Interactions with Chiral Mobile Phases

The enantiomeric separation with chiral mobile phases consists of the addition of an active compound in the mobile phase which is constantly pumped though the chromatographic system. The active ingredient contributes to a specific secondary chemical equilibrium, interacting with the enantiomers in the mobile phase as well as in the stationary phase, leading to the formation of diastereomeric complexes potentially in both phases. This affects the overall distribution of the analyte between the stationary phase and the mobile phase, affecting its retention and the overall enantiomeric separation. The rates of formation of the diastereomeric complexes should be similar to the diffusion rates to minimize excessive chemical contribution to the band-broadening. 

For solutes meeting the requirements for use with a standard or derivatized p-CD CSP, the NEC-p-CD CSP can be used with aqueous mobile phases modified with acetonitrile or another organic modifier. Since chiral recognition is a function of the interaction of the solute with the chirality of the CD as a whole, the configuration about the stereogenic... 

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. 

CMPA-based enantiomer separation techniques appear attractive from the viewpoints of conceptual simplicity and flexibility as they operate with relatively inexpensive achiral stationary phases and easy-to-prepare chiral mobile phases. In practice, the development of CMPA-based analytical assays may pose a considerable challenge for a number of reasons. In the course of the development and optimization of CMPA-based enantiomer separations a set of conditions must be identified that favor CMPA-analyte interactions and simultaneously maximize... 

A second approach to Isomer separation by HPLC Is to use a non-optlcally active stationary phase and an optically active solvent. If the amino acids can Interact with both the stationary and mobile phases, but one of the Isomers Interacts more strongly with the mobile, optically active phase, separation of the Isomers Is possible (49). In 1979, several laboratories reported methods Involving the use of chiral mobile-phases containing zlnc(II) or copper (II) complexed to an L-amlno acid (51-53). A distinct advantage of these methods Is that they do not require derlvatlza-tlon of the sample prior to analysis. However, separation of a complete mixture of amino acids (such as that obtained from a protein hydrolysate) has not been reported. 

Each enantiomer carried by the mobile phase interacts in a different manner with the stationary phase which contains an enantiomerically pure chiral element. There again, it is diastereomeric interactions that are translated into different elution rates through the column. In principle, whatever the detector (ultraviolet-visible spectrometer, refractometer, etc.) the response factor is identical for the two enantiomers being analysed. As a result, integration of the peak areas corresponding to each enantiomer leads to a measure of the ee by the simple relationship ee = (Si — S2)/(Si + S2) (Figure 2.66). 

Nonpolar organic mobile phases, such as hexane with ethanol or 2-propanol as typical polar modifiers, are most commonly used with these types of phases. Under these conditions, retention seems to foUow normal phase-type behavior (eg, increased mobile phase polarity produces decreased retention). The normal mobile-phase components only weakly interact with the stationary phase and are easily displaced by the chiral analytes thereby promoting enantiospecific interactions. Some of the Pirkle-types of phases have also been used, to a lesser extent, in the reversed phase mode. 

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