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The pH of the aqueous mobile phase is most often near neutral, with or without a few percent of added organic modifier, usually propanol; but acetonitrile and methanol have also been used . Besides pH, temperature, buffer components, and added charged, organic modifiers may influence the enantioselectivity and retention .
E. Cyclodextrin Stationary Phases
/Ç-Cyclodextrin, in intact or derivatized form, has been employed as a chiral selector bonded to silica. Armstrong and co-workers have made major contributions in the development and
Pettersson and Persson
Figure 5 Enantioselective separation of omeprazole on protein-based columns at pH 7.0 (a) CHIRAL-AGP; (b) BSA-DSC; (c) Ultron ES-QVM. (From Ref. 38.)
exploitation of this kind of stationary phases [108-110]. Columns with native cyclodextrin are used under reversed-phase conditions with a polar aqueous mobile phase, and both normal and reversed-phase modes are used with stationary phases of derivatized cyclodextrin on silica [108-112]. Applications in the drug field concern amines, such as chlorpheniramine , terbutaline , scopolamine , terodiline , nicotine and analogues , and /3-adrenoceptor blockers, such as propranolol, metoprolol, atenolol, and timolol [116,117]. Weakly acidic drugs, such as ibuprofen, ketoprofen, mephenytoin, and hexobarbital , and more neutral compounds, such as nisoldipine and chlorthalidone , oxazepam, bendroflu-methiazide, glutethimide, and indapamide , complete the picture of a versatile chiral stationary phase, for which, however, the enantioselectivity generally seems to be lower, com-pared with the protein phases. Coupled column liquid chromatography combined with mass-spectrometric detection was employed for the enantioselective assay of terbutaline in biological samples [113,117].
Chiral Pharmaceutical Analysis
F. Pirkle Phases
Pirkle and co-workers introduced yV-(3,5-dinitrobenzoyl) amino acids as chiral selectors ion-ically or covalently linked to aminopropyl silica . Through the years this group has made several contributions to this type of chiral stationary phases, but also others have developed useful and commercially available material [121-123]. These selectors are based on charge-transfer complexation, and the retention is mediated by òã-òò interaction along with hydrogen bonding in the nonpolar solvent used as mobile phase. To improve the chiral recognition of the solutes, derivatization by acylation or carbamoylation and introduction of a 3,5-dinitrophenyl or a naphthyl group were often used.
Applications within the pharmaceutical field are numerous (e.g., mephenytoin , ben-zodiazepinones , propranolol , pindolol , nadolol , and ibutilide ). The /Ç-adrenoceptor-blocking agents and ibutilide were separated after derivatization with phosgene  or alkyl or aryl isocyanates [127-129]. Other applications are dihydropyri-dines , /Ç-adrenoceptor blockers without prior derivatization [131,132] and naproxen . Most recently, an extensive survey on the applicability of Pirkle-concept chiral stationary phases in the pharmaceutical field has been presented .
There are not many applications of the Pirkle-type columns to the enantioselective assay of drugs in biological samples. Phenprocoumon, an anticoagulant, was determined in human plasma and urine , albruterol in serum , and ibuprofen  and fenfluramine  in biological material after derivatization.
III. CHIRAL MOBILE-PHASE ADDITIVES (CMPAs)
Direct separation of enantiomers can be obtained by adding a chiral molecule to the mobile phase; a chiral mobile-phase additive (CMPA). The mechanisms of chiral recognition by CMPAs have previously been reviewed [139-142]. In principle, the CMPA gives labile diastereomeric complexes with the enantiomeric analytes in the mobile phase. The two diastereomeric complexes differ in energy content and in physical and chemical properties; they can be separated by an achiral solid phase. A chiral separation obtained by a CMPA is due to (1) an enantioselective complexation in the mobile phase, (2) a selective distribution of labile diastereomeric complexes to the stationary phase, or (3) because the CMPA is dynamically coated on the solid phase and forms a chiral stationary phase with the possibility to discrim-inate between enantiomers (i.e., the stationary phase is operating as an ordinary CSP.) Clearly, a combination of these different separation mechanisms is also possible. The chromatographic enantioselectivity (a = k2/k\) may be improved or impaired by the presence of several si-multaneously operating enantioselective mechanisms [see 142,143]. A drawback of the separation technique based on a CMPA is that the presence of a chiral additive in the mobile phase may restrict the choice of detection systems and the labile diastereomeric complexes may have different detector response .