| Summary: | 碩士 === 國立成功大學 === 化學系 === 88 === In this study, the parameters affecting protein separation on reversed-phase liquid chromatography, such as organic solvent、ion-pairing agent and the pressure were investigated. For the effect of pressure, the thermodynamic principals were applied to infer the cause of observed results.
For organic solvent effect, three model proteins, ribonuclease A、lysozyme and α-lactalbumin which have the similar molecular weight and number of amino acids but different hydrophobicity, were judiciously chosen for this investigation. It was found that the separation efficiency using methanol as organic modifier is always worse than using acetonitrile. Moreover the efficiency difference between methanol and acetonitrile were smaller for the more hydrophobic proteins. It could be that the hydrophobic proteins were easily solvated by organic solvents such that less interaction with stationary phase once desorbed. For ion-pairing agent effect, two homologous series, poly-L-phenylalanine and poly-L-lysine, in addition to the three model proteins were used to investigate the effect of four ion-pairing agents : phosphoric acid、formic acid、trifluoroacetic acid and heptafluorobutyric acid. It was found that the retention time was increased with the hydrophobicity of ion-pairing agents. Moreover, the retention time of the hydrophilic peptides was more sensitive to the change of ion-pairing agents. The more hydrophilic peptides resulted in a better separation efficiency when paired with a more hydrophobic ion-pairing agent. Furthermore, the separation of lysozymes from chicken and turkey which are only different in 7 amino acids out of total 129 amino acids, had a better resolution and efficiency ehen under an isocratic elution using the mobile phase composed of acetonitrile and trifluoroacetic acid.
For pressure effect, it was demonstrated that the retention time of the lysozyme was increased by as much as two to three times as the absolute pressure on the viewing window was increased from 23 to 318 bar. Since many factors such as conformational changes of proteins, ionization, and hydrophobic interactions may all contribute to the protein retention and are subjective to change with the pressure, it is desirable to find out the major cause of the pressure effect. In this study, a homologous series of poly-aminoacid which have no secondary structures was investigated. It was found that the more hydrophobic peptides resulted in a more pronounced pressure-induced retention and consequently, a greater volume change ( △V＝Vsta－Vmob ) which was calculated from the first derivative of the lnk'' versus pressure polts. For example, the volume change of hydrophobic poly-L-phanylalanine was determined to be around minus 10 cm3/mol per phenylalanine, and the volume change of hydrophilic poly-L-lysine is about minus 1.5 cm3/mol per lysine. It is believed that perturbations in solute ionization、conformation change and stationary phase structural change have a minor impact under the investigated conditions, and the pressure-induced shift of the equilibria regarding hydrophobic ad-desorption is the major cause of the observed increase of protein retention. Assuming a linear relationship between the volume change and the hydrophobicity of amino acids, the volume change of other amino acids could be estimated. Moreover from comparing the volume changes of lysozyme and phenylalanine, it was predicted that about tens phenylalanine-equivalent residues on the lysozyme surface were involved in the hydrophobic association with the chromatographic ligands.
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