A three-dimensional stochastic simulation has been developed for electrophoretic separations. This simulation follows the trajectories of individual molecules by applying the fundamental equations of motion for diffusion, electroosmotic flow, and electrophoretic migration. The molecular zone profile may then be examined and characterized at any specified time or distance. The stochastic simulation has been validated by comparison with classical transport models, having average relative errors for the mean zone distance and variance of 0.01-0.04% and 2.67-4.36%, respectively. The simulation also shows excellent agreement with experimental results for the electrophoretic separation of the nucleotide monophosphates in phosphate buffer solution, having average relative errors for the mean zone distance and variance of 1.65% and 12.99%, respectively. This stochastic simulation provides an accurate and versatile means to examine and characterize transport processes in complex electrophoretic systems.Electrophoresis is an important analytical technique for the separation of strong and weak electrolytes as well as large polyelectrolytes of biological and industrial importance. Our understanding of the fundamental processes that comprise electrophoresis has been achieved through comparison of experimental results with the predictions of theoretical models. The classical models used to describe mass transport in electrophoretic separations are usually developed to address the conditions of a specific mode of separation, such as zone electrophoresis (ZE), 1-6 moving boundary electrophoresis (MBE), 7 isotachophoresis (ITP), 5,8-12 and isoelectric focusing (IEF). 13-17 However, unified models that can be used to describe all of the electrophoretic separation modes have also been developed. 18-21 Descriptions of these and other electrophoretic models can be found in several reviews. [22][23][24] These classical transport models require the formulation of detailed differential equations of mass and charge balance, together with the associated boundary conditions. The solution of these equations yields the concentration profile as a function of time and/or the steady-state concentration profile in the cases of ITP 10,11 and IEF. 15-17 A comprehensive model must include all of the physical and chemical processes that contribute to the migration and dispersion of the solute zone in the electrophoretic system. In practice, however, these comprehensive differential equations cannot be solved analytically in closed form and are generally solved by numerical methods after simplifying assumptions are invoked. 25 For example, some models may consider electrophoretic migration alone, 6,26 whereas others may include additional processes such as molecular diffusion and electrodiffusion, 27 convection, 4,5,28 chemical equilibria, 12 and various combinations thereof. 1,2,29 As a consequence of these simplifying assumptions, the classical models do not provide a completely general description of transport phenomena in electrophoretic systems...