Hydrophobic interaction chromatography of homo-oligonucleotides on derivatized Sepharose CL-6B: Application of the solvophobic theory

Abstract

This work describes the hydrophobic interaction chromatography of homo-deoxyoligonucleotides polyA, polyT and polyU with sizes up to 30 bases on a Sepharose gel derivatized with the 1,4-butanediol diglycidyl ether. The oligonucleotides interacted differently with the column according to the molecular mass, the hydrophobic character of the individual bases, the secondary structure of the molecule and the concentration of ammonium sulphate in the eluent. The retention factor, k′, was determined from the chromatographic profiles at different concentrations of ammonium sulphate. A linear relationship between log k′ and the concentration of ammonium sulphate in the eluent was found for all oligonucleotides at the higher concentrations (>1.0 M) of ammonium sulphate. The slope of these plots, termed the hydrophobic interaction parameter, was found to be an increasing function of the number of nucleotides. The same plots reveal that polyA molecules with high molecular mass have lower retention factors when compared with polyT, an observation that was not expected since the hydrophobicity of adenine is higher than that of thymine. This behaviour was due to the existence of secondary structures in polyA, which decrease the exposed hydrophobic area of the molecule.