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Part OneSpecial Liquid Chromatography Modes

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1Chiral Liquid Chromatography: Recent Applications withSpecial Emphasis on the Enantioseparation of AminoCompoundsIstván Ilisz

1.1Introduction

The phenomenon of optical isomerism discovered by Pasteur has been knownfor many years. Nowadays, it is evident that the physical, biological, and chemi-cal properties determined by the molecular symmetry and asymmetry playimportant role in nature. The physiological environment within a living orga-nism is chiral, and the biological activities of enantiomeric forms of moleculescan differ dramatically. With the exception of glycine, all of the 20 proteinogenicα-amino acids have a chiral carbon atom adjacent to the carboxyl group. Thischiral center allows the existence of enantiomers, that is, two chemically identi-cal molecular species differing only in optical activity (i.e., their ability to rotatethe plane of polarized light). The stereoisomers (epimers) of the peptides inwhich all these amino acids are to be found may possess differences in biologicalactivity in living systems.The past 20 years has seen an explosive growth in the field of chiral technolo-

gies, as illustrated by the rapid progress in the various facets of this intriguingfield. The impetus for advances in chiral separation has been highest in the pastdecade and this still continues to be an area of high focus. Many chemical com-pounds such as drugs, fertilizers, and food additives have been commercializedas racemic mixtures, although in most cases only one of the isomers possessesdesirable properties. As the understanding of the biological actions of com-pounds with respect to stereochemistry has grown, the necessity to investigatethe pharmacological and toxicological properties has become more important.In light of the increased awareness concerning biologically important isomers,the US Food and Drug Administration has issued certain guidelines for the mar-keting of racemic compounds [1,2]. Chirality is now a major theme in the phar-maceutical industry in the design, discovery, and development, and launchingand marketing of new drugs. Therefore, there is considerable pressure to developanalytical methods for enantiomeric purity control, pharmacological studies,pharmacodynamic investigations, clinical studies, and so on. The advances instereoselective bioanalysis have led to a new awareness of the importance of

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Analytical Separation Science, First Edition. Edited by Jared L. Anderson, Alain Berthod,Verónica Pino Estévez, and Apryll M. Stalcup. 2015 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2015 by Wiley-VCH Verlag GmbH & Co. KGaA.

stereoselective pharmacodynamics and pharmacokinetics, enabling differentia-tion of the relative contributions of enantiomers to the overall drug process. It isworth to mention that aspects of chirality are also very important in the environ-ment, food and beverages, agrochemical, and petrochemical industries.Initially chiral analyses were relatively difficult and most separations were car-

ried out with derivatization prior to analysis. The main methodologies used forenantioseparation are enzymatic digestion, crystallization, membrane-basedspectroscopic, capillary electrophoretic, and chromatographic methods. At ana-lytical level nowadays, the chromatographic techniques are the most popularones. Various strategies have been devised for the differentiation of enantiomersby chromatography: (i) indirect methods involving sample derivatization by achiral derivatizing agent (CDA) prior to injection into achiral chromatographycolumns, (ii) direct methods that use chiral mobile-phase additives with standardstationary phases, and (iii) direct methods where chiral separations are achievedby using a chiral stationary phase (CSP). High-performance liquid chromatogra-phy (HPLC) with CSPs seems one of the most promising tools for the chiralresolution of different racemates. The design and development of a CSP capableof effective chiral recognition of a wide range of enantiomers is the key point ofthe chiral HPLC technique. A number of CSPs for HPLC have been prepared,consisting of either small chiral molecules or chiral polymers as selectors, andmany of them have been commercialized.This chapter focuses on the direct HPLC separations applying different types

of CSPs; however, indirect methods are also briefly discussed. Even in thisrestricted field, the limitations do not permit the survey of the large number ofstudies published on the enantioseparation of various chiral molecules recently.Since two excellent review papers surveying the literature [3,4] and a book onchiral separations [5] have been published within a short period of time, in thischapter publications appeared in the past 2 years (2013–2014) are considered asrecent applications with special emphasis on the enantioseparation of com-pounds containing amino groups.

1.2Indirect Methods

The reaction of enantiomers with enantiomerically pure reagent-form diastereo-meric derivatives that could be separated on achiral columns was the first widelyused general method in the field of chiral analysis. The indirect methods are stillquite efficient techniques for the enantioseparation of several chiral compounds,including amino acids. The advantages include the commercial availability of alarge number of CDAs and a relatively wide choice of chromatographic condi-tions. The enantiomer molecule and the CDA must possess easily derivatizableand compatible functional groups. The reaction should be comparatively fast, asotherwise a difference in the rate of the formation of diastereomers may causekinetic resolution. However, it is essential that the chiral derivatization reaction

302 1 Chiral Liquid Chromatography

should proceed quantitatively for both enantiomers and that racemizationshould not occur. Resolution via diastereomer formation is usually improvedwhen bulky (chromophore or fluorogenic) groups are attached to the chiral cen-ters. Furthermore, if the chiral purity of the CDA is not known and/or is nottaken into consideration, the chiral purity of the analyte will not be determinedprecisely. Thus, the indirect method is difficult to apply for the analysis of a stan-dard sample or in pharmaceutical preparations, where a low amount of antipode(at a level of 0.1 or 0.05%) is to be determined. Furthermore, it is difficult toapply for preparative purposes. However, it is suitable for the trace analysis ofenantiomers in complex matrices such as biological samples because of theintroduction of a highly sensitive UV-visible or fluorescence tag.Following the introduction of new chromatographic and electrophoretic tech-

niques, the importance of chiral derivatization has naturally decreased to someextent. However, this general method is still a method of choice that is widelyused, especially in HPLC. The reasons include the large number of commerciallyavailable homochiral reagents, well-established reactions, and (if the R and Sforms of the reagent are available) the peak of the enantiomeric impurity can beeluted before the main peak.Many biochemically important compounds have at least one functional group

in their structure. The major types of reactions for chiral amines, involvingamino acids, are mainly based on the formation of amides, carbamates, ureas,and thioureas. The reactions of acid chloride and chloroformate reagents pro-ceed rapidly to furnish the corresponding amides and carbamates. The mostimportant chloroformate possessing a reactive functional group is 1-(9-fluorenyl)ethyl chloroformate. Chiral isocyanates and mainly isothiocyanates aregood labels with which to produce stable ureas and thioureas. Among the chem-ically most selective CDAs are isothiocyanates such as 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate and 2,3,4,6-tetra-O-benzoyl-β-D-glucopyranosylisothiocyanate. Marfey’s reagent, 1-fluoro-2,4-dinitrophenyl-5-L-Ala amide andits chiral variants, in which the L-Ala amide is replaced by some amino acidamide, are among the most important CDAs. Ortho-phthalaldehydes with chiralthiols give highly fluorescent adducts with the primary amino groups of aminoacids, ensuring a very low detection limit in enantiomer analysis. CDAs devel-oped earlier are still in use for different new applications.Further information on general chiral HPLC derivatization may be found in

the original papers in numerous reviews and monographs [6–9].

1.3Direct Methods

The first CSP for gas chromatography was described in 1966 [10]. Davankov andRogozhin [11] introduced chiral ligand-exchange chromatography (CLEC) in1971, and CLEC became the first direct HPLC method for the resolution ofamino acids. The breakthrough in HPLC technology in the 1970s led to the

1.3 Direct Methods 303

commercial availability of liquid chromatographs appropriate for fast, efficient,and reliable separations. The development of the hardware techniques wasaccompanied in the 1970s and 1980s by advances related to packing materials.Polymeric materials are not suitable for high-pressure operation, while the rela-tive instability of the physical coating of the support materials proved to be oneof the main drawbacks of the early CSPs. With increasing experience and knowl-edge and continuous development in the field, the disadvantageous propertieswere progressively overcome. The appearance of mechanically stable, porous,and small-diameter silica particles led to the commercialization of chemicallybonded CSPs. The chiral separation techniques have subsequently become avery sophisticated field of analytical chemistry. The number of commercializedCSPs now exceeds 200, and studies focusing on the development of new types ofchiral selectors are published virtually daily. Although a large number of CSPsare available nowadays, they tend to be derived from relatively few classes ofcompounds. Most chiral selectors are based on amino acids (native or deriva-tized), proteins, oligosaccharides (e.g., cyclodextrins, cyclofructanes, etc.), deriva-tized linear or branched polysaccharides (e.g., cellulose or amylose), andmacrocyclic selectors, such as macrocyclic antibiotics, chiral crown ethers, ion-exchangers, and synthetic polymers. Their advantages lie in the ability to operatein different modes of HPLC: in reversed phase (RP), normal-phase (NP), polarorganic (PO), or polar ionic (PI) modes.

1.3.1

Ligand-Exchange-Based CSPs

Since Davankov and Rogozhin [11] introduced CLEC for the direct separationof amino acid enantiomers, metal chelate additives have been frequently usedin the mobile phase. The mechanism of the chiral recognition process inCLEC is assumed to be based on the reversible formation of diastereomericcomplexes between the stereoisomers of analytes, chiral selector, and a metalion. On the basis of this concept, it can be noted that in case of CLEC, exclu-sively among the chromatographic techniques, the separation does not requirethe direct contact between the analyte and the chiral selector; the metal ion,acting as a Lewis acid, coordinates the selector and the analyte through dativebonds leading to the formation of a ternary complex. Chromatographic sepa-ration occurs if the complexes of enantiomers have different rates of forma-tion and/or thermodynamic stabilities or different adsorption behaviors.Several chiral ligand-exchange stationary phases have been applied so far withdifferent selectors, mostly chiral amino acids and chiral amino alcohols andtheir derivatives, for example, proline, leucinol, cysteine, phenylalanine, andpenicillamine. Applying copper(II) as metal ion, the formation of five-membered chelate ring is normally expected. Retention and separation areinfluenced by the concentration and nature of the mobile-phase components,together with other variables, such as pH and temperature. In all CLEC sys-tems, the eluent pH and ionic strength, specifically the concentration of


copper(II), markedly define the stability of the transient metal complexes andhence the chromatographic retention of analytes.New selectors for CLEC, O-benzyl-(S)-Ser, S-benzyl-(R)-Cys, S-diphenylmethyl-

(R)-Cys, and S-trityl-(R)-Cys were synthetized by Natalini et al. [12,13]. The newselectors have proved to be effective in the enantioseparation of some nonprotei-nogenic underivatized amino acids, such as phenylglycine, and amino acids con-taining thienyl-, cyclopentyl-, and tetrahydrofurenyl-rings with fair to goodseparation and resolution factors.

1.3.1.1 Recent ApplicationsTo develop CLEC-based CSPs for the resolution of chiral drugs, the residualsilanol groups on the silica surface of a CSP based on sodium N-[(S)-1-hydrox-ymethyl-3-methylbutyl]-N-undecylarninoacetate, a (S)-leucinol derivative, wereprotected with n-octyl groups [14]. Proton pump inhibitors (omeprazole, panto-prazole, lansoprazole, and rabeprazole) were enantioresolved with improved effi-ciency. The observed chiral recognition ability of the CSP was assumed to beoriginated from the protection of the nonenantioselective interaction sites onthe silica surface and the improved lipophilicity of the stationary phase. Chiralionic liquids (CILs) with amino acids as cations were applied as novel chiral lig-ands coordinated with copper(II) for the separation of tryptophan enantiom-ers [15]. The applicability of CILs with amino acids as cations for chiralseparation was demonstrated and a comparative study was also conducted forexploring the mechanism of the CILs as new ligands in CLEC. Indirect chiralligand-exchange chromatography (ICLEC) was facilitated by substituting chiralligands from the bidentate Cu(II) complex via racemic analytes in reactionvial [16]. For the resulting Cu(II)–analyte–ligand complexes, higher stabilityconstants and better chromatographic resolutions were found than in the con-ventional CLEC. The new technique was found to be successful in determiningenantiomeric purity of drugs in pharmaceutical formulations. A new chromato-graphic procedure was developed for the separation of atenolol enantiomersbased upon CLEC [17]. The separation was carried out on a C8 column withL-alanine and Cu(II) applied as a chiral selector and central bivalent complexingion. The optimized HPLC method was utilized in some synthetic and humanblood plasma samples. By applying cinchona alkaloids as chiral selectors alongwith Cu(II) ions, good enantioseparation was obtained for several amino acidsusing equimolar amounts of Cu(II) and cinchonidine, quinine, or quinidine [18].The molecular geometry of the diastereomeric complexes formed was modeledand energetic differences between both compounds were calculated by methodsbased on semiempirical force field. CILs containing imidazolium cations andL-proline anions were applied as chiral selector to separate tryptophan enan-tiomers on a C18 column [19]. Factors influencing Trp enantiomer separation,such as alkyl chain length of CILs, concentrations of Cu(II) and CILs, pH of themobile phase, flow rate, organic solvent, and temperature, were studied andsome thermodynamic parameters were calculated. Chromatographic propertiesof isoxazoline-fused 2-aminocyclopentanecarboxylic acid analogs with a sodium


N-(R)-2-hydroxy-1-phenylethyl)-N-undecylaminoacetate–Cu(II) complex as achiral selector were investigated under various conditions [20]. The effects oftemperature were studied at constant mobile-phase compositions and thermo-dynamic parameters were calculated. Some mechanistic aspects of the chiralrecognition process were discussed with respect to the structures of the analytes.

1.3.2

Protein-Based CSPs

The stereoselective interactions of proteins with chiral compounds can be a basisfor their applications as efficient CSPs. Due to the complexity of the protein-based selectors, numerous interactions might be used (e.g., hydrogen bonds,π–π, dipole–dipole, and ionic interactions) for achieving enantioselectiverecognition for a wide range of chiral compounds. However, even small changesunder the applied conditions may cause drastic variations in their enantiosepara-tion capacity [21]. The most important commercially available protein-basedselectors are human serum albumin (HSA), α1-acid glycoprotein (AGP), ovomu-coid (OVM), and cellobiohydrolase I (CBH). HSA is proved to be a useful CSPfor acidic and CBH for basic chiral compounds, while AGP and OVM possesswide enantiorecognition ability for a variety of neutral, basic, and acidic pharma-ceuticals [3]. Protein-based CSPs played an important role in the 1980s, but thedecreased number of publications in the last couple of years indicates a signifi-cantly reduced interest. (It is important to note that the protein-based CSPs canserve as a model environment in drug–protein binding studies.)

1.3.2.1 Recent ApplicationsChicken α1-acid glycoprotein was immobilized on aminopropyl silica particlesand the effects of silica particle diameters on column performances were investi-gated [22]. Applying different chiral model compounds, the column filled withparticles with lowest diameter gave the best performance. Retention of severalstructurally diverse drugs on α1-acid glycoprotein column was investigatedunder different chromatographic conditions [23]. AGP retention mechanismwas found to be sensitive to changes produced by the percentage and nature ofthe organic modifier as a result of different shielding degree of AGP chargedsites.

1.3.3

Polysaccharide-Based CSPs

The utilization of the ability of polysaccharides to resolve racemic mixtures datesback to 1951, when Kotake et al. reported the resolution of some amino acids bypaper chromatography [24] using cellulose as CSP. Early works from Lüttring-haus, Hess, and Rosenbaum [25], Hesse and Hagel [26], and others reported theutilization of different cellulose derivatives without supporting beads. Derivatives


of cellulose as beads can be applied for CSPs, offering theoretically higher load-ing capacity and efficiency, but higher mechanical stability and wider solventcompatibility require the application of support materials. Coating cellulosederivatives onto the surface of silica beads was reported by Okamoto Kawa-shima, and Hatada in 1984 [27]. Further developments in coating proceduresand column technology have led to commercially available, mechanically stable,and state-of-the-art polysaccharide-based CSPs.Among the various polysaccharides, cellulose and amylose have been used for

the preparation of commercialized CSPs. Because of their difficult handling andresolution problems, cellulose and amylose could not be utilized as efficientCSPs without modification; however, their ester and carbamate derivativesproved to be very suitable selectors for HPLC applications. Recent developmentsof the polysaccharide-based CSPs have been focused on the optimization of thesubstituents (position and quality) on the aromatic moiety of the derivatives.The polymeric chains are built from D-glucose units through ß-1,4 linkage in

cellulose and α-1,4 linkage in amylose. In case of polysaccharide-based CSPs, thechiral recognition properties originate from several factors: (i) the presence ofchiral centers in the glucose units (molecular chirality), (ii) conformational chi-rality due to the structure of the separate chains, and (iii) the supramolecularchirality that stems from the alignment of the polymeric chains forming highlyordered structures [28].The enantiorecognition ability of the polysaccharide-based CSPs is generally

assumed to be based on hydrogen bonding and dipole–dipole interactions. Forthese interactions to take place, the presence of water as a strongly competingspecies should be avoided. Thus, applying alkanes (e.g., hexane, heptane, etc.)and alcohol (e.g., propan-2-ol) in NP mode can be a good choice as a startingmobile phase for a polysaccharide-based CSP.Recent innovations have led to the availability of immobilized polysaccharide-

based CSPs with extended solvent compatibility, and even nonstandard solvents(such as dichloromethane, chloroform, dioxane, toluene, etc.) can be applied.Polysaccharide CSPs recently commercialized can also be applied under RP con-ditions, but it is important to note that varying the chromatographic modes (col-umn not dedicated to a specific mode) may result in extended equilibration timesupon the change of mobile phases and reduction of the observed efficiency.

1.3.3.1 Recent ApplicationsThe enantioseparations of 3,5-disubstituted hydantoins were examined underNP mode using Chiralpak IA containing an immobilized amylose tris-(3,5-dime-thylphenylcarbamate) CSP [29]. The effects of polar alcoholic modifier and col-umn temperature on retention and enantioseparation were determined, whereboth solvent- and temperature-induced reversal of elution order was observed.The enantioseparation of three pyroglutamide derivatives were investigated onamylose and cellulose tris-(3,5-dimethylphenylcarbamate), amylose tris-(S)-α-methylbenzylcarbamate), and cellulose tris-(4-methylbenzoate) with various


mobile phases under both HPLC and SFC conditions [30]. Performances atsemipreparative and analytical levels were compared and validation parameterswere calculated. Development of chromatographic methods based on the use ofan amylose derivative CSP was achieved to enable enantioresolution of four fullyconstrained ß-amino acids in NP mode [31]. The results obtained with the poly-saccharide-based phase were compared with the results achieved with a glyco-peptide-based column employed in PI mode. Chiralpak IC3 and Chiralcel OCcolumns were applied for the investigation of the enantiomeric composition ofoxaliplatin [32]. Experimental results demonstrated the benefits arising from theenantioselective hydrophilic interaction liquid chromatography based on poly-saccharide phases. A simple and sensitive HPLC method was developed and vali-dated to determine the enantiomers of propranolol in rat serum [33]. Molecularmodeling studies were performed, and binding affinities and interaction dis-tances between propranolol enantiomers and chiral selector were calculated too.Enatioseparations of Koga bases were investigated applying polysaccharide-basedCSPs under NP conditions [34]. As a result of temperature changes, alteration inCSP conformation and/or enantioseparation mechanism was observed. A silica-based cellulose 3,5-dimethylphenylcarbamate derivative hybrid material wasdeveloped and characterized as CSP [35]. Compared to a commercial ChiralpakIB column, better separation ability was observed for ß-blocker drugs as chiralprobes. A systematic study of the effect of basic and acidic additives on HPLCseparation of enantiomers of some basic chiral drugs on polysaccharide-basedchiral columns in PO mode was conducted [36]. It was concluded that additivesmay serve, on the one hand, as useful tools for the adjustment of the enantiomerelution order and, on the other hand, they may help in better understanding thechiral recognition mechanisms. A comparative study was done for the evaluationof teicoplanin-based and polysaccharide-based columns applying unusual aminoacids as chiral probes [37]. The applied phases were found to be complementaryto each other to some extent, while regarding the CSP used the change of elutionorder of enantiomers was observed.

1.3.4

Cyclodextrin-Based CSPs

Cyclodextrins (CDs) are naturally occurring, nonreducing oligosaccharides.Three of the most widely utilized CDs are composed of 6, 7, and 8 D-glucopyr-anose units bonded through α-1,4 glycosidic linkages, called α-, β-, and γ-CD,respectively. CDs can be described as toroids with larger and smaller openings.The arrangement of the carbon backbone and the primary and the secondaryhydroxyl groups leads to a lipophilic cavity and a hydrophilic surface. Thanksto this unique property, CDs are water soluble and are able to form inclusioncomplexes. The hydroxyl groups can be easily derivatized forming a large vari-ety of modified CDs possessing uncharged or ionic substituents. Due to theirrelatively simple production, natural and modified CDs have found severalapplications.


Since the introduction of the first high-coverage stable bonded phaseCDs [38], they have been successfully applied as CSPs for the separation of vari-ous chiral compounds. The CDs are some of the more popular materials used asselector molecules in the field of chiral analysis. One of their advantages lies intheir solvent compatibility, they have the ability to operate in different modes ofHPLC: RP, NP, PO, and PI modes (they are multimodal CSPs).For the mechanism of the separation on CD selectors, it is generally accepted

that under RP conditions, lipophilic solutes interact with CD selectors via inclu-sion complexation; the hydrophobic part of the guest molecule penetrates theCD cavity and leads to the release of solvent molecules. Van der Waals interac-tions inside the cavity and additional hydrophilic interactions may also takeplace. For aromatic CD derivatives π–π stacking increments with analytes con-taining aromatic moiety may exist as well. In the PO mode, hydrogen bonding,dipolar interactions, and steric repulsion can lead to chiral recognition. In theNP mode, enantioseparation can be achieved via a combination of hydrogenbonding, steric effects, π–π interactions (in the presence of aromatic moieties),and dipole–dipole stacking.

1.3.4.1 Recent ApplicationsA facile approach to construct a novel native CD CSP bearing cationic imidazo-lium on the linking bridge via thiol-ene reactions was reported [39]. Theenhanced electrostatic interaction of the prepared cationic CSPs with oppositelycharged chiral analytes were demonstrated applying dansyl amino acids. Twomultiurea-bound ß-CD CSPs were prepared through the Staudinger reactionsand the HPLC enantioseparation behaviors were investigated under multimodalelution [40]. The prepared phases exhibited good separation performances forthe analytes investigated and also showed some complementary enantioselectiv-ity to each other, due to different electron-donating/withdrawing groups in thephenylcarbamate moieties. Perphenylcarbamoylated CD-clicked CSP was pre-pared with improved column efficiency and CD surface loading [41]. The enan-tioselectivity of the prepared CSP was explored with 26 racemates and thecharacteristics of the column were evaluated in terms of linearity, limit of detec-tion, and limit of quantification.

1.3.5

Cyclofructan-Based CSPs

The cyclofructans (CFs) consist of six or more (usually seven or eight) ß-(2-1)-linked D-fructofuranose units. Common abbreviations for these compounds areCF6, CF7, CF8, and so on. Each of the fructofuranose units contains four chiralcenters and three hydroxyl groups. These selectors are members of the macro-cyclic oligosaccharides such as the cyclodextrins, which are the best knownmembers of this class. CF6, with six D-fructofuranose units, contains 18-crown-6ether core, similar to the respective crown ethers. The 18-crown-6 ring servesas the skeleton core of CF6, with six fructofuranose units attached on its rim,


where the fructofuranose units are alternatively pointing toward and away fromthe molecular center. The application of native and derivatized CF6 for theenantioresolution of primary amines was reported in 2009 by Armstrong andcoworkers [42]. They found that isopropyl-carbamate-functionalized CF6 mayserve as an efficient CSP to separate compounds containing primary aminogroups (e.g., amino acids and their derivatives), while CF6 functionalized witharomatic moieties can be applied for the enantioseparation of a wide spectra ofother chiral compounds. Such columns can be used in NP and PO modes.

1.3.5.1 Recent ApplicationsR-naphthylethyl-derivatized CF6 was used for direct enantioseparation of novelchiral analogs of spiroindoline phytoalexins under NP conditions [43]. It wasfound that analytes with electron-withdrawing substituents showed better chiralrecognition ability on this CSP. Biaryl atropisomers were screened with CF6-based CSPs in NP and PO mode to investigate the interactions governing reten-tion and enantioselectivity [44]. Preparative-scale enantioseparations were alsostudied. Methods were developed for newly synthesized functional ethano-Tröger base racemates applying CD and CF-based CSPs and capillary electropho-retic determinations [45]. CF-based CSPs were most successfully applied underNP conditions in HPLC. The chromatographic performance of a superficiallyporous CF6-based column was compared with that of the columns packed with5 μm and 3 μm fully porous particles [46]. Under constant mobile-phase condi-tions, selectivity and resolution of the separations were found to be comparablebetween fully porous and superficially porous columns, even though the columnfilled with superficially porous particles contained lower absolute amounts ofchiral selector.

1.3.6Crown Ether-Based CSPs

Crown ethers with a cavity of specific size are macrocyclic polyethers, where theoxygen atoms can serve as electron donors. Due to this property, alkali metalions and ammonium ion may be incorporated into their cavity. As a conse-quence, crown ethers can be used to resolve enantiomers that contain a primaryamine functional group. The generated chiral ammonium ions can bind to themacrocyclic crown by inclusion complexation. Enantioselectivity is governedprobably by steric factors of the substituents of the chiral ammonium ions andthe residues attached to the chiral moieties incorporated into the crown ether.Sogah and Cram introduced the first crown ether-based CSP by immobilizing

bis-(1,19-binaphthyl)-22-crown-6 on polystyrene or silica gel [47]. Two decadeslater Hyun, Jin, and Lee [48] and Machida et al. [49] prepared the first chemi-cally bonded crown ether-based CSPs. Since then, several applications applyingcrown ether-based CSPs have been published, but because of their ratherrestricted applicability, these CSPs could not gain a really important position inthe field of chiral HPLC analysis.


1.3.6.1 Recent ApplicationsThe enantioseparation ability of CSPs containing acridino-18-crown-6 etherselectors was studied by HPLC [50]. The newly prepared CSPs separated theenantiomers of selected protonated primary aralkylamines efficiently. Mexiletineand its analogs were resolved on three different crown ether-based CSPs [51].The chromatographic behaviors of mexiletine analogs containing a substitutedphenyl group at the chiral center were found to be quite dependent on the phe-noxy group of analytes. A doubly tethered N-CH3 amide CSP based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid was applied to the resolution of tocai-nide and its analogs [52]. The obtained results were compared with those on asingle-tethered N-H amide CSP, a single-tethered N-CH3 amide CSP, and a dou-ble-tethered N-H amide CSP. Crown ether-based CSP was utilized for the enan-tioresolution of aminophosphonic acids and their aminocarboxylic acidanalogs [53]. Computer modeling and H1-NMR analyses were performed to gaina better understanding of interactions of the analytes with the CSP.

1.3.7

Macrocyclic Glycopeptide-Based CSPs

The concept of using macrocyclic antibiotics as CSPs was initiated by Armstrongin 1994 [54]. Macrocyclic antibiotics possess several characteristics that allowthem to interact with analytes and serve as chiral selectors. There are hundredsof these compounds and, unlike other classes of chiral selectors, they comprise alarge variety of structural types. In general, these compounds have molecularmasses greater than 600 but less than 2200. There are acidic, basic, and neutraltypes, and they may display little or no UV-Vis absorbance. The macrocyclicantibiotics used for chiral separations in HPLC include the ansamycins (rifamy-cins), the glycopeptides (avoparcin, teicoplanin, ristocetin A, vancomycin, andtheir analogs), and the polypeptide antibiotic thiostrepton. In the past two deca-des, they have had a rapid and significant impact on the field of enantiosepara-tion. They have unique structural features and functionalities that allow variouschiral interactive sites and interactions (i.e., electrostatic, hydrophobic, H-bond-ing, steric repulsion, dipole stacking, π–π interactions, etc.) between the analyteand the stationary phase.The macrocyclic glycopeptide-based Chirobiotic phases are the latest class of

chiral selectors created by the bonding of macrocyclic antibiotics to silica.Because of their favorable properties, these CSPs are able to work in the NPmode with apolar mobile phases, in the RP mode with hydro-organic mobilephases, in the PO mode with 100% polar nonaqueous solvent, and in the PImode with nonaqueous solvent containing some acid and/or base to adjust theselector (and analyte) ionization state. Most of the time, higher efficiencies areobserved in the NP and PO mode; however, applying mass spectrometric (MS)detection PI or RP mode would be more advantageous to start with. The enan-tioselectivity was found to be different in each of these chromatographic modes.The popularity of Chirobiotic phases is being proven by several hundred papers


dealing with the separation of various amino acids, amines, amides, β-blockers,β-agonists, nonsteroidal anti-inflammatory drugs, antineoplastics, and variousother biologically important compounds.One of the most characteristic features of the antibiotic-based selectors is their

chiral ionic character. All Chirobiotic phases possess analogous ionizable groupsthat have been proven to play important role in the chiral recognition. Theirspecific structures and the variety of functionalities give them the power of effi-cient resolution of almost all types of neutral, acidic, and basic racemates. Beforechoosing an appropriate glycopeptide-based CSP, it is advisable to examine thestructure of the analyte.In chiral chromatography, there are several types of interactions that can

occur between solute and CSP. Naturally, not all of these are active or decisivein all mobile phases, so mobile-phase choice can be driven by the type of interac-tion that is available on a particular CSP or is available on a particular solute.Each Chirobiotic phase has a peptide backbone that provides hydrogen bondingand dipole–dipole interactions, and each, uniquely, has an ionic site of sometype. The sugar moieties, if present, may provide hydrogen bonding and somesteric effects. Last but not least, the glycopeptide has internal ring structuresthat will provide inclusion complexation in RP mode. Because of the structuralsimilarities, the macrocyclic glycopeptides are to some extent complementary toone another: where partial enantioresolution is obtained with one glycopeptide,there is a high probability that baseline or better separation can be obtained withanother. Each type of interaction has different strength in different mobilephases, so by going from one mobile-phase type to another, on the same col-umn, the mechanism is changing, giving another opportunity for efficientseparation.

1.3.7.1 Recent ApplicationsThe chromatographic retention and thermodynamics of the adsorption of enan-tiomers of α-phenylcarboxylic acids on an eremomycin-based CSP under RPconditions were studied [55]. Relationships between the retention characteristicsof the acids, the enantioselectivity, and the concentration of organic modifierwere explored. The distribution of L- and D-amino acids in Antarctic lakes weremonitored applying a teicoplanin aglycone-based CSP by HPLC–MSmethod [56]. A vancomycin-based CSP in PI mode was utilized to achieve theenantioseparation of chiral pharmaceuticals from activated sludge inoculumoriginated from a wastewater treatment plant [57]. The developed method wassuccessfully validated and the enantioselectivity of the biodegradation processwas characterized by its application. Macrocyclic glycopeptide-based CSPs wereapplied for the separation of four bicyclo[2.2.2]octane based 2-amino-3-carbox-ylic acid enantiomers [58]. The effects of the mobile-phase composition, thestructure of the analytes, and the temperature on the separations were investi-gated. Enantiomeric resolution of atenolol was achieved applying a vancomycin-based CSP [59]. The proposed method was validated and utilized for the deter-mination of atenolol from plasma samples.


1.3.8

Ion-Exchanger-Based CSPs

In case of CSPs based on the ion-exchange procedure, retention relies on ionicinteractions forming between ionic (or ionizable) solutes and the ionic functionalgroup(s) of the selector. Lindner and coworkers have been particularly interestedand active in the development of cinchona alkaloid-based CSPs. CSPs based onquinine, quinidine, epiquinine, and epiquinidine tert-butylcarbamate selectorswere synthesized and evaluated under ion-exchange HPLC conditions with a setof racemic N-acetylated and N-oxycarbonylated proteinogenic and nonproteino-genic amino acids [60]. The enantioseparation of quinine- and quinidine-derivedCSPs proved to be far superior to that of their C9-epimeric congeners.Cinchona alkaloid-based chiral weak anion-exchangers (WAX) and aminosul-

fonic acid-based chiral strong cation-exchangers (SCX) have been fused in acombinatorial synthesis approach into single, zwitterionic, and chiral selectormotifs [61]. The corresponding zwitterionic ion-exchange-type CSPs allow enan-tioseparations of chiral acids and amine-type solutes in liquid chromatographyusing PO mode with largely rivaling separation factors compared to the parentWAX and SCX CSPs. Furthermore, the application spectrum could be remark-ably expanded to various zwitterionic analytes such as α- and ß-amino acids andpeptides. The new CSP provided strong unequivocal evidence for synergisticeffects of the two oppositely charged ion-exchange subunits being involved inmolecular recognition of zwitterionic analytes by zwitterionic selectors driven bydouble ionic coordination.Cinchona alkaloid-based zwitterionic CSPs were recently marketed under the

trademark Chiralpak ZWIX(+)TM and ZWIX(�)TM by Chiral TechnologiesEurope. Due to their zwitterionic nature, the chiral selectors in principle allowthe enantiomeric separation of a remarkably broad scale of ionizable chiral ana-lytes, ranging from acidic to basic and zwitterionic compounds. Anion-, cation-,and zwitterion-exchange processes have been confirmed to be predominantlyinvolved in selector–selectand interactions and in the enantioselective retentionof the analytes. Nonaqueous polar organic solvents in combination with acid andbase modifiers (PI mode) proved to be the preferential mobile phase for the sep-aration of zwitterionic solutes on the cinchona alkaloid-based zwitterionic CSPs.The use of MeOH as a protic solvent (which can suppress H-bonding interac-tions) and MeCN as an aprotic solvent to permit ionic interactions, but to inter-fere with aromatic π–π interactions, seemed to be the best due to thesuppression of nonspecific hydrophobic interactions with the CSP, therebyenhancing enantioselectivity.

1.3.8.1 Recent ApplicationsA quinine- and a quinidine-based zwitterionic CSPs were applied for the enan-tioseparation of 27 unusual cyclic secondary α-amino acids [62]. The effects ofthe nature and concentration of the bulk solvent, the acid and base additives,the structures of the analytes, and the temperature on the enantioresolution


were investigated. A large series of diverse amino acids and analogs were appliedfor elucidation on the chromatographic conditions to be used for generic samplescreening and for specific separations [63]. The zwitterionic CSPs were found tooffer the features not only of a double ion-pairing with chiral ampholytes inretaining and resolving them but also of a single ion-pairing with acid- or base-type analytes. The enantioseparation of four bicyclo[2.2.2]octane-based 3-amino-2-carboxylic acids was reported in PI mode with zwitterionic CSPs [64]. Experi-ments were performed at constant mobile-phase compositions in order to studythe effects of temperature, and thermodynamic parameters were calculated. Theeffects of temperature on the chiral recognition of cyclic ß-amino acid enan-tiomers on zwitterionic CSPs were investigated [65]. Unusual temperaturebehavior was observed, especially on the ZWIX(�)TM column, where the appli-cation of MeOH/MeCN (50/50 v/v) containing 25 mM triethylamine and50 mM formic acid as mobile phase led to nonlinear van’t Hoff plots andincreasing retention time with increasing temperature. Several free amino acidsand analogs were directly resolved into enantiomers by HPLC on zwitterionicCSPs [66]. The systematic investigation was undertaken to gain an insight intothe influence of the structural features on the enantiorecognition. Racemic ami-nophosphonic acids were used as chiral probes for the evaluation of the chiralrecognition abilities of zwitterionic CSPs [67]. Mobile-phase characteristics suchas acid-to-base ratio, type of counterion, and solvent composition were system-atically varied in order to investigate their effect on the separation performance.The enantioseparation of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate(AQC)-tagged amino acids and other zwitterionic compounds was tested ontert-butyl-carbamate-modified quinine- and quinidine-based CSPs employingPO conditions [68]. The enantioselectivity of proteinogenic AQC-tagged aminoacids was found to strongly depend on the chemistry and stereochemistry of theside chain of the amino acid.

1.3.9

Brush-Type (Donor–Acceptor Type, Pirkle-Type) CSPs

When individual synthetic chiral molecules with low molecular mass areattached to a solid support, a brush-type, donor–acceptor phase, or inrecognition of his pioneering work [69], Pirkle-type CSP is obtained. In case ofPirkle-type CSPs, the selector molecules are covalently bonded to the chromato-graphic support, and regularly covering the surface of the inert matrix, they areeasily accessible for the solute molecules. Hydrogen bonding, π–π interactions,and dipole–dipole stacking are usually the most important interactions responsi-ble for chiral recognition. These donor–acceptor phases are usually run in NPmode, with mixtures of alkane and alcohol solvents applying basic (e.g., diethyl-amine) and/or acidic (e.g., trifluoroacetic acid) modifiers. CSPs based on thisconcept are in widespread use nowadays because of their advantageous charac-teristics (e.g., good kinetic performance and quite often thermal inertness).Besides these advantageous properties, it is important to note that these CSPs


are usually available in both enantiomeric forms, namely, the elution sequencefor two enantiomers can be changed by exchange of the column for the onepacked with CSP having opposite configuration. Their advantageous character-istics make them a good choice for preparative applications also.

1.3.9.1 Recent Applications1,2-Diaminocyclohexane and 1,2-diphenyl-1,2-ethylene-diamine were used asselectors for new brush-type CSPs containing a stable bidentate urea-type struc-ture [70]. The totally synthetic CSPs were prepared and characterized in terms ofretentivity, selectivity, and permeability, while their efficiency was determinedthrough van Deemter plot analysis. The enantioselectivity of the new columnswas tested for a wide range of racemates both in NP and in PO mode.

1.3.10

Molecularly Imprinted CSPs

One of the most attractive synthetic approaches to mimic nature is molecularimprinting. This is a concept of preparing substrate-selective recognition sites ina matrix, based on the application of a molecular template in a casting proce-dure. Molecularly imprinted polymers (MIPs) can be prepared conceptuallyapplying three steps: (i) prearrangement of the functional monomers with thetemplate molecule in solution phase, (ii) copolymerization of the prepolymercomplex forming a rigid network, which holds the templates, and (iii) creatingbinding cavities in the polymer that are complementary in shape by removingthe templates. After grinding and sieving the polymeric material, the obtainedparticles can be packed into columns and utilized for enantioselectiveseparations.

1.3.11

Synthetic Polymer-Based CSPs

CSPs based on synthetic polymers can also be applied as chiral selectors, andthey can mimic the enatiorecognition abilities of the semisynthetic polysaccahr-ides. These polymer phases are commonly prepared by two different approaches:(i) “grafting-to” approach, in which the chiral monomer is applied to synthesizethe initiator polymeric chains in solution phase and then the polymeric chainsthus formed are grafted to the surface-activated support by a copolymerizationprocess; (ii) “grafting-from” approach, in which the initiator is immobilized onthe surface of the support, thus the growth of the polymer chains starts from thesurface, resulting in a well-ordered, surface-confined polymer layer [71]. Thesynthetic polymer-based CSPs are typically used under NP conditions, wherethe chiral recognition abilities are mainly due to the hydrogen bonding, π–πinteractions, and steric factors.


1.4Summary

Before the availability of stable and efficient CSPs, chiral analyses could beaccomplished mainly through derivatization prior to analysis. The developmentsin column technologies led to the appearance and commercialization of chemi-cally bonded CSPs. By the 1990s, the rapid resolution of optical isomers becameroutine and commonplace. The number of CSPs nowadays exceeds 200, andenantioseparations based on direct HPLC methods are the most popular ones.The continuous growth and evolution of the knowledge in the field of chiralanalysis in the near future will probably allow the design of new chiral stationaryphases offering more efficient separations in a shorter analysis time with higherrobustness.

Acknowledgments

The author wishes to express his thanks to Prof. Antal Péter for all the usefulcomments and suggestions.

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part one special liquid chromatography modes€¦ · chiral liquid chromatography: recent...

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