Name: PENG ZANGUO Degree: DOCTOR OF PHILOSOPHY Dept.: CHEMICAL AND BIOMOLECULAR ENGINEERING Thesis Title: SEPARATION OF ORGANIC AND BIOMOLECULES USING NANO-SIZED MAGNETIC PARTICLES Abstract Separation of organic and biomolecules by nano-sized magnetic particles was studied Nano-sized magnetic particles were prepared using Fe2+, Fe3+ salts and ammonium hydroxide by chemical precipitation method in nitrogen atmosphere Extraction of bovine serum albumin (BSA) and lysozyme (LSZ) were carried out either in single component or in binary mixture Surface modifications of magnetic particles by coating double layer surfactants were carried out for the separation of 2-hydroxyphenol (2-HP) and 2-nitrophenol (2-NP) Characterizations of adsorption process were carried out using different methods Effect of pH and salt concentration on adsorption were studied Adsorption equilibrium was fitted with Langmuir model Adsorption kinetics was fitted with linear driving force model Desorption of target molecules from magnetic particles were carried out using different desorption agents Conformational change of desorbed proteins and enzymatic activity were measured Experimental results show nano-sized magnetic particles are effective tools for the separation of organic and biomolecules Keywords: Magnetic separation; Nano-sized magnetic modification; Adsorption; Desorption; Conformational change particles; Surface SEPARATION OF ORGANIC AND BIOMOLECULES USING NANO-SIZED MAGNETIC PARTICLES PENG ZANGUO NATIONAL UNIVERISTY OF SINGAPORE 2004 SEPARATION OF ORGANIC AND BIOMOLECULES USING NANO-SIZED MAGNETIC PARTICLES PENG ZANGUO (M ENG., TIANJIN UNIV.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMICAL & BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2004 ACKNOLEDGEMENT First, I would like to take this opportunity to express my deepest gratitude and appreciation to my supervisors, Associate Professor M S Uddin and Associate Professor K Hidajat, for their guidance, support, help and encouragement throughout the research program Thanks are also given to all staff members in the Department of Chemical and Biomolecular Engineering and to all my colleagues in the lab, who have given me great help in my research work I am extremely grateful to my family members for their selfless love and full support throughout the time of the Ph.D course Finally, I would like to thank the National University of Singapore for providing the research scholarship during my postgraduate studies Peng Zanguo June, 2004 i TABLE OF CONTENTS ACKNOLEDGEMENT i TABLE OF CONTENTS ii SUMMARY vii NOMENCLATURE ix LIST OF FIGURES xii LIST OF TABLES xvi Chapter 1 1.1 Research Background 1.2 Research Objectives 1.3 Organizations of Thesis Chapter Introduction Literature Review 2.1 Magnetic Separation 2.1.1 Principle of Magnetic Separation 2.1.2 Advantages and Disadvantages of Magnetic Separation 2.1.3 Types of Magnetic Separation 2.1.4 Applications of Magnetic Separation 2.2 Magnetic Particles 10 19 2.2.1 Type of Magnetic Particles 20 2.2.2 Preparation of Magnetic Particles 21 2.2.3 Interaction between Magnetic Particles and Magnetic Field 23 2.3 Surface Modification of Magnetic Particles 24 2.3.1 Coated with metal chelating agent 25 2.3.2 Coated with thermosensitive polymer 25 2.3.3 Coated with functional ligands 25 ii 2.4 Adsorption and Desorption 27 2.4.1 Adsorption Equilibrium 27 2.4.2 Adsorption Kinetics 28 2.4.3 Parameters Affecting Adsorption 31 2.4.4 Desorption study 32 2.5 Evaluation of the Separated Targets 2.6 Scope of the Thesis Chapter 33 33 Materials and Methods 36 3.1 Materials 36 3.2 Experimental Procedures 38 3.2.1 Synthesis of Magnetic Particles 38 3.2.2 Coating of Surfactants on Magnetic Particles 38 3.2.3 Adsorption Experiments 41 3.2.4 Desorption Experiments 44 3.3 Analytical Methods 44 3.3.1 Transmission Electron Microscopy (TEM) 45 3.3.2 Vibrating Sample Magnetometer (VSM) 45 3.3.3 X-ray Diffraction Analysis (XRD) 45 3.3.4 Brunauer-Emmett-Teller (BET) Method 46 3.3.5 Zeta Potential Analyzer 46 3.3.6 Thermogravimetric Analysis (TGA) 46 3.3.7 Total Organic Carbon Analyzer (TOC) 47 3.3.8 X-ray Photoelectron Spectroscopy (XPS) 47 3.3.9 Fourier Transform Infrared Spectroscopy (FTIR) 48 3.3.10 Circular Dichroism (CD) 48 iii 3.3.11 Fluorescence 3.3.12 Differential scanning calorimetry (DSC) 49 3.3.13 SDS-PAGE 50 3.3.14 Lysozyme Activity Measurement Chapter 49 51 Extraction of Bovine Serum Albumin (BSA) by Magnetic Particles 53 4.1 Introduction 53 4.2 Results and Discussion 55 4.2.1 Characterization of Magnetic Particles 55 4.2.2 Adsorption of BSA on Magnetic Particles 57 4.2.2.1 Adsorption at different pH 58 4.2.2.2 Adsorption at different salt concentration 61 4.2.2.3 Adsorption Equilibrium 62 4.2.2.4 Characterization of Adsorption of BSA 63 4.2.2.5 Adsorption Kinetics 65 4.2.2.6 Effect of Carbodiimide on Adsorption 67 4.2.3 Desorption of BSA 68 4.2.4 Conformational Changes of BSA in Adsorption/Desorption 70 4.2.4.1 UV-Spectrum Measurement 70 4.2.4.2 CD-spectrum Measurements 71 4.2.4.3 Fluorescence Measurements 74 4.2.4.4 DSC Measurements 75 4.2.4.5 Effect of BSA Structure on Adsorption Behavior 78 4.3 Conclusions Chapter 79 Extraction of Lysozyme (LSZ) by Magnetic Particles 81 5.1 Introduction 81 iv 5.2 Results and Discussion 5.2.1 Adsorption of LSZ on Magnetic Particles 83 83 5.2.1.1 Adsorption Equilibrium 83 5.2.1.2 Characterization of Adsorption of LSZ 84 5.2.2 Desorption of LSZ 86 5.2.3 Evaluation of LSZ 87 5.2.3.1 UV-spectrum Measurements 87 5.2.3.2 CD-spectrum Measurements 89 5.2.3.3 Fluorescence Measurements 90 5.2.3.4 DSC Measurements 92 5.2.3.4 Enzymatic Activity Assay 94 5.3 Conclusions Chapter 94 Magnetic Separation for Binary Mixture of BSA and LSZ 95 6.1 Introduction 95 6.2 Results and Discussion 97 6.2.1 Selective Adsorption of BSA and LSZ 97 6.2.2 Sequential Adsorption of BSA and LSZ 102 6.2.3 Desorption of BSA and LSZ 106 6.2.4 Evaluation of Desorbed Protein 107 6.3 Conclusions Chapter 108 Magnetic Separation for Binary Mixture of 2Hydroxyphenol and 2-Nitrophenol 109 7.1 Introduction 109 7.2 Results and Discussion 111 7.2.1 Characterization of Magnetic Fluids 111 v 7.2.2 Adsorption of 2-Hydroxyphenol and 2-Nitrophenol 114 7.2.2.1 Effect of pH 115 7.2.2.2 Adsorption Equilibrium 117 7.2.2.3 Adsorption Kinetics 118 7.3 Conclusions Conclusions and Recommendations 121 8.1 Conclusions 121 8.2 Recommendations for Further Work Chapter 119 124 References of the Thesis 126 List of Publications 151 vi SUMMARY The magnetic separation method utilizes magnetic particles to bind the target molecules via intermediates (such as surfactant, ligand, and so on) and thereafter form a complex, which can be separated from the bulk solution by a magnetic field The interacting mechanisms between magnetic particles and target could be hydrophobic interactions, electrostatic interactions and ligand bonding interactions Due to its simplicity and effectiveness, magnetic separation is drawing more and more attention, and has been used in a wide range of applications such as removal of metal ions from waste water, isolation of proteins from cell lysate, and extraction of nucleic acids Many published work focused on the synthesis of micro-sized polymer matrixes containing magnetic particles and its application in the separation of protein with the aid of specific ligand coated particles Only limited work has been published on the application of nano-sized magnetic particles in the separation of proteins Nano-sized magnetic particles produce a larger specific surface area and therefore, may result in high adsorption capacity 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Physical and chemical properties of magnetite and magnetite-polymer nanoparticles and their colloidal dispersions, J Colloid Interf Sci., 212: pp.49-57, 1999 150 List of Publications Peng, Z.G., K Hidajat and M.S Uddin, Extraction of 2-hydroxyphenol by Surfactants Coated Nanosized Magnetic Particles in Aqueous Suspension, Korean J Chem Eng., 20, pp.896-901, 2003 Peng, Z.G., K Hidajat and M.S Uddin, Adsorption of Bovine Serum Albumin on Nanosized Magnetic Particles, J Coll Inter Sci., 271, pp.277-283, 2004 Peng, Z.G., K Hidajat and M.S Uddin, Conformational Change of Adsorbed and Desorbed Bovine Serum Albumin on Nano-sized Magnetic Particles, Coll Surf B, 33, pp.15-21, 2004 Peng, Z.G., K Hidajat and M.S Uddin, Adsorption/Desorption of Lysozyme on Nano-sized Magnetic Particles and its Conformational Change, Coll Surf B, 35, pp.169-174, 2004 Peng, Z.G., K Hidajat and M.S Uddin, Selective and Sequential Adsorption of Bovine Serum Albumin and Lysozyme from a Binary Mixture on Nano-sized Magnetic Particles, J Coll Inter Sci., 281, 11-17, 2005 .. .SEPARATION OF ORGANIC AND BIOMOLECULES USING NANO- SIZED MAGNETIC PARTICLES PENG ZANGUO NATIONAL UNIVERISTY OF SINGAPORE 2004 SEPARATION OF ORGANIC AND BIOMOLECULES USING NANO- SIZED MAGNETIC PARTICLES. .. application of nanosized magnetic particles in the separation of organics and biochemical molecules, and the evaluation of the effectiveness of the separation method The desired goals of different... topics such as background of magnetic separation, types of magnetic particles and separating target 2.1 Magnetic Separation 2.1.1 Principle of Magnetic Separation Magnetic separation is a recent