Selectivity with Chiral Mobile-Phase Additives

Two mechanisms foe chiral separations using chiral mobile-phase additives, analogous to models developed for ion-pair chromatography, have been proposed lo explain the chiral selectivity obtained using chiral mobile-phase additives. In one model, the chiral mobile-phase additive and the analyle enamiomers form diaslereomeric complexes in solution. As noted previously, diaslereomers may have slightly different physical properties such as mobile phase solubilities or slightly different affinities for the stationary phase. Thus, the chiral separation can be achieved with conventional columns. 

The numerator in equation (22-26) represents the processes occurring in the mobile phase, while the denominator represents the processes occurring in the stationary phase. Such a situation can be realized by combining a chiral stationary phase in a push-pull mode with a chiral mobile phase of opposite con-hguration, where two enantiomers of the chiral selector are involved, one for the chiral stationary phase and the other for the chiral mobile phase. The most selective chiral chromatographic system should be encountered when one enantiomer binds to the immobilized chiral selector in the stationary phase, whereas the other enantiomer predominantly associates with the chiral mobile-phase additive [158]. The above treatment is applicable to all applications regarding the use of chiral mobile phases. 

As mentioned above, enantioseparations in EKC rely on a chromatographic separation principle. Despite this fact, there are significant differences between these techniques. Responsible for all differences between chromatographic and electrophoretic enantioseparations is the property of the electrophoretic mobility to be selective for the analytes residing in the same physical phase [2]. Another important point is that in chromatographic techniques, except in the case of a chiral mobile phase additive (CMPA), the analyte is virtually immobile when associated with a chiral selector. In EKC the analyte selector complex is commonly mobile. 

An enantioseparation in this mode is based on the formation of non-covalent diastereomer-ic complexes between the enantiomers of an analyte and the chiral additive in the mobile phase (CAMP). Compared with indirect enantioseparations, the CAMP technique has advantages such as the absence of a derivatization step or a higher flexibility (easier change of a chiral additive than a chiral or an achiral packing material). As documented by Davan-kov [105], the enantiomer migration order with CAMP most likely will be opposite to that observed with the same chiral selector as the stationary phase. The complementary enantio-selectivity of enantioseparation with CAMP compared with CSPs is a significant advantage. 

ABSTRACT. The copper(n) complexes of p-cyclodextrins functionalized with aliphatic or pseudoaromatic amines were used for the chiral recognition of unmodified amino acids. Molecular recognition, assisted by non-covalent interactions, was proved by means of thermodynamic and spectroscopic (c.d., e.p.r. and fluorescence) measurements. A cis-disposition of amino groups seems to assist enantiomeric selectivity. The copper(II)-p-cyclodextrin complexes can be used as mobile phase additives in HPLC to separate enantiomeric mixtures of unmodified aromatic amino acids. 

Typical NP conditions involve mixtures of n-hexane or -heptane with alcohols (EtOH and 2-propanol). In many cases, the addition of small amounts (<0.1%) of acid and/or base is necessary to improve peak efficiency and selectivity. Usually, the concentration of alcohols tunes the retention and selectivity the highest values are reached when the mobile phase consists mainly of the nonpolar component (i.e., n-hexane). Consequently, optimization in NP mode simply consists of finding the ratio n-hexane/alcohol that gives an adequate separation with the shortest possible analysis time [30]. Normally, 20% EtOH gives a reasonable retention factor for most analytes on vancomycin and TE CSPs, while 40% is more appropriate for ristocetin A-based CSPs. Ethanol normally gives the best efficiency and resolution with reasonable backpressures. Other combinations of organic solvents (ACN, dioxane, methyl tert-butyl ether) have successfully been used in the separation of chiral sulfoxides on five differenf glycopepfide CSPs, namely, ristocetin A, teicoplanin, TAG, vancomycin, and VAG CSPs [46]. 

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