On-line dynamic titration: determination of dissociation constants for noncovalent complexes using Gaussian concentration profiles by electrospray ionization mass spectrometry.

A new method for determination of dissociation constants (Kd) using on-line titration by electrospray ionization mass spectrometry is presented. Unlike in common titration experiments, where a set of discrete solutions with a fixed concentration of host and increasing concentration of guest is measured, here a continuous Gaussian concentration profile of guest, formed by band-broadening dispersion during passage through a long tubing, is utilized. An equation allowing access to dissociation constant values from experimental data fit to a 1:1 binding model was derived and incorporated into an in-house-written computer program for automated data processing. The new method is demonstrated for noncovalent complexes of cinchona alkaloid carbamate chiral selectors with N-dinitrobenzoylleucine enantiomers and a series of cyclodextrins with sulfonated azo dyes.