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1 Novel Nanomaterials and Chromatographic System for Enhanced Separation and Characterization of Biomacromolecules and Nanoparti Novel Nanomaterials and Chromatographic System for Enhanced Separation and Characterization of Biomacromolecules and Nanoparticles Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Yanhui Wang, M.S. Graduate Program in Chemistry The Ohio State University 2018 Dissertation Committee Dr. Susan V. Olesik, Advisor Dr. Philip Grandinetti Dr. Abraham Badu-Tawiah 1 Copyrighted by Yanhui Wang 2018 2 Abstract With recent advances in technologies and methodologies, proteomics, which is the large-scale analysis of proteins, has been continuously developed in the field of bioinformatics, biotherapeutics and biomarker discovery. Top-down proteomics, which focuses on the analysis of intact proteins, has emerged within the last decade with significant advantages over the traditional bottom-up approach, such as the characterization of labile protein structures and the universal detection of all existing modifications. The front-end separation technologies for intact proteins are of the primary importance for the successful implementation of top-down proteomics. The work reported herein focuses the development of miniaturized liquid chromatography (LC) system and an effective and eco-friendly solvent system to address the challenges faced in intact protein separation and characterization. Electrospun nanofibers featuring effective chromatographic performance as the stationary phase of the ultrathin layer chromatography (UTLC) was developed in this work for the separation of amino acids and intact proteins. Nafion, a synthetic perfluorinated cationic polymer, was incorporated into a carrier polymer, polyacrylonitrile (PAN), to fabricate the nanofibrous stationary phase via electrospinning method. The separation of charged amino acids and proteins on the Nafion-PAN UTLC was based on the ion exchange mechanism (IEX). Design of experiments (DOEs) ii methods were applied to optimize the Nafion-PAN stationary phase and separation conditions. The nanofibers exhibited excellent mechanical stability and solvent compatibility. The separation of amino acids confirmed the feasibility of Nafion-PAN nanofibers as the ion exchange UTLC stationary phase. The separation of intact proteins has illustrated that Nafion-PAN stationary phase is also suitable for separation of large biomolecules. The retention of proteins on the Nafion-PAN UTLC largely depends on the properties of proteins including the net charge, hydropathicity, molecular size and structure. The Nafion-PAN UTLC demonstrated high separation efficiency for both amino acids and intact proteins. In addition to intact protein separation, a micellar liquid chromatography (MLC) system was developed using the polyacrylonitrile UTLC device for the size characterization of polyethylene glycol (PEG)ylated gold nanoparticle (AuNP), an emerging agent in cancer therapeutics, of which the cellular uptakes and cytotoxicity are highly dependent on its size. PEGylated AuNPs with different sizes in the range of 10-80 nm were well separated from each other. The developed method also permitted the separation of AuNPs capped with different molecular weight of PEG in the range of 2-20 kDa. Micellar mobile phases were adopted to provide a highly biodegradable chromatographic system. This method exhibited excellent separation performance with smallest plate heights < 2 µm and resolution of each pair of AuNPs > 1.5. Decent separations for all PEGylated AuNPs could be achieved within 5 min. This method was applied to monitor the transformation of AuNPs in serum protein, serving as a rapid and iii convenient tool for characterization of size distribution and modification of PEGylated AuNPs. Efforts towards improving the mobile phase system of intact protein separation were also made by adopting enhanced fluidity liquid chromatography (EFLC), which involves the addition of liquefied gas, such as carbon dioxide, to the conventional liquid mobile phases. The addition of liquefied CO2 provides increased diffusivity and decreased viscosity of the mobile phase, which inherently leads to more efficient separation. Herein, EFLC was first time applied to hydrophobic interaction chromatography (HIC) to study the impact of liquefied CO2 on the chromatographic behaviors of proteins. Since conventional HIC is known to preserve the native structure of proteins, the effects of liquefied CO2 on protein structures, charge state distributions (CSD) and ionization efficiencies would be more pronounced by adapting EFLC to HIC online coupled to electrospray ionization mass spectrometry (ESI-MS). In this work, EFLC offered improved chromatographic behaviors including a shorter analysis time, better peak shapes and a higher plate number. Liquefied CO2 proved to be an ESI friendly and “supercharging” reagent without sacrificing chromatographic performance, which can be used to improve peptide and protein identification in the large-scale application. The EFLC system was also applied to reversed phase chromatography (RPC) for intact protein separation. The EFLC solvents utilizing liquefied CO2/methanol/water mixture could be considered as a “greener” alternative to traditional water/acetonitrile mobile phase in LC with better separation efficiency and peak symmetry. Various mobile iv phase additives were compared under RPC mode to provide the optimum condition for integrated EFLC-MS system for intact protein separation and characterization. v Dedication To my beloved grandma, I miss you everyday vi Acknowledgments The completion of this dissertation would have not been possible without the support, guidance and efforts of many special individuals. I would like to extend my sincere gratitude to all of them. First, I would like to express my special thanks to my advisor, Dr. Susan Olesik for her generous support, invaluable guidance and mentorship over the past five years. Thank you for encouraging my research and allowing me to grow as a scientist. You have led me way farther than I thought I could go. I would also like to thank every member from the Olesik research group for their dedicated efforts and cooperative attitude. I would especially like to recognize Michael Beilke, Martin Beres, Raffeal Bennett, Jiayi Liu, Juan Bian, Brian Fitch and Rebekah Gibson for helping me both professionally and personally. I’m grateful to have all of you who could share laughter, dreams and frustrations with me all along the way. Finally, I would like to express my deepest gratitude to my family. Thank you to my parents for always believing in me and teaching me the most important job in life is to learn how to understand myself and others, to give out love and to be happy. Thank you to my big brother, Yanfeng, for taking good care of Mom and Dad. Grandma, I hope I have made you proud. Words cannot express how much I miss all of you. To my husband-to-be, Wey Jian, this journey could never be possible without you. Thank you vii for always being there for me through thick and thin. Thank you for never letting me give up on myself and pushing me to be a better person day by day. viii Vita 2010 to 2013 ..........................B.S. Medicinal Chemistry, University at Buffalo, SUNY, Buffalo, NY 2013 to 2015 ..........................M.S. Chemistry, The Ohio State University, Columbus, OH 2014 to 2018 ..........................Graduate Teaching Assistant and Graduate Research Assistant, Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH Publications Wang, Y.; Olesik, V. S. Separation of PEGylated Gold Nanoparticles by Micellar Enhanced Electrospun Fiber Based Ultrathin Layer Chromatography. Analytical Chemistry 2018, 90 (4), pp 2662–2670. Wang, Y.; Olesik, V. S. Electrospun Nafion/PAN as Ion Exchange Ultrathin Layer Chromatography Stationary Phase. Analytica Chimica Acta 2017, 970, 82-90. ix Deuro, R. E.; Lieker, K. M.; Wang, Y.; Caras, C. A.; Milillo, T. M.; Bright, F. V. Denim Fiber Characterization Using Multispectral Luminescence Imaging. Applied Spectroscopy 2015, 69,103-114. Fields of Study Major Field: Chemistry x Table of Contents Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... vi Acknowledgments............................................................................................................. vii Vita ..................................................................................................................................... ix List of Tables .................................................................................................................... xv List of Figures ................................................................................................................. xvii Chapter 1. Introduction ....................................................................................................... 1 1.1 Overview ................................................................................................................... 1 1.2 Basic chromatography fundamentals ........................................................................ 2 1.2.1 Retention Factor and Retardation Factor ........................................................... 2 1.2.2 Selectivity .........................................................................................................
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