Effect of alkyl length and density on the retention of proteins and amino acids in hydrophobic interaction chromatography

• Main Authors: Yen Ming Yang, 楊晏銘
• Other Authors: Rocky Ruaan
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/29768821950382611766

碩士 === 中原大學 === 化學工程學系 === 87 === Hydrophobic interaction chromatography (HIC) has been widely accepted as one of the major techniques for preparative protein purification. However, the elution order of different species out of different hydrophobic columns was usually unexpected. A protein exhibiting high hydrophobicity when it was applied on one column may not be that hydrophobic when it was on another. There are only two possible factors responsible for such an order reverse. One was the density, the other was the type of hydrophobic ligands. We chose alkyl chains as the hydrophobic ligands. The purpose of this study was to understand how the density and chain length of the ligands affected the retention of various amino acids and proteins. It was found that the retention of amino acids and most of the proteins increased linearly with the increase of ligand density. However, the amino acids could not be retained when the chain length was less than 2.5 carbons and it needs an alkyl length more than 3 carbon long to retain the proteins. As the chain length increased, the retention factor first increased rapidly and then reached finite values. A simple mathematical model was proposed and described very well the effect of chain length. The model suggested that the retention was affected by the size and the hydrophobic strength of the hydrophobic region of proteins and amino acids. According to this model, the retention factor was proportional to the size of the hydrophobic region when the alkyl was long. The elution order would then be similar to that obtained form reverse phase chromatography. When the alkyl chain was short, the retention factor was proportional to both the size and the hydrophobic strength of the hydrophobic region. The elution order would be similar to the hydrophilic order obtained from the aqueous two-phase system.