Mechanistic Principles in Chiral Separations Using Liquid Chromatography and Capillary Electrophoresis

2006, 63, 295307

I. Ali1, K. Kumerer2, H. Y. Aboul-Enein3,&

1 2 3

National Institute of Hydrology, Roorkee 247 667, India Institute of Environmental Medicine and Hospital Epidemiology, University Hospital Freiburg, Hugstetter Strasse 55, 79106, Freiburg, Germany Pharmaceutical and Medicinal Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki, Cairo 12311, Egypt; E-Mail: hyaboulenein@yahoo.com

Received: 4 January 2006 / Accepted: 20 February 2006 Online publication: 10 April 2006

Abstract
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.

Keywords
Liquid chromatography Capillary electrophoresis Chiral separation Mechanisms in CE

Introduction
In a non-chiral environment, the enantiomers of a racemate possess the same physico-chemical properties but into a chiral situation they behave as entirely dierent molecules leading to dierent distribution rates, metabolism, excretion, toxicity and pharmacodynamic activity. In early 1930s, Easson and Stedman introduced a three points attachment model which requires a minimum of three simultaneous interactions between the chiral stationary phase (CSP) and the analyte; which laid the basis for the initial understanding of stereochemical dier-

ences in pharmacological activity [1]. However, Mesecar and Koshland [2] urged the importance of four points attachment model for the chiral discrimination on protein CSPs. It is well known that one of the enantiomers of racemic drug may be ballast or, sometimes, toxic leading to various serious side aects on the health [311]. In spite of these facts, the bioactive synthetic compounds; most of the chiral drugs; are administered as racemates [12], which is a matter of great serious concern from the health point of view. In 1993 Witte et al. [13] and in 1994 Rauws and Groen [14] reviewed the regulatory aspects on the chiral medicinal

products with pharmaceutical industries in USA, Japan and some other European countries. In these countries, distinct authorities take the responsibility for the controlling and approving the newly developed drugs (both chiral and nonchiral). In Unites States, the Food and Drug Administration (FDA) is the institution which controls the new drugs applications. Many pharmaceutical industries started to market the pure enantiomer of some drugs [710, 15]. Most important drugs of whose optically active pure form is demanding are anti-inammatory, analgesics, antiviral, anticancer, cardiovascular, dermatological, gastrointestinal, ophthalmic, respiratory diseases and other pharmaceuticals used for the central nervous system [16]. The debate of racemic versus enantiomers resulted in a new market strategy, so called racemic switch. The economic interest is very important in the development of the drugs. Therefore, the separation methods are more economical than the asymmetric synthetic methods and, hence, most of the pharmaceutical companies are preparing single enantiomers by separation methods. Therefore, the development of rapid, reproducible and inexpensive resolution methods is still a demanding area. It is worthy to mention here that the development of more ecient methods requires the detailed knowledge of the existing separation technologies specially the mechanisms of the chiral separations. Therefore, it is very important to discus the mechanisms of the chiral separation

Review DOI: 10.1365/s10337-006-0762-5 0009-5893/06/04

Chromatographia 2006, 63, April (No. 7/8) 2006 Friedr. Vieweg & Sohn/GWV Fachverlage GmbH

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Fig. 1. Graphical representation of the mechanisms of the chiral separation

methods so that scientists can apply these principles to improve the chiral resolution methods in future.

Chiral Separation Technologies

Various methodologies have been used for the resolution of the enantiomers at analytical and preparative scales which can be categorized into two classes i.e. the classical approach using enzymatic degradation of one of the enantiomers and preferential crystallization and the modern technologies including spectroscopic, electrophoretic and chromatographic methods. In the enzymatic method, one enantiomer is destroyed during resolution

process while the crystallization is carried out by diastereomeric salts formation followed by their manual separation. These classical methods could not achieve the status of routine laboratory practice due to the degradation of one of the enantiomers in enzymatic method and the limitations of crystallization method. Moreover, these methods can not be used at analytical scale. Nowadays, chromatographic, electrophoretic, spectroscopic, biosensors and membranes techniques are most common in this concern. Among these technologies chromatographic methods have been used frequently as they are rapid, eective, reproducible and preparative in nature. Mostly high performance liquid chromatography (HPLC) has been used for the chiral separation but reports are also available using other modalities of liquid chromatography such as sub- and supercritical uid chromatography, capillary electro-chromatography, micellar electrokinetic chromatography and thin layer chromatography. Recently, capillary electrophoresis (CE), a versatile technique of high speed, high sensitivity and lower limit of detection, is a major trend in analytical science and numerous publications have increased exponentially in recent years of its application in the eld of chiral separation [17]. But it is necessary to mention here that capillary electrophoresis could not achieve a status of routine analysis into the chiral separations due to poor reproducibility. Moreover, the limited application of CE is due to the lack of the development of the modern chiral stationary phases. However, capillary electrophoresis is under its development stage. In chromatographic methods, two approaches are used i.e. indirect and the direct. The indirect chromatographic separation of racemic mixtures can be achieved by diastereomeric salt formation (with chiral reagent) followed their separation by using achiral chromatographic modalities. On the other hand, the direct chromatographic approach involves the use of the chiral selectors either in mobile phase called mobile phase additives (CMPAs) or on stationary phase known as chiral stationary phases (CSPs). In the later case, the chiral selector is chemically bonded or coated or allowed to adsorbed on some suitable solid support. The use of chiral selectors as CMPAs is limited due to the high running cost of the experiment since the chiral selector is Chromatographia 2006, 63, April (No. 7/8)

wasted and could not be recovered from the mobile phase. Moreover, this approach is not successful at preparative separation of the enantiomers. Contrarily, CSPs have achieved a great reputation in the chiral separation of enantiomers by chromatography and, today, are the tools of the choice of almost all analytical, biochemical, pharmaceutical and pharmacological laboratories. Most important and useful CSPs are available in the form of open and tubular columns. However, some chiral capillaries and thin layer plates are also available for use in capillary electrophoresis and thin layer chromatography respectively. The chiral columns and capillaries are packed with several chiral selectors such as polysaccharides, cyclodextrins, macrocyclic antibiotics, Pirkle type, ligand exchangers, crown ethers among other types. Briey, the chiral selectors are necessary for the enantiomeric resolution since these chiral selectors interact and bind with enantiomers at dierent magnitudes, which result into their resolution. To understand the chiral resolution phenomena and design of a new experiment, the discussion of the mechanisms of the chiral resolution using dierent chiral selectors is essential into the present scenario. In view of these points the possible mechanisms of the enantiomeric resolution using dierent chiral selectors have been presented herein.

Mechanisms in the Chiral Separations

As discussed above chiral selectors are used to resolve mixtures of racemic compounds using chromatographic and capillary electrophoretic methods. Various chiral selectors have dierent structures and capabilities to interact with enantiomers stereochemically which resulted into the resolution of antipodes. The chiral selectors have special type of structures such as grooves, baskets, cavities etc. in which enantiomers get trapped stereospecically. The trapping of enantiomers is stabilized by various interactive forces. Among the most important interactive forces are hydrogen bonding, coordination bonding, electrostatic force of attraction, p-p interactions, van der Waal forces, steric aects, dipole induced dipole attraction, dispersive forces. The combination of these forces is Review

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entirely dierent for each chiral selectors. The specic chiral recognition mechanisms on dierent chiral selectors are explained in the forthcoming sections of this review. However, a general schematic representation of the chiral resolution mechanisms is shown in Fig. 1.

Polysaccharide Based Chiral Phases

Polysaccharides are naturally occurring polymers having the capabilities of chiral separations because of their asymmetrical structures (grooves). Most commonly used polymers are cellulose, amylose, chitosan, xylan, curdlan, dextran and inulin, which have been used as native or after their derivatization. The chiral recognition mechanisms at a molecular level on the polysaccharide based CSPs is still unclear because of the diculties in the spectroscopic studies of these selectors. Even though some experimental eorts were made to solve this riddle. Hesse and Hagel [18] and later on Francotte et al. [19] proposed an inclusion mechanism by which enantiomers may be adsorbed in the chiral grooves of the cellulose triacetate I (CTA-I) matrix. Other theoretical [20] and X-ray studies of the model compound, fully acetylated D-glucopyronose-(R)-phenylethyl amine inclusion complex [21] also support the inclusion mechanism. It has been established that the main chiral sites of bonding are the polar carbonyl groups of esters, which can interact with racemic compounds through hydrogen bonding and dipoledipole interactions for chiral discrimination [22]. Wainer et al. [23] proposed a similar mechanism based on the separation properties of a series of aromatic amides [22] and alcohols [24] on cellulose tribenzoate (CTB) phase; the mechanism of retention is an attractive binding-steric t formulation involving hydrogen bonding and dipole-dipole interactions. Furthermore, Wainer et al. [24] explained the mechanism by the formation of complexes between enantiomers and CSP through a hydrogen bonding and steric t which are stabilized by insertion of the aromatic portion of the solute into a chiral groove of the CSP. Most important adsorbing sites on phenylcarbamate derivatives are probably the polar carbamate groups which interact with racemic compound via hydrogen bonding through NH- and Review
Fig. 3. Optimized structure (left) and possible interaction sites (right) of a cellulose triphenylcarbamate derivative [26]

Fig. 2. Schematic interactions between polycarbamate residue and racemates [25]

>C=O groups and the dipole-dipole interaction on >C=O (Fig. 2) [25]. Therefore, the nature of the substituents on phenyl ring aects the polarity of phenyl groups, which resulted into dierent chiral recognition capacities. Yashima [26] carried out molecular dynamics studies and predicted the optimized structure and interaction sites of cellulose triphenylcarbamate, which showed the stable structure (Fig. 3). X-ray analysis of cellulose tris(phenylcarbamate) (CTPC) (Fig. 4) was also carried out showing a left handed three fold (3/2) helix with glucose units arranged in helical fashion [27]. A chiral helical groove with polar carbamate residues exists along the main chain. The polar carbamate groups are favorably located inside so that polar enantiomers may be inserted into the grooves and interact with the carbamate residues via hydrogen bonding. This interaction results into an ecient chiral discrimination. Lipkowitz et al. [28,29] carried out computational studies on the chiral discrimination mechanism using cellulose triphenylcarbamate and have extensively studied the mechanisms of chiral recognition from a theoretical viewpoint. Yashima et al. [30] has concluded that NH- and >C=O groups are most important bonding sites. Recently, the same group extended their work [31] and compared the chiral recognition between cellulose tris (phenylcarbamate) (CTPC) and cellulose tris (3,5-dimethylphenylcarbamate) (CDMPC) using trans-stilbene oxide and benzoin as the racemates. The calculations of interaction energies between cellulose tris (phenylcarbamate) (CTPC) or cellulose tris (3,5-dimethylphenylcarbamate) (CDMPC) and transstilbene oxide or benzoin were performed Chromatographia 2006, 63, April (No. 7/8)

Fig. 4. The structure of cellulose tris(phenylcarbamate) developed by X-ray studies [27]

by various methods using force elds. The calculations were divided into two methods i.e. (1) enantiomers were generated and tumbled around the NH- proton and the >C=O oxygen of the carbamoyl group of CTPC and CDMPC which are considered to be most important adsorption sites, and then the interaction energy was calculated and (2) enantiomers were randomly generated by the Monte Carlo method on the surface of CTPC and CDMPC dened by the particular van der Waal radius using the reported technique of blowing up the atomic radii [32] and then the interaction energy was calculated. The results, for the chiral resolution of trans-stilbene oxide or benzoin, obtained from both methods were in good agreement with the results obtained by chromatographic studies for both CTPC and CDMPC CSPs. However, signicant dierences of interaction energies between enantiomers appeared only in case where enantiomers were generated inside of CTPC or CDMPC. These results indicate that the polar carbamate residues of cellulose derivatives may be most important adsorbing site for polar racemates and may play a crucial role in the chiral

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Fig. 5. Method for calculating the interactions between cellulose tris(phenylcarbamate) or cellulose tris(3,5-dimethylphenylcarbamate) derivatives and enantiomers of trans-stilbene or benzoin [31]

Aboul-Enein and Ali [35] have observed that coordination bonding also plays an important role for the chiral resolution of the racemates having sulpher atom. Finally, a look on the structures of polysaccharides derivatives (cellulose and amylose) (Fig. 6) clearly shows the presence of chiral groove/grooves on these CSPs. The electronegative atoms such as oxygen, nitrogen and halogens of racemates form hydrogen bonding and dipole-dipole induced interactions with polysaccharides based CSP. Besides, p-p interactions also occur between the phenyl rings of aromatic racemates and the CSP. During the chiral resolution the enantiomers stereogenically t in dierent fashions into the chiral grooves of the CSP which is stabilized by various types of bonding (as discussed above) of dierent magnitudes and, hence, the resolution of enantiomers takes place. In addition to these bonding, steric eect also governs the chiral resolution on polysaccharide CSPs. Besides, some other achiral weak bonding like Van der Waal forces and ionic bonding may also contribute in the chiral resolution.

Cyclodextrin Based Chiral Phases

Cyclodextrins are cyclic oligosaccharides comprising six to twelve D-(+)-glucopyranose units in a-(1,4) linkage with chair conformation. There are three types having dierent number of glucopyranose units i.e. a-, b- and c-CDs containing six, seven and eight glucopyranose units respectively. The primary C-6 hydroxyl groups, free to rotate, can partially block CD cavity from one end. The mouth of the opposite end of the CD cavity is enriched due to the presence of C-2 and C-3 secondary hydroxyl groups. This arrangement of CD favors the complex formation with a variety of racemates. The presence of stereospecic glucopyronose units, restricts the conformational freedom and orientation of the secondary hydroxyl groups which are supposed to be responsible for the chiral recognition capacities of CDs [36]. Therefore, dierent diastereoisomeric inclusion complexes are formed by the enantiomers which was supported by Armstrong et al. [37] by reporting the computer projections of inclusion complexes of dextro- and levorotatorypropranolol with b-CD (Fig. 7). This fact Review

Fig. 6. Three dimensional structures of amylose and cellulose polymers

recognition. The method of energy calculation is shown in Fig. 5 [31]. These methods are useful for a qualitative understanding of the chiral recognition mechanisms of cellulose phenylcarbamate, although the use of molecular dynamics calculations will be needed to simulate the dynamic behavior of the interactions occurring in chromatography. Recently, Aboul-Enein and Ali [33] have calculated the energies of interaction of nebivolol enantiomers on polysaccharide phases under normal and reversed modes. The authors have observed reversal order of elution of nebivolol on normal phase using dierent alcohols as the mobile phases. Furthermore, these authors also studied the eect of mobile phase on the conformations of these phases and suggested that dierent conformations were responsible for the chiral

resolution of enantiomers. Furthermore, these authors [34] carried out the chiral resolution of methylphenidate on derivatives of cellulose and amylose. They observed that p-p interactions are also important binding forces for the chiral resolution of aromatic racemates. The best resolution of methylphenidate (MPH) on Chiralcel OB column was achieved when phenol or benzoic acid, separately, were used as mobile phase additives. Phenol or benzoic acid form MPH-phenol or MPH-benzoic acid pairs in which the possibility of p-p interaction between these pairs and CSP is greater than the possibility of p-p interactions between MPH and CSP alone. Therefore, an improved resolution of MPH enantiomers occurred, when using phenol or benzoic acid as the mobile phase additives due to an enhancement of p-p interactions. In another study, Chromatographia 2006, 63, April (No. 7/8)

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has been proven by various data obtained from crystallography, NMR, mass spectrometric studies [3845]. Basically, in the chiral recognition process, the enantiomers enter into the cavity of the CD molecules and the diastereoisomeric complexes are formed which are called inclusion complexes. Therefore, even a slight variation in the chromatographic conditions, such as change into mobile phase, derivatization of CDs aects the chiral resolution greatly. Because of this, many research groups, synthesized different derivatives of CDs and utilized them for the chiral resolution of dierent racemates. Therefore, the selection of CD phase is very important for the stable complex formation with a specic racemic compound. Lipkowitz and Stoehr [40] carried out NMR study of methyl mandelate on bCD. The authors conducted molecular dynamics simulations and predicted the guest (molecule)-host (CD) interactions. It was observed that the short range dispersion forces rather than long range coulombic forces were responsible for both complexation and enantio-discrimination. To explain the chiral resolution on CDs, several models and approaches of guest-host formation were proposed and discussed [43, 4648]. The stabilities of the inclusion complexes are controlled by a number of interactions between enantiomers and CSPs. Most important interactions are hydrogen bonding, dipole induced dipole interactions and p-p forces [4951]. Besides, steric aect also plays a crucial role into the chiral resolution as the complexation is governed by the presence of several groups the on enantiomers [52, 53]. However, some other forces such as van der Waal, ionic interactions, solvation aect are also responsible for the chiral resolution. These dierent types of interactions occurred between the hydroxyl groups of CD and various groups of the enantiomers. These interactions may be enhanced by introducing electronegative or phenyl groups in CDs (by derivatization) and, hence, CD derivatives are better CSPs than the CSPs obtained from native CDs. It has also been reported that the magnitudes of these interactions varied in normal, polar organic and reversed phase modes and, hence, the chiral recognition mechanisms dier slightly in all the three mobile phase modes. However, the exact mechanism of chiral resolution on these phases is still obscure. The schematic Review

Fig. 8. Schematic representation of solute inclusion into cyclodextrin cavity Fig. 7. Computer projected structures of the diastereoisomeric inclusion complexes of (a) dextro-propranolol and (b) levo-propranolol with b-CD; dashed lines are hydrogen bondings [37]

representation of the chiral resolution on these phases is shown in Fig. 8.

Macrocylic Glycopeptide Antibiotics Based Chiral Phases

Macrocyclic antibiotics are one of the newest and perhaps most varied class of chiral selectors [54]. The concept of utilizing macrocyclic glycopeptide, as chiral stationary phase, for HPLC was rst introduced in 1994 [55]. Since then their use for chiral resolution by liquid chromatography is increasing exponentially [56, 57]. Antibiotics used for the chiral resolution are vancomycin, vancomycin aglycon, teicoplanin, teicoplanin aglycon, ristocetin A, thiostrepton, rifamycin, fradiomycin, streptomycin, kanamycin and avoparcin. However, vancomycin, teicoplanin and ristocetin A are supposed to be the best chiral selectors due to the presence of an aglycon (fused macrocyclic rings) portion which can exhibit dierent morphological characteristics such as the openness of the aglycon cavity and the degree of helical twist. The twist degree does not seem to depend on the molecular size. In fact vancomycin, which possesses the smallest macrocyclic ring, has the highest twist degree. Their relatively small size, stability in 0100% organic modier, high sample capacity and the fact that their structures are known, basic studies on chiral recognition can be done easily and exactly. They are often complimentary in the type of compounds they can separate. The selectivities of antibiotics in normal, reversed and new polar organic mobile phase modes make them ideally suitable chiral selectors. The macrocyclic antibiotics stationary phases have similarities and dierences with both the cyclodextrin and protein phases from the mechanistic point of view. All antibiotics contain ionisable groups and their Chromatographia 2006, 63, April (No. 7/8)

charges and possibly their conformations may vary with pH of the mobile phase. Three dimensional complex structures and dierent spatial stereochemical arrangements of the functional groups of these antibiotics; containing dierent chiral centers, inclusion cavities, phenyl rings, pyranose, furanose, rings, several hydrogen donor and acceptor sites, sugar moieties, and other groups; are responsible for their surprising chiral selectivities in dierent mobile phases. This allows for an excellent potential to resolve a greater variety of racemates. The normal functioning of these interactions are well known [7, 55, 58] and their discussion in detail is out of the scope of this review. However, these interactions take place individually or in combinations with one another that can result into very high chiral recognition capacities for these antibiotics. The strength of these interactions depends upon the type of mobile phases used. The reversed phase condition favors the ionic interactions, hydrophobic inclusion, hydrogen bonding and steric interaction. The normal phase favors p-p complexation, hydrogen bonding, dipole stacking and steric interaction. On the other hand, the new polar organic phase mode enhances hydrogen bonding, dipole stacking, p-p interaction and steric interaction. Recently, Lee and Beesley [59] presented the dierent interactions such as p-p, hydrogen bonding, dipole-dipole and steric repulsion, between racemates and the glycopeptide antibiotics CSPs (vancomycin, teicoplanin and ristocetin A) under aqueous versus organic mobile phases. The electronegative atoms such as oxygen, nitrogen and halogens of racemates form hydrogen bonding and dipole-dipole induced interactions with the groups of antibiotics. Besides, p-p interactions also occur between phenyl ring of aromatic racemates and the CSP. In addition to these bonding, steric aect also governs the chiral resolution on antibiotic CSPs. Besides, some other weak bonding like van der Waal forces and ionic bonding may also contribute in the chiral resolution. During the chiral resolution, the enantiomers t stereogen-

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Fig. 10. The structural features of the binding of pheniramine molecule on AGP protein with protonated aliphatic nitrogen guiding the drug towards the anionic region into the binding site. Hydrophobic moieties provide anchoring to drug into the lipophilic region(s) of the site [65] Fig. 9. Graphical representation of the chiral recognition mechanism of phenylalanine on teicoplanin CSP [60]

ically in the dierent fashions into the chiral baskets of the CSP which is stabilized by various types of bonding (as discussed above) of dierent magnitudes resulting in the resolution of enantiomers. A graphical representation of chiral recognition mechanisms of phenylalanine amino acid on teicoplanin CSP is presented in Fig. 9 [60]. It may be observed from this Figure that D-enantiomer of phenylalanine is more strongly bonded to CSP (because of lower steric hindrance) than L-enantiomer and, hence, L-enantiomer eluted rst followed by D-enantiomer.

Protein Based Chiral Phases

The proteins are complex in structure and enantiospecic in nature with dierent functional groups, alkyl chains, bridges and chiral loops. Therefore, the chiral recognition by proteins is a complex process. Most commonly used proteins for chiral resolution are bovine serum albumin (BSA), human serum albumin (HSA), rat serum albumin (RSA), guinea pig serum albumin (GPSA), glycoproteins such as a1-acid glycoprotein (AGP), ovomucoid (OVM), ovotransferin, avidin, trypsin (CT), chymotrypsin, riboavin, lysozyme, pepsin, amyloglucosidase, lactoglobulin, cellobiohydrase-I (CBH-I) etc. Recently, Hage [61] and Haginaks [62] reviewed the applications of protein phases in liquid chromatography with the discussion of certain studies carried out on chiral mechanisms. Generally, proteins have specic chiral sites for a pair of enantiomers with a few exception where the two antipodes bind to the dierent

sites [63]. Most common interactions involved into chiral recognition on protein CSPs are non-polar, dipole or coulombic interactions, hydrogen bonding and steric aects [61, 64]. Of course, the protein loops provide the chiral environment to the enantiomers; if the enantiomers bind at the same site. If the enantiomers bind at dierent sites the contribution of chiral loops may or may not be involved into chiral recognition process. Ultimately, the combination of the above cited forces resulted into enantiospecic binding of enantiomers and the chiral resolution is achieved. An important structural features with interaction forces; identied by a qualitative structure relationship; for the binding of basic drug (pheniramine) to AGP protein is shown in Fig. 10 [65]. As discussed earlier, two binding sites of HSA i.e. warfarin (I) and indole (II) are responsible for the chiral resolution of a variety of racemates. Besides, other binding sites on this protein have also been identied [62]. Recently, He and Carter [66] established three dimensional structure of HSA which showed that I and II are the binding sites and are located in hydrophobic cavities. It has also been reported that the chemistry of binding site I is homologous to that of II. Yang and Hage [6769] indicated that D- and L- tryptophan bind to single but distinct sites of HSA while R- and S- enantiomers of ibuprofen and warfarin had one common binding site. Peyrin et al. [70, 71] described II as the binding site for dansyl amino acids on HSA. Kremer et al. [72] observed the hydrophobic pockets as the binding sites on AGP protein. However, more than one binding sites were reported. Haupt et al. [73] presented a retention model for the chiral resolution of uncharged solutes, felodipine, on AGP and the model has assumed the presence of two Chromatographia 2006, 63, April (No. 7/8)

dierent stereoselective sites for dierent enantiomers. In another study, Waters et al. [74] carried out certain thermodynamic experiments for the elucidation of chiral separation mechanisms on AGP protein. The authors reported two different binding sites for two enantiomers. Sometimes, sialic acid residues are supposed to take part in the chiral resolution of enantiomers [7577]. In order to gain information on the chiral recognition mechanisms on ovomucoid and ovoglycoproteins, various studies were carried out [7884]. It has been observed that all three domains present on the protein took part into chiral resolution individually or in dierent combinations. It has also been concluded that sialic acid or sialic acid galactose groups, sometimes, play a role into the chiral recognition process on the reported proteins. Oda et al. [85] carried out certain studies on modied avidin protein and they have reported that avidin has multiple chiral recognition sites. Recently, Felix and Descorps [86] observed the enantioselectivity of a-chymotrypsin protein and the authors reported that enantioresolution was related to structures of enantiomers, hydrophobicity and electrostatic interactions. These relationships determine the recognition mechanisms and the position of each enantiomers onto the enzymatically active sites [87]. Cellobiohydrolase-I protein was found to have the binding site on both portions [88]. Recently, three dimensional structure of CBH-I was determined by x-ray studies [89,90] and it was found that the binding site was about long tunnel. Similarly, specic sites, 40A responsible for chiral resolution on other proteins, were determined and discussed by Haginaka in his review [62]. In spite of various studies on the chiral recognition mechanisms on protein CSPs, the exact mechanism is not known due to the lack of tertiary structure of proteins. Besides, the change in protein conformation in dierent solvents and environment may be another hindrance in the determination of the chiral recognition mechanisms.

Crown Ethers Based Chiral Phases

The crown ethers are synthetic macrocyclic polyethers with the appearance of the crown. The appearance of their molecular Review

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structures and ability to crown selectively with the cations give them crown ethers as their names. Most important chiral crown ethers used for chiral resolution are binaphthyl, biphenanthryl, hericene derivatives, tartaric acid derivatives, carbohydrate moiety, chiral carbon atom with a bulky group directly incorporated in crown ring, aromatic bicyclo derivatives, nonane derivatives, hexahydrochrysese or tetrahydroindenoinden and 9,9-spiro-biuorene groups. Only racemic compounds having primary amino groups can be resolved on these CSPs. Hyun reported no resolution for proline in which primary amino group is not free [91]. It means that primary amino group is essential for the chiral resolution and it plays an essential role in the chiral resolution and in the formation of guest-host complex. Recently, Steeck et al. [92] resolved secondary amines on these chiral CSPs, which conrmed that racemates containing secondary amino groups can also be separated. Pedersen [93] calculated the values of distribution constants and free energies of the enantiomers of some amino acids on a variety of chiral crown ethers (CCEs). The author reported dierent values of these parameters for two enantiomers. Furthermore, Cram et al. [9497] determined the stability of guest-host complex formation and postulated the importance of hydrogen, p-p and steric forces in the guest-host complex formation. Pocsfalvi et al. [98] described the guest-host complex formation of the enantiomers of two ammonium cations on CCE and observed dierent stabilities of the complexes formed between two enantiomers and CCE. The authors also described hydrogen, p-p and steric interactions as the responsible forces for the complex formation. Recently, Machida et al. [99] studied the guest-host complex formation by X-ray analysis. They have reported the presence of above cited forces between the chiral selectors and the analytes. It has also been indicated that the presence of carboxylic groups on analytes enhanced hydrogen bonding, which resulted into an improved chiral resolution [100]. Besides, greater chiral resolution has been observed when the functional and alkyl groups were located at the chiral centre of the analyte. Therefore, CCEs involve a simple chiral recognition mechanism. The two enantiomers t stereogenically at dierent extents into the chiral cavity of CCE which are stabilized by the various Review

interactions (as cited above) at dierent magnitudes and, hence, the chiral resolution occurs. The primary amino group of analyte ionizes in the presence of acid and forms ammonium ion (-NH3+). The ammonium ions form strong hydrogen bonding with the oxygen atoms of CCE. Therefore, the presence of an acid in the mobile phase is essential to achieve the chiral resolution on these CSPs. But, recently, Aboul-Enein et al. [101] observed very interesting results for the chiral resolution of thyroxine and tocainide racemates on (+)-(18-crown-6)-2, 3, 11, 12-tetracarboxylic acid CCE. The authors reported the chiral resolution of these molecules without using acid into the mobile phase. Moreover, they have also achieved the chiral resolution of thyroxine using triethylamine (a base) as the organic modier. Therefore, acid is not essential for the chiral resolution of all the racemates containing primary amino groups. Furthermore, it may be concluded that the acid is not required for the chiral resolution of the molecules who have high hydrogen bonding capacity through primary amino group while it is required for those molecules having low ability of hydrogen bonding. Therefore, acid is used to convert primary amino group into ammonium ion as the later (-NH3+) has stronger hydrogen ability than the former (-NH2). Therefore, the concept of essentiality of acid in the mobile phase is not always true. However, the presence of primary or secondary amino groups is an essential feature for the chiral resolution on CCEs based CSPs. To make this concept clear, the structure of guest-host complex is shown in Fig. 11.

Fig. 11. Graphical representation of the guesthost diastereoisomeric complex formation (a) into the presence of an acid into the mobile phase and (b) into the absence of an acid into the mobile phase. (c): Three dimensional structures of the guest-host diastereoisomeric complexes formed between R- and S- enantiomers of a-phenylethylamine and chiral 18crown-6-ether CSP

Pirkle Type Based Chiral Phases

Pirkle type CSPs are molecules attached to the silica gel containing p electron donor or p electrons receptor or both types of groups. These are three types i.e. p-acidic (with p electrons acceptor groups), p-basic (with p electrons donor groups) and p acidic-basic (with p electrons acceptor and donor groups). The main advantage of these phases is to have the choice of the chiral molecule to be attached to the silica gel. Pirkle type CSPs contain a chiral moiety having phenyl ring and, therefore, the formation of p-p charge transfer diastereoisomeric Chromatographia 2006, 63, April (No. 7/8)

complex of the enantiomers (with phenyl group) with CSP is supposed to be essential. In view of this, p-acidic CSPs are suitable for the chiral resolution of p donor solutes and vice versa. However, the newly developed CSPs containing both p-acidic and p-basic groups are suitable for the chiral resolution of both types of solutes i.e. p-donor and p acceptor analytes. Dierent studies were carried out to predict the chiral recognition mechanisms on these CSPs. Most important methodologies include the use of chromatography [102106], X-ray [107], NMR [102, 108, 109], molecular modeling [102, 107, 110] and computer aided chemistry [102, 111113]. Initially, Pirkle et al. [103, 105] obtained the chiral resolution of a series of homologous compounds on dierent CSPs. The authors advocated two antinomic mechanisms involving either hydrogen bonding or dipole stacking phenomenon. In another study, Pirkle and Da ppen [114], starting from the reciprocality concept, established the presence of hydrogen bonding and dipole stacking mechanisms. Wainer et al. [115] observed the reverse order of elution of some amides by changing the dipole group and the authors reported the existence of hydrogen bonding and dipole stacking processes. Vinkovic et al. [105] studied the chiral resolution of 2arylpropionic acid enantiomers on different CSPs. The authors reported that hydrogen bonding and pp interaction forces are responsible for the chiral resolution. Even chromatographic studies provide adequate evidence for the chiral

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Fig. 12. The chiral recognition model showing three simultaneous bondings between (S)methyl-N-(2-naphthyl)alaninato and (S)-N(3,5-dinitrobenzoyl)leucine n-propylamine [109]

Fig. 13. Graphical representation of the chiral recognition of naproxen enantiomers on (S,S)Whelk-O1 CSP [116]

recognition mechanisms, but are also limited as they give an overall view of the mechanisms. Altomare et al. [107] carried out X-ray and circular dichroism measurements of 3-phenyl-4-(1-adamantyl)-5-Xphenyl-D2-1,2,4-oxadiazolines on N,N(3,5-dinitrobenzoyl)-1(R),2(R)-diaminocyclohexane CSP. The authors claimed the chiral recognition due to p-basicity and hydrophobicity of the analytes. Pirkle and Pochapsky [109] studied the chiral binding of 3,5-dinitrobenzoyl leucine propylamide on (S)-methyl(N)-(2naphthyl)alaninate CSP by NMR. The chiral recognition model of this study is shown in Fig. 12, which indicates the presence of hydrogen bonding and p-p interactions. Similarly, Fukushima et al. [108] postulated the presence of p-p interactions and hydrogen bonding in the diastereoisomeric complexes of the enantiomers and CSP. Besides, computational aided chemistry and molecular modeling calculations were carried out and applied to the chromatographic experiments. The combination of these calculations and the results obtained from the experimental methodologies (X-ray, NMR, Chromatography) indicated that p-p interactions, hydrogen bonding, steric aects and dipole-dipole forces are responsible for the chiral resolution on Pirkle type CSPs. Briey, Pirkle type CSPs contain chiral moieties, which provide the chiral environment to the enantiomers. Therefore, enantiomers interact with the chiral moieties with dierent ttings (due to dierent spacial conguration of the enantiomers). In this way, two enantiomers form diastereoisomeric complexes

having dierent binding energies. The dierent binding energies of the diastereoisomeric complexes are due to various interactions, as mentioned above, of different magnitude. Therefore, two enantiomers eluted at dierent retention times with the ow of the mobile phase and, hence, the chiral resolution occurs. A general graphical representation of the chiral resolution of naproxan enantiomers on (S,S)-Whelk-O1 CSP is shown in Fig. 13 [116].

Ligand Exchange Based Chiral Phases

In chiral ligand exchange chromatography, the separation occurs due to the exchange of the ligands and enantiomers on a metal ion. The ligand exchange involves the breaking and formation of coordinate bonds among metal ion, the ligands and the enantiomers. Metal ions may be grafted onto CSP or dissolved into mobile phase. Copper has been used frequently as the liganding metal ion but other metal ions such as nickel and zinc have also been tested. Therefore, the ligand exchange CSPs are useful for the chiral resolution of the molecules containing electrons donating atoms such as oxygen, nitrogen and sulphur. These types of molecules include amino acids, their derivatives, amines, hydroxy acids, peptides and other drugs. The two enantiomers have dierent exchange capacities because of the stereospecic nature of ligand exchange process and, hence, chiral resolution takes place. Davankov et al. [117119] suggested a theoretical model for the mechanisms of the chiral resolution on Chromatographia 2006, 63, April (No. 7/8)

these CSPs. In this model, the enantiomers were coordinated to Cu(II) ions into the dierent ways depending on their interactions with the ligands bonded to the stationary phase acting as chiral selectors. The authors explained that the chiral resolution is due to dierent bonding along with the steric eects. In this way, diastereoisomeric complexes are formed by the two enantiomers which are stabilized at dierent magnitudes by dipole-dipole interactions, hydrogen bonding, van der Waal forces and steric eects. The diastereoisomeric complexes of phenylalanine enantiomers and Cu(II)L-proline are shown in Fig. 14a [120]. It is clear from this gure that the diastereoisomeric complexes having D-enantiomer is less stable, due the presence of steric forces, and, hence, eluted rst followed by L-enantiomer. To make the concept more clear, an example of the work carried out by Aboul-Enein and Ali [121] is presented herein. The authors reported the chiral resolution of econazole, miconazole and sulconazole (antifungal agents) on Chiralpak WH column. The expected structures of the diastereoisomeric complexes of these antifungal agents with the CSP are shown in Fig. 14b [121]. All the three antifungal agents contain electrons donating atoms such as nitrogen, oxygen and sulphur. The Chiralpak WH CSP is a L-proline-Cu(II) complex attached to silica gel. The copper is a transition metal ion and contains an empty d orbitals which are essential for coordinate bonding. Therefore, nitrogen, oxygen and sulphur atoms of antifungal agents are coordinated with copper atom and, hence, the ternary complexes of L-proline-Cu(II)-enantiomers are formed. The enantiomeric resolution of these antifungal agents may be explained on the basis of ligand exchange mechanisms through coordination bonding. Besides, some other interactions such as hydrogen bonding, dipole induced dipole interactions, steric eect and van der Waal forces also participate in the chiral recognition phenomenon. Therefore, (+) and ()) enantiomers of the reported antifungal agents formed diastereoisomeric complexes of dierent stabilities with WH CSP in stereo-specic way which resulted into the complete resolution of the enantiomers. Fig. 14b [121] shows that, for (+) isomers of antifungal agents, the substituents on the chiral carbon of antifungal Review

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agents are located far from the pyrrolidine group of L-proline while the substituent on the chiral carbon of ()) enantiomers are located near the pyrrolidine group of L-proline. Therefore, Lproline-Cu(II)-())-enantiomer complexes feel some steric force while the L-prolineCu(II)-(+)-enantiomer complexes do not face any steric eect and, hence, the Lproline-Cu(II)-())-enantiomer complexes are less stable than L-proline-Cu(II)-(+)enantiomer complexes. Because of this steric eect, ())-enantiomers of all three antifungal agents eluted rst followed by the (+)-enantiomers. It is also interesting to observe that the coordination capacity of nitrogen, oxygen and sulphur is in the order of S>N>O. Therefore, it may be concluded that the stability of the L-proline-Cu(II)-enantiomers of sulconazole is greater than the complexes of the enantiomers of econazole and miconazole. This fact supports better resolution of sulconazole as compared to the resolution of econazole and miconazole enantiomers.

Miscellaneous Chiral Phases

Besides the above cited CSPs, some other molecules have been used as the chiral selectors for the resolution of variety of racemates. These molecules includes re alkaloids, amides, amines, acids and synthetic polymers. The miscellaneous types of CSPs have dierent types of structures containing various groups and atoms. There are only few reports dealing with the determination of chiral recognition mechanisms on these CSPs. However, Allenmark et al. [122] attempted to explain the chiral recognition mechanisms of the separation of amino alcohols, profens, b-blockers, benzodiazepinones and benzothiadiazines on CSP based on N,Ndiallyl-(R,R)-tartaric acid diamide. The authors reported the involvement of various types of interactions such as hydrogen bonding, dipole-dipole stacking and charge transfer complexes between the CSP and the racemic compounds. In another study, Franco et al. [123] synthesized nine new quinine carbamate dimers and immobilized onto silica gel. The chiral recognition mechanisms were ascertained on these phases using FT-IR and X-ray analysis and it was observed that hydrogen bonding and p-p interactions are responsible for the chiral resolution. Therefore, the chiral recognition Review

Fig. 14. Graphical representation of the chiral recognition of (a) phenylalanine and (b) antifungal agents on the chiral ligand-exchange CSPs [120, 121]

on these CSPs depends on the structures of CSPs and racemic compounds. The chiral resolutions on these CSPs are controlled by dierent types of forces and interactions such as hydrogen bonding, pp interactions, formation of charge transfer and inclusion complexes, dipole interactions, coordination bonding and steric forces. Besides, the weak interactive forces such as van der Waals and dispersion also play crucial role for the chiral resolution on miscellaneous types CSPs. Briey, the presence of the chiral moiety on these CSPs is essential as they provide the chiral environment to the racemic compounds. The racemic compounds t onto the chiral moiety in dierent fashion and are stabilized, with dierent values of binding energies, through the above cited forces. As a result of the ow of the mobile phase the two enantiomers elute at dierent retention times and, hence, the chiral resolution occur. Chromatographia 2006, 63, April (No. 7/8)

Imprinted Polymers Based Chiral Phases

Of course, above cited CSPs are very eective in chiral resolution but the limited predictability of elution orders and separation capacities; making screening of stationary phase libraries a necessary step in the method development; are the serious drawbacks. Polymers imprinted with chiral templates promises to alleviate these drawbacks and oering a new generation of tailor made CSPs with predictable selectivities [124126]. Molecularly imprinted polymers (MIPs) can be prepared by a number of approaches that are dierent in the way the template is linked to the functional monomer and subsequently to the polymeric binding sites. First example of molecular imprinting of organic network polymers was introduced by Wulf et al. [126], which was based on a covalent

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attachment strategy i.e. covalent monomer template and covalent polymer template. Most commonly used imprinted polymers are based on vinylic, acrylic and methacrylic monomers. The important applications of these CSPs are the chiral resolution of amino acids, amino acid derivatives, amines, carboxylic acids, peptides, b-blockers, profens, alkaloids and other pharmaceuticals [125]. The chiral recognition selectivity on these polymers is aected greatly by the composition of mobile phase. Sellergren [125] reviewed the retention mechanisms on these polymers under organic and aqueous mobile phases. The interactions of racemates with imprinted polymers have been reported through hydrogen bonding [127], ion exchange processes [128], hydrophobic interactions [129, 130] etc. Recently, Huang et al. [131] described the chiral recognition mechanisms of enantiomers of amino acid derivatives and diastereomers of cinchona alkaloids using chiral molecularly imprinted monolithic stationary phase. The authors advocated the presence of hydrophobic interactions, ionic and hydrogen bonding interactions to be responsible for the recognition mechanisms. The detailed studies on the chiral recognition mechanisms on these CSPs have not been fully investigated yet but it seems that other forces such as steric, van der Waals may also play crucial role into the chiral separation processes.

depends on the type and nature of the CMPAs and racemates. In case of cyclodextrins the inclusion complexes are formed while in case of other CMPAs simple chiral diastereoismeric complexes are formed. Again as in case of CSPs the diastereoismeric complexes formation is controlled by a number of interactions such as p-p complexation, hydrogen bonding, dipole-dipole interactions, ionic bindings, and steric eects. La mmerhofer and Lindner [132] explained the chiral resolution of N-derivatized amino acids by capillary electrochromatography. The authors explained the formation of the transient diastereomeric ion-pairs between negatively charged enantiomers and positively charged chiral selector by multiple intermolecular interactions, which might be dierentially adsorbed to the ODS-stationary phase. Furthermore, they claimed that the enantio-separation was achieved due to dierent mobilities of the enantiomers originating from different ion-pair formation rates of the enantiomers and/or dierential adsorption of the diastereoisomeric ion-pairs to the ODS-stationary phase [132].

Mechanisms in Capillary Electrophoresis

As in case of liquid chromatography the chiral environment is essential for the enantiomeric resolution in capillary electrophoresis too. In CE chiral situation is provided by the chiral compound used in background electrolyte (BGE) and is called as chiral selector or chiral BGE additive. Various chiral selectors have been used in CE such as polysaccharides, cyclodextrins, proteins, Pirkle types, alkaloids, macrocyclic antibiotics and crown ethers, [133135]. However, the best chiral selectors in CE are CDs followed by macrocyclic antibiotics, proteins and polysaccharides. Basically, chiral recognition mechanisms in CE are similar to those in liquid chromatography using chiral mobile phase additive mode except that the resolution occurs through dierent migration velocities of the diastereoisomeric complexes in CE. Chiral resolution takes place due to the formation of diastereomeric complexes between the enantiomers and the chiral selector. The formation of diastereomeric complexes depends on the type and nature of the chiral selectors used and enantiomers. Gu bitz and Schmidt [134] reviewed the Chromatographia 2006, 63, April (No. 7/8)

Chiral Mobile Phase additives

It is well known fact that the chiral environment is essential in liquid chromatography for the chiral resolution. The chiral situation is provided by using chiral selector into mobile phase, which is called as mobile phase additive (CMPA). Basically, the chiral recognition mechanisms on this sort of liquid chromatography are similar to those of on CSPs. The dierences between the chiral recognition mechanisms between CMPAs and CSPs lies in the fact that the diastereoismeric complexes of the racemic compounds are formed into the mobile phase in case of CMPAs while these complexes are formed on stationary phase in case of CSPs mode. These diastereoisomeric complexes having dierent physicochemical properties are separated on some solid support (achiral column). The formation of diastereoisomeric complexes

chiral recognition mechanisms in CE. Indirect resolution is achieved by diastereoisomeric complex formation followed by their resolution by CE. Contrarily, direct resolution is achieved by using chiral selectors in BGE. The theoretical consideration of chiral recognition mechanisms in CE was reviewed by Vespalec and Bocek [136]. In case of cyclodextrins, the inclusion diastereomeric complexes are formed which are controlled by a number of interactions such as p-p complexation, hydrogen bonding, dipole-dipole interactions, ionic bindings and steric effects [134]. It is also considered that electrostatic interactions contribute substantially to the chiral recognition processes. Therefore, the selector-enantiomers with a net charge of the same sign must be unfavorable for the chiral separations. Hence, in most studies in CE, oppositely charged selector-enantiomers or charged selector-neutral enantiomers and vice versa have been used. Zerbinati et al. [137] used ethylcarbonate-b-CD, hydroxypropyl-b-CD and native a-CD for the chiral resolution of mecoprop and dichlorprop. The authors calculated the performances of these chiral selectors by means of two-level full factorial design and calculating inclusion constants from CE migration time data. Furthermore, they have proposed the possible structures of inclusion complexes on the basis of molecular mechanics simulations. Recently, Chankvetadze et al. [138] explained the chiral recognition mechanisms in cyclodextrin using UV, nuclear magnetic resonance spectroscopy (NMR) and electrospray ionization mass spectrometric methods. Furthermore, the authors determined the structures of the distereomeric complexes by X-ray crystallographic method. Mostly macrocyclic antibiotics containing ionizable groups, chiral baskets, fused rings and sugar moieties change in their charge and possibly three dimensional conformation with dierent pH and the concentrations of BGE. This allows for an excellent potential to resolve a greater variety of racemates. The possible interactions involved in the formation of diastereomeric complexes are p-p complexation, hydrogen bonding, inclusion complexation, dipole interactions, steric interactions and anionic and cationic binding [139]. Accordingly, the diastereomeric complexes possessing different physical and chemical properties Review

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separated on the capillary path (achiral phase). The dierent migration times of the formed diastereomeric complexes depend on their sizes, charges and interaction with the capillary wall and as a result these complexes eluted at dierent time intervals. In 1992, Kuhn et al. [140] used a chiral crown ether in CE for the chiral resolution of several amino acids. In case of chiral crown ethers, host-guest complexes of enantiomers are formed which are stabilized by hydrogen bonding, electrostatic interactions and steric effects. The carboxylic groups of crown ether; perpendicular to the plane of the ring; form a chiral barrier which divides the space available for the substituents at the chiral centre of the enantiomers into two domains. In this way, two different diastereoisomeric complexes are formed which separated by CE [141]. Kuhn et al. [142] proposed that the chiral recognition mechanism was based on space availability for the substituents of both the chiral carbon atoms; adjacent to amine functional groups; into two cavities. Koide and Ueno [143] proposed a model and theoretical equations to investigate the enantiomeric recognition of primary amino acids using achiral crown ether with cyclodextrins by capillary electrophoresis and NMR studies. The association constants were calculated by using CE and NMR techniques. The authors reported the formation of diastereoisomeric complexes of amino acid enantiomers-CD-crown ether, which resulted into their resolution by CE. Lin et al. [144] introduced HPLC principle of imprinted polymers in CE using Dphenylalanine as the print molecule loosely bound to a methacrylate polymer. The polymer was found to have imprints of D-phenylalanine after its removal, which shows high enantioselectivity for the same or closely related molecules. The chiral mechanisms were almost similar as discussed in case of HPLC. The chiral recognition mechanisms in CE using other chiral selector such as ligand exchange, polysaccharides, alkaloids were similar to HPLC [134].

chiral selectors provide a chiral surface to the enantiomers and the formation of transient complexes, of dierent bonding energies. Dierent binding energies of the enantiomers result from their dierent ttings onto the structures of chiral selectors. The dierent stereo-congurations of the enantiomers are responsible for the dierent ttings onto the chiral selectors. These transcient complexes are stabilized by a number of interactions such as hydrogen, p-p, dipole induced dipole, ionic and steric interactions. However, the other weaker forces such as van der Waals, charge transfer and dispersion may also play a crucial role into the chiral recognition mechanisms. The chiral recognition mechanisms are based on key and lock arrangement system. In spite of all these eorts on the chiral resolution mechanisms, the complete information of the chiral recognition mechanisms are not available which need more attention. The knowledge of the chiral mechanisms involving various bonding may be useful to design new chiral selectors, which may contain maximum interactions and capable for the resolution of a wide range of racemates. Briey, it is essential to have detailed knowledge of the chiral recognition mechanisms before designing a routine or new chiral separation operations.

12. 13. 14. 15.

16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

29.

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Conclusion
The chiral recognition mechanisms using all the chiral selectors are almost similar except on ligand exchangers. All the

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