Abstract: An ampholyte separation apparatus and method which utilize a thermally engendered pH gradient to separate amphoteric molecules according to their isoelectric points and/or mobilities. Two independently temperature controllable thermal masses located on opposing ends of a plurality of barriers situated transverse to a flow channel create a pH gradient along the length of the transverse barriers. A pair of electrodes create an electric field along the length of the transverse barriers so as to cause ampholytes traveling down the flow channel to migrate into the transverse barriers and stabilize at points corresponding to their isoelectric points. Alteration of the pH gradient causes the separated amphoteric molecules to reenter the flow stream at different times and to be separately eluted and collected.