Enantioseparation of planar chiral ferrocenes on cellulose-based chiral stationary phases: Benzoate versus carbamate pendant groups

In this study, the enantioseparation of 14 planar chiral ferrocenes containing halogen atoms, and methyl, iodoethynyl, phenyl, and 2-naphthyl groups, as substituents, was explored with a cellulose tris(4-methylbenzoate) (CMB)-based chiral column under multimodal elution conditions. n-Hexane/2-propanol (2-PrOH) 95:5 v/v, pure methanol (MeOH), and MeOH/water 90:10 v/v were used as mobile phases (MPs). With CMB, baseline enantioseparations were achieved for nine analytes with separation factors (α) ranging from 1.24 to 1.77, whereas only three analytes could be enantioseparated with 1.14 ≤ α ≤ 1.51 on a cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC)-based column, used as a reference for comparison, under the same elution conditions. Pendant group–dependent reversal of the enantiomer elution order was observed in several cases by changing CMB to CDMPC. The impact of analyte and chiral stationary phase (CSP) structure, and MP polarity on the enantioseparation, was evaluated. The two cellulose-based CSPs featured by different pendant groups were also compared in terms of thermodynamics. For this purpose, enthalpy (ΔΔH°), entropy (ΔΔS°) and free energy (ΔΔG°) differences, isoenantioselective temperatures (T_iso), and enthalpy/entropy ratios (Q), associated with the enantioseparations, were derived from van ’t Hoff plots by using n-hexane/2-PrOH 95:5 v/v and methanol/water 90:10 v/v as MPs. With the aim to disclose the functions of the different substituents in mechanisms and noncovalent interactions underlying analyte–selector complex formation at molecular level, electrostatic potential (V) analysis and molecular dynamics simulations were used as computational techniques. On this basis, enantioseparations and related mechanisms were investigated by integrating theoretical and experimental data.     

β-环糊精键合手性固定相分离苯并恶嗪类对映体

研究了在反相高效液相色谱模式下,基于点击化学的β-环糊精手性固定相对苯并恶嗪类对映体的手性分离情况。考察了流动相中有机改性剂的类型和比例、缓冲盐的浓度和pH值对分离的影响。考察结果表明: 乙腈作为有机改性剂比甲醇更有利于苯并恶嗪对映体的分离;乙酸三乙胺缓冲盐体积分数从0.1%增大到1.0%时,苯并恶嗪对映体的保留时间和分离度都随之减小,在pH 4.1时苯并恶嗪对映体具有最大分离度。因此确定乙腈和体积分数为0.1%的乙酸三乙胺缓冲盐流动相(pH 4.1)为最佳分离条件。分离机理研究结果表明,固定相和样品之间的包容络合相互作用以及样品和固定相之间的氢键作用,是样品得以分离的基础。本研究为进一步深入研究β-环糊精固定相提供了实验基础,同时也证明了点击化学在手性环糊精固定相制备中具有极大潜力。     

Enantioseparations of polyhalogenated 4,4’-bipyridines on polysaccharide-based chiral stationary phases and molecular dynamics simulations of selector–selectand interactions

2’-(4-Pyridyl)- and 2’-(4-hydroxyphenyl)-TCIBPs (TCIBP = 3,3’,5,5’-tetrachloro-2-iodo-4,4’-bipyridyl) are chiral compounds that showed interesting inhibition activity against transthyretin fibrillation in vitro. We became interested in their enantioseparation since we noticed that the M-stereoisomer is more effective than the P-enantiomer. Based thereon, we recently reported the enantioseparation of 2’-substituted TCIBP derivatives with amylose-based chiral columns. Following this study, herein we describe the comparative enantioseparation of both 2’-(4-pyridyl)- and 2’-(4-hydroxyphenyl)-TCIBPs on four cellulose phenylcarbamate-based chiral columns aiming to explore the effect of the polymer backbone, as well as the nature and position of substituents on the side groups on the enantioseparability of these compounds. In the frame of this project, the impact of subtle variations of analyte and polysaccharide structures, and mobile phase (MP) polarity on retention and selectivity was evaluated. The effect of temperature on retention and selectivity was also considered, and overall thermodynamic parameters associated with the analyte adsorption onto the CSP surface were derived from van ’t Hoff plots. Interesting cases of enantiomer elution order (EEO) reversal were observed. In particular, the EEO was shown to be dependent on polysaccharide backbone, the elution sequence of the two analytes being P-M and M-P on cellulose and amylose tris(3,5-dimethylphenylcarbamate), respectively. In this regard, a theoretical investigation based on molecular dynamics (MD) simulations was performed by using amylose and cellulose tris(3,5-dimethylphenylcarbamate) nonamers as virtual models of the polysaccharide-based selectors. This exploration at the molecular level shed light on the origin of the enantiodiscrimination processes.     

Polysaccharide‐based chiral stationary phases as halogen bond acceptors: A novel strategy for detection of stereoselective σ‐hole bonds in solution

In the last few years, halogen bonds have been exploited in a variety of research areas both in the solid state and in solution. Nevertheless, several factors make formation and detection of halogen bonds in solution challenging. Moreover, to date, few chiral molecules containing electrophilic halogens as recognition sites have been reported. Recently, we described the first series of halogen‐bond‐driven enantioseparations performed on cellulose tris(3,5‐dimethylphenylcarbamate) by high‐performance liquid chromatography. Herein the performances of amylose tris(3,5‐dimethylphenylcarbamate) as halogen bond acceptor were also investigated and compared with respect to cellulose tris(3,5‐dimethylphenylcarbamate). With the aim to explore the effect of polysaccharide backbone on the enantioseparations, the thermodynamic parameters governing the halogen‐dependent enantioseparations on both cellulose and amylose polymers were determined by a study at variable temperature and compared. Molecular dynamics were performed to model the halogen bond in polysaccharide‐analyte complexes. Chiral halogenated 4,4′‐bipyridines were used as test compounds (halogen bond donors). On this basis, a practical method for detection of stereoselective halogen bonds in solution was developed, which is based on the unprecedented use of high‐performance liquid chromatography as technical tool with polysaccharide polymers as molecular probes (halogen bond acceptors). The analytical strategy showed higher sensitivity for the detection of weak halogen bonds.     

Monolithic chiral stationary phases for liquid‐phase enantioseparation techniques

This short overview summarizes the development in the field of enantioselective monolithic chromatographic media and their application for pressure‐driven and electrokinetic separations. The major emphasis is put on the currently existing problems and the author's vision for their solution is provided. Due to the author's personal experience silica‐based monoliths are discussed in more detail although the key developments in the field of organic monolithic materials for separation of enantiomers are also discussed.     

Enantiomeric analysis of simendan on polysaccharide‐based stationary phases by polar organic solvent chromatography

Herein, the enantiomeric separation of simendan by high‐performance liquid chromatography with ultraviolet detection using polysaccharide‐based chiral stationary phases in polar organic mode is described. Three chiral columns (Chiralpak AD‐H, Chiralcel OD‐H, and Chiralpak AS) were screened using pure methanol and acetonitrile without additives under isocratic conditions. A reversed elution order was observed on the Chiralpak AD‐H column when the methanol content in the mobile phase (methanol–acetonitrile mixtures) was above 10%, whereby levosimendan eluted prior to dextrosimendan. Further, it was found that increasing temperature effectively improved the enantioresolution on the Chiralpak AD‐H column. Van't Hoff analysis was performed to evaluate the contribution of enthalpy and entropy to the chiral discrimination process. The best enantioseparation (α = 3.00, Rs = 12.85) was obtained on the Chiralpak AD‐H column with methanol as the mobile phase at 40°C. Thus, a quantitative method for the resolution of dextrosimendan was established and validated, which could be used as a reference for the determination of dextrosimendan in levosimendan products.     

Recent development trends for chiral stationary phases based on chitosan derivatives,cyclofructan derivatives and chiral porous materials in high performance liquid chromatography

The separation of enantiomers by chromatographic methods, such as gas chromatography, high‐performance liquid chromatography and capillary electrochromatography, has become an increasingly significant challenge over the past few decades due to the demand of pharmaceutical, agrochemical, and food analysis. Among these chromatographic resolution methods, high‐performance liquid chromatography based on chiral stationary phases has become the most popular and effective method used for the analytical and preparative separation of optically active compounds. This review mainly focuses on the recent development trends for novel chiral stationary phases based on chitosan derivatives, cyclofructan derivatives, and chiral porous materials that include metal‐organic frameworks and covalent organic frameworks in high‐performance liquid chromatography. The enantioseparation performance and chiral recognition mechanisms of these newly developed chiral selectors toward enantiomers are discussed in detail.     

手性固定相AD、AS和OD的拆分性能

手性固定相-高效液相色谱法在手性药物、手性农药等的分离分析中应用广泛。本文采用3种多糖衍生物的手性固定相(即EnantioPak AD、AS和OD)对20种手性化合物开展手性分离研究,进而探讨样品分子结构、多糖骨架和衍生基团对手性分离的影响。结果表明,除化合物13外,其余化合物在EnantioPak AD上均实现基线分离,分离度多在2.0以上,在正己烷-醇流动相中加入酸碱添加剂可改善和优化酸性或碱性化合物的分离效果;芳香醇(化合物13~16)随着侧链碳数增加在色谱柱上的保留减弱,其分离度呈现增加的趋势;对比8种化合物在3种手性固定相上的分离结果可知,EnantioPak AD表现出更优的分离性能。这为深入研究和了解多糖手性固定相、拓展其手性分离应用提供了参考。     

Enantioselective separation of twelve pairs of enantiomers on polysaccharide‐based chiral stationary phases and thermodynamic analysis of separation mechanism

The enantioseparation of twelve pairs of structurally related 1‐aryl‐1‐indanone derivatives was studied in the normal‐phase mode using three different polysaccharide‐type chiral stationary phases, namely Chiralpak IB, Chiralpak IC, and Chiralpak ID. n‐Hexane/2‐propanol and n‐hexane/ethanol were employed as mobile phases. Among all the investigated chiral columns, Chiralpak IC exhibited the most universal and the best enantioseparation ability toward all the racemates, particularly with the mobile phase composed of n‐hexane/2‐propanol (90/10, v/v). Then the effects of column temperature on retention and enantioselectivity were examined in the range of 25–40°C. Satisfactory enantioseparation was obtained at ambient temperature. The natural logarithm of retention and separation factors (ln k and ln α) versus the reciprocal of absolute temperature (1/T) (Van't Hoff plots) were found to be linear for all racemates, indicating that the retention and separation mechanisms were independent of temperature in the range investigated. Then, the thermodynamic parameters (ΔΔH°, ΔΔS°, and ΔΔG°) were calculated from Van't Hoff plots. These values indicated that the solute transfer from the mobile to stationary phase was enthalpically favorable, and the process of enantioseparation was mainly enthalpy controlled. At last, the impact of small changes in molecular structures of the tested 1‐indanone derivatives on enantioseparation was also discussed.     

HPLC separation of dihydropyridine derivatives enantiomers with emphasis on elution order using polysaccharide‐based chiral columns

The separation of enantiomers of five chiral dihydropyridine derivatives was studied on five different polysaccharide‐based chiral HPLC columns with various normal‐phase (NP), polar organic, and reversed‐phase eluents. Along with the successful separation of analyte enantiomers, the emphasis of this study was on enantiomer elution order (EEO) with various columns and mobile phase composition. The interesting phenomenon of reversal of EEO, recently reported in the case of amlodipine (AML) depending on the concentration of formic acid in acetonitrile, was also confirmed with NP eluents. Under RP conditions at relatively low water content, the EEO of AML could also be reverted by varying the concentration of formic acid in the mobile phase. However, at higher water content the same parameter did not affect the EEO, but only induced gradual decrease in resolution up to complete co‐elution of enantiomers. Additionally, in organic‐aqueous mobile phases retention factors decreased with increasing water content but only up to 20% (v/v), while above this concentration the expected typical RP behavior was observed. The presence of the commonly used additive diethylamine in the mobile phase seems important for observing a reversal in EEO with increasing concentration of formic acid. The reversal of the EEO was characteristic of AML only and was not observed for any of other dihydropyridines included in this study.     

Novel chiral stationary phases based on peptoid combining a quinine/quinidine moiety through a C9‐position carbamate group

By connecting a quinine or quinidine moiety to the peptoid chain through the C9‐position carbamate group, we synthesized two new chiral selectors. After immobilizing them onto 3‐mercaptopropyl‐modified silica gel, two novel chiral stationary phases were prepared. With neutral, acid, and basic chiral compounds as analytes, we evaluated these two stationary phases and compared their chromatographic performance with chiral columns based on quinine tert‐butyl carbamate and the previous peptoid. From the resolution of neutral and basic analytes under normal‐phase mode, it was found that the new stationary phases exhibited much better enantioselectivity than the quinine tert‐butyl carbamate column; the peptoid moiety played an important role in enantiorecognition, which controlled the elution orders of enantiomers; the assisting role of the cinchona alkaloid moieties was observed in some separations. Under acid polar organic phase mode, it was proved that cinchona alkaloid moieties introduced excellent enantiorecognitions for chiral acid compounds; in some separations, the peptoid moiety affected enantioseparations as well. Overall, chiral moieties with specific enantioselectivity were demonstrated to improve the performance of peptoid chiral stationary phase efficiently.     

Chiral separation of lenalidomide by liquid chromatography on polysaccharide‐type stationary phases and by capillary electrophoresis using cyclodextrin selectors

Complementary techniques were applied for the investigation of the chiral recognition and enantiomeric resolution of lenalidomide using various cyclodextrins and polysaccharides as chiral selectors. The high‐performance liquid chromatography enantioseparation of the anticancer drug was achieved using polysaccharide‐type chiral stationary phases in polar organic mode. Elution order and absolute configuration were elucidated by combined circular dichroism spectroscopy and time‐dependent density functional theory calculations after the isolation of pure enantiomers. Chiral selector dependent and mobile‐phase dependent reversal of the enantiomer elution order was observed, and the nonracemic nature of the lenalidomide sample was also demonstrated. Eight anionic cyclodextrins were screened for their ability to discriminate between the uncharged enantiomers by using capillary electrophoresis. Only two derivatives presented chiral interactions, these cases being interpreted in terms of apparent stability constants and complex mobilities. The best results were delivered by sulfobutylether‐β‐cyclodextrin, where quasi‐equal stability constants were recorded and the enantiodiscrimination process was mainly driven by different mobilities of the transient diastereomeric complexes. The optimized high‐performance liquid chromatography (Chiralcel OJ column, pure ethanol with 0.6 mL/min flow rate, 40°C) and capillary electrophoresis methods (30 mM sulfobutylether‐β‐cyclodextrin, 30 mM phosphate pH 6.5, 12 kV applied voltage, 10°C) were validated for the determination of 0.1% (R)‐lenalidomide as a chiral impurity, which could be important if a racemic switch is achieved.     

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.     

Evaluation of differences between Chiralpak IA and Chiralpak AD‐RH amylose‐based chiral stationary phases in reversed‐phase high‐performance liquid chromatography

Polysaccharide‐based chiral stationary phases can be used for the enantioselective separation of a wide range of structurally different compounds. These phases are available with chiral selectors coated or immobilized on silica gel support. The means of attachment of the chiral selector to the carrier can influence the separation performance of these stationary phases. This paper deals with evaluation of differences in the separation abilities of coated Chiralpak AD‐RH versus immobilized Chiralpak IA amylose‐based stationary phases in the reversed–phase mode of high–performance liquid chromatography. A set of chiral analytes was separated under acidic and basic conditions. Differences were observed in the enantioseparation potential of the tested phases. The linear‐free energy relationship and additional evaluation of ionic interactions were used to ascertain whether the interactions that participate in retention and enantioseparation are affected by the means of preparation of these phases. All the interactions covered by the linear‐free energy relationship were significant for the studied phases and their absolute values were almost always higher for the coated phase. Ionic interactions were found to be more important on the immobilized stationary phase but did not contribute to any improvement in the enantioselective separation performance.     

Liquid chromatographic enantioseparation of limonene‐based carbocyclic β‐amino acids on zwitterionic Cinchona alkaloid‐based chiral stationary phases

The eight stereoisomers of limonene‐based carbocyclic β‐amino acids containing three chiral centers have been directly separated on chiral stationary phases containing Cinchona alkaloid‐based zwitterionic selectors. The effects of bulk solvent composition of the mobile phase, the nature of base additives, counterion concentration, and the structure of selector on the enantiorecognition were studied. Experiments were performed at constant mobile phase composition in the temperature range 5–40°C to study the effect of temperature. Thermodynamic parameters were calculated on the basis of the plots of ln α versus 1/T curves. The enthalpically or entropically driven enantioseparations were found to depend strongly on the structures of analyte and selector. The eight stereoisomers of limonene‐based carbocyclic β‐amino acids could be differentiated as well‐separated peaks in a traditional 1D chromatographic system in two runs by applying the two complementary ZWIX(+)™ and ZWIX(–)™ columns.     

Enantiomers separation by capillary electrochromatography using polysaccharide‐based stationary phases

The separation of chiral compounds is an interesting and important topic of research because these compounds are involved in some biological processes, fundamentally in human health. Among the various application fields where enantiomers are remarkable, drug analysis has to be considered. Most of the drugs contain enantiomers and very often one of the two isomers could be pharmacologically more active or even dangerous. Therefore, the separation of these compounds is very important. Among the different analytical techniques usually employed, capillary electrochromatography has demonstrated great capability in enantiomers resolution. The great potential of this electromigration technique stands mainly in its high efficiency due to the use of an electrosmotic flow (flat flow profile) and on the high selectivity because of the use of a stationary phase. Chiral separation can be obtained utilizing several chiral stationary phases including a polysaccharide derivative. The aim of this review paper is to summarize the main features of capillary electrochromatography and polysaccharide derivatives of chiral stationary phase. It also report examples of practical applications utilizing this approach.     

Preparation and evaluation of novel chiral stationary phases covalently bound with chiral pseudo-18-crown-6 ethers

Novel chiral stationary phases consisting of silica gel covalently bound with chiral pseudo-18-crown-6 type hosts, which possess either an OH or OMe group as a binding functionality, were prepared for enantiomer-separation of lipophilic amines.     

Evaluation of chlorine containing cellulose-based chiral stationary phases for the LC enantioseparation of basic pharmaceuticals using polar non-aqueous mobile phases

The discrimination ability of three cellulose-based chiral stationary phases (CSPs) was evaluated towards the enantiomers of basic drugs, using ACN as the main solvent in polar organic mobile phases. The study was focused on CSPs containing cellulose tris(3-chloro-4-methylphenylcarbamate) (3-Cl-4-MePC), cellulose tris(4-chloro-3-methylphenylcarbamate) (4-Cl-3-MePC) or cellulose tris(3,5-dichlorophenylcarbamate) (3,5-diClPC) as the chiral selector. The behaviour of these CSPs was studied systematically in order to investigate the influence of the presence and position of the chlorine substituents on the phenylcarbamate moieties on the retention and resolution of the enantiomers. The evaluation was made with three different generic mobile phases, namely ACN/0.1%DEA/0.1% TFA (DEA, diethylamine), ACN/0.1%DEA/0.2% FA and ACN/0.1%DEA/0.2%AcA, deduced from the previous study. The nature of the acidic additive and of the chiral selector was found to be particularly important for the retention and enantioresolution of these basic compounds. High-resolution values could be obtained for most studied enantiomers with these CSPs, clearly demonstrating the interest of using them in combination with polar organic mobile phases. However, significant differences in enantioresolution between the CSPs have been observed for many compounds, indicating that these phases seem to be quite complementary.     

Enantioseparation characteristics of the chiral stationary phases based on natural and regenerated chitins

Natural and regenerated chitins were derivatized with 3,5‐dimethyphenyl isocyanate. The corresponding chiral stationary phases were prepared by coating the resulting chitin derivatives on 3‐aminopropyl silica gel. The swelling capacity of the chitin derivatives, enantioseparation capability, as well as eluents tolerance of the chiral stationary phases were evaluated. The results demonstrated no remarkable difference in enantioseparation capability between natural and regenerated chitins based chiral stationary phases. The similar enantioseparation characteristics of two chiral stationary phases could be understood by comparing the IR spectra of related chitin derivatives. The one of the two chiral stationary phases prepared by coating the chitin derivative with a lower molecular weight generally provided better enantioseparations. All chiral stationary phases can work in 100% chloroform, 100% ethyl acetate, 100% acetone, and the mobile phases containing a certain amount of tetrahydrofuran. The chiral stationary phase prepared from the chitin derivative with the highest swelling capacity exhibited better enantioseparations than others. This chiral stationary phase was damaged by flushing with 100% tetrahydrofuran, however, the enantioseparation capability was recovered again after the column was allowed to stand for 1 month. Furthermore, the recovered chiral stationary phase provided better enantioseparations for some chiral analytes than before.     

Chiral stationary phases based on cinchona alkaloids for the HPLC resolution of racemates

Summary The employment of three chiral stationary phases (CSPs) obtained by derivatizing γ-mercaptopropyl-silanized silica gel with quinine, quinidine and N-methyl-quinium iodide, for the separation of organic racemates, is presented. They are quite useful in the resolution of alkylarylcarbinols, binaphthyl derivatives, amides and other substances of pharmaceutical interest.