In this work, affinity chromatography was used to remove endotoxin from human hemoglobin preparations with a Sterogene Acticlean Etox column. The effects of solution conditions on endotoxin removal efficiency and protein recovery have been investigated. It has been found that cations Na(+) or Ca(2+) reduced endotoxin removal efficiency from 73% (sample prepared with endotoxin-free water) to 31% (sample prepared with 0.15 M NaCl, ionic strength, I = 0.15 M), and from 73% sample prepared with endotoxin-free water) to 9% (sample prepared with 0.05 M CaCl2, ionic strength, I = 0.15 M). It has also been found that the protein recovery was increased from 90% to 99%, respectively.