Electrochromatography is a form of gradient liquid chromatography in which an axial electric potential is applied to columns packed with gel-filtration media. Experimental methodology and a mechanistic model are further developed for a system that minimizes Joule heating at electric field strengths of 100 V/cm by dissipating heat through a cooling jacket and use of a cooled, low ionic strength eluting buffer. Focusing of proteins can be achieved in a 15-mm-dia. column by the interplay of eluent velocity electrophoretic migration rate, and electrically induced concentration polarization when the stationary phase is more conductive than the mobile phase. Voltage gradients of up to 125 V/cm for eluent velocities at 18-25 cm/h separate binary protein mixtures of Bhb-alpha-lactalbumin, BSA-myoglobin, and alpha-lactalbumin-myoglobin over Sephadex G-100 and G-50. Retention times are consistent with values obtained from a mechanistic nonlinear model.