle-type chiral stationary phase in analytical and preparative subcritical fluid chromatography of pharmaceutical compounds

Good results have been obtained with use of the new bonded chiral stationary phase Whelk-O 1 in analytical and preparative subcritical fluid chromatography. A wide variety of enantiomeric pairs of compounds with different functional groups that are of pharmaceutical and biological interest have been resolved. This Pirkle-concept CSP appears to be more rugged than cellulosic phases (e.g., Chiralcel) with regards to solvents and pressure. In comparing the usefulness of the column for SFC versus HPLC chiral analysis, we have observed a clear superiority of SFC in terms of higher speed and efficiency of analysis, and faster method development. This is consistent with our experience with Chiralcel CSPs. With the Whelk-O 1 we have shown that the effects of temperature and modifier on SFC separations are similar to what has been reported for most other CSPs. We also observed a unique selectivity advantage of SFC for verapamil. We had good success with using a 1-in. diameter column packed with Whelk-O 1 to perform preparative SFC separations of a number of enantiomeric mixtures. The advantages of preparative SFC over preparative HPLC will be discussed. The feasibility of preparative SFC is dependent on how well we meet the practical challenges such as sample introduction issues, special hardware requirements due to the high pressure, and fraction collection issues. © 1994 Wiley-Liss, Inc.     

Liquid Chromatography Enantioseparations of Halogenated Compounds on Polysaccharide‐Based Chiral Stationary Phases: Role of Halogen Substituents in Molecular Recognition

Halogenated chiral molecules have become important in several fields of science, industry, and society as drugs, natural compounds, agrochemicals, environmental pollutants, synthetic products, and chiral supports. Meanwhile, the perception of the halogen moiety in organic compounds and its role in recognition processes changed. Indeed, the recognition of the halogen bond as an intermolecular interaction occurring when the halogen acts as a Lewis acid had a strong impact, particularly in crystal engineering and medicinal chemistry. Due to this renewed interest in the potentialities of chiral organohalogens, here we focus on selected recent applications dealing with enantioseparations of halogenated compounds on polysaccharide‐based chiral stationary phases (CSPs), widely used in liquid chromatography (LC). In particular, recently the first case of halogen bonding‐driven high‐performance LC (HPLC) enantioseparation was reported on a cellulose‐based CSP. Along with enantioseparations performed under conventional HPLC, representative applications using supercritical fluid chromatography (SFC) are reported. Chirality 27:667–684, 2015. © 2015 Wiley Periodicals, Inc.     

Synthesis and Enantioseparation Ability of Xylan Bisphenylcarbamate Derivatives as Chiral Stationary Phases in HPLC

Ten novel xylan bisphenylcarbamate derivatives bearing meta‐ and para‐substituents on their phenyl groups were synthesized and their chiral recognition abilities were evaluated as the chiral stationary phases (CSPs) for high‐performance liquid chromatography (HPLC) after coating them on macroporous silica. The chiral recognition abilities of these CSPs depended on the nature, position, and number of the substituents on the phenyl moieties. The introduction of an electron‐donating group was more attractive than an electron‐withdrawing group to improve the chiral recognition ability of the xylan phenylcarbamate derivatives. Among the CSPs discussed in this study, xylan bis(3,5‐dimethylphenylcarbamate)‐based CSP seems to possess the highest resolving power for many racemates, and the meta‐substituted CSPs showed relatively better chiral recognition than the para‐substituted ones. For some racemates, the xylan bis(3,5‐dimethylphenylcarbamate) derivative exhibited higher enantioselectivity than the CSP based on cellulose tris(3,5‐dimethylphenylcarbamate). Chirality 27:518–522, 2015 © 2015 Wiley Periodicals, Inc.     

Analysis and purification of alcohol-sensitive chiral compounds using 2,2,2-trifluoroethanol as a modifier in supercritical fluid chromatography

2,2,2-Trifluoroethanol (TFE) is evaluated as an alternative modifier for the analysis and purification of alcohol-sensitive chiral compounds using supercritical fluid chromatography (SFC). Four chiral compounds, selected for their sensitivity to alcohols, in addition to a variety of standard chiral compounds were analyzed by SFC using TFE with polysaccharide and Pirkle-type chiral stationary phases (CSPs) to produce selectivities (alpha) and resolutions (Rs) as high as 1.4 and 7.2. A preparative isolation of 2-phenylglutaric anhydride was achieved using TFE as the mobile phase modifier to produce clean enantiomers.     

Enabling chiral separations in discovery chemistry with open‐access chiral supercritical fluid chromatography

Work from this paper details a novel walk‐up open‐access (OA) approach to enable chiral analytical method development and preparative separation of enantiomers in early discovery chemistry using supercritical fluid chromatography (SFC). We have demonstrated the success of this OA approach using immobilized chiral stationary phases (CSPs). After screening a diverse set of racemic drug candidates, we have concluded that a simplified OA chiral SFC platform can successfully purify approximately 60% of the analysed racemates. This streamlined OA workflow enables medicinal chemists with limited expertise in chiral method development to successfully and rapidly purify enantiomers for their projects using Waters UPC² and Prep100‐SFC instrumentation.     

Polar organic phase liquid chromatography with packed capillary columns using a vancomycin chiral stationary phase

Vancomycin immobilized on silica served as the chiral stationary phase (CSP) in this investigation with polar organic solvents as the mobile phase in liquid chromatography (LC). It was shown that trace amounts of water were beneficial for improving peak shape and efficiency. To regulate the retention and selectivity an acid and/or base were added to the mobile phase where an excess of acid was shown to be preferential for enantioseparation. An unusual increase in selectivity with increasing temperature was shown for the acidic drug, thalidomide. Additionally, nonlinear van't Hoff plots were obtained for metoprolol enantiomers that showed increased retention with increasing temperature. Metoprolol also showed unusual behavior in the polar organic phase when water was added to resemble reversed-phase chromatography, with minimum retention observed at high water or high methanol concentrations. In both instances a high degree of electrostatic interaction between metoprolol and vancomycin was concluded. Metoprolol and ten of its analogs were examined on this CSP to evaluate the enantiorecognition process. A comparison in enantioselectivity for a number of acidic and basic drugs using this CSP was also carried out using the polar organic phase, reversed phase, and normal phase LC which were all compared to the results obtained in supercritical fluid chromatography (SFC). Polar organic phase LC offered a better separation of basic molecules while reversed phase LC was preferred for the resolution of acids. SFC showed the broadest enantioselectivity overall and normal phase LC indicated similar properties, as expected, to SFC but with lower column efficiency. Copyright 2000 Wiley-Liss, Inc.     

Evaluation of π-acid chiral stationary phases deriving from tyrosine and related amino acids for the chromatographic resolution of racemates: Specific requirements for enantiorecognition ability

Chromatographic applications of three novel chiral stationary phases (CSPs) deriving from (S)-(N)-(3,5-dinitrobenzoyl)tyrosine are reported, under liquid chromatographic (LC) and subscritical fluid chromatographic (SubFC) conditions. Two grafting modes of the chiral moiety have been experimented starting either from γ-mercaptopropyl-silanized (type 1) or γ-aminopropyl-silanized (type 2) silica gels. For type 2 CSPs an evaluation of the stability of the amide linkage was achieved by means of SubFC; the relative contriution of ionic and covalent bindings to the ciral recognitio aility was then outlined. The chromatographic properties of these CSPs were compared with those of the corresponding CSPs deriving from phenylglycine, p-hydroxyphenylglycine, and phenylalanine for the resolution of some tertiary phosphine oxide, naphthoyl amide, and α-methylene γ-lactam enantiomers. Some simple requirements regarding the solute and CSP structures for chiral recognition ability can be inferred from these results. In addition, the resolutio of π-acid α-N-(3,5-dinitrobenzoyl)amino esters was investigated on these π-acid CSPs. An example of preparative scale chromatography is also presented.     

Reversed-phase liquid chromatographic enantioseparation by cycloalkylcarboxylates of cellulose and amylose

Three novel cycloalkylcarboxylates, cyclopentyl, cyclohexyl, and 1-adamantylcarboxylates of cellulose and amylose were prepared and their chiral recognition abilities as chiral stationary phases (CSPs) for high-performance liquid chromatography (HPLC) were evaluated using a methanol-water mobile phase. Among these esters, cellulose tris(cyclohexylcarboxylate) showed a relatively high chiral recognition ability. The 1-adamantylcarboxylates of cellulose and amylose showed dissimilar chiral recognition abilities from the other two, probably due to the low degree of substitution and the high hydrophobicity of this group.     

Recent progresses in protein-based chiral stationary phases for enantioseparations in liquid chromatography

Chiral stationary phases (CSPs) based on proteins orglycoproteins have been developed for the enantioseparations of various compounds. In 2001, a review article [J. Haginaka, J. Chromatogr. A, 906 (2001) 253] dealing with CSPs based on proteins and glycoproteins was published. After that serum albumin from other species, penicillin G-acylase, antibodies, fatty acid binding protein and streptavidin were newly introduced as the chiral selectors in CSPs. This review article deals with recent progresses in CSPs based on protein or glycoproteins in LC after 2001, focusing on their enantioselective properties and chiral recognition mechanisms.     

Enantioseparation by HPLC using phenylcarbonate, benzoylformate, p-toluenesulfonylcarbamate, and benzoylcarbamates of cellulose and amylose as chiral stationary phases

Phenylcarbonate, benzoylformate, and p-toluenesulfonylcarbamate of cellulose and five new benzoylcarbamate derivatives of both cellulose and amylose were synthesized and their chiral recognition abilities were evaluated as chiral stationary phases (CSPs) for high-performance liquid chromatography (HPLC). Cellulose benzoylcarbamate has a higher chiral recognition ability compared to phenylcarbonate, p-toluenesulfonylcarbamate, and benzoylformate of cellulose. The benzoylcarbamate derivatives exhibited a characteristic chiral recognition for the racemates, which bear a hydrogen atom capable of hydrogen bonding to the carbonyl group of the benzoylcarbamates. The structures of the benzoylcarbamates were investigated by CD spectroscopy.     

Evaluation of a ristocetin bonded stationary phase for subcritical fluid chromatography of enantiomers

A stationary phase derived from ristocetin was evaluated for chiral separation in subcritical fluid chromatography. Separation of various enantiomers having different structures and pK(a) values were investigated using carbon dioxide and polar modifiers. The influence of modifiers, additives, temperature, and mobile phase flow rate on separations is presented. It is concluded that this stationary phase can be used for SFC despite its structural similarity with protein-derived stationary phases that can only be used in HPLC. The separation mechanisms could not be elucidated or predicted using these initial experiments. The separations of warfarin and, especially, efavirenz demonstrate the potential of this type of stationary phase for rapid SFC chiral separations.     

Evaluation of enantioselective gas chromatography for the determination of minute deviations from racemic composition of alpha-amino acids with emphasis on tyrosine: accuracy and precision of the method

Whereas the determination of high enantiomeric fractions (EF) of chiral compounds is very well established, the accurate determination of small deviations from racemic compositions has not yet received much attention despite its relevance to studies dealing with the origin of homochirality, where only small initial enantiomeric bias is expected. Racemic samples of representative alpha-amino acids were derivatized as N-(O,S)-trifluoroacetyl/ethylesters and analyzed by enantioselective gas chromatography (GC) on fused silica capillaries coated with the chiral stationary phases (CSPs) Chirasil-D-Val, Chirasil-L-Val, and Lipodex E with GC/FID and GC/MS detection. The validation (accuracy and precision) of the determination of the enantiomeric fraction EF of the D-enantiomer in racemic or near-racemic compositions for 10 DL-alpha-amino acids obtained from commercial sources has been carried out. Emphasis is given to DL-tyrosine, the enantiomers of which have recently been claimed to show different crystallization properties. Values of EF obtained from GC measurements using CSPs were compared with those from CE using chiral mobile phase additives. While the precision of the GC method is generally better than 0.08% for all DL-alpha-amino acids studied, accuracy (trueness) of determination of amino acids with polar side chains is poorer than expected from the precision as a result of systematic errors. The accuracy determined relied on measurements on two oppositely configurated CSPs.     

Dichloro-, dimethyl-, and chloromethylphenylcarbamate derivatives of cyclodextrins as chiral stationary phases for high-performance liquid chromatography

New dichloro-, dimethyl-, and chloromethylphenylcarbamate derivatives of cyclodextrins (CDs) were prepared and their enantiomeric recognition abilities were evaluated as chiral stationary phases (CSPs) in normal phase high-performance liquid chromatography (HPLC). The effects of the type of cyclodextrins, the nature and position of the substituents on the phenyl ring, binding mode and spacer on the chiral recognition were studied in detail. No marked change of chiral recognition abilities was established by reversing the binding side of CDs (i.e., by the narrower [primary] opening of the cone-shaped CD to silica gel with the wider [secondary] opening sides). This result indirectly proves the previously drawn conclusion about the minor role of inclusion phenomena in chiral recognition in this case. Nevertheless, chiral recognition of these CSPs toward some compounds critically depends on the type of CDs used. All CD derivatives described in this study show rather low enantiomeric resolving abilities compared with corresponding polysaccharide (cellulose and amylose) derivatives, although very high enantioselectivity of separation was observed for a few compounds, such as racemic flavanone and cyclopropanedicarboxilic acid dianilide. © 1996 Wiley-Liss, Inc.     

Supercritical fluid chromatography comparison of the poly(trans-1,2-cyclohexanediyl-bis acrylamide) (P-CAP) column with several derivatized polysaccharide-based stationary phases

The poly(trans-1,2-cyclohexanediyl-bis acrylamide) (P-CAP) column has so far been primarily used with normal phase and polar organic mobile phase chromatography. Its use in supercritical fluid chromatography (SFC) was investigated via the analysis of 40 commercial and 100 proprietary compounds using a 12-min gradient with methanol as a modifier. Results were then compared against those obtained from the popular derivatized polysaccharide-based chiral stationary phases (CSPs) such as Chiralpak AD-H and Chiralpak AS-H as well as Chiralcel OD-H and Chiralcel OJ-H columns. P-CAP demonstrated separation of 25% of the 140 total compounds, while each of the derivatized polysaccharide-based CSPs separated at least 46%. A study that compared the loading of 1,1'-bi-2-naphthol with P-CAP and Chiralpak AS columns indicated a similar trend in resolution vs. amount injected, though AS appeared capable of allowing a greater loading of material. The P-CAP column was found to be beneficial in the separation of a complex mixture of enantiomers and achiral impurities, where the derivatized polysaccharide-based columns did not show as desirable of a separation. A key advantage of this type of chiral stationary phase is the fact that it is available in both enantiomeric forms, allowing manipulation of elution order of enantiomers, which is especially helpful for preparative applications. P-CAP also demonstrated that it could resolve an achiral impurity from the desired compound in a different mixture, while the same impurity co-eluted on the Chiralpak AD-H column. Overall, the synthetic polymer-based P-CAP showed less chiral discrimination power compared to the derivatized polysaccharide-based CSPs under the conditions explored in this study.     

Effects of Hexane in Supercritical Fluid Chromatography for the Separation of Enantiomers

Supercritical fluid chromatography (SFC), operated in conventional mode, is normally recognized as normal phase chromatography, and uses a solvent combination of supercritical CO₂ and alcohols to separate compounds. Hexane, a commonly used solvent in normal phase liquid chromatography (NP‐LC), is rarely used in SFC and, in some cases, is added to the organic modifiers to increase liquid content in order to achieve better efficiency in preparative SFC for poorly retained compounds. Although hexane is believed to have similar solvent strength to that of supercritical CO₂, its effects on the enantioseparation in SFC is largely unknown. To understand the chromatographic effects of an apolar solvent, such as hexane in SFC, we compared the chromatographic behaviors of 35 chiral compounds using a parallel SFC method under traditional SFC mode of only “pure” alcohol‐CO₂ to that of hexane‐assisted SFC (HA‐SFC), which uses mixtures of alcohol and hexane (as cosolvents) and CO₂. We observed that, in some cases, hexane behaves just like supercritical CO₂, where replacement of a portion of CO₂ with hexane does not significantly change retention times or resolution of the peaks. In many cases, however, addition of hexane in mobile phases does affect chromatographic behavior of one or both enantiomers. Such effects might provide opportunities for separation of some enantiomers. Chirality 28:192–198, 2016. © 2016 Wiley Periodicals, Inc.     

HPLC with cellulose Tris (3,5‐DimethylPhenylcarbamate) chiral stationary phase: Influence of coating times and coating amount on chiral discrimination

Coating cellulose tris (3,5‐dimethylphenylcarbamate) (CDMPC) on silica gels with large pores have been demonstrated as an efficient way for the preparation of chiral stationary phase (CSP) for high‐performance liquid chromatography (HPLC). During the process, a number of parameters, including the type of coating solvent, amount of coating, and the method for subsequent solvent removing, have been proved to affect the performance of the resultant CSPs. Coating times and the concentration of coating solution, however, also makes a difference to CSPs' performance by changing the arrangement of cellulose derivatives while remaining the coating amount constant, have much less been studied before, and thereby, were systematically investigated in this work. Results showed that CSPs with more coating times exhibited higher chiral recognition and column efficiency, suggesting that resolution was determined by column efficiency herein. Afterwards, we also investigated the effect of coating amount on the performance of CSPs, and it was shown that the ability of enantio‐recognition did not increase all the time as the coating amount; and four of seven racemates achieved best resolution when the coating amount reached to 18.37%. At the end, the reproducibility of CDMPC‐coated CSPs were further confirmed by two methods, ie, reprepared the CSP‐0.15‐3 and reevaluated the effect of coating times.