Thermodynamic Fundamentals of Enantioselective HPLC

As discussed above, chiral recognition in HPLC (and all other kinds of enantioselective chromatography) rehes on the formation of intermediate diastereomeric complexes. For a racemic sample (identical amounts of (R)- and (S)-enantiomers), the two association constants (K and KjJ have to be different in order to observe an enantiomer separation (see Fig. 3) [3]. Both enantiomers are in solution and can interact with the selector, which is attached to the silica. If, for example, the complexation constant is higher for the (S)-enantiomer, it is bound more strongly to the chiral stationary phase and is eluted later. 

Since the capacity factor of a compotmd is connected via the phase ratio with the association constant (Eqs. 4 and 5), the ratio of the retention factors k of the two enantiomers can be used to determine the ratio of the complexation constants (Eq. 6). 

This ratio allows estimation of the difference in free energy of the enantiomer separation, - AAGg r, and provides an insight into the order of magnitude of the interaction energies (see Fig. 4).  

The interaction of a chiral analyte with a selector requires a temporary adsorption of the enantiomers on the surface of the stationary phase (see Fig. 4). Different models have been proposed to explain the reversible formation of energetically different quasi-diastereomeric molecular associates however, none of them can explain all of the observed retention mechanisms. Association and dissociation constants for enantiomers interacting with a CSP can be different, since the rates at which the diastereomeric associates are formed can differ. Analogously, the dissociation rates can be different 

If the interaction of one of the enantiomers is only small, it will not be adsorbed and, in extreme cases, can be eluted within the void time. However, in most cases both enantiomers show some retention, requiring an equilibrium of adsorption and desorption. In some cases, one might observe that one or both of the enantiomers are strongly adsorbed and are not eluted during the run time of the 

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