A Method for the Measurement of Site-Specific Tautomeric and Zwitterionic Microspecies Equilibrium Constants

We describe a method for the individual measurement of simultaneously occurring, unimolecular, site-specific “microequilibrium” constants as in, for example, prototropic tautomerism and zwitterionic equilibria. Our method represents an elaboration of that of Nygren et al. (Anal. Chem. 1996, 68, 1706−10), which thereby becomes generalized and improves the accuracy. Specifically, by making spectral measurements as a function of temperature, we demonstrate the ability to determine site-specific microenthalpies unambiguously. Analysis proceeds via multivariate nonlinear regression modeling of, for example, the Gibbs−Helmholtz relation. Additional determinations of macroscopic equilibrium constants as a function of temperature, in combination with the previously determined microenthalpies, in turn enables the determination of all remaining site-specific thermodynamic parameters, i.e., microentropies, micro free energies, and/or microequilibrium constants, and moreover allows us to resolve and measure the spectra of tautomeric isocoulombers. To our knowledge, we have hereby devised the first such universally applicable and accurate measurement method on record.