Small Molecules as Chromatographic Tools for HPLC Enantiomeric Resolution: Pirkle-Type Chiral Stationary Phases Evolution

This review focuses on the evolution of Pirkle-type chiral stationary phases (CSPs), based on chiral recognition mechanism of small molecules and applications directly related with Medicinal Chemistry. Therefore, the strategies to plan these chiral selectors for enantioseparation of diverse therapeutic classes of chiral drugs and the understanding of the recognition mechanism are emphasized. The planning of Pirkle and co-workers to design different classes of CSPs was initially based on NMR studies, following the principle of reciprocity together with chromatographic results and studies of chiral recognition phenomena. All those features are described and critically discussed in this review. Finally, based on general principles established by Pirkle’s work it can be inferred that diverse chiral small molecules can be successfully used as chromatographic tools for enantiomeric resolution. In this context, several research groups were inspired on Pirkle’s design to develop new CSPs. Xanthone derivatives bonded to chiral groups were also exploited as selectors for CSPs and are briefly reported.

     

Chiral Stationary Phases Based on Small Molecules: An Update of the Last 17 Years

A literature survey covering the report on Pirkle-type chiral stationary phases (CSPs) from January 2000 to March 2017 is presented in this review. More than 200 CSPs comprising small molecules as chiral selectors covalently bound to the chromatographic support have been reported in this period. The chemical nature of these new chiral selectors, new insights into the development strategies and their applications in liquid chromatography were emphasized.     

Polysaccharide- and β-Cyclodextrin-Based Chiral Selectors for Enantiomer Resolution: Recent Developments and Applications

Polysaccharides, oligosaccharides, and their derivatives, particularly of amylose, cellulose, chitosan, and β-cyclodextrin, are well-known chiral selectors (CSs) of chiral stationary phases (CSPs) in chromatography, because they can separate a wide range of enantiomers. Typically, such CSPs are prepared by physically coating, or chemically immobilizing the polysaccharide and β-cyclodextrin derivatives onto inert silica gel carriers as chromatographic support. Over the past few years, new chiral selectors have been introduced, and progressive methods to prepare CSPs have been exploited. Also, chiral recognition mechanisms, which play a crucial role in the investigation of chiral separations, have been better elucidated. Further insights into the broad functional performance of commercially available chiral column materials and/or the respective newly developed chiral phase materials on enantiomeric separation (ES) have been gained. This review summarizes the recent developments in CSs, CSP preparation, chiral recognition mechanisms, and enantiomeric separation methods, based on polysaccharides and β-cyclodextrins as CSs, with a focus on the years 2019–2020 of this rapidly developing field.     

双手性选择单元手性固定相的研究进展

手性固定相（chiral stationary phase,CSP）作为手性色谱分离的核心技术,在手性化合物的识别和分离中得到广泛应用。以双手性选择单元结合作为CSP是近些年的研究热点,研究表明,两种手性选择单元相结合的CSP可增加手性识别位点,显著提高分离效果。本文介绍了近几年双手性选择单元手性固定相在手性分离中的研究进展,并对其发展前景进行了展望。     

Advances in chiral separations of small peptides by capillary electrophoresis and chromatography

Many chemical and biological processes are controlled by the stereochemistry of small polypeptides (di‐, tri‐, tetra‐, penta‐, hexapeptides, etc). The biological importance of peptide stereoisomers is of great value. Therefore, the chiral resolution of peptides is an important issue in biological and medicinal sciences and drug industries. The chiral resolutions of peptide racemates have been discussed with the use of capillary electrophoresis and chromatographic techniques. The various chiral selectors used were polysaccharides, cyclodextrins, Pirkle types, macrocyclic antibiotics, crown ethers, imprinted polymers, etc. The stereochemistry of dipeptides is also discussed. Besides, efforts are made to explain the chiral recognition mechanisms, which will be helpful in understanding existing and developing new stereoselective analyses. Future perspectives of enantiomeric resolution are also predicted. Finally, the review concludes with the demand of enantiomeric resolution of all naturally occurring and synthetic peptides.     

High-performance liquid chromatography chiral stationary phases based on low-molecular-mass selectors

A review of HPLC chiral stationary phases (CSPs) based on low molecular mass selectors is given. The review is focused on brush- and monomeric-type CSPs obtained by covalent linkage of chiral selectors, with emphasis on those obtained by total synthesis. Emphasis is given to new, emerging aspects like enantioseparations on receptor-like chiral stationary phases and dynamic enantioselective chromatography of stereolabile compounds.     

Recent developments in the HPLC enantiomeric separation using chiral selectors identified by a combinatorial strategy

Two kinds of chiral stationary phases (CSPs) can be distinguished: (i) the "conventional" CSPs for which the selectivity is not pre-determined and (ii) the CSPs which are characterized by a predictable elution order, depending on the target enantiomer used for the selection of the chiral selector. At the present time, three general methodologies have been described to create chiral selectors specifically designed against the racemate to resolve: the molecular imprinting technology, the production of antibodies and the combinatorial approach. The latter methodology involves two categories of procedures to develop CSPs: an approach from a small library of low-molecular weight selectors and an approach from a very large library of single-stranded oligonucleotides (DNA and RNA aptamers). In this review, the recent advances in the HPLC applications of the chiral selectors identified through these two combinatorial procedures are addressed.     

The Evolution of Chiral Stationary Phases for Liquid Chromatography

The development of effective chiral stationary phases (CSPs) and separation strategies for the liquid chromatographic (LC) resolution of enantiomers has been beneficial to many scientific disciplines. Over the last decade the number and type of CSPs has expanded tremendously. Not only have new classes of chiral selectors been introduced, but also many of the first CSPs have been changed and/or improved. The second or third generation of a CSP often can be different from the original. This can be confusing and intimidating to someone just entering the area of LC enantiomeric separations. Fortunately, all CSPs can be categorized in one or another of a few classes. There are generally one or two columns that can accomplish the majority of separations in each class. In this work we look at the different classes of CSPs and how they have expanded and changed over the last decade.     

Effect of the presence of a free hydroxyl group on the enantiodiscrimination properties of cholic acid based CSPs bearing 2-naphthylcarbamate and 3,5-dinitrophenylcarbamate moieties in the HPLC resolution of racemic compounds

Two new chiral selectors, obtained by derivatizing two of the three hydroxyl groups of cholic acid with 2-naphthylisocyanate and 3,5-dinitrophenylisocyanate, have been prepared and linked to silica gel to obtain chiral stationary phases (CSPs) for the HPLC separation of enantiomers. The enantiodiscriminating capability of the two CSPs has been compared to that of the analogous CSP obtained from an exhaustively derivatized cholic acid based selector, in order to establish the effect of the presence of a free hydroxyl group on the enantiodiscrimination properties of this kind of selector. The chromatographic results demonstrate that the enantioselectivity of these selectors strongly depends on the position of the hydroxyl group on the cholestanic backbone.     

Separation of enantiomers by gas chromatography

The separation of enantiomers by gas chromatography is performed on chiral stationary phases (CSPs) via hydrogen bonding, coordination and inclusion. Thus, typical chiral selectors are amino acid derivatives, terpene-derived metal coordination compounds and modified cyclodextrins. In Chirasil-type stationary phases the chiral selector is anchored to a polysiloxane backbone improving gas chromatographic performance. The present review article describes the state-of-the-art, scope and limitations, applications and mechanistic considerations at the advent of the millennium incorporating 16 figures and 168 references.     

外消旋萘普生酯类在(S,S)-Whelk-O1与CDMPC手性柱上的对映体分离及手性识别机理的比较

在Pirkle型的(S,S)-Whelk-O 1与纤维素衍生物型的CDMPC两种手性柱上对六种 外消旋萘普生酯进行了对映体分离，通过研究烷氧基结构上的差异以及流动相中不 同的醇类添加剂对手性识别的影响，探讨和比较了外消旋萘普生酯在两种手性固定 相上手性识别的机理。对于 (S,S)-Whelk-O 1， 溶质与固定相之间的吸引作用于 手性识别的主要因素，而对于CDMPC，溶质在手性空腔中的空间适应性很可能是手 性识别的关键。     

Rational optimization of the Whelk-O1 chiral stationary phase using molecular dynamics simulations

Rational in silico optimization of the Whelk-O1 chiral stationary phase (CSP) has been carried out based on the chiral recognition mechanism extracted from previous molecular dynamics simulations [C.F. Zhao, N.M. Cann, Anal. Chem. 80 (2008) 2426] of this CSP. Three modified CSPs have been examined. The first two are designed to increase selectivity by reducing the docking probability of the less retained analyte. The third modified selector reverses the amide bridge to introduce a structural motif found in the popular carbamate-derivatized polysaccharide CSPs [Y. Okamoto, M. Kawashima, K. Hatada, J. Am. Chem. Soc. 106 (1984) 5357]. For each modified selector, an atomistic model has been obtained through extensive ab initio calculations. The effect of selector modification is then evaluated via simulations of the modified interface in the presence of target analytes. Simulation results show that the separation factors are increased for the modified CSPs but in some cases elution orders are reversed. The Whelk-O1 CSP was originally designed to separate naproxen [W.H. Pirkle, C.J. Welch, B. LAmm, J. Org. Chem. 57 (1992) 3854]. With this in mind, molecular dynamics simulations of naproxen are compared for the original, and the modified, selectors.     

双选择体手性固定相中选择体的构型对分离性能的影响

为研究选择体的构型对双选择体固定相手性识别的影响,以(1S,2S)-(~)-二苯基乙二胺及L-(~)-二苯甲酰酒石酸为手性源,合成了一种新的双选择体固定相,并用不同结构的手性样品测试了其手性分离能力。结果表明,这种固定相与以(1R,2R)-(+)-二苯基乙二胺及L(~)-二苯甲酰酒石酸为手性源制备的双选择体固定相有相当的手性分离能力,但这两种固定相所能分离的化合物不尽相同。对双选择体固定相中两个选择体的构型对固定相手性识别的影响进行了探讨。在手性识别中,以不同手性源制备的两个选择体的立体构型不能同时与一个手性样品的立体构型相匹配,从而导致相应的双选择体固定相手性分离能力的下降。     

Mechanistic Principles in Chiral Separations Using Liquid Chromatography and Capillary Electrophoresis

Due to the importance of chiral separations of drugs, pharmaceuticals, agrochemicals and xenobiotics by high performance liquid chromatography (HPLC) and capillary electrophoresis (CE), it is important to have the knowledge of the enantiomeric recognition mechanisms so that scientists may design and module the new chiral selectors for rapid, inexpensive and reproducible chiral separations; specially at preparative scale. The mechanisms of the chiral separation by HPLC and CE using polysaccharides, cyclodextrins, macrocyclic glycopeptide antibiotics, Pirkle type, ligand exchangers, crown ethers and other several types of chiral selectors have been discussed. Various complex formation and different types of interactions responsible for chiral resolution have been presented in detail.     

Novel strong cation-exchange type chiral stationary phase for the enantiomer separation of chiral amines by high-performance liquid chromatography

The preparation of novel brush-type chiral cation-exchange materials based on de novo designed synthetic low molecular mass selectors (SOs) and their evaluation for enantioselective separation of chiral amines by HPLC are presented. The SO as the functional unit for enantioselectivity contains a beta-aminocyclohexanesulfonic acid moiety and is readily accessible via straightforward synthesis in both enantiomeric forms yielding chiral stationary phases (CSPs) with opposite configurations, CSPs 1 and 2, and reversed elution orders. For the evaluation of these novel CSPs by HPLC a sound set of chiral amines, mainly amino-alcohol type drug molecules, was selected. The chromatographic evaluations were carried out using polar organic mobile phase conditions. All of the analytes could be baseline separated, compared to common CSPs in parts with excellent peak efficiencies (up to 70000 theoretical plates per meter for the second eluted enantiomer). A number of experimental parameters have been varied to look at and prove the underlying ion-exchange process on CSPs 1 and 2, and to reveal suitable conditions for their operation. In this context, the influence of proton activity in the mobile phase and the effects of varying concentration and type of the counterion as well as type of co-ion and of bulk solvent components were thoroughly investigated.     

Evaluation and comparison of a methylated teicoplanin aglycone to teicoplanin aglycone and natural teicoplanin chiral stationary phases

HPLC enantiomeric separations of a wide variety of racemic analytes was evaluated using chiral stationary phases (CSPs) based on the macrocyclic glycopeptides teicoplanin (T), teicoplanin aglycone (TAG), and methylated teicoplanin aglycone (Me-TAG) in two different mobile phase modes, i.e., the RP mode and the polar organic (PO) mode. Comparison of the enantiomeric separations using Chirobiotic T, Chirobiotic TAG, and the methylated form of TAG were conducted in order to gain a better understanding of the roles of the polar functional groups on the CSP. Substantial effects due to the cleavage of saccharides and/or methylation on chiral separations were observed in both separation modes. Improved separation efficiencies for many acidic analytes were obtained by methylating the H-bonding groups of TAG. These groups were believed to be a contributing factor to band broadening on TAG due to their negative effect on mass transfer between the stationary phase and mobile phase. Ionic/dipolar interactions between the carboxylate group of the analytes and the amine groups on T, TAG, or Me-TAG are important for chiral discrimination. Therefore, analytes possessing a carboxyl group are good candidates for successful separations on these CSPs. Hydrophobic interactions are important for enantiomeric separations in the RP mode where the H-bonding interactions between analytes and the chiral selectors are relatively weak. Me-TAG offers higher hydrophobicity, which can accentuate the interactions of analytes with hydrophobic moieties, but these interactions are not necessarily stereoselective. In the PO mobile phase, electrostatic/dipolar interactions between polar functional groups are the dominating interactions in chiral recognition. Another important factor is steric fit, which could be changed with every modification of the T structure. Therefore, substantial changes of enantioseparations were obtained within this studied group of CSPs. The PO mode was shown to be the most powerful mobile phase mode for enantiomeric separations on T-based stationary phases, mainly due to the improved efficiency. Methylation of the TAG proved to be a very useful tool for investigating the chiral recognition mechanism for this group of chiral selectors.     

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.     

Synthesis of a new family of four deoxycholic acid derived chiral stationary phases and their evaluation in the HPLC resolution of racemic compounds

Four new chiral selectors obtained by suitable derivatization of the hydroxyl groups of the deoxycholic acid with two identical (homoderivatized) or different (heteroderivatized) arylisocyanates have been prepared and linked covalently to silica gel to obtain new chiral stationary phases (CSPs) for the HPLC separation of enantiomers. The CSPs containing two identical substituents are able to enantiodiscriminate different classes of racemic compounds, or the same racemates to a different extent, a property which depends on the different electronic character of the arylcarbamate moieties. The heteroderivatized CSPs retain the character of the two homoderivatized phases: however, the relative position of the two different arylcarbamate moieties on the deoxycholic backbone strongly affects the enantiodiscrimination capability of these two CSPs.     

Covalently bonded polysaccharide derivatives as chiral stationary phases in high-performance liquid chromatography

Polysaccharide derivatives have been extensively used as chromatographic chiral selectors in chiral stationary phases (CSPs) for the separation of enantiomers by HPLC. When coated onto a silica matrix, they represent nowadays one of the most popular type of CSPs. However, they are only compatible with a limited choice of solvents. The main drawback of these CSPs is related to the solubility of the chiral selector in a number of solvents, which limits their applicability. The different attempts which have been described up to now to overcome this problem by covalently fixing the chiral selector to a matrix are reviewed in this paper.     

Bile Salts in Chiral Micellar Electrokinetic Chromatography: 2000–2020

Bile salts are naturally occurring chiral surfactants that are able to solubilize hydrophobic compounds. Because of this ability, bile salts were exploited as chiral selectors added to the background solution (BGS) in the chiral micellar electrokinetic chromatography (MEKC) of various small molecules. In this review, we aimed to examine the developments in research on chiral MEKC using bile salts as chiral selectors over the past 20 years. The review begins with a discussion of the aggregation of bile salts in chiral recognition and separation, followed by the use of single bile salts and bile salts with other chiral selectors (i.e., cyclodextrins, proteins and single-stranded DNA aptamers). Advanced techniques such as partial-filling MEKC, stacking and single-drop microextraction were considered. Potential applications to real samples, including enantiomeric impurity analysis, were also discussed.