Capillary electrokinetic fractionation (CEkF) is investigated as a new, simple, and robust approach for semipreparative and analytical sample analysis based on pK(a)-dependant pH-driven electrophoretic mobility. CEkF was optimized with contactless conductivity detection and conducted with 10 kV reverse voltage for 10 min, then coupled on/at-line to ESI/MS. We propose a semi-empirical model with 14 representative compounds based on the correlation between sample/medium pH regulating the partial charge, the electrokinetic loading of the capillary and intensity (I) of analytes. According to the model, an empirical function (I = f (pH)) could be derived to calculate the acid dissociation constant (pK(a)) of various model compounds based on their pH-dependant MS intensity profiles with the RSD < 4.05. Using the ultrahigh-resolution of ion cyclotron resonance Fourier transform MS, the pK(a) model was further illustrated in real samples into the structure prediction of important compounds in wine over two vintages. The established CEkF was successfully used to selectively fractionate sulfur compounds from the complex wine samples at pH 1.66. The proposed CEkF approach should allow in the future the simultaneous pK(a) evaluation of multiple constituents without complicated separation out of a complex mixture in metabolomics or environmental chemistry.