The relative hydrophobicity of human serum alpha1-antitrypsin (AAT) and alpha1-acid glycoprotein (AGP) in comparison to various reference proteins was determined by hydrophobic interaction chromatography (HIC). Apolar character of glycoproteins was generated using three different cosmotropic salts, ammonium sulfate, sodium sulfate, sodium citrate and isocratic, or reversed linear gradient elution techniques. Human serum AAT and AGP showed different apolar properties on Fractogel EMD phenyl and propyl columns modulated either by the type and concentration of cosmotropic salts, or by the pH of the mobile phase. According to its higher carbohydrate content AGP proved to be more polar than AAT. Human serum AAT and AGP were pre-separated by Cibacron Blue F3G-A dye ligand affinity chromatography and based on their different hydrophobicity were fractionated and purified by HIC.