The authors have studied the influence of ligand d., temperature, eluent pH and Triton X-100 on the separation performance of Bu Sepharose 4 Fast Flow. The proteins used during the study were α-chymotrypsinogen, cytochrome C, RNase A and lysozyme. Small adjustments to the chromatog. conditions can affect the retention times of the proteins. The effects from changes in temperature prove that the separation process is entropy-driven. The functional stability of Bu Sepharose 4 Fast Flow was studied by testing the separation of a protein mixture during repeated cleaning-in-place (CIP) treatments with 1.0 μ sodium hydroxide solution and 0.1 M hydrochloric acid. The separation behavior of the protein mixture was unaltered after the medium had been treated for a total contact time of 4 wk with 1.0 M sodium hydroxide solution However, a slight decrease in retention times was observed in acidic conditions. The study shows that Bu ligands are released due to hydrolysis of the agarose support in acidic conditions. This explains the effect on retention time observed after CIP at pH 1. For this study, a method for determining the ligand content was developed. The amount of Bu groups coupled via ether linkage to the agarose matrix was determined by gas chromatog. after cleavage of ether bonds by boron tribromide. The authors have also investigated the clearance of ethanol, 2-propanol and Triton X-100 from an HR 10/10 column packed with Bu Sepharose 4 Fast Flow. For a column treated with Triton X-100, a regeneration procedure with 2-propanol was necessary in order to retain the original retention behavior of the medium.