Enantioselective mobile phase modes

The use of antibiotic-based CSPs has been reported in capillary electrochromatography (CEC) for chiral resolution [60]. Teicoplanin CSP covalently bonded to silica gel was used to resolve the enantiomers of tryptophan and dinitrobenzoyl leucine by CEC [61]. Good levels of enantioselectivity were obtained with optimized separations. Vancomycin covalently bonded to silica gel was also evaluated in CEC for the chiral resolution of thalidomide and jS-adrenergic blocking agents under all the three mobile phase modes. The... 

Table 3. Mobile phase modes In enantioselective HPLC.

The enantioselectivity of the macrocyclic CSPs are different in each of the operating modes, probably because of different separation mechanisms functioning in the different solvent modes. The possible chiral recognition mechanisms for three mobile phase compositions on glycopeptide phases are listed in Table 2-3 in descending order of strength. 

As a general rule, in the case of CSPs featuring hydrophobic pockets, a decrease of mobile phase flow-rate results in an increase of chromatographic resolution (Rs), as a consequence of better stationary phase mass transfer [78]. This change has significant impact mostly in RP mode [17]. In the NP enantioselective separations of two test solutes (4-hexyl-5-cyano-6-methoxy-3,4-dihydro-2-pyridone and... 

Avery recent study [128] deals with the comparison of two commercially available vancomycin-based CSPs with different surface coverage of the chiral selector in the enantioseparation of P-blockers and profens, by RP and POM separation modes. Higher retention and better resolution were obtained on the CSP with higher coverage of vancomycin in both the separation modes. However, in the case of pro fens, higher retention was not always accompanied by an improvement of the enantioselectivity in the RP mode. An accurate study of the influence of the mobile phase composition was also performed in both the separation modes. 

Generally, CD-based chiral stationary phases have been used in the reversed-phase mode. Earlier, it was assumed that in the normal phase mode, the more nonpolar component of the mobile phase would occupy the CD cavity, thereby blocking inclusion complexation between the chiral analyte and CD [4,11], But with the development of CD derivatives, it has become possible to use the normal phase mode too [45,74], Among the various CSPs based on CD derivatives, one based on a naphthylethyl carbamoylated derivative has shown excellent enantioselectivity in the normal phase mode [46,59]. Armstrong et al. [45] synthesized several /CCD derivatives and had them tested in the normal phase mode to resolve the enantiomers of a variety of drugs hexane-2-propanol (90 10, v/v) served as the mobile phase. The authors discussed the similarities and differences of the enantioselectivities on the native and derivatized CD phases. 

Lammerhofer and Lindner [62] reported on the enantiomer separation of deriva-tized amino acids on an ODS-packed capillary with a chiral quinine-derived selector as buffer additive in two different modes (i) in an electrophoretically dominated mode at high electrolyte concentration and (ii) in an electroosmotically dominated mode at a low electrolyte concentration. Enantiomer separation in the electrophoretically dominated mode (i) leads to high efficieny (about two to three times higher than in LC) but to a moderate enantioselectivity (about the same as in LC). In the electroosmotically dominated mode (ii) a higher enantioselectivity but a lower efficiency (even inferior to LC) occurs. The separations can also been performed in a non-aque-ous buffered mobile phase. Pressurization (8-10 bar) of the flow system on both ends of the separation capillary was applied. 

The teicoplanin CSP (Chirobiotic T) exhibits enantioselectivity for underivatized and Mderivatized (FMOC or Z) amino acids, hydroxy carboxylic acids and other chiral acids including chiral phenols, small peptides, neutral aromatic analytes and cyclic aromatic and aliphatic amines [285] (see also Table 9.11). Selection of the mobile phase mcxle (reversed-phase, normal-phase, or polar-organic phase mode) follows the same criteria as described for vancomycin CSP. 

In this mode of separation, active compounds that form ion pairs, metal complexes, inclusion complexes, or affinity complexes are added to the mobile phase to induce enantioselectivity to an achiral column. The addition of an active compound into the mobile phase contributes to a specific secondary chemical equilibrium with the target analyte. This affects the overall distribution of the analyte between the stationary and the mobile phases, affecting its retention and separation at the same time. The chiral mobile phase approach utilizes achiral stationary phases for the separation. Table 1 lists several common chiral additives and applications. 

For enantioseparation on CSPs in CEC, nonstereospecific interactions, expressed as 4>K, contribute only to the denominator as shown in Eq. (1), indicating that any nonstereospecific interaction with the stationary phase is detrimental to the chiral separation. This conclusion is identical to that obtained from most theoretical models in HPLC. However, for separation with a chiral mobile phase, (pK appears in both the numerator and denominator [Eq. (2)]. A suitable (f)K is advantageous to the improvement of enantioselectivity in this separation mode. It is interesting to compare the enantioselectivity in conventional capillary electrophoresis with that in CEC. For the chiral separation of salsolinols using /3-CyD as a chiral selector in conventional capillary electrophoresis, a plate number of 178,464 is required for a resolution of 1.5. With CEC (i.e., 4>K = 10), the required plate number is only 5976 for the same resolution [10]. For PD-CEC, the column plate number is sacrificed due to the introduction of hydrodynamic flow, but the increased selectivity markedly reduces the requirement for the column efficiency. 

Tesarova and Bosakova [58] proposed an HPLC method for the enantio-selective separation of some phenothiazine and benzodiazepine derivatives on six different chiral stationary phases (CSPs). These selected CSPs, with respect to the structure of the separated compounds, were either based on b-CD chiral selectors (underivatized (J>-CD and hydroxypropyl ether (3-CD) or on macrocyclic antibiotics (vancomycin, teicoplanin, teicoplanin aglycon and ristocetin A). Measurements were carried out in a reversed-phase separation mode. The influence of mobile phase composition on retention and enantio-selective separation was studied. Enantioselective separation of phenothiazine derivatives, including levopromazine (LPZ), promethazine and thioridazine, was relatively difficult to achieve, but it was at least partly successful with both types of CSPs used in this work (CD-based and glycopeptide-based CSP), except for levomepromazine for which only the [CCD-based CSP was suitable. 

Chiral mobile phase additives provide a more versatile and cost-effective approach for enantiomer separations in thin-layer chromatography. Typically, chemically bonded layers with cyclodextrin and its derivatives, bovine serum albumin, or macrocyclic glycopeptides are used as chiral additives in the reversed-phase mode [59,60,172-178]. For [5- and y-cyclodextrins and their derivatives, a 0.1 to 0.5 M aqueous methanol or acetonitrile solution of the chiral selector is used as the mobile phase. Bovine serum albumin is generally used at concentrations of 1-8 % (w/v) in an aqueous acetate buffer of pH 5 to 7 or in a 0.5 M acetic acid solution, in either case with from 3-40 % (v/v) propan-2-ol (or another aliphatic alcohol), added to control retention. Enantioselectivity usually increases with an increase in concentration of the chiral selector, and may be non existent at low concentrations of the chiral selector. 

Figure 11 Chromatogram of the imprinted stationary phase. This imprinted polymer does work in aqueous solvents and chromatographic characterizations are performed using an aqueous mobile phase (25 mM sodium citrate, pH 3.0) containing 10% MeCN at a flow rate of 1 mL/min and chromatograms, run in isocratic mode and recorded at 280 nm. Injections of 20 luL of 2 mM racemic isoproterenol HCl (40 pmol) dissolved in the mobile phase are done in order to evaluate the enantioselectivity towards its imprinted print molecule isoproterenol. Eluent used was a sodium citrate buffer (pH 3.0, 25 mM citrate, 10% MeCN) flow rate 1 ml/min, peak detection at 280 nm, injection of 20 pL of a racemic isoproterenol hydrochloride solution (2 mM), acetone was used as void marker. The structures of + and - isoproterenol are given in Fig. 8.

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