This article is Part II of a series describing a newly-developed gradient chromatofocusing high-performance liquid chromatography (HPLC) technique. Theoretical aspects of the technique are discussed. In gradient chromatofocusing, the column pH gradient with respect to column distance can be varied without necessarily affecting the outlet pH gradient with respect to time. Factors influencing the value of the slope of the column pH gradient are identified through derived equations and a computer simulation model. A newly-identified parameter is introduced, column travel time, which can be uniquely varied in gradient chromatofocusing. Experiments show increased conversion of fibrinogen to denatured forms with increased column travel time. Another unique aspect of gradient chromatofocusing is that the mobile phase buffer concentration can be manipulated without necessarily affecting the outlet pH gradient slope, giving the technique expanded versatility for optimizing the separation. In the present work, the pIapparent for fibrinogen is found to increase with increased mobile phase buffer concentration.