Mobility difference between enantiomers

In general, charged CDs have shown superior discrimination abilities, especially the highly-sulfated (HS-CDs) ones. Furthermore, the separation mechanism is altered by the introduction of electrostatic interactions. Finally, the use of chiral selectors carrying a charge opposite to that of the analytes can greatly improve the mobility difference between the two enantiomers. The use of mixtures of CDs in chiral separation is also possible. ... 

In order to find the concentration at which the mobility difference between two enantiomers reaches a maximum, one must differentiate Eq. (17) according to the concentration, i.e., to find a partial differential 3 AijlrsI d[C], After differentiating Eq. (17) and simplification of the obtained result, one may obtain the equation that relates the maximal mobility difference between the enantiomers to the concentration of a chiral selector as follows ... 

Thus, based on Eq. (18) it becomes possible to determine the concentration of the chiral selector resulting in a maximal mobility difference between the enantiomers, assuming that the binding constants of both enantiomers with chiral selector are known. This method for determination of the optimal CD concentration has been used by several groups. 

Analyzing Eq. (1), one realizes that when the requirement Kk + Kg is not necessary for obtaining A/x 0. This means that in CE, a chiral recognition in the classical meaning of this definition Kk + Kg) is not always necessary for a chiral separation. This is theoretically feasible but has not been undoubtedly evidenced yet experimentally. More common is the case when ixqr = ixc,s and then Kk + Kg is the necessary requirement for enantioseparation. A combined contribution of both a stereoselective binding of the enantiomers to a chiral selector Kk Ks) and a mobility difference between the transient di-astereomeric complexes ixqr + mc,s) is also possible. 

Another difference between enantioseparations in EKC and HPLC is the fact that an enantioseparation is, in principle, feasible in EKC even in the case when the binding constants of the enantiomers with the chiral selector are identical [2, 3, 7], This conclusion can be made from Eq. (3). According to this equation, for the generation of a mobility difference between the enantiomers, e.g., an enantioseparation in EKC, the following are required ... 

The model described in [14,179] allows to optimize the concentfation of a chiral selector which may result in a maximum mobility difference between the enantiomers. Although the model proposed by Wren and Rowe allows optimizing only one separation parameter, in particular the concentration of a chiral selector, among many variables, it attracted much attention by the researchers perhaps most likely... 

Two critical points should be mentioned when applying the aforementioned model for optimization purposes in chiral CE (1) the maximum mobility difference between the enantiomers does not a priori mean the maximum resolution and (2) the model does not cover several important parameters affecting chiral CE separations. However, this model without any doubt markedly contributed to the development of chiral CE and good correlations between the values of the optimal chiral selector concentrations calculated based on this model and observed experimentally have been reported [182-185]. 

In recent years, for analytical purposes the direct approach has become the most popular. Therefore, only this approach will be discussed in the next sections. With the direct approach, the enantiomers are placed in a chiral environment, since only chiral molecules can distinguish between enantiomers. The separation of the enantiomers is based on the complex formation of labile diastereoisomers between the enantiomers and a chiral auxiliary, the so-called chiral selector. The separation can only be accomplished if the complexes possess different stability constants. The chiral selectors can be either chiral molecules that are bound to the chromatographic sorbent and thus form a CSP, or chiral molecules that are added to the mobile phase, called chiral mobile phase additives (CMPA). The combination of several chiral selectors in the mobile phase, and of chiral mobile and stationary phases is also feasible. 

The existence of the aforementioned difference between the mobilities of transient diastereomeric complexes of the enantiomers with the chiral selector may have some important consequences in chiral CE. For instance, the enantioseparation can, in principle, be possible even in those cases when the binding constants of both enantiomers to a given chiral selector are the same. On the other hand, this may allow, in certain cases, observation of the reversal of the enantiomer migration order, depending on the concentration of the chiral selector (17). 

The effect of surface polarity is even more important in separations where two or more simultaneous interactions must occur in order to achieve the desired selectivity. This is particularly true in chiral separations. Since aqueous buffer systems are almost universally used as CEC mobile phases, enantioseparations are often run under re-versed-phase conditions as opposed to the normal-phase mode typically used in chiral HPLC. Therefore, non-specific hydrophobic interactions would be highly detrimental to the discrimination process that involves subtle differences between the enantiomers. 

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. 

Chiral separation can be observed when there is a suitable difference between free energies (AG) of diastereomeric associates formed by R- and S-enantiomers of selectand and a chiral selector (SO). The energy differences can be directly attributed to the chiral recognition phenomena, involving complementarity of size, shape, and molecular interaction of SO and SA molecules. These factors are also controlled by experimental conditions such as temperature, mobile phase... 

The conventional C-18 and the CD columns do interact differently with solutes of certain classes of isomers. For example, C-18 columns cannot separate enantiomers unless special additives are introduced into the mobile phase. The cyclodextrin bonded phases, however, can easily separate enantiomeric species as illustrated in Figure 9. The D and L enantiomers of dansyl-DL-leucine and of dansyl-DL-norleucine are resolved using a beta-CD column, but attempts to separate these isomers were unsuccessful using a C-18 column. The nature of the interactions between enantiomers and the cyclodextrin cavity has been described elsewhere (20. 21). 

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 . 

Contrary to above-mentioned assumption, a few earlier studies indicated that the mobilities of the diastereomeric associates of two enantiomers with the chiral selector are not always equal to each other." In 1997, a theoretical assumption was made that two enantiomers can be resolved in CE even when their association constants with the chiral selector are equal to each other (ATr = Ks = Th prerequisite for enantiomer separation in this particular case is the non-zero difference between the mobilities of temporary diastereomeric associates (Mcr Mcs)- Under these conditions Eq. 1 transforms toEq. 3." " ... 

In chiral chromatography, the two diastereomeric adducts ArEr and ArEs are formed during elution, rather than synthetically, prior to chromatography. The adducts differ with respect to their stability in the use of chiral stationary phases (CSPs) or chiral-coated stationary phases (CCSPs) and/or in their interphase distribution ratio with the addition of a chiral selector to the mobile phase (CMP). The difference between the interactions of the chiral environment with the two enantiomers is called enantioselectivity. 

Enantiomeric compounds R and S may be separated by capillary electrophoresis by complexing them with a chiral selector compound B. Here the equilibrium binding constants for the R and S enantiomers are, respectively, Xr and Ks (in a molar concentration based expression). Develop expressions for the effective mohUity of the R and S species, assuming that the complexes continue to have the same charge as the uncomplexed compounds. Obtain an expression for the mohUity difference between the effective mobilities of the R and S species. What contributes to a nonzero value of this mohUity difference ... 

For many years the elecBophoretic mobility of analyte (/rep) was considered to be a selective transport able to differentiate between enantiomers, while the electroos-motic mobility (/reo) was considered to be a non-selective transport. This is not correct for chiral EKC, although it applies without any limitation for true electrophoretic separations, i.e., for the separations which are based on a different electric charge density of the sample components [2],... 

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