The Analytical and Surface Chemistry Program supports Professor Jason Coym of the University of South Alabama to explore novel additives (e.g. cholesterol) for varying selectivity and retention in reversed-phase liquid chromatography (RPLC). Perhaps the most commonly used form of liquid chromatography, RPLC is a key method for separating analytes in solution, and is used heavily in pharmaceutical and biochemical settings. The work entails dynamically coating cholesterol onto the stationary phase, as a simpler and more flexible alternative to typical covalent linkage. A parallel aim is to use cholesterol-enhanced and lipid-coated phases as membrane mimics for studies of biopartitioning.
The development of unique chromatographic selectivities, and the ability to easily modulate selectivity, is of broad interest to analytical and bioanalytical chemists. Students working on this project will gain knowledge and practice working with chromatography, physical chemistry, and biochemistry, thereby coming to realize the interdependence of the various disciplines of chemistry.
Intellectual Merit High Performance Liquid Chromatography (HPLC) is an analytical technique used to separate components of a mixture so that they can be individually quantified. Solutes are eluted through a column containing a stationary phase, using a high-pressure pump which delivers a solvent called the mobile phase. Different solutes have differing affinities for the two phases, which causes them to exit the column at different times. In reversed-phase liquid chromatography, the stationary phase is typically a non-polar surface, and the mobile phase is a mixture of water and an organic modifier such as methanol or acetonitrile. This project investigated the addition of various additives to the mobile phase, and the effect of these additives on separation. Some additives were selected to coat the stationary phase, changing its nature, while others were selected to remain in the mobile phase and interact with solutes directly. The first additive to be investigated was cholesterol. We were able to quantitatively coat a layer of cholesterol onto a C18 stationary phase, and observe the changes in chromatographic selectivity provided by the modified stationary phase. We proved that the cholesterol coated phases were stable by examining chromatographic selectivity--no change was seen after over 250 column volume of neat mobile phase had been flushed through the column, indicating stablity of the cholesterol coating. In addition to directly observing changes in selectivity, changes in retention mechanism were investigated using van’t Hoff and LSER analysis. This study resulted in a paper in Journal of Chromatography A. One problem encountered with the cholesterol coating was the limited solubility of cholesterol in polar solvents. We solved this problem by solubilizing cholesterol with methyl-β-cyclodextrin (MBCD). Cyclodextrins are used in cell culture to remove cholesterol from and deliver cholesterol to lipid bilayers, and we used this same idea to deliver cholesterol to the lipid-like C18 stationary phase. This similar behavior between our cholesterol-cyclodextrin-C18 system and cholesterol-cyclodextrin-bilayer systems suggests the ability to use C18 phases as membrane mimics in some scenarios. This study of the cholesterol-cyclodextrin-C18 system resulted in a publication in Journal of Chromatography A. In addition to using cyclodextrin to solubilize cholesterol, we investigated its use as a mobile phase additive on its own. We used chromatographic experiments to determine binding constants of MBCD to various steroids, and examined changes in chromatographic selectivity when MBCD was used as an additive. We found that when certain solutes in a solution bind strongly to MBCD the selectivity of the separation drastically changes. For example, we were able to completely reverse the elution order of a set of terphenyl isomers by including MBCD in the mobile phase, due to the differing binding constants of the different isomers to the cyclodextrin. Our development of cholesterol-coated stationary phases lead to an investigation of other biologically-inspired stationary phases. We preformed van’t Hoff and LSER analysis of retention on our cholesterol-coated phases, as well as it base C18 phase, a commercially available bonded cholesterol phase, and a commercially available Immobilized Artificial Membrane phase. The final aspect of this project was development of concomitant coating of both cholesterol and a lipid on a C18 stationary phase. While we have been able to qualitatively achieve this goal, we are still working on developing a procedure to quantify the amount of lipid and cholesterol coated on a phase. Broader Impacts This project supported the undergraduate research experiences of eight students in the Chemistry Department at the University of South Alabama. To date, two peer reviewed publications in Journal of Chromatography A with undergraduate student co-authors have resulted from this award. In addition, five students were afforded the opportunity to present their work on this project, at either the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy (2012) or at a National Meeting of the American Chemical Society (2013). This project supported the Undergraduate Honors Thesis projects of two students at the University of South Alabama. This project supported infrastructure development at South Alabama by allowing for the acquisition of a modern HPLC instrument.
