Master of Science
James T. Hsu
The primary goal of this thesis is to present a fundamental understanding of ion exchange chromatography and enzyme kinetics. Both techniques are explored through simple examples such as the inversion of sucrose by invertase as well as the separation of fructose and glucose by ion exchange chromatography. Enzymes are biocatalysts that allow biological systems to function, which can also be utilized for a variety of industrial reactions. Ion ex-change chromatography is widely implemented in the pharmaceutical and biotechnology industries as well as by environmental industry, or for water filtering by consumers in their homes.
The hydrolysis of sucrose is catalyzed by the enzyme invertase. An enzymatic assay was used to determine the Michaelis-Menten constant and the maximum reaction rate. Initial reaction rates were determined by plotting glucose concentration versus time. These rates were then used to create a double reciprocal, or Lineweaver Burke, plot to determine the Michaelis-Menten constant and the maximum reaction rate. The Michaelis-Menten constant of invertase was found to be 9.97 mM and the maximum velocity was found to be 17.57 mMol/(L*min).
Ca-Cation exchange chromatography is useful for separating mixtures of glucose and fructose. The basis of separation is the stronger interaction between fructose and the calcium ion complex than glucose and the calcium ion complex. Physical parameters such as temperature and pH were varied to determine their effects on separation. The data show that lower temperatures increase the retention time of fructose and do not affect the retention time of glucose.
McDermott, Jack Wade, "Inversion of Sucrose by Invertase and the Separation of Fructose and Glucose by Ion Exchange Chromatography" (2020). Theses and Dissertations. 5685.