Measurement of Binding Constants by CE

This linearized form allows the straightforward determination of binding constants from the slopes of plots of effective mobilities vs. the term (ma — Meff )/ct. (Note The mobility of the free tBuCQN selector ma can be easily measured without additive and conversely, Meff of the selector is measured at various DNB-Leu concentrations.) The intercepts of such plots represent the effective mobilities of the complex, a figure 

It becomes obvious from the clustered data points that the binding constant for the 7 -enantiomer is too small to be accurately determined by this method. Hence, indirect affinity CE (resolution method) was utilized to determine the binding constant for the 7 -enantiomer. Indirect affinity CE makes use of the knowledge of the constant for one enantiomer (here, A s) and in addition of experimental separation data as obtained with the racemate of DNB-Leu in presence of the tBuCQN selector as BGE additive. By use of Equation 1.10, an enantioselectivity factor may be defined as the ratio of the binding constants of S- and 7 -enantiomers yielding the following equation  

Mobilities of free S- and 7 -enantiomers of DNB-Leu (u) are identical (readily measurable in absence of the tBuCQN additive, which gave under given conditions 

Finally, the effective mobilities Metr for R- and 5-enantiomers of DNB-Leu, at any concentration of tBuCQN as additive, have been determined accurately by the so-called constant time method [88], For example, at 5 mM tBuCQN in the BGE the effective mobilities were Meg(5) = —6.79 0.02 x 10 m V s and UesiR) = -12.15 0.07 x 10 m V s , which after substitution into Equation 1.12 gave Ks/Kr = 49.8 and thus a binding constant Kr of 2.5 Lmol  

Overall, it is seen that the binding constants in presence of counterions (total buffer concentration of 20 mM) are lower by a factor of about 10 than in a plain methanolic medium without counterions (see Table 1.4). In contrast, the intrinsic enantioselecitivity is still in the same order of magnitude or even higher (A s/A r 17 by ITC and ca. 50 under CE conditions) because of a more proper balance of the otherwise dominating ionic interaction. 

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