The Thermodynamics Study On The Retention Of Proteins And Aromatic Alcohol Homologues In Hydrophobic Interaction Chromatography

The thesis is composed of four parts as follows:1. Effect of composition of mobile phases on the retention behavior of solutes in hydrophobic interaction chromatography (HIC)The retention behavior of aromatic alcohol homologues in the solutions of different salts in HIC was firstly investigated, and the retention behavior of proteins was also studied. It was found that the retention of aromatic alcohol homologues and proteins obeys to the stoichimetric displacement model for retention (SDM-R) in the temperature range of 0-SO癈 and 0-50癈, respectively, and the former also conforms to the homologue rule. The composition of mobile phase has different influence on the elution range and ability to the two types of solutes. By the comparison of the changes of Z for the two types of solutes in the solutions of different kinds of salts, it was found that the salt types only change the hydration degree of small molecules, while for proteins, different salts not only change the hydration degree of proteins and the stationary phase, but also affect the conformation of the protein molecules at the contact regions between the proteins and the surface of the stationary phase.2. Carbon-number and concentration convergences of the retention of aromatic alcohol homologues in HICThe retention of aromatic alcohol homologues in HIC changes with the molecular structure of elutes and the chromatographic condition, and has a phenomenon of carbon number and concentration convergences under the temperature range from 0癈 to 80 癈. in the four kinds of mobile phases. The coordinates and their physical meaning of carbon-number and concentration convergent points were obtained, and elucidated, respectively.3. Thermodynamics of the retention of solutes in HICThe effect of temperature on the retention of aromatic alcohol homologues in HIC was firstly investigated. It was found that the retention of aromatic alcohol homologues increase firstly, and then decrease with the raising temperature. The relationships between the retention and temperature can be expressed by the Van't Hoff quadratic equation, and the thermodynamic parameters can be also obtained. It was found that the retention of aromatic alcohol homologues in HIC is entropy driven at low temperatures and enthalpy driven at high temperatures. The thermodynamic parameters of the proteins were also obtained by the same manner. However, the retention of proteins in HIC was found only entropy driven at the temperature rangeinvestigated. In addition, the other thermodynamic parameters (enthalpy A//?^ entropy AS ?and heat capacity AC^ ?) except free energy AG ?for all solutes correlated linearly with the reciprocal of absolute temperature.4. Enthalpy-entropy compensation of proteins and aromatic alcohol homologues in HICIsothermodynamic temperatures were firstly obtained with (Murphy-Privalov-Gill) MPG enthalpy and entropy plots of exothermodynamic theory. The retention of aromatic alcohol homologues in HIC was found to have the same mechanism in different mobile phases. The compensation temperatures for proteins and aromatic alcohol homologues were calculated at different temperatures, respectively, The compensation temperatures were found to be a function of temperatures, the difference in the functions, TC(T), between proteins and aromatic alcohol homologues indicates that they have different retention mechanism. The compensation temperature of proteins at 298K was calculated to be 296K, which is very close to that of the compensation temperature (293K) determined by the microcalorimetry for 11 proteins in literature.