The Solubility and Secondary Structure of Zein in Imidazolium- Based Ionic Liquids

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The Solubility and Secondary Structure of Zein in Imidazolium- Based Ionic Liquids THE SOLUBILITY AND SECONDARY STRUCTURE OF ZEIN IN IMIDAZOLIUM- BASED IONIC LIQUIDS by SEAN R. TOMLINSON B.S., Kansas State University, 2005 AN ABSTRACT OF A DISSERTATION Submitted in partial fulfillment of the requirements for the degree DOCTOR OF PHILOSOPHY Department of Chemical Engineering College of Engineering KANSAS STATE UNIVERSITY Manhattan, Kansas 2013 Abstract Ionic liquids are low melting salts composed of an organic cation and an inorganic or organic anion. Ionic liquids are of interest for their wide range of applications and unique properties, such as the negligible vapor pressure of some types of ionic liquids, and the ability to modify ionic liquid properties by selection of the cation or anion. It has been hypothesized that over one million binary ionic liquids (meaning a single cation/anion pair) are possible. Due to the vast number of potential combinations, it should be possible to design ionic liquids specifically for an application of interest. One potential application is their use as protein solvents. However there is little understanding of how ionic liquids affect proteins. This research examined the solubility and secondary structure of the hydrophobic corn protein zein in seven ionic liquids and three conventional solvents as a function of temperature and solvent properties. Zein’s solubility in the solvents was measured gravimetrically from 30 to 60 degrees Celsius. Solubility was then related to solvent properties to gain an understanding of what solvent properties are important, and how to design an ionic liquid to dissolve zein. It was found that a good solvent for zein has a small molecular volume, a low polarity, and is a weak hydrogen bond acceptor. Infrared spectroscopy with curve fitting was used to examine the secondary structure of zein as a function of both solvent and temperature from 25 to 95 degrees Celsius. It was found that most of the ionic liquids change zein’s secondary structure, but those secondary structure changes were not affected by temperature. Aprotic ionic liquids increase the amount of β-turn secondary structure through non-polar interactions between the mixed aromatic-alkyl imidazolium cations and the non-polar portions of the zein. Strong hydrogen bond accepting molecules were found to increase the amount of β-turn secondary structure. It is hypothesized from this research that suitable solvents for zein will have a small molar volume, low polarity, and be poor hydrogen bond acceptors. This combination of properties will enhance zein’s solubility and limit secondary structure changes that can harm protein properties. THE SOLUBILITY AND SECONDARY STRUCTURE OF ZEIN IN IMIDAZOLIUM- BASED IONIC LIQUIDS by SEAN R. TOMLINSON B.S., Kansas State University, 2005 A DISSERTATION Submitted in partial fulfillment of the requirements for the degree DOCTOR OF PHILOSOPHY Department of Chemical Engineering College of Engineering KANSAS STATE UNIVERSITY Manhattan, Kansas 2013 Approved by: Major Professor Jennifer L. Anthony Copyright SEAN TOMLINSON 2013 Abstract Ionic liquids are low melting salts composed of an organic cation and an inorganic or organic anion. Ionic liquids are of interest for their wide range of applications and unique properties, such as the negligible vapor pressure of some types of ionic liquids, and the ability to modify ionic liquid properties by selection of the cation or anion. It has been hypothesized that over one million binary ionic liquids (meaning a single cation/anion pair) are possible. Due to the vast number of potential combinations, it should be possible to design ionic liquids specifically for an application of interest. One potential application is their use as protein solvents. However there is little understanding of how ionic liquids affect proteins. This research examined the solubility and secondary structure of the hydrophobic corn protein zein in seven ionic liquids and three conventional solvents as a function of temperature and solvent properties. Zein’s solubility in the solvents was measured gravimetrically from 30 to 60 degrees Celsius. Solubility was then related to solvent properties to gain an understanding of what solvent properties are important, and how to design an ionic liquid to dissolve zein. It was found that a good solvent for zein has a small molecular volume, a low polarity, and is a weak hydrogen bond acceptor. Infrared spectroscopy with curve fitting was used to examine the secondary structure of zein as a function of both solvent and temperature from 25 to 95 degrees Celsius. It was found that most of the ionic liquids change zein’s secondary structure, but those secondary structure changes were not affected by temperature. Aprotic ionic liquids increase the amount of β-turn secondary structure through non-polar interactions between the mixed aromatic-alkyl imidazolium cations and the non-polar portions of the zein. Strong hydrogen bond accepting molecules were found to increase the amount of β-turn secondary structure. It is hypothesized from this research that suitable solvents for zein will have a small molar volume, low polarity, and be poor hydrogen bond acceptors. This combination of properties will enhance zein’s solubility and limit secondary structure changes that can harm protein properties. Table of Contents List of Figures ................................................................................................................................ ix List of Tables ............................................................................................................................... xiv Acknowledgements ...................................................................................................................... xvi Dedication ................................................................................................................................... xvii Chapter 1 - Introduction .................................................................................................................. 1 Hypothesis .................................................................................................................................. 1 Objective ..................................................................................................................................... 2 References ................................................................................................................................... 4 Chapter 2 - Background .................................................................................................................. 6 Ionic liquid introduction ............................................................................................................. 6 Ionic liquid applications .......................................................................................................... 7 Ionic liquid syntheses .............................................................................................................. 8 Introduction to protein structure analysis ................................................................................... 9 An introduction to infrared spectroscopy ................................................................................. 12 Attenuated total reflectance IR spectroscopy........................................................................ 13 Application of IR spectroscopy to protein secondary structure ............................................ 16 Introduction to zein ................................................................................................................... 19 Solvent properties ..................................................................................................................... 22 Linear solvation energy relationship ..................................................................................... 23 Temperature effects on solubility ............................................................................................. 24 References ................................................................................................................................. 27 Chapter 3 - Experimental Methods ............................................................................................... 39 Ionic liquid synthesis ................................................................................................................ 39 Solvent density and molar volume ............................................................................................ 40 Zein solubility ........................................................................................................................... 41 Infrared analysis ........................................................................................................................ 41 Linear solvation energy relationship ......................................................................................... 45 References ................................................................................................................................. 46 vi Chapter 4 - Solubility of Zein ....................................................................................................... 48 Introduction ............................................................................................................................... 48 Results and Discussion ............................................................................................................. 48 The solubility of
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