In HPLC enantioseparations

This review provides an overview of the literature published to date on macrocyclic antibiotics exploited for enantioselective separations in high-performance liquid chromatography (HPLC). It was not intended as a comprehensive issue on the applications of such antibiotics in sub- and supercritical fluid chromatography (SFC), thin layer chromatography (TLC), capillary electrophoresis (CE), and capillary electrochromatography (CEC). A number of structural properties of the most important macrocyclic antibiotics applied in HPLC enantioseparations are listed in Table 2.1. 

Structural Properties of the Most Important Macrocyclic Antibiotics Applied in HPLC Enantioseparations... 

CyDs have been used as major chiral mobile phase additives (CMPAs) for enantio-separations in HPLC. The first application of 8-CyD as a CMPA in combination with an achiral reversed-phase material for HPLC enantioseparations was reported by Sybilska and co-workers in 1982 [27]. These authors could achieve partial resolution of the enantiomers of mandelic acid and derivatives. The CMPA method played an important role in HPLC enantioseparations before the development of effective chiral stationary phases (CSPs) but is now rarely used. The major disadvantage of this technique, together with difficulties associated with the isolation of resolved enantiomers, is the rather large consumption of chiral selector. 

Lindner W. Recent development in HPLC enantioseparation—a selected review. Chromatographia 1987 24 97-107. 

In HPLC enantioseparation, the mobile phase not only provides the control of eluting time for analytes but also acts in a specific way on the enantiorecognition process. For certain CSPs, the mobile phase provides the particular environment in which recognition can be produced. This fact has promoted the popularization of elution practices alternative to those most commonly used in conventional HPLC. Again there are not universally applicable conditions. On the contrary, the choice of mobile phase composition often depends on the kind of CSP used. 

Another common practice in HPLC enantioseparation is the use of organic solvents of low polarity as mobile phase components. Although, from the point of view of lipophilic-ity, the organic material bonded or coated onto the chromatographic matrix makes CSPs similar to C8, C18, or phenyl standard stationary phases, normal phase mode is often preferred over reversed-phase conditions. The use of a lipophilic solvent in a lipophilic environment favors dipolar interactions such as hydrogen bonding, dipole-dipole interactions, and ir-stacking, while nonselective van der Waals interactions are minimized. As a result, the selective association CS-enantiomer is favored. 

Note that not all enantioseparations in SFC are better than in HPLC [34], Bernal et al. [62] described the enantiomeric separation of several pharmaceutical-related compounds on a polysaccharide-based column using HPLC and SFC. They showed that most of the separations obtained by SFC are better, in terms of resolution and analysis time, than the separations obtained by HPLC. However, one compound could not be resolved using SFC, but LC provided baseline resolution. 

Since one or more of the interactions in these systems might originate from the stationary phase, only a two- or a one-point interaction between the solute and the selector is necessary for mechanisms (2) and (3) to occur [50]. However, some of the CMPAs used in HPLC [37,40,51,52] have also been used as chiral selectors in CE [53-56], which indicates that at least one of the separation mechanisms between the selector and enantiomers is selective complex formation in the mobile phase in these cases, since there is no stationary phase present in CE. A recent example by Yuan et al. [57] is presented in Eigure 17.1. The authors introduced the use of (R)-A,A,A-trimethyl-2-aminobutanol-bis(trifluoromethane-sulfon)imidate as the chiral selector for enantioseparation in HPLC, CE, and GC. This chiral liquid serves simultaneously as a chiral selector and a co-solvent. 

H. Kaga, Precision synthesis of (1 —> 6)-o -D-glucopyranan by cationic ring-opening polymerization of l,6-anhydro-tri-0-allyl-/8-D-glucopyranose, Macromol. Symposia, 181 (2002) 101-106 (b) A. Kusuno, M. Mori, T. Satoh, M. Miura, H. Kaga, and T. Kakuchi, Enantioseparation properties of (1 - 6)- -i)-glucopyranan and (1 - 6)-a-D-mannopyranan tris(phenylcarbamate)s as chiral stationary phases in HPLC, Chirality, 14 (2002) 498-502. 

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. 

Figure 4. Enantioseparations of propranolol in HPLC using capillary column (100 pm x 25 cm) packed with Chiralcel-OD material [120].

Direct HPLC enantioseparation techniques, which are free of many disadvantages of GC, indirect and chiral mobile phase HPLC methods, have gained unequivocal prevalence in bio-analytical studies. Several methods have been advanced so much that they allow enantiose-lective determination not only of the parent chiral drugs but also of their pharmacologically relevant metabolites [121]. As already mentioned above, a direct injection of biofluids offers several advantages in terms of analysis time and sample recovery. Precolumns packed with achiral or chiral packings, or with the recently developed so-called restricted-access packing materials, may be useful in this case. 

In addition to the miniaturization of HPLC enantioseparations, another current trend occurs in the opposite direction, namely the scaling-up of separations. The techniques of preparative-scale enantioseparations using liquid chromatography (LC) are described below. 

CyD-based columns for HPLC enantioseparations are well established and several tens of these columns are commercially available at present. Although highly suitable for analytical-scale enantioseparations these columns hardly compete with polysaccharide and macrocydic antibiotic-based CSPs for preparative-scale enantioseparations. More research is needed in this area. 

Although native CDs show acceptable enantioselectivity toward a large number of enantiomer pairs in many HPLC systems, their separation capacity is not always enough for the baseline separation of enantiomers, which is the prerequisite for reliable quantitative analysis. To overcome this difficulty, a considerable number of CD derivatives were synthesized and their separation capacities were tested using enantiomer pairs that are not well separated by native CDs. Unfortunately, the theoretical basis of the inclusion formation of new CD derivatives and the effect of new inclusion complexes on the enantioseparation of a given isomer pair are not well understood. This type of theoretical investigation is urgently needed for further development of the application of CDs and CD derivatives in HPLC. 

The reversal of enantiomeric elution order for the polysaccharide CSP was first reported by Okamoto et al. in 1991. They found that the reversal of the elution order of the enantiomers on a modified cellulose column was associated with changes in the mobile phase modifiers during the investigation of the direct chromatographic enantioseparation of pyriproxyfen, an insect growth regulator. If one can find such phenomena, although very rare in HPLC, it will be important to understand the reasons for this behavior and to anticipate when such inversions of elution order are likely to occur. 

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