Fast protein separation by reversed-phase high-performance liquid chromatography on octadecylsilyl-bonded nonporous silica gel. II. Improvement in recovery of hydrophobic proteins. |
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Authors: | H Itoh N Nimura T Kinoshita N Nagae M Nomura |
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Institution: | School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan. |
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Abstract: | 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 microns 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 apo-ferritin (443 kDa) and thyroglobulin (669 kDa) as well as egg white proteins. |
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