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Controlling Protein-Surface Interactions in Chromatography

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Controlling Protein-Surface Interactions in Chromatography CONTROLLING PROTEIN-SURFACE INTERACTIONS IN CHROMATOGRAPHY USING MIXED SELF-ASSEMBLED MONOLAYERS By Chrysanty Tedjo Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Chemical Engineering May, 2011 Nashville, Tennessee Approved: Professor Paul E. Laibinis Professor David E. Cliffel Professor Scott A. Guelcher Professor G. Kane Jennings Professor M. Douglas LeVan Copyright © 2011 by Chrysanty Tedjo All Rights Reserved To my parents, for their continuous support and endless love iii ACKNOWLEDGEMENTS I would first like to acknowledge my advisor Professor Paul Laibinis for his guidance during my research at Vanderbilt University. I am grateful for all the valuable lessons that I have learnt from him. His critical advice and analytical skills have helped me in solving many research problems. I would like to thank the members of my thesis committee Professor Doug LeVan, Kane Jennings, Scott Guelcher, and David Cliffel, who have assisted me since the beginning of my research. Their critical advice from their own research background has provided me with different perspectives on how to approach my research. I would also like to thank Professor Bridget Rogers for her assistance with the x-ray photoelectron spectroscopy (XPS) in her laboratory. The staff of the Department of Chemical and Biomolecular Engineering also deserves to be recognized for their contribution to my research. I would like to thank Mark Holmes for his assistance in setting the equipments in the laboratory and for various technical advice. I am grateful to know Mary Gilleran and am thankful for her assistance and enjoyable discussions about life in general. I would also like to thank Rae Uson for her very diligent work in helping me with administrative issues. I am grateful to know fellow graduate students and researchers in the Department of Chemical and Biomolecular Engineering. I would first like to thank Zhou Xu, my cohort in the Laibinis group for his assistance in using some of the equipments in our laboratory. I would like to thank Chris Faulkner, Brandon Booth, Steve Vilt, Juan Carlos Tuberquia from the Jennings group for their help in one way or another and valuable research discussions. I iv would like to thank Ben Schmidt from the Rogers group for teaching me how to operate XPS and for his insightful knowledge about XPS. I wish to thank Katarzyna Zienkiewicz for her advice in operating HPLC. I would also like to thank the Young group for letting me to use their centrifuge, pH meter and hood. It is also necessary that I acknowledge my family and friends for their support during my period at Vanderbilt University. I will be forever grateful to my parents who taught me that I can accomplish any goal with continual hard work and high expectations. I cherish my sisters for their never ending love and support. I am thankful for my wonderful roommate here in Nashville, who has been through this process with me for the last one and a half year. Finally, none of this would be possible without the continual support from my husband. v TABLE OF CONTENT Page ACKNOWLEDGEMENTS .............................................................................................. iv LIST OF TABLES ............................................................................................................ ix LIST OF FIGURES ............................................................................................................x I. INTRODUCTION ...........................................................................................................1 1.1. Chromatography of Proteins using Hydrophobic Interactions .................................4 1.2. Self-Assembled Monolayers (SAMs) .......................................................................7 1.3. Motivation ...............................................................................................................14 1.4. Thesis Overview .....................................................................................................15 1.5. References ...............................................................................................................17 II. SELF-ASSEMBLY OF TRI(ETHYLENE GLYCOL)-TERMINATED SILANES IN PURE AND MIXED MONOLAYERS ON SiO2/Si SURFACES ...................................24 2.1. Introduction .............................................................................................................24 2.2. Materials and Methods.............................................................................................26 2.2.1. Materials ..........................................................................................................26 2.2.2. Synthesis of EG3OMe .....................................................................................27 2.2.3. Formation of Self-Assembled Monolayers (SAMs) on SiO2/Si .....................28 2.2.4. Characterizations of SAM ..............................................................................28 2.2.5. Protein Adsorption Experiments ....................................................................29 2.3. Results and Discussion ............................................................................................30 2.3.1. Self-Assembled Monolayers of EG3OMe and Octadecyltrichlorosilanes ......30 2.3.2. Mixed Self-Assembled Monolayers of EG3OMe and C8 ..............................40 2.4. Conclusions .............................................................................................................52 2.5. References ...............................................................................................................53 III. MIXED SELF-ASSEMBLED MONOLAYERS OF TRIETHYLENE GLYCOL TERMINATED SILANE AND OCTYLTRICHLOROSILANE ON POROUS SILICA PARTICLES .....................................................................................................................56 3.1. Introduction .............................................................................................................56 3.1.1. Flotation as a Method to Determine Critical Surface Tensions of Particles ..57 3.2. Materials and Methods ............................................................................................60 3.2.1. Materials .........................................................................................................60 3.2.2. Self-Assembled Alkylsiloxane Monolayers ...................................................60 vi 3.2.3. Characterizations of SAMs .............................................................................61 3.3. Results and Discussion ............................................................................................64 3.3.1. Self-Assembled Alkylsiloxane Monolayers on Silica Particles .....................64 3.3.2. XPS Characterizations ....................................................................................70 3.3.3. Floatability Measurements of Mixed SAM-Coated Silica Particles ..............73 3.3.4. Determination of Critical Surface Tension from Floatability Data ................76 3.3.5. Determination of Critical Surface Tension from Zisman’s Plot ....................76 3.3.6. Floatability of Particles of Different Sizes .....................................................77 3.4. Conclusions .............................................................................................................80 3.5. References ...............................................................................................................80 IV. PREPARATION AND CHARACTERIZATION OF CHROMATOGRAPHIC COLUMNS .......................................................................................................................84 4.1. Introduction .............................................................................................................84 4.2. Materials and Methods ............................................................................................86 4.2.1. Particles Functionalization and Characterization ...........................................86 4.2.2. Column Packing and Characterization ...........................................................87 4.3. Results and Discussion ...........................................................................................88 4.3.1. Thermogravimetric Analysis of Coated Silica Particles ................................88 4.3.2. Column Characterization ................................................................................90 4.4. Conclusions .............................................................................................................94 4.5. References ...............................................................................................................94 V. INFLUENCE OF SURFACE HYDROPHOBICITY OF MIXED SELF- ASSEMBLED MONOLAYERS (SAMS)-COATED SUPPORTS ON PROTEIN RETENTION IN CHROMATOGRAPHY .......................................................................96 5.1. Introduction .............................................................................................................96 5.2. Theory .....................................................................................................................99 5.3. Materials and Methods ..........................................................................................105 5.3.1. Proteins and Chemicals ................................................................................105
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