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Development of Monolithic Stationary Phases for Cation-Exchange Capillary Liquid Chromatography of Peptides and Proteins" (2011)

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Development of Monolithic Stationary Phases for Cation-Exchange Capillary Liquid Chromatography of Peptides and Proteins Brigham Young University BYU ScholarsArchive Theses and Dissertations 2011-02-22 Development of Monolithic Stationary phases for Cation- Exchange Capillary Liquid Chromatography of Peptides and Proteins Xin Chen Brigham Young University - Provo Follow this and additional works at: https://scholarsarchive.byu.edu/etd Part of the Biochemistry Commons, and the Chemistry Commons BYU ScholarsArchive Citation Chen, Xin, "Development of Monolithic Stationary phases for Cation-Exchange Capillary Liquid Chromatography of Peptides and Proteins" (2011). Theses and Dissertations. 2467. https://scholarsarchive.byu.edu/etd/2467 This Dissertation is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. DEVELOPMENT OF MONOLITHIC STATIONARY PHASES FOR CATION-EXCHANGE CAPILLARY LIQUID CHROMATOGRAPHY OF PEPTIDES AND PROTEINS Xin Chen A dissertation submitted to the faculty of Brigham Young University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Milton L. Lee, Chair Steven W. Graves H. Dennis Tolley Matthew R. Linford Adam T. Woolley Department of Chemistry & Biochemistry Brigham Young University April 2011 Copyright © 2011 Xin Chen All Rights Reserved ABSTRACT DEVELOPMENT OF MONOLITHIC STATIONARY PHASES FOR CATION-EXCHANGE CAPILLARY LIQUID CHROMATOGRAPHY OF PEPTIDES AND PROTEINS Xin Chen Department of Chemistry & Biochemistry Doctor of Philosophy This dissertation focuses on the preparation of polymeric monolithic capillaries for ion exchange chromatography of peptides and proteins, since polymeric monoliths have shown promise for providing improved protein separations. Characteristics of monolithic columns include low back pressure, simplicity of fabrication and biocompatibility. Preparation of strong and weak cation-exchange monolithic stationary phases in 75 μm I.D. capillaries by direct in situ copolymerization was achieved using various functional monomers including sulfopropyl methacrylate, phosphoric acid 2-hydroxyethyl methacrylate, bis[2-(methacryloyloxy)ethyl] phosphate and 2-carboxyethyl acrylate with polyethylene glycol diacrylate and other PEG materials. The resulting monoliths provided excellent ion exchange capillary LC of peptides and proteins with good run-to-run [relative standard deviation (RSD) < 1.99%] and column-to- column (RSD < 5.64%) reproducibilities. Narrow peaks were obtained and peak capacities of over 20 were achieved. Dynamic binding capacities of over 30 mg/mL of column volume for lysozyme were measured. A single monomer was used to synthesize a phosphoric acid containing monolith to improve its stability and reproducibility. The monolith was synthesized from only BMEP in 75 μm I.D. UV transparent fused-silica capillaries by photo-initiated polymerization. A dynamic binding capacity (lysozyme) of approximately 70 mg/mL of column volume was measured. Efficiencies of 52,900 plates/m for peptides and 71,000 plates/m for proteins were obtained under isocratic conditions. Good reproducibilities were achieved. Zwitterionic monolithic columns based on photo-initiated copolymerization of N,N- dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl)ammonium betain and poly(ethylene glycol) diacrylate were prepared in 75 μm I.D. fused silica capillaries for hydrophilic interaction chromatography. Inverse size exclusion chromatography was used to characterize the pore structure of the resulting monolith. A typical hydrophilic interaction chromatography mechanism was observed when the organic content in the mobile phase was higher than 60%. Good separations of amides, phenols, and benzoic acids were achieved. The effects of mobile phase pH, salt concentration, and organic modifier content on retention were investigated. Keywords: monolith, ion exchange, hydrophilic interaction, liquid chromatography ACKNOWLEDGMENTS First and foremost, I gratefully acknowledge and thank my advisor, Dr. Milton L. Lee, for not only providing me an opportunity to study in his group, but also for providing me a model of what a successful scientist should be. His breadth and depth of knowledge in the field of separation science was a constant source of support to encourage my independent and original thought. With a sense of humor and a love of life, he has been successful in both academics and life. I am honored that I could spend five years in his group. I can not image that anyone could graduate from his group without a life-long admiration and respect for him. I would like to thank my graduate committee members, Dr. H. Dennis Tolley, Dr. Matthew R. Linford, Dr. Adam T. Woolley, and Dr. Steven W. Graves for their critical evaluation and invaluable suggestions during my research. I wish to acknowledge my fellow graduate students in Dr. Lee’s group. Cooperation and friendship form the foundation of his group, where I learned from each of them. Each member in this group contributes immensely in a complementary way. In particular, I would like to thank Dr. Binghe Gu, from whom I learned to prepare polymeric monoliths and operate several instruments. I also would like to thank Dr. Yun Li, Dr. Yuanyuan Li, Dr. Yan Fang, Dr. Shu-ling Lin, Dr. Nosa Agbonkonkon, Dr. Xuefei Sun, Dr. Yansheng Liu, Dr. Jikun Liu, Dr. Miao Wang, Dr. Jesse Contreras, Dr. Jacolin Murray, Tai Truong, Kun Liu, Jie Xuan, Dan Li, Xiaofeng Xie, Anzi Wang, Pankaj Aggarwal, and other friends in both Dr. Lee’s group and other groups. I appreciate their friendships and help at Brigham Young University. I would like to thank the staff in the department instrument shop for helping me check and repair instruments. I give thanks to Susan Tachka for her service. I thank the Department of Chemistry & Biochemistry at Brigham Young University for offering me the opportunity and financial support to study here. Financial support from Pfizer is also gratefully acknowledged. Finally, I must thank my family and Ying’s family. The help and love given by both families during the past five years is indescribable. My deepest gratitude belongs to my wife, Ying, who is a graduate student as well in the Department of Chemistry & Biochemistry. It was a challenge to bear two children and take care of them while pursuing graduate studies during the past five years. Without her excellent work at home and without her personal sacrifice, it would have been impossible for me to finish this project. Deep gratitude also belongs to my parents-in- law and to my parents. Bearing great loneliness, they traveled to America, which was unfamiliar to them, to help take care of our children. Their unselfish love and understanding were the greatest impetus for me to finish this dissertation. This dissertation is dedicated to my parents-in- law, my parents, my wife, and my two children, Elvin and Kaelyn. TABLE OF CONTENTS LIST OF ABBREVIATIONS ................................................................................................... vii LIST OF TABLES .......................................................................................................................x LIST OF FIGURES .................................................................................................................. xii CHAPTER 1 BACKGROUND AND SIGNIFICANCE .............................................................1 1.1 Liquid Chromatography .................................................................................................... 1 1.1.1 History of Column Liquid Chromatography.............................................................1 1.1.2 Classification of LC Modes ......................................................................................2 1.1.3 Ion-Exchange Chromatography (IEC) ......................................................................3 1.2 Monoliths .......................................................................................................................... 5 1.2.1 General Introduction of Monoliths ...........................................................................5 1.2.2 Monolith Preparation ................................................................................................7 1.2.2.1 Polymerization Methods ..................................................................................... 7 1.2.2.2 Monolith Preparation Methods ......................................................................... 14 1.2.3 Applications of Monoliths in LC ............................................................................21 1.2.3.1 Applications in IEC........................................................................................... 21 1.2.3.2 Applications in RPLC ....................................................................................... 25 1.2.3.3 Applications in HIC .......................................................................................... 30 1.2.3.4 Applications in Hydrophilic Interaction Chromatography (HILIC) ................. 31 1.3 Dissertation Overview .................................................................................................... 33 1.4 References ....................................................................................................................... 36 i CHAPTER 2 STRONG CATION-EXCHANGE MONOLITHIC COLUMNS CONTAINING SULFONIC ACID FUNCTIONAL GROUPS ..........................48 2.1 Introduction ....................................................................................................................
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