Electrophoretic mobility chiral selectors

In a solution without chiral selectors, enantiomers cannot be distinguished from each other through their electrophoretic mobility. Separation can, however, be achieved when the buffer solution contains certain chiral compounds. The chiral compounds used to distinguish enantiomers are referred to as selectors. 

The separation mechanism is based on stereoselective ion-pair formation of oppositely charged cationic selector and anionic solutes, which leads to a difference of net migration velocities of the both enantiomers in the electric field. Thus, the basic cinchona alkaloid derivative is added as chiral counterion to the BGE. Under the chosen acidic conditions of the BGE, the positively charged counterion associates with the acidic chiral analytes usually with 1 1 stoichiometry to form electrically neutral ion-pairs, which do not show self-electrophoretic mobility but... 

Thus, as shown earlier, CE represents a suitable technique for the determination of enantioselective binding constants between chiral drugs and cyclodextrins. The results obtained under appropriate conditions are reasonable and can be applied for optimization purposes as well as for a better understanding of the fine nuances of chiral CE separations. On the other hand, some care must be taken for the proper application of CE methods for the determination of the binding constants as well as when applying these data. A critical review of the calculation of stability constants for the chiral selector-enantiomer interactions from electrophoretic mobilities has been published by Vespalec and Bocek (40). 

R Vespalec, P Bocek. Calculation of constants for the chiral selector-enantiomer interactions from electrophoretic mobilities. J Chromatogr A 875 431-445, 2000. 

Chankvetadze et al. have demonstrated the potential of flow-counterbalanced capillary electrophoresis (FCCE) in chiral and achiral micropreparative separations [27], Unlimited increase of separation selectivity can be achieved for binary mixtures, such as (+ )-chlorpheniramine with carboxymethyl-(3-cyclodextrin chiral selector, or a- and (3-isomers of a asparatame dipeptide. The carrier of the chiral selector or pseudo-stationary phase, electroosmotic flow (EOF), pressure-driven flow, or hydrodynamic flow can be used as a counterbalancing flow to the electrophoretic mobility of the analyte or vice versa, resulting in dramatic changes of the effective mobilities of the sample mixture components [28], This approach can be used for micropreparative CE, stepwise separations, and fraction collection of multicomponent mixtures [27],... 

The first electrically driven enantioseparations involved the addition of a chiral selector to the mobile phase in CE. This selector is usually a complexing agent and acts as a pseudo-stationary phase. The separation is accomplished by the difference in the distibution equilibria between the pseudo-stationary phase and the enantiomers [134], The most common additives incorporated into these CE experiments were cyclodextrins and cyclodextrin derivatives [135-138], However, these experiments required the replacement of the chiral selector after each electrophoretic run. 

Additional substances (buffer additives) are often added to the buffer solution to alter selectivity and/or to improve efficiency, and the wall of the capillary may be treated to reduce adsorptive interactions with solute species. Organic solvents, surfactants, urea and chiral selectors are among the many additives that have been recommended (table 4-24). Many alter or even reverse the EOF by affecting the surface charge on the capillary wall, whilst some help to solubilize hydrophobic solutes, form ion-pairs, or minimize solute adsorption on the capillary wall. Chiral selectors enable racemic mixtures to be separated by differential interactions with the two enantiomers which affects their electrophoretic mobilities. Deactivation of the capillary wall to improve efficiency by minimizing internet ions. with solute species can be achieved by permanent chemical modification such as silylaytion or the... 

Conceptually. CE enantioseparations are mainly applied to charged SAs. Micellar electrokinetic chromatography (MEKC) (introduced by Terabe et al. in 1984 488 ), in contrast, permits the separation of electrically neutral compounds. In enantiomer separation by MEKC. ionic pseudo-stationary phases, such as chiral micelles composed of chiral SO moieties, which migrate according to their electrophoretic mobility, may interact stereoselectively with the solutes to be separated. MEKC with synthetic (e.g. A-dodecoxycarbonylvalines, commercialized as SDVal by Waters) 1489.490) or naturally occurring chiral surfactants (e.g. bile salts) 1491-494). and cyclodextrin-moditied MEKC (most often SDS/CD combinations) 1495-498) are the mo.st widely used selector systems in MEKC. The topic of MEKC enantioseparation has been reviewed by Nishi )499). 

The first applications of CDs as chiral selectors in CE were reported in capillary isotachophoresis (CITP) [2] and capillary gel electrophoresis (CGE) [3]. Soon thereafter, Fanali described the application of CDs as chiral selectors in free-solution CE [4] and Terabe used the charged CD derivative for enantioseparations in the capillary electrokinetic chromatography (CEKC) mode [5]. It seems important to note that although the experiment in the CITP, CGE, CE, and CEKC is different, the enantiomers in all of these techniques are resolved based on the same (chromatographic) principle, which is a stereoselective distribution of enantiomers between two (pseudo) phases with different mobilities. Thus, enantioseparations in CE are commonly based on an electrophoretic migration principle and on a chromatographic separation principle [6]. 

The EOF contributes significantly to the mobility of analytes in CE. The EOF is considered to be a nonse-lective mobility. However, for enantiomers, both the EOF and the electrophoretic mobility of the analyte are inherently nonenantioselective. The stereoselective analyte-selector interactions may turn both of these mobilities into a selective transport with equal success. This is the principal difference between the roles of the EOF in true electrophoretic separations and in chiral CE separations. 

A different but experimentally simpler solution is to use a chiral selector (called often in such a case chiral additive) dissolved directly in the mobile phase. The diastereomeric associates thus formed differ in their mobility or adsorption, and in favorable cases they can be separated on an achiral phase. This approach was applied in the 1980s by Pettersson et al. who used native cinchona alkaloids in ion-pairing chromatography [ 51 ]. N owadays, ion-pairing technique is of considerable interest in a number of electrophoretic techniques (see below). 

Chiral Additive A chiral selector added as a component of a mobile phase or electrophoretic medium. 

At equilibrium, the effective mobility of an enantiomer in the presence of a chiral selector in capillary electrophoresis is the sum of the electrophoretic mobility of each species containing the enantiomer, weighted by the mole fraction of each species. Assuming for a given separation the enantiomers exist as either the free enantiomer with a mobility, ixr, or the enantiomer-chiral selector complex, p,c,R, then the effective mobility for the enantiomer is given by... 

While the migration principle, i.e., the driving forces moving the analytes through the separation capillary, is based on electrophoretic mechanisms the chiral separation is based on enantioselective interactions between the analyte enantiomers and a chiral selector and is, therefore, a chromatographic separation principle. The fact that the selector is in the same phase as the analytes in CE and not part of a stationary phase that is immiscible with the mobile phase as found in chromatography does not represent a conceptional difference between both techniques. The chiral selector in CE is also called pseudophase as it is not a physically different phase and may also possess an electrophoretic mobility. Enantioseparations in CE have also been termed capillary electrokinetic chromatography . 

Chiral separation is one of the major outstanding advantages for CE compared to other separation techniques. As enantiomers have identical electrophoretic mobilities, some chiral complexing reagents, called chiral selectors, must be added to the separation buffer to form diastero-meric complexes in dynamic equilibrium. One of the most... 

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