Dynamic field gradient focusing (DFGF) uses an electric field gradient opposed by a counter-flow of buffer to separate milligrams of proteins according to their electrophoretic mobilities. A nonlinear model of protein separation in a preparative-scale DFGF device was developed to aid in re ning the instrument's design and finding optimal run conditions prior to performing experiments. The model predicted the focal points of bovine serum albumin (BSA), and bovine hemoglobin (Hb) to within the 95% confidence intervals about the means of the experimental values. The resolution between the proteins in the model was 2.08, which was 3% less than the lower limit of the 95% confidence interval about the experimental value. The model predicted 67% more dispersion than was present in the experimental device, which made the simulated BSA peak 22% wider than the experimentally measured width. (C) 2008 American Institute of Chemical Engineers AIChE J, 55: 63-74, 2009