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V·M·! University Microfilms International a 8Ell & Howell Information Company 300 North Zeeb Road, Ann Arbor, MI 48106-1346 USA 313 761-4700 800521-0600 Electrophoresis of solutes in aqueous two-phase systems. Item Type text; Dissertation-Reproduction (electronic) Authors Levine, Mark Louis. Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 08/10/2021 02:52:32 Link to Item http://hdl.handle.net/10150/186087 INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. V·M·! University Microfilms International A 8ell & Howell Information Company 300 North Zeeb Road, Ann Arbor, MI 48106-1346 USA 313 761-4700 800521-0600 Order Number 9310596 Electrophoresis of solutes in aqueous two-phase systems Levine, Mark Louis, Ph.D. The University of Arizona, 1992 V·MaI 300 N. Zccb Rd. Ann Arbor, MI 48106 ELECTROPHORESIS OF SOLUTES IN AQUEOUS TWO-PHASE SYSTEMS by Mark Louis Levine A Dissertation Submitted to the Faculty of the DEPARTMENT OF CHEMICAL ENGINEERING In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY In the Graduate College THE UNIVERSITY OF ARIZONA 1 9 9 2 2 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE As members of the Final Examination Committee, we certify that we have read the dissertation prepared by Mark Louis Levine ~~~~~~=-~~-------------------- entitled __~E~l~e~c~t~r~o~p~h~o~r~e~s~is~o~f~S~o~l~u~t~e~s~i~n~A~q~u~eo~u~s~T~w~o~-~p~h~a~s~e~S~y~s~t~em~s ____ __ and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy l{-'?Z.-f'- Date q/~2/q:( Date ~. Thomas W. Peterson Date 7 William R. Monfort Dade I Date Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College. I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement. Dissertation Director Milan Bier Date ' t I /IS /q."J, Dissertation Director Heriberto Cabezas, Jr. Date 3 STATEMENT BY AUTHOR This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the maj or department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. SIGNED:_fi-L-.:...v_1_J__ ~_~ __' ______ _ 4 ACKNOWLEDGMENTS I would very much like to thank my advisors on this dissertation, Dr. Milan Bier and Dr. Heriberto Cabezas for their advice, support, and encouragement on this work. Special thanks must also go to Drs. Richard Mosher, Glyn Roberts, and Dudley Saville for their assistance in the development of the computer models and Dr. Ned Egen, Mr. Terry Long, and Mr. Garland Twitty for their help with experimental work. I am also indebted to Dr. Subhas Sikdar and the National Institute of Standards and Technology for time spent there learning about aqueous two-phase systems. Of course, I would never have even begun work on this dissertation without a lifetime of support and motivation from my family, friends, and teachers. The financial support of the NASA Graduate Student Researchers Program, grant number NGT-50270, as well as NASA grant number NAGW-693 is also gratefully acknowledged. 5 TABLE OF CONTENTS List of Figures . · 10 List of Tables • 14 ABSTRACT 18 INTRODUCTION 20 BACKGROUND • 24 Aqueous Two-Phase Systems • 24 The Thermodynamics of Phase Separation . · 25 Flory-Huggins Theory . • . • • • 29 Partitioning in Phase Systems • 34 Diffusion of Solutes in Two-Phase Systems • 35 Analytical Solution of the Diffusion Equation for Two Phases • . • . • . 37 More on Diffusion in Two-Phase Systems . · 45 Davies Work and the Diffusion Equation ..... 45 Equilibrium and Mass Transfer Boundary Conditions . • • • 47 Related Work with Membranes . · 48 Electrophoresis • • 49 Modeling of Electrophoresis · 52 Complete Model for Electrophoresis in Single-Phase Systems . .... ... 52 Boundary Conditions . • • 56 Non-Dimensionalization 57 Solution of the Complete Model for Electro­ phoresis in a Single Phase . 60 6 TABLE OF CONTENTS (Continued) significance of the Complete Model for Single Phase Systems . • . • . .• •... 61 Other studies Involving Electrophoresis and Phase SystenlS . • .. ..•..••.•.•... 62 Electrophoresis across the Interface between an Aqueous and an Organic Phase . 63 Application of Electric Fields across the Interface between Two organic Phases . 69 Demixing Agueous Two-Phase Systems by Electro- phoresis . 70 EXPERIMENTAL STUDIES OF ELECTROPHORESIS IN AQUEOUS TWO­ PHASE SYSTEMS . • . • . • • . .. 72 Characterization of Two-Phase System for Free Fluid Experiments • • . 73 Materials . 73 Preparation of Phase Systems 74 Methods for the Characterization of Phase Systems . · . 75 Results from the Characterization of Phase Systems . · . 77 Partition Coefficient Measurement for Hemoqlobin in Ammonium Acetate Buffer . .. 77 Electrophoretic Mobility Measurements 83 Moving Boundary Electrophoresis U-tube Apparatus. 87 Moving Boundary Electrophoresis of Solutes in Polymer Systems . • . 93 Single Phase Experiments 93 Two-Phase Experiments with Hemoglobin as Solute • . · . 95 7 TABLE OF CONTENTS (Continued) Two-Phase Experiments with Proteins (Including Hemoglobin) ...•..•.•... 101 Two-Phase Systems with Small Molecules as Solute • . 108 Bulk Phase Mobility Effects 116 Effect of pH Changes at the Interface 120 Two-Phase Polyacrylamide Gel Electrophoresis . 122 Materials . 122 Preparation of Gels . 123 Zone Electrophoresis Experiments in Poly- acrylamide Gels 123 CONSTRUCTION OF THE MODELS FOR ELECTROPHORESIS IN TWO- PHASES . 127 Simple Eauilibrium Model for Electrophoresis in Two-Phase Systems . 128 Results from the Simple Model for Electrophoresis with Equilibrium at the Interface . .. 134 Simple Model for Electrophoresis with Mass Transfer Resistance at the Interface . .. 139 Results from the Simple Model for Electrophoresis in Two-Phase Systems with Mass Transfer Resistance at the Interface . .. 140 Assumptions Made for the Complete Two-Phase Electrophoresis Models 145 Complete Model for Moving Boundary Electrophoresis with Equilibrium at the Interface . .. 146 Complete Model for Moving Boundary Electrophoresis with Mass Transfer Resistance at the Interface . • .• •........ 150 Comparison of the Simple and Complete Models 152 8 TABLE OF CONTENTS (Continued) Modeling Zone Electrophoresis in Two-Phase Systems • . 156 Demonstration of the relationship between equilibrium and transfer models . 160 Further Mathematical Analysis of the Two-Phase Problem 160 Estimating the Effect of a Limiting Case - No Transport across the Interface • . 162 Flux Interpretation of the Equilibrium Model 163 Energy Required for Electrophoresis . 165 Stability and Convergence of the Complete Simula- tions . .. 166 RESULTS FROM SIMULATION OF TWO-PHASE ELECTROPHORESIS 169 Simulations of MBE with Equilibrium Boundary Condition . 171 Changing the Supporting Buffer 171 comparing Reversing the Electric Field with the Effect of Inverting the Partition Coefficient . .. 174 Extended Simulation Time 174 Magnitude of the Partition Coefficient 180 Changing Preferred Phase to Non-Preferred Phase . 182 Bulk Phase Mobility Differences . 184 Other Solutes - Weak Bases and Proteins . 187 High Solute Concentration Interactive Systems 193 Resul ts from Moving Boundarv Electrophoresis Simulations with Mass Transfer Boundary Condition . .. 197 Magnitude of the Mass Transfer Coefficient 197 9 TABLE OF CONTENTS (Continued) Simulation of Zone Electrophoresis with Equilibrium Boundary Condition • • • • • . • • • . 199 CONCLUSIONS AND RECOMMENDATIONS
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