Recent advances in peptide chiral selectors for electrophoresis and liquid chromatography

The application of peptides in chiral separations using techniques such as capillary electrophoresis (CE), electrokinetic capillary chromatography (EKC) and liquid chromatography is the focus of this review. Methods for finding peptide selectors using combinatorial library approaches are discussed, as well as recent advances in the use of peptides as general chiral selectors for electrophoresis and liquid chromatography. One example shows the effectiveness of polymeric dipeptide surfactants as general chiral selectors for electrophoresis. Another example shows the versatility of oligoproline chiral stationary phases, exhibiting resolution for a number of racemic analytes comparable to other well-established chiral stationary phases.     

Chiral recognition by enantioselective liquid chromatography: Mechanisms and modern chiral stationary phases

An overview of the state-of-the-art in LC enantiomer separation is presented. This tutorial review is mainly focused on mechanisms of chiral recognition and enantiomer distinction of popular chiral selectors and corresponding chiral stationary phases including discussions of thermodynamics, additivity principle of binding increments, site-selective thermodynamics, extrathermodynamic approaches, methods employed for the investigation of dominating intermolecular interactions and complex structures such as spectroscopic methods (IR, NMR), X-ray diffraction and computational methods. Modern chiral stationary phases are discussed with particular focus on those that are commercially available and broadly used. It is attempted to provide the reader with vivid images of molecular recognition mechanisms of selected chiral selector–selectand pairs on basis of solid-state X-ray crystal structures and simulated computer models, respectively. Such snapshot images illustrated in this communication unfortunately cannot account for the molecular dynamics of the real world, but are supposed to be helpful for the understanding. The exploding number of papers about applications of various chiral stationary phases in numerous fields of enantiomer separations is not covered systematically.     

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.     

Chiral recognition: design and preparation of chiral stationary phases using selectors derived from ugi multicomponent condensation reactions and a combinatorial approach

Combinatorial approaches together with high-throughput screening have been used to develop highly selective stationary phases for chiral recognition. Libraries of potential chiral selectors have been prepared by the Ugi multicomponent condensation reactions and screened for their enantioselectivity using the reciprocal approach involving a chiral stationary phase with immobilized model target compound N-(3,5-dinitrobenzoyl)-alpha-l-leucine. The best candidates were identified from the library of phenyl amides of 2-oxo-azetidineacetic acid derivatives. This screening also enabled specification of the functionalities of the selector desired to achieve the highest level of chiral recognition. The substituents of the phenyl ring adjacent to the chiral center of the selector candidates exhibited the most profound effect on the chiral recognition. The best candidate was then synthesized on a larger scale, resolved into single enantiomers using preparative enantioselective HPLC, and attached to porous poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) beads via an ester linkage to afford the desired stationary phase. Selectivities alpha as high as 3.2 were found for the separation of a variety of amino acid derivatives.     

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。     

Enantiomer separation of chiral pharmaceuticals by capillary electrochromatography

Enantiomer separation of chiral pharmaceuticals by capillary electrochromatography (CEC) is achieved with open-tubular capillaries (o-CEC), with packed capillaries (p-CEC) or with monolithic capillaries. In o-CEC, capillaries are coated with a thin film containing cyclodextrin derivatives, cellulose, proteins, poly-terguride or molecularly imprinted polymers as chiral selectors. In p-CEC, typical chiral HPLC stationary phases such as silica-bonded cyclodextrin or cellulose derivatives, proteins, glycoproteins, macrocyclic antibiotics, quinine-derived and 'Pirkle' selectors, polyacrylamides and molecularly imprinted polymers are used as chiral selectors. Chiral monolithic stationary phases prepared by in situ polymerization into the capillary were also developed for electrochromatographic enantiomer separation.     

Recent advances in chiral separation principles in capillary electrophoresis and capillary electrochromatography

This review summarizes recent developments in chiral separation in capillary zone electrophoresis (CZE), electrokinetic chromatography (EKC), and capillary electrochromatography (CEC) covering literature published since the year 2000. New chiral selectors and innovative approaches for CE and CEC are introduced. Recent progress in column technology for CEC is highlighted and the development of new chiral stationary phases is discussed. This review is not dedicated to list applications but will focus on new developments.     

Chiral Separation of Four Piperidinic Benzoxazolinone Compounds by CE and LC

Capillary electrophoresis with negatively charged cyclodextrins and high performance liquid chromatography on polysaccharide chiral stationary phases have been explored for the enantioseparation of chiral piperidinic benzoxazolinone compounds. Operational parameters such as the nature and the concentration of the chiral selectors in CE and the nature of the polysaccharide stationary phase and the composition of the mobile phase in LC were varied in order to achieve the separation of the all four concerning compounds. Limits of detection and quantification of both methods were evaluated under the optimal conditions.     

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.     

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.

     

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.     

Comparative enantioseparations of pharmaceuticals in capillary electrochromatography on polysaccharide-based chiral stationary phases containing selectors with or without chlorinated derivatives

The screening conditions of an existing chiral strategy in CEC were tested for their applicability on four chlorine-containing polysaccharide-based stationary phases. The selectors of these phases are cellulose tris(3-chloro-4-methylphenylcarbamate), amylose tris(5-chloro-2-methylphenylcarbamate), cellulose tris(4-chloro-3-methylphenylcarbamate) and cellulose tris(3,5-dichlorophenylcarbamate). The enantioselectivity of these phases was compared with those of the four phases without chlorine (Chiralpak? AD-RH, Chiralcel? OD-RH, Chiralpak? AS-RH and Chiralcel? OJ-RH) used in the earlier defined strategy. A test set of 48 structurally diverse drug compounds was analyzed using the screening conditions of the strategy. These results led to possibilities to upgrade the current screening strategy so that improved success rates are obtained. The chlorine-containing chiral stationary phases demonstrated an added value to the screening process since they showed enantioresolution for compounds not resolved by the chiral stationary phases not containing chlorine in their structure.     

HPLC separation of amino acid enantiomers and small peptides on macrocyclic antibiotic-based chiral stationary phases: a review

The search for new and effective chiral selectors capable of separating a wide variety of enantiomeric compounds is an ongoing process. In the past decade, macrocyclic antibiotics have proved to be an exceptionally useful class of chiral selectors for the separation of enantiomers of biological and pharmacological importance by means of HPLC, TLC and electrophoresis. More chiral analytes have been resolved through the use of glycopeptides than with all the other macrocyclic antibiotics combined (ansamycins, thiostrepton, aminoglycosides, etc.). The glycopeptides avoparcin, teicoplanin, ristocetin A and vancomycin have been extensively used as chiral selectors in the form of chiral bonded phases in HPLC, and HPLC stationary phases based on these glycopeptides have been commercialized. Teicoplanin, vancomycin, their analogs and ristocetin A seem to be the most useful glycopeptide HPLC bonded phases for the enantioseparation of proteins and unusal native and derivatized amino acids. In fact, the macrocyclic glycopeptides are to some extent complementary to one another: where partial enantioresolution is obtained with one glycopeptide, there is a high probability that baseline or better separation can be obtained with another. This review sets out to characterize the physicochemical properties of these antibiotics and their application in the enantioseparations of amino acids. The mechanism of separation, the sequence of elution of the stereoisomers and the relation to the absolute configuration are also discussed.     

Enantiomeric Resolution of Benzoxazolinonic Aminoalcohols, Potential Adrenergic Ligands, by LC and CE

Benzoxazolinone amino alcohols are chiral derivatives that carry one asymmetric center. Both capillary electrophoresis and high-performance liquid chromatography (LC) techniques were used to separate enantiomers with high resolution and thus permitted the determination of enantiomeric excess. Highly sulfated and anionic cyclodextrins were used as chiral selectors under CE conditions. Amylose and cellulose chiral stationary phases were used for LC for the enantioseparation of five racemic compounds. Separations were carried out on a Chiralpak AS column with various polar organic modifiers. The HPLC method appeared more to be more sensitive and was subsequently validated in terms of limits of detection and quantification. LC detection was based on UV and evaporating light scattering.     

Capillary electrochromatographic chiral separations with potential for pharmaceutical analysis

The use of capillary electrochromatography as a chiral separation technique for pharmaceutical applications is reviewed. Publications of the past 10 years that provide a potential practical application in pharmaceutical analysis are considered. Method development or validation, separation strategies, and potential routine analysis by the methods/applications cited are the main subjects on which we focused our attention. The indirect chiral separation method was only used once in CEC mode. In the direct chiral separations, the use of chiral stationary phases was obviously preferred over the use of chiral mobile phases with non-chiral stationary phases. Amongst the chiral stationary phases, those based on macrocyclic antibiotics and polysaccharide selectors were the most frequently used. Monolithic stationary phases also have several applications, but not so extended as those with packed capillary electrochromatography. The considered papers not only describe the applicability of the technique for relatively large sets of chiral analytes, they also showed that various types of stationary phases can be produced in-house in a simple manner. However, to survive as a mature separation technique, considerable time and effort are still needed to solve some disadvantages currently characterizing capillary electrochromatography.     

Efficient resolution of racemic 1,1'-bi-2-naphthol with chiral selectors identified from a small library

Efficient resolution of racemic 1,1'-bi-2-naphthol, a well-studied analyte in chiral separation, was achieved using selectors developed from a small library. Separation factors (up to 7.2) obtained are significantly higher than the ones reported previously for this analyte. The library consists of 121 members and it does not contain the pi deficient 3,5-dinitrobenzoyl (Dnb) group. These highly efficient stationary phases may lead to the practical large-scale chromatographic resolution of enantiomers of 1,1'-bi-2-naphthol, which are widely used as chiral auxiliaries and ligands in asymmetric synthesis.     

Ionic Liquids as Modifiers of Chromatographic and Electrophoretic Systems

The application of ionic liquids to chromatography and capillary electrophoresis as modifiers of background electrolytes, chiral selectors, and components of mobile, stationary, and pseudostationary phases in the separation of different classes of organic compounds is discussed; the effect of ionic liquids on the efficiency of separation and selectivity is considered.     

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.     

Enantioselective Liquid Chromatographic Separations Using Macrocyclic Glycopeptide-Based Chiral Selectors

Numerous chemical compounds of high practical importance, such as drugs, fertilizers, and food additives are being commercialized as racemic mixtures, although in most cases only one of the isomers possesses the desirable properties. As our understanding of the biological actions of chiral compounds has improved, the investigation of the pharmacological and toxicological properties has become more and more important. Chirality has become a major issue in the pharmaceutical industry; therefore, there is a continuous demand to extend the available analytical methods for enantiomeric separations and enhance their efficiency. Direct liquid chromatography methods based on the application of chiral stationary phases have become a very sophisticated field of enantiomeric separations by now. Hundreds of chiral stationary phases have been commercialized so far. Among these, macrocyclic glycopeptide-based chiral selectors have proved to be an exceptionally useful class of chiral selectors for the separation of enantiomers of biological and pharmacological importance. This review focuses on direct liquid chromatography-based enantiomer separations, applying macrocyclic glycopeptide-based chiral selectors. Special attention is paid to the characterization of the physico-chemical properties of these macrocyclic glycopeptide antibiotics providing detailed information on their applications published recently.