The antibodies and fragments represent an important class of molecules employed in clinical diagnosis in vitro and in vivo, and in the area of biosensors. The need for research development in this area is great, aiming an efficient purification, using low cost techniques. This work studies the separation of Fab fragments of human immunoglobulin G (IgG) through pseudoafinity chromatography using ortho-phosphotyrosine (OPT) as the ligand.