Digitized Honors Theses (2002-2017)
Date of Award
5-2009
Document Type
Undergraduate Thesis
Degree Name
BS
Department
Chemistry
Faculty Mentor
Jason Coym, Ph.D.
Advisor(s)
Sandra Stenson, Ph.D., Scott Miller, Ph.D.
Abstract
Drug compounds are only effective if they can cross the plasma membrane of the target cell. Determination of this aspect of drug behavior has proven difficult due to costly and/or moderately inaccurate techniques. One technique that has been developed uses Reversed Phase-High Performance Liquid Chromatography (RP-HPLC). While the 18 carbon chains of this technique's stationary phase mimics the strong hydrophobics and basic structure of the cell's phospholipid bilayer, it lacks an integral component that provides the membrane with its structural rigidity. This compound is known as cholesterol and its incorporation into the stationary phase should improve the accuracy of the RP-HPLC technique as a membrane mimic by providing the stationary phase with rigidity analogous to the bilayer. This addition can be accomplished by coating the stationary phase with cholesterol via stationary phase addition. This coating process involves dissolving the additive into the mobile phase, pumping this mobile phase through the stationary phase of the column, and waiting for a breakthrough curve in the absorbance UV-vis spectrometer chromatogram. The breakthrough point indicates that the additive has become maximally loaded into the stationary phase for the current coating conditions of mobile phase composition and temperature. The stationary phase can become coated and remain stable due to the limited solubility of cholesterol in polar mobile phases.
This technique was found to coat from 5 mg up to 50 mg of cholesterol into the stationary phase of a Phenomenex Luna C 18(2) HPLC chromatographic column with dimensions 150 x 4.6 mm containing 5 micron particles. When cholesterol was coated into the stationary phase, it was found that the hydrophobicity of the stationary phase remained unchanged but the shape selectivity was improved from a value of 1.08 to 1.44 when the stationary phase was coated with 21.76 mg of cholesterol. The cholesterol coated stationary phase remained stable for 250 column volumes of 70/30 methanol/water mobile phase. The addition of cholesterol was also found to change the thermodynamics of retention from a qualitatively constant enthalpy to a variable enthalpy that decreases with temperature from endothermic to exothermic around a temperature of 25°C. The initial study of the biopartitioning of analytes through the coated stationary phase was not found to be statistically different from the uncoated stationary phase.
Recommended Citation
Ogden, Phillip, "Chromatographic Properties of a Cholesterol-Coated C 18 Stationary Phase in High Performance Liquid Chromatography: Toward Development of a Biomembrane Mimic" (2009). Digitized Honors Theses (2002-2017). 86.
https://jagworks.southalabama.edu/honors_theses-boundprint/86
Comments
© 2009 Phillip Bradford Ogden ALL RIGHTS RESERVED