The authors have studied the influence of ligand density, temperature, eluent pH and Triton X-100 on the separation performance of Butyl Sepharose 4 Fast Flow. The proteins used during the study were alpha-chymotrypsinogen, cytochrome C, ribonuclease A and lysozyme. Small adjustments to the chromatographic 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 Butyl Sepharose 4 Fast Flow was studied by testing the separation of a protein mixture during repeated cleaning-in-place (CIP) treatments with 1.0 M sodium hydroxide solution and 0.1 M hydrochloric acid. The separation behaviour of the protein mixture was unaltered after the medium had been treated for a total contact time of 4 weeks with 1.0 M sodium hydroxide solution. However, a slight decrease in retention times was observed in acidic conditions. The study shows that butyl 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 butyl groups coupled via ether linkage to the agarose matrix was determined by gas chromatography 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 Butyl 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 behaviour of the medium.