| Summary: | 碩士 === 國立成功大學 === 化學系 === 89 === Abstract
For standard C18 column , it is known that when the pressure rises from 23 bar to 318 bar, the retention time of protein lysozyme will increase two or three times. Also, we can get the same outcome if we proceed to experiment with pressure for solutes using a homologous series hydrophobic peptides with different amounts of phenylalanine. Hence, we may inference that the pressure interaction relates to hydrophobic interaction. On the other hand, the chromatographic elution has relations with chemical equilibrium of the thermodynamics. We can figure out a 10cm3 decrease of volume for per molar phenylalanine molecules in the stationary phase than in the mobile phase. To realize more about the effect caused by the pressure, we choose five different temperatures in the range from 24℃ to 87℃ for both high-pressure condition and low-pressure one. Then we choose suitable mobile phase for different solutes and proceed with isocratic elutions. We also calculate heat parameters, including enthalpy and entropy basing on Van’t Hoff formula. In addition to the temperature and the pressure, we change the ratio of organic solvent and observe how it will effect the serious of experiments.
In this experiment, we take three homologous series of hydrophobic amino acids, including phenylalanine, alanine, and lysine as model compounds. By diagramming, we can find it exists nice linear relation between the amount of amino acids and log k’. It shows that k’ can be additive to the amount of amino acids. Under the room temperature, k’ increases as the amount of amino acids increase at 318 bar and 23 bar. Comparing the k’ value’s increasing rate of three homologous series of amino acids, the phenylalanine, which is the most hydrophobic, increases most and its increasing rate is between 1.359 and 2.152. The alanine is the second and increases about 1.339 to 1.359 time. However, the increase of lysine is the most unobvious and is about 1.078 to 1.054. Plotting of the reciprocal of the absolute temperature (1/ T) with log k’, we can find that the slope is -H/R and the intercept is S + log. Also, we can get the change of enthalpy (（H）and the change of entropy（S）by deducting H values of the same solute with different amount of amino acids. It is shown that the value of H is always negative in both high-pressure condition and low-pressure one because the ad-desorption behavior of the solute in the column is an exothermic reaction. The ACN ratio of the organic solvent decreases in the mobilie phase, however, the k’ value, H, and S raise. According to the Van’t Hoff equation, the decrease of H and the increase of S both are helpful for the increasing of k’. We inference that the retention time increase of solute’s being held up caused by the decreasing of the organic solvent is entropy-driven, but is not relative with the enthalpy. Because the phenylalanine is hydrophobic and is unsolvable by the water, the existence of organic solvents is contributive for the solvation made by mobile phases to the hydrophobic character of solutes, and is conducive to the misoibility of solutes and the mobile phases. The less is the content of the organic solute, the weaker is the misoibility with the mobile phase. and its S increases . When the pressure raises, we find that the increase of k’ value is also caused by the expanding of the S. However, the negative value of H decreases, and its decreasing trend is similar with the decreased ratio of the organic solvent. I t was concluded that the pressure-induced retention of hydrophobic peptides or proteins is major due to an entropy-driven process rather than the enthalpy-driven process , because the pressure cause the misoibility between solutes and stationary phase rather than mobile phase. According to the experiment that we just took, the volume of a hydrophobic solute is smaller in the stationary phase than in the mobile phase. High pressure reduced the distance among molecules, thus the interaction seems to increase. However, smaller volume made the surface that can react with the solute reduced at one time and made the overall heat release decreased. On the contrary, high pressure is help for the misoibility between hydrophobic materials. Under high pressure, the solute that in more hydrophobic stationary phase has higher misoibility and entropy, and increases its k’ value. Comparing with the phenylalanine, which is very hydrophobic, the alanine is less hydrophobic and its k’ value is effected less by pressure. Therefore, changes of alanine’s enthalpy and entropy are smaller. The k’ of hydrophilic lysine is almost not effected by pressure, the enthalpy and entropy of hydrophilic lysine change little both in high pressure condition or in low pressure one.
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