FAST PROTEIN SEPARATION BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY ON OCTADECYLSILYL-BONDED NONPOROUS SILICA-GEL .2. IMPROVEMENT IN RECOVERY OF HYDROPHOBIC PROTEINS

Recovery of hydrophobic proteins from an RP-HPLC column was improved using a fast-separation RP-HPLC system operated at room temperature. Hydrophobic proteins such as ovalbumin could be adequately eluted from a nonporous octadecylsilyl (C18) spherical silica gel with a particle diameter of 20 μm using steep gradient elution with a 0.1% aqueous trifluoroacetic acid-acetonitrile system at a constant flow rate of 4 ml/min. Recoveries improved under fast separation since the protein sample suffered only a slight amount of irreversible denaturation on the hydrophobic surface of the stationary phase. The fast-separation system was also applied to the separation of larger proteins such as apoferritin (443 kDa) and thyroglobulin (669 kDa) as well as egg white proteins. © 1991.