Type of Document Dissertation Author Coym, Jason William Author's Email Address email@example.com URN etd-04192004-122753 Title The Use of Composition, Density, Pressure, and Temperature as Mobile Phase Variables in Reversed-Phase Chromatography Degree Doctor of Philosophy Department Chemistry and Biochemistry, Department of Advisory Committee
Advisor Name Title John G. Dorsey Committee Chair Alan G. Marshall Committee Member Gregory B. Dudley Committee Member Joel E. Kostka Committee Member William T. Cooper Committee Member Keywords
- Chromatographic Retention
- Supercritical Fluid Chromatography
- Mobile Phase
Date of Defense 2004-04-13 Availability unrestricted AbstractThis work consists of various studies of a “unified” theory of chromatography. Typically, liquid chromatography and gas chromatography are considered two separate techniques. In liquid chromatography, retention is controlled by the composition of the mobile phase, while in gas chromatography, retention is controlled by the temperature of the carrier gas. However, when the mobile phase variables of temperature, composition, and pressure are all considered, one observes that it is possible to traverse the entire phase space from gas to liquid without undergoing a phase transition. This is done by going through the supercritical fluid region. In this work, supercritical fluid chromatography was used as a “bridge” between gas and liquid chromatography.
Using mobile phases of either neat carbon dioxide, or CO2 mixed with an organic modifier, the entire phase space can be examined by setting appropriate pressure, temperature, and composition values. Retention and selectivity are examined as functions of mobile phase composition, temperature, and density. Continuous trends as the phase space is traversed suggest a unified description of chromatography. In addition, work was performed examining the effect of temperature and stationary phase chemistry on the phaes ratio and gradient reequilibration volume in reversed-phase chromatography. After a solvent gradient is run, it takes a significant amount of time to reequilibrate the column to the initial mobile phase composition before the next run is performed. It was found that for some stationary phases, temperature has a significant influence on gradient reequilibration time, while for others there is little or no temperature dependence.
Shape selectivity in the context of unified chromatography was examined. Shape selectivity refers to the ability of a chromatographic system to separate shape isomers. Using CO2/acetonitrile mobile phases, Shape selectivity trends as a function of mobile phase composition, as the mobile phase goes from supercritical fluid to subcritical fluid to enhanced fluid to liquid, are examined. Changes in selectivity as the mobile phase traverses the phase diagram were continuous, suggesting that the mechanism for shape selectivity was the same, regardless of the “type” of chromatography being performed. A study of the effect of mobile phase density on shape selectivity was performed, using neat carbon dioxide as the mobile phase. As density is decreased, shape selectivity increases. This is due to the an increase in stationary phase interactions as the mobile phase density is reduced.
Methylene selectivity in supercritical fluid chromatography was studied. Methylene selectivity is a measure of the hydrophobic selectivity of a chromatographic system; its value is indicative of the thermodynamics of retention of a methylene group. By measuring methylene selectivity as a function of mobile phase density at a given temperature and extrapolating to a mobile phase density of zero, the stationary phase contribution to methylene selectivity can be determined. These experiments were performed at a variety of temperatures, with both C18 and C30 stationary phases. It was found that, in the temperature range of 75° to 150° C, the enthalpy of transfer of a methylene group from the mobile phase to the stationary phase was proportional to the length of the stationary phase alkyl chain. This supported a partition mechanism for retention of non-polar solutes in supercritical fluid chromatography.
In addition to examining CO2/organic mobile phases, work was perfomed using superheated water as a mobile phase for reversed-phase separations. Superheated water is water heated to between 100° and 225° C, but pressurized to maintain the liquid state. The dielectric constant of water decreases from around 80 at 25° C to around 35 at 200° C. This is similar to the change in dielectric constant as organic modifier is added to water at room temperature. Thermodynamic analysis of retention with pure-water mobile phases are both ambient and superheated temperature revealed that retention at ambient temperature with a pure water mobile phase was entropically driven, but at superheated temperature retention was enthalpically driven. This change in thermodynamic signature was attributed to the change in the hydrogen bond structure as temperature is raised. At superheated temperatures, the hydrogen bond structure of water is nearly completely disrupted. This thermodynamic shift is similar to what is observed at room temperature as organic modifier is added to a mobile phase.
An investigaton of the effect of temperature on the phase ratio and gradient reequilibration volume in reversed-phase chromatography was performed. Experiments were performed on both traditional C18 stationary phases, and on newer polar embedded group stationary phases. Polar embedded group phases are designed to be more robust in highly aqueous environments. I found that, for stationary phases with polar-embedded groups, gradient reequilibration volume did not change much with temperature. However, for normal C18 stationary phases, the gradient reequilibration time was more than cut in half at 60° C as compared to 15° C. This suggests that the two types of phases are solvated differently, and that on normal C18 phases, the amount of solvation is strongly a function of temperature. These findings have both practical and theoretical importance. Any method to reduce reequilibration time would be beneficial in industry. Reequilibration time is “wasted time” that is, the time spent reequilibrating a stationary phase is time not spent performing an analysis. Reducing this time would make gradient chromatography more cost-effective. From a theoretical standpoint, my results suggest significant differences in the way that normal vs. polar embedded group stationary phases are solvated. The solvation structure of the stationary phase influences both retention and selectivity, and is important in examining the thermodynamics of retention in chromatography.
My doctoral work contributed to the understand of retention and selectivity in chromatography. By utilizing a “unified” approach to chromatography, the entire phase space is opened up for use in method development. Utilizing temperature, pressure, and composition variables, chromatographers should find the best combination for their separation, regardless of its position in the phase diagram.
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