The effect of pH and temperature on separating a mixture of similar proteins, namely -lactoglobulin A (LGA) and -lactoglobulin B (LGB) in anion-exchange chromatography is explored. The proteins carry a slight difference in negative charge at basic pH, providing a separation basis on an Q Sepharose Fast Flow anion-exchange resin. They were separated at different temperatures and pH values, and the separation factor was evaluated. The experimental results were matched to a theoretical model to compute the equilibrium constant K-A. The data shows that an increase in temperature and pH leads to an increase in the retention time of the proteins. The results were correlated with the net charge of the molecule for the separation so that the elution can be simulated for any condition that was studied. The tertiary structures of LGA and LGB are analyzed to illustrate the structure effect on the separation. (c) 2018 American Institute of Chemical Engineers AIChE J, 64: 1928-1937, 2018