Protein losses in ion-exchange and hydrophobic interaction HPLC were examined. The supports were all non-porous, packed in columns of identical dimensions. Two ion-exchange chromatography (IEC), anion and cation, as well as a hydrophobic interaction chromatography (HIC) columns were tested. Proteins included cytochrome c, bovine serum albumin (BSA), immunoglobulin G and fibrinogen. Temperature effects on HIC supports were studied for cytochrome c and BSA. Both retention times and recoveries of the proteins were measured. The influence of column residence time on the recovery of proteins was also investigated. We found a linear relationship between the amount of protein recovered and the log of the molecular mass. Retention times also generally increased with temperature for both HIC and IEC. Other trends in retention behavior and recoveries are discussed.