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Temperature may influence both retention and selectivity  and must be controlled. Because, often, only minor effects are needed to improve an insufficient resolution or to change retention, temperature is a useful variable. The pH of the mobile aqueous phase can be varied between 2 and 7 and will affect both retention and selectivity. A lower pH will elute bases earlier, as in reversed-phase liquid chromatography. The choice of pH buffer components may also influence the elution of both the acid anion and the cation . Reten-tion by ion-exchange and ion-pair formation has been suggested, in addition to chiral recognition [64,78]. Therefore, charged additives may displace analytes of the same charge and increase retention of analytes of opposite charge; as in reversed-phase chromatography. Charged organic modifiers may have a significant effect on the enantioselectivity, as shown both for cationic and anionic solutes [51,52] and illustrated in Fig 3.
Uncharged organic modifiers, such as propanols and acetonitrile, are used to regulate retention. For hydrophilic or moderately hydrophobic compounds low, if any, content is
Pettersson and Persson
S-2-phenylbutyric acid (mM)
Figure 3 Influence by addition of (S)-2-phenylbutyric acid on capacity factors for the enantiomers of atropine (?, H) and homatropine (A, A) on CHIRAL-AGP. (From Ref. 64.)
needed, whereas for hydrophobic calcium channel blockers, 20% or more organic modifier has been added. Besides the effect on retention, selectivity will naturally be influenced, for it must be anticipated that the chiral recognition structure of the protein will be affected by most additives. Gradient elution may be advantageous in terms of peak sharpness and is preferably performed by a successive increase of the content of the organic modifier . However, a pH gradient may be as useful if the pH buffer capacity of the stationary phase is taken into account (Fig. 4). The application of the AGP phases to enantioselective assay of drugs in
Figure 4 Influence on pH by isocratic and gradient elution of metoprolol enantiomers on CHIRAL-AGP: (a) pH 7.5, (b) pH 6.40, (c and d) pH-gradients. (From Ref. 80.)
Chiral Pharmaceutical Analysis
biological material often demands careful sample preparation and selective detection (e.g., by fluorescence) if low concentrations have to be measured . Coupled column systems have also been employed . Guard columns and prevention of particles from entering the packing will prolong the life of the column and, at least a few hundred, biological extracts can be run provided precautions are taken.
C.Ovomucoid-Bonded Stationary Phase
Ovomucoid protein-based chiral columns have shown about the same application field in drug analysis as the AGP-phase. That means these columns can be tested and used for most kinds of drugs whether basic, neutral, or acidic . Drugs with amino function, such as verapamil [84-86] or chlorpheniramine [87-89]; /Ç-adrenoceptor-blocking agents, such as oxprenolol, pindolol, propranolol [83,90-91], and homochlorocyclizine , have been resolved on Ultron ES-OVM, which is the trade name for the ovomucoid column. Neutral drug compounds are represented by warfarin , oxazepam , nimodipine, nicardipine [93,94], econazole , and omeprazole ; and acidic ones by mephenytoin, thalidomide, hexobarbital, ibuprofen, ketoprofen; and ketorolac [87-89,96]
The enantiomeric resolution on the ovomucoid column is greatly influenced by pH of the mobile aqueous phase. Basic drugs are most often separated at neutral pH, whereas acids are run at pH 4-5. The content of an organic modifier (e.g., methanol, acetonitrile, or propanol) is used to regulate retention and may also influence the resolution of an enantiomeric pair. Certain hydrophobic drugs, such as dihydropvridine calcium channel blockers, have strong affinity for the ovomucoid phase, and a high concentrations 25-30% of the organic modifier will be required for reasonable retention times [93,94]. In other respects there are similarities, but also differences, between the performance of the ovomucoid and AGP chiral phases, not the least of which is the enantioselective properties. Verapamil [84,85] and propranolol  have been assayed in biological samples using column-switching during which the ovomucoid-bonded phase has enabled the enantioselective separation. Drug enantiomers of chlorpheniramine, ketoprofen, and oxazepam in serum were injected directly on to this kind of stationary phase .
D. Serum Albumin Phases
Bovine serum albumin (BSA), was the first protein to be immobilized on silica and used for enantiomer separation. It was introduced as a stationary phase by Allenmark and co-workers in 1982-1983 [97,98]. In recent years, human serum albumin (HSA) has also been covalently bonded to silica and used as a chiral stationary phase . The albumin phases have found use for enantioselective separation of acidic and neutral compounds; however, these phases are not as well suited for basic drugs with aliphatic amino function. Ketoprofen and suprofen are carboxylic acids that have been resolved  and, among neutral compounds, warfarin , methaqualone , oxazepam and other benzodiazepinones [102,103], tryptophan , and sulfoxide-substituted benzimidazoles, such as omeprazole , will illustrate the applicability of this kind of chiral stationary phase in the drug field. The resolution of ome-prazole on an albumin phase and two other protein columns is shown in Fig. 5.