Introduction to Modern Liquid Chromatography, Third Edition part 76 doc

706 ENANTIOMER SEPARATIONS 14.6.6 Chiral Crown-Ether CSPs Stereoselective CSP-analyte complexation with chiral crown-ether CSPs and their first application as CSPs were pioneered by Cram and coworkers [127]. In this early work, two 1,1  -binaphthyl units were incorporated into a crown ether as replacement elements of two ethylene groups of the well-known 18-crown-6. Structural analogues of such 1,1  -binaphthyl-derived chiral crown-ether based CSPs were later developed by Shinbo’s group using (3,3  -diphenyl-1,1  -binaphthyl)- or (6,6  -dioctyl-3,3  -diphenyl-1,1  -binaphthyl)-20-crown-6 dynamically coated onto octadecyl-silica [128]; a related CSP has been commercialized as Crownpak CR by Daicel and Chiral Technologies (Fig. 14.24a). Since they are of synthetic ori- gin, both enantiomeric forms, with opposite elution orders, are available—denoted as Crownpak CR(+) and CR(−). Structural analogues are available, based on (18-crown-6)-2,3,11,12-tetracarboxylic acid (i.e., tartaric acid incorporated into crown ether) that is bivalently immobilized via two carboxylic functionalities onto 3-aminopropylated-silica [129]. These are commercially available as Chi- roSil RCA(+)andSCA(−) (from Regis, Morton Grove, IL) and ChiralHyun-CR-1 (from K-MAC, Korea) (Fig. 14.24b). The applications of such chiral crown-ether-based CSPs are essentially restricted to primary amines comprising mainly amino acids, amino acid esters, amino alcohols, and chiral drugs with free primary amino functionality. Typically aqueous mobile phases with pH between 1 and 3.5 are required to ensure full protonation of the solute. The resulting chiral ammonium ions can bind to the macrocyclic crown by inclusion complexation, driven by triple hydrogen-bond formation between the ammonium ion and the three oxygens of the crown (Fig. 14.25). Enantioselectivity may be governed by steric factors arising from the substituents of the chiral ammonium ions and the residues attached to the chiral moieties that are incorporated into the 18(20)-crown-6. Maintaining strongly acidic conditions appears also important to suppress silanol interactions that can be formed non-enantioselectively. This can be achieved by employing, for example, 5 mM perchloric acid in water or methanol-water mixtures (up to 15% methanol) Crownpak CR ChiroSil RCA ChiroSil (a) (b) Silica silica O O O O O O Si O O O O O O COO H C O N H O SiO O O O O O HOOC silica N H Figure 14.24 Commercially available chiral crown-ether based CSPs (adapted from online application notes provided by the suppliers). 14.6 CHIRAL STATIONARY PHASES AND THEIR CHARACTERISTICS 707 Si NH O O O O O O O O H H COOH H N + R O O O Si HN O O O HOOC Figure 14.25 Molecular-recognition mechanism for chiral crown-ether CSPs: Schematic rep- resentation of solute-selector interaction driven by triple hydrogen bonding (adapted from the Regis webpage). as mobile phase. Such harsh conditions can prove harmful for both the equipment and CSP, which has contributed to the limited popularity of these CSPs. Newer work on crown-ether-based CSPs can be found in [130–133]. 14.6.7 Donor-Acceptor Phases The first silica-bound CSPs with entirely synthetic selectors were developed in the late 1970s [134, 135]. Subsequent work by Pirkle and coworkers led in 1981 to the first commercialized CSP with a DNB-phenylglycine derivative immobilized ionicly onto silica. Later this synthetic chiral selector was grafted onto silica via a covalent amide linkage; this chiral packing material is still commercially available from Regis, Machery Nagel, and Merck as DNBPG (Fig. 14.26a). Such donor-acceptor-type CSPs (Brush-type CSPs) are based on chiral, low-molecular-weight selectors that are neu- tral, synthetic or semi-synthetic, and used in the NP mode. They are capable of gener- ating enantioselectivity based on complementary, non-ionic attractive binding forces [27]. Hydrogen bonding, face-to-face or face-to-edge π–π interaction (between electron-rich and electron-poor aromatic groups), and dipole–dipole stacking play important roles in stabilizing the selector-analyte complex and enantiorecognition. Enantioselectivity is often supported by steric interactions of bulky groups, which can represent effective steric barriers to a close selector-solute contact for one enan- tiomer. Due to the relative importance of hydrogen-bonding and other non-ionic electrostatic interactions, such CSPs are less effective in polar protic media, including the RP and PO modes. Because of the important contribution of Pirkle’s group in this field, such donor-acceptor-type CSPs are now often referred to as Pirkle-type CSPs. A number of powerful CSPs evolved early on from Pirkle’s group as a result of systematic chromatographic [136] and spectroscopic [137–139] studies of chiral recognition phenomena, as well as the consistent exploitation of the reciprocity principle of chiral recognition [140, 141]. This reciprocity recognizes that the roles 708 ENANTIOMER SEPARATIONS N H HN O Si O 2 N NO 2 O H 3 C H 3 C H 3 C H 3 C CH 3 CH 3 DNBPG NH Si O NO 2 NO 2 WHELK-O 1 N H HN O O O 2 N NO 2 Si ULMO (a) (b) (c) Figure 14.26 Structures of popular Pirkle-type donor-acceptor phases. (a)DNBPG;(b) WHELK-O 1; (c)ULMO. of selector and analyte are interchangeable. Hence a single enantiomer of an analyte that is well resolved by a CSP with a given chiral selector will (after its immobilization at positions that are not involved in the chiral recognition process) be able to separate the racemate of this selector. Such concepts and tools have been used for the rational design of new advanced CSPs [136, 142]. As noted above, such donor-acceptor-type CSPs usually have been designed to exploit π–π-stacking interactions between electron-rich and electron-deficient aromatic systems as the primary attractions. Initially developed were either CSPs with π-acidic groups (with electron-deficient aromatic moieties, usually 3,5-dinitrobenzoyl) for π-basic solutes (with electron-rich aromatic groups) or CSPs with π-basic residues (e.g., naphthalene) for π-acidic solutes. The latter CSPs (e.g., N-2-naphthylalanine undecylester-derived CSP) [143] seemed to have less broad application and therefore disappeared form the market. Several of the early-invented π-electron acceptor phases from the Pirkle group, in contrast, are still available from Regis (e.g., DNBLeu, DNBPG, β-Gem 1, α-Burke 2, PIRKLE 1-J; see Table 14.3). Eventually CSPs with both π-electron donor and acceptor moieties incorporated into a single selector turned out to be more powerful in terms of broader applicability. Along this line, the Whelk-O 1 phase was developed that has pre-organized clefts for solute insertion and allows for simultaneous face-to-face 14.6 CHIRAL STATIONARY PHASES AND THEIR CHARACTERISTICS 709 Table 14.3 Commercially Available Donor-Acceptor (Pirkle-Type) CSPs Chiral Selector Column Trade Name Supplier π-electron acceptor/π-electron donor phases 3-[1-(3,5-dinitro benzamido)-1,2,3,4- tetrahydrophenanthrene- 2-yl]-propyl-silica Whelk-O 1 Regis 11-[2-(3,5-dinitroben- zamido)-1,2- diphenylethylamino]- 11-oxoundecyl-silica ULMO Regis 3-[N-(3,5-dinitrobenzoyl)- (R) − 1-naphthyl- glycine-amido]propyl-silica Chirex 3005 (Sumichiral 2500) Phenomenex (Sumitomo) π-electron acceptor phases 3-{3-{N-[2-(3,5- dinitrobenzamido-1-cyclohexyl)]- 3,5-dinitrobenzamido}- 2-hydroxy-propoxy}- propyl-silica DACH-DNB Regis 3-[3-(3,5-dinitrobenzamido)- 2-oxo-4-phenyl- azetidine-1-yl]- propyl-silica PIRKLE 1-J Regis 5-(3,5-dinitrobenzamido)- 4,4-dimethyl-5-dimethyl phosphonyl-pentanyl-silica α-Burke 2 Regis 11-[N-(3,5-dinitrobenzoyl)-3- amino-3-phenyl-2- (1,1-dimethylethyl) propanoyl]- undecyl-silica β-Gem 1 Regis 3-[N-(3,5-dinitrobenzoyl) leucine-amido]propyl-silica Leucine (DNBLeu) Regis 3-[N-(3,5-dinitrobenzoyl) phenylglycine-amido] propyl-silica Phenylglycine (DNBPG) Regis (Merck) π-electron donor phases 3-{N-[(R)-(α-naphthyl) ethylcarbamoyl]-(S)- indoline-2-carboxamido} propyl-silica (urea linkage) Chirex 3022 (Sumichiral OA 4900) Phenomenex (Sumitomo) 3-{N-[(R)-1-(α - naphthyl) ethylcarbamoyl]- (S)-tert-leucine-amido} propyl-silica (urea linkage) Chirex 3020 (Sumichiral OA 4700) Phenomenex (Sumitomo) 710 ENANTIOMER SEPARATIONS and face-to-edge π–π-interactions to facilitate chiral recognition [144, 145] (Fig. 14.26b). Inspired by the work of Pirkle, several other research groups followed this concept of CSPs based on synthetic, low-molecular-weight selectors. Among others, Oi and coworkers developed amide-type and urea-type CSPs, now commercialized as Sumichiral OA columns from Sumitomo or as Chirex columns from Phenomenex. A number of structural variants have been made accessible; the one denoted as Chirex 3005 (amide-type π-electron donor-acceptor phase) (see Table 14.3) appears to have the broadest applicability, followed by the Chirex 3022 and to minor degree Chirex 3020 [urea-type π-electron donor-phases derived from (S)-indoline-2-carboxylic acid and (R)-1-[α-naphthyl]ethylamine as well as (S)-tert-leucine and (R)-1-[α-naphthyl]ethylamine, respectively] (Table 14.3). Other powerful π-donor-acceptor-type CSPs utilized C 2 -symmetric diamine scaffolds such as bis-N , N  -(3,5-dinitrobenzoyl)-1,2-diamino cyclo- hexane from Gasparrini’s group [146, 147] (DNB-DACH ® ,Regis)and N-3,5-dinitrobenzoyl-N  -undecanyl-1,2-diphenyl-1,2-diamine from Uray et al. (ULMO ® , Regis; Fig. 14.26c) [148]. The latter CSP allows, for instance, the chromatographic separation of underivatized arylcarbinols as depicted in Figure 14.27. The evolution of CSPs in the Pirkle laboratory, as well as some design considerations and strategies that have lead to the modern donor-acceptor phases, have been comprehensively reviewed by Welch [142]. More recently some of the newer developments in this field were summarized and discussed by Gasparrini [149]. Since donor-acceptor phases are almost always used in the NP mode, method development is straightforward. It usually starts with a mixture of hexane or heptane/2-propanol (1–10% polar solvent). For basic solutes, 0.1% of a basic modifier such as diethylamine is added to the mobile phase; for acidic solutes, 0.1% of an acidic additive such as trifluoroacetic acid. After an initial separation, the polar-solvent content is adjusted to achieve a reasonable retention factor (1< k< 10). If no baseline separation results, 2-propanol can be substituted by other polar solvents such as ethanol, dichloromethane, dioxane, methyl tert-butyl ether, R S S R S R S R 0 5 10 15 ( min ) t 0 OH OH OH OH Figure 14.27 Separation of chiral alcohols on ULMO. Conditions: (R, R)-ULMO; 250 × 4-mm column; 99.5:0.5 n-heptane-isopropanol; 1 mL/min; 254 nm; 25 ◦ C. Reprinted with permission from [148]. 14.6 CHIRAL STATIONARY PHASES AND THEIR CHARACTERISTICS 711 or ethyl acetate. If RP conditions are required, enantioselectivity values usually drop significantly, since the retention- and selectivity-driving polar interactions are effectively nullified (or at least extremely weakened) by such strong, protic solvents. It should be noted that Regis offers a screening service for the Pirkle phases. A number of characteristic benefits arise from the use of Pirkle-type CSPs. Since the building blocks of the selectors are available in both enantiomeric forms, CSPs can be developed in both configurations, allowing an opposite elution order for enan- tiomers (Section 14.3). The low molecular weight of these selectors, with their limited molecular dimensions, yields high surface concentrations of the CSP. As a result the sample loading capacities are much higher than for protein phases, macrocylic antibiotic CSPs, and cyclodextrin-based CSPs [61]. Synthetic donor-acceptor phases have also proved to be valuable tools for SFC enantiomer separation [150, 151]. 14.6.8 Chiral Ion-Exchangers Chiral ion-exchangers utilize ionizable selectors to exploit ionic interactions between oppositely charged selectors and analytes. Although a number of these CSPs are based on large molecules (e.g., protein CSPs, glycopeptide CSPs), we refer here to low-molecular-weight selectors that are similar to classical ion-exchangers yet have a chiral backbone. These CSPs can also be regarded as a subset of Pirkle phases, but carrying ionizable functional groups—thereby departing from the non-ionic interaction mode of the Pirkle-type CSPs. Several chiral ion-exchangers have been developed for the enantiomer separation of ionizable chiral compounds: chiral anion-exchangers based on cinchona alkaloid derivatives for chiral acids [152], chiral cation exchangers based on chiral amino sulfonic acids, and amino carboxylic acids for the separation of chiral bases [153], and zwitterionic ion-exchangers for the separation of both acids, bases, and zwitterionic solutes such as amino acids and peptides [154]. Only the chiral anion-exchangers with cinchonan carbamate selectors were commercially available at the time this book was published (under the tradename Chiralpak QN-AX and Chiralpak QD-AX; from Chiral Technologies) (Fig. 14.28a). The abbreviation AX refers to their anion-exchanger characteristics, while QN and QD denote the type of cinchona alkaloid employed as backbone of the selectors—quinine (QN) and quinidine (QD). The selectors of these columns are highly enantioselective, as a result of five stereogenic centers. While configurations in position N 1 ,C 3 ,C 4 are fixed as 1S,3R,4S, those of carbon C 8 and C 9 are opposite in quinine (8S,9R) and quini- dine (8R,9S) as well as separation materials derived therefrom. The experimental behavior of these cinchona-alkaloid derived CSPs is often under the stereocontrol of the stereogenic center of C 9 ; this gives them pseudoenantiomeric characteristics as a result of an opposite configuration of the two alkaloids at this chiral center. Aside from this peculiar configurational arrangement of the natural alkaloids, the exceptional enantiorecognition capability of the cinchonan carbamate-based chiral stationary phases arises also from several features: the bulky quinuclidine, the planar quinoline ring, and the semi-flexible carbamate group with a bulky t-butyl residue. These functionalities serve as potential binding sites, and they are structurally assembled to form a semi-rigid scaffold with predefined binding clefts for analyte insertion. Much is known about the principal molecular recognition mechanisms of these semi-synthetic CSPs from various chromatographic [156–158], FTIR and 712 ENANTIOMER SEPARATIONS N O O N SH H N O silica CH 3 H 9 8 1S 4S 3R H X CHIRALPAK ® CHIRALPAK ® min0 4 8 12 (R) (S) min0 4 8 12 (S) (R) CH 3 N COOH O H CHIRALPAK ® QD-AX Quinidine-derived CHIRALPAK ® QN-AX Quinine-derived (a) (b) QN-AX: (8S,9R) QD-AX: (8R,9S) Figure 14.28 Commercially available cinchona alkaloid-derived chiral anion-exchangers. (a) Structure; (b) illustration of a reversal of elution order by change from the quinine-derived CSP to the corresponding pseudoquinidine-derived CSP. Experimental conditions: Column dimen- sion, 150 × 4-mm column; mobile phase, 1% acetic acid in methanol; temperature, 25 ◦ C; flow rate, 1 mL/min; UV detection at 230 nm. Adapted from [155]. NMR spectroscopic [159–163], thermodynamic [163, 164], molecular model- ing [161, 163], and X-ray diffraction studies [161–163,165]. If complementary H-donor-acceptor sites and aromatic moieties are incorporated into the guest molecule, favorable intermolecular H-bonding and π–π-interactions may result in stable complexes and exceptionally high enantioselectivities. Targeted optimiza- tion based on knowledge from the mechanistic studies mentioned above has led to a number of powerful CSPs [166], of which the commercially available ones provide broad applicability. The cinchona alkaloid-based, anion-exchange columns offer excellent chi- ral resolving power for chiral carboxylic, sulfonic, phosphonic, and phosphoric acids [166], preferably by way of the PO or RP mode. Their applicability covers N-derivatized α-, β-, and γ -amino acids (Fig. 14.28b), their corresponding phospho- nic, phosphinic, and sulfonic acid analogues, as well as many other pharmaceutically relevant chiral acids (e.g., arylcarboxylic acids, aryloxycarboxylic acids, hydroxy acids, pyrethroic acids, and a few underivatized amino acids). If the cinchona-alkaloid based CSP is used with (weakly) acidic mobile phases, the quinuclidine nitrogen becomes protonated and acts as the fixed charge of the chiral anion-exchanger. Acidic analytes are then primarily retained by anion exchange, and retention can be explained by a stoichiometric displacement model [166]. Linear plots of log k versus the log of the counter-ion concentration [Z] (i.e., of the buffer anion) then result (Section 7.5.1 and Eq. 7.13). As discussed in Chapter 7, the slope of log k–log counter-ion concentration will, for a given 14.6 CHIRAL STATIONARY PHASES AND THEIR CHARACTERISTICS 713 column, vary with the charge on the analyte and the counter-ion, being steeper for a larger analyte charge and less steep for a larger counter-ion charge. A change in counter-ion concentration can be used to vary retention, often without much effect on enantioselectivity. The eluotropic strength (competitor effectiveness) increases in the order acetate ≤ formate < phosphate < citrate. A series of counter-ions (acid additives) in the PO mode have been tested confirming these trends for nonaqueous polar solvent-based mobile phases as well [167]. Other variables have a significant effect on enantioselectivity and allow for flexible method development: pH (RP mode), acid-base ratio (PO mode), and type and content of organic solvent(s) (RP and PO modes). Preferred mobile phases are composed of methanol plus 0.5–2% glacial acetic acid, as well as 0.1–0.5% ammonium acetate (PO-mode), or methanol-ammonium acetate buffer (total buffer concentration in the mobile phase between 10 and 100 mM, pH 5–6) (RP mode). Methanol may be replaced by acetonitrile or methanol-acetonitrile mixtures, which are to some extent complementary (dif- ferent enantioselectivities and elution orders) regarding their enantiorecognition capabilities. As noted above, quinine and quinidine CSPs are actually diastereomers, but they behave like enantiomers. Therefore they usually (but not always) show opposite elution orders, as illustrated in Figure 14.28b. This complementarity in their chiral recognition profile can be systematically exploited in enantiomeric impurity-profiling applications and preparative enantiomer separations—since it is desirable to have the enantiomeric impurity elute first (Section 14.5). Cinchona carbamate-type CSPs also show great promise for preparative enantiomer separations, by virtue of their remarkable sample loadabilities. For example, adsorption isotherm measurements for FMOC-α-allylglycine on the O−9-tert-butylcarbamoylquinidine-CSP revealed a close to homogeneous adsorption mechanism with mass loading capacities of 20 mg/g CSP [168]. Although the primary application of these cinchonan carbamate CSPs are for chiral acids, recent studies showed that they can be used for neutral and basic compounds as well, via either RP [169] or NP mobile phases [170]. 14.6.9 Chiral Ligand-Exchange CSPs (CLEC) Chiral ligand-exchange CSPs allowed the first complete separation of a racemate by chromatography in the late 1960s. Davankov immobilized proline onto a polystyrene support and used this enantioselective matrix in combination with Cu(II)-ion con- taining mobile phases for the enantiomer separation of amino acids [171]. The basic principle of chiral ligand-exchange chromatography (CLEC) is the reversible coor- dination of immobilized selectors and analytes within the metal-ion coordination sphere that forms a mixed ternary metal-ion/selector/analyte complex (Fig. 14.29) [172]. Depending on the steric and functional properties of the analytes, these diastereomeric complexes result in enantioselectivity. During the chromatographic process the coordinated ligands are reversibly replaced by other ligands from the mobile phase such as ammonia and water. An important aspect of these separations is that the exchange of ligands at the metal center is fast; otherwise, column efficiency would be compromised. An essential prerequisite for CLEC is the presence of metal-chelating function- alities in both the selector and analyte [172]. Suitable structures feature bidentate 714 ENANTIOMER SEPARATIONS N X O Cu N (S) (S) O O H 2 O O (S) (R) N X O Cu N O O H 2 O O Pol y st y rene-su pp ort Pol y st y rene-su pp ort (S, S)(R, S)(a)(b) Figure 14.29 Principle of chiral ligand-exchange chromatography. Ternary diastereomeric Cu(II)-complexes of immobilized S-enantiomer of proline (X = H) (or hydroxyproline X = OH) ligand with S-andR -proline analytes, (a)and(b), respectively. Adapted from [173]. OH O OH OH O OH H 3 C OH O OH H 3 C OH O OH H 3 C CH 3 H 3 C Time ( min ) 02040 Figure 14.30 Enantiomer separation of hydroxy acids by chiral ligand-exchange chromatog- raphy (CLEC). Experimental conditions: column, CHIRALPAK MA; mobile phase, 10% ACN/H 2 Oplus2-mMCuSO 4 . Adapted from [8]. or tridentate ligands with two or three electron-donating functional groups, such as hydroxyl, amino, and carboxylic functionalities. Such structural prerequisites are typically found in α-amino acids, amino alcohols, and α-hydroxy acids (Fig. 14.30), representative compounds that have been separated by CLEC. Cu(II) is the preferred chelating metal ion, but Zn(II) and Ni(II) are suitable alternatives. As selectors for 14.7 THERMODYNAMIC CONSIDERATIONS 715 CLEC-type CSPs, rigid cyclic amino acids, such as proline and hydroxyproline, have been shown to give the best results in combination with Cu(II). These chelating selec- tors are either (1) covalently anchored onto the surface of silica and organic polymer particles, respectively, or (2) dynamically coated onto reversed-phase materials (usu- ally immobilized adsorptively by hydrophobic interactions based on the long alkyl chain substituents of the selectors; only a low %-organic in the mobile phase is tolerated). Because of the polar nature of the analytes for separation by CLEC, as discussed above, the mobile phase is aqueous or aqueous based. The mobile phase is usually doped with small quantities of metal ion, in order to compensate for loss of metal from the column packing during chromatography, thereby rendering the separation more stable. The detection of nonchromophoric amino acids and hydroxy acids is possible as a result of their enhanced UV absorbance in the presence of Cu ++ , while the presence of metal ions in the mobile phase may hamper mass spectrometric detection. Experimental conditions that can be varied in method development include mobile phase pH, type and concentration of buffer salts, nature and content of organic solvent, temperature, and the mobile-phase metal-ion concentration. A number of covalently anchored and coated CSPs for CLEC are commercially available, including Chiralpak MA+ (based on N, N-dioctyl- L-alanine coated onto RP18) from Chiral Technologies, Nucleosil Chiral-1 (based on L-hydroxyproline chemically bonded to silica) from Macherey-Nagel, and Chirex 3126 (based on N, S-dioctyl-penicillamine coated onto RP18) from Phenomenex. In the past, CLEC was the only procedure that enabled the direct enantiomer separation of amino acids without derivatization. However, today the importance of chiral ligand-exchange chromatography is reduced, as a result of more favorable alternatives. More details can be found in a recent review [174]. 14.7 THERMODYNAMIC CONSIDERATIONS Analyte retention and enantioselectivity are, of course, based on the thermodynamics of the retention process—similar to the separation of achiral solutes, as discussed in preceding chapters. However, enantiomer separations are subject to some additional thermodynamic considerations. 14.7.1 Thermodynamics of Solute-Selector Association The equilibrium constant K i (see Fig. 14.9) of the solute-selector association can be related to thermodynamic parameters G 0 i =−RT ln K i = H 0 i − TS i 0 (14.3) Here G 0 i , H 0 i , and S 0 i refer to the standard free energy, enthalpy, and entropy changes upon the solute-selector complexation, R is the universal gas constant, T the absolute temperature (in K), and subscript i denotes the corresponding species (i.e., . donor-acceptor phases have also proved to be valuable tools for SFC enantiomer separation [150, 151]. 14.6.8 Chiral Ion-Exchangers Chiral ion-exchangers utilize ionizable selectors to exploit. process) be able to separate the racemate of this selector. Such concepts and tools have been used for the rational design of new advanced CSPs [136, 142]. As noted above, such donor-acceptor-type CSPs. Table 14.3). Eventually CSPs with both π-electron donor and acceptor moieties incorporated into a single selector turned out to be more powerful in terms of broader applicability. Along this