Separation of organic and biomolecules using nano sized magnetic particles

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 Therefore, there is a need to synthesize nano-sized magnetic particles with large surface area and to develop a suitable adsorption/desorption process for the separation of protein Some previous works have been done to extract metal ions using magnetic separation method But there is still a need to utilize magnetic particles for the extraction of other chemicals such as organics In the present study, nano-sized magnetic particles (Fe3O4), either naked or coated with a double layer of surfactants, are used to extract organics (2-hydroxyphenol and 2nitrophenol) and biochemicals (bovine serum albumin and lysozyme) Firstly, nano- vii References Kang Y S., S Risbud, J F Rabolt, P Stroeve, Synthesis and characterization of nanometer-size Fe3O4 and gamma-Fe2O3 particles, Chem Mater., 8: pp.2209-2211, 1996 Khalafalla S E., G W Reimers, Preparation of dilution-stable aqueous magnetic fluids, IEEE Trans Magn., MAG-16: pp.178-183, 1980 Khng H P., D Cunliffe, S Davies, N A Turner, E N Vulfson, The synthesis of submicron magnetic particles and their use for preparative purification of proteins, Biotechnol Bioeng., 60: pp.419-424, 1998 Kim D K., Y Zhang, W Voit, K V Rao, M Muhammed, Synthesis and characterization of surfactant-coated superparamagnetic monodispersed iron oxide nanoparticles, J Magn Magn Mater., 225: pp.30-36, 2001 Kim Y., B Lee, J Yi, Preparation of functionalized mesostructured silica containing magnetite (MSM) for the removal of copper ions in aqueous solutions and its magnetic separation, Separ Sci Technol., 38: pp.2533-2548, 2003 Kimata M., D Nakagawa, M Hasegawa, Preparation of monodisperse magnetic particles by hydrolysis of iron alkoxide, Powder Technol., 132: pp.112-118, 2003 Knox J H., 1982 High Performance Liquid Chromatography Edinburgh: Edinburgh University Press, 137 References Kobayashi H., T Matsunaga, Amino-Silane Modified Superparamagnetic Particles with Surface-Immobilized Enzyme, J Colloid Interf Sci., 141: pp.505-511, 1991 Kondo A., H Fukuda, Preparation of thermo-sensitive magnetic hydrogel microspheres and application to enzyme immobilization, J Ferment Bioeng., 84: pp.337-341, 1997 Kondo A., S Oku, K Higashitani, Structural-Changes in Protein Molecules Adsorbed on Ultrafine Silica Particles, J Colloid Interf Sci., 143: pp.214-221, 1991 Kondo A., S Oku, F Murakami, K Higashitani, Conformational changes in protein molecules upon adsorption on ultrafine particles, Colloid Surface B, 1: pp.197-201, 1993 Koneracka M., P Kopcansky, M Antalik, M Timko, C N Ramchand, D Lobo, R V Mehta, R V Upadhyay, Immobilization of proteins and enzymes to fine magnetic particles, J Magn Magn Mater., 201: pp.427-430, 1999 Koneracka M., P Kopcansky, M Timko, C N Ramchand, Direct binding procedure of proteins and enzymes to fine magnetic particles, J Magn Magn Mater., 252: pp.409-411, 2002 Koneracka M., C N Ramchand, R V Mehta, P Kopcansky, Immobilization of a neutral protease in magnetic particles using direct binding procedure, J Phys Iv, 7: pp.671-672, 1997 138 References Koseko S., M Hisamatsu, T Yamada, Preparation of Magnetic Support for Glutaminase Immobilization - (a Study on the Conversion of Glutamine to GlutamicAcid by an Immobilized Glutaminase on a Support Having Magnetic Sensitivity 1.), J Jpn Soc Food Sci., 41: pp.31-36, 1994 Kubal B S., K Sankaran, S F Dsouza, Immobilization of Yeast by Invertase Adsorption on Magnetite, J Microbial Biotech., 4: pp.98-102, 1989 Leao A M A C., E A Oliveira, L B Carvalho, Immobilization of Protein on Ferromagnetic Dacron, Appl Biochem Biotech., 31: pp.53-58, 1991 Lee J., T Isobe, M Senna, Preparation of ultrafine Fe3O4 particles by precipitation in the presence of PVA at high pH, J Colloid Interf Sci., 177: pp.490-494, 1996 Lee W K., J S Ko, H M Kim, Effect of electrostatic interaction on the adsorption of globular proteins on octacalcium phosphate crystal film, J Colloid Interf Sci., 246: pp.70-77, 2002 Leun D., A K Sengupta, Preparation and characterization of magnetically active polymeric particles (MAPPs) for complex environmental separations, Environ Sci Technol., 34: pp.3276-3282, 2000 Levison P R., S E Badger, J Dennis, P Hathi, M J Davies, I J Bruce, D Schimkat, Recent developments of magnetic beads for use in nucleic acid purification, J Chromatogr A., 816: pp.107-111, 1998 139 References Lewus R K., G Carta, Binary protein adsorption on gel-composite ion-exchange media, AIChE J., 45: pp.512-522, 1999 Li G S., R L Smith, H Inomata, K Arai, Preparation and magnetization of hematite nanocrystals with amorphous iron oxide layers by hydrothermal conditions, Mater Res Bull., 37: pp.949-955, 2002 Liao M H., D H Chen, Fast and efficient adsorption/desorption of protein by a novel magnetic nano-adsorbent, Biotechnol Lett., 24: pp.1913-1917, 2002a Liao M H., D H Chen, Preparation and characterization of a novel magnetic nanoadsorbent, J Mater Chem., 12: pp.3654-3659, 2002b Liu H S., Y C Wang, The Sorption of Lysozyme and Ribonuclease onto Ferromagnetic Nickel Powder Desorption and Competitive Adsorption, Colloid Surface B, 5: pp.35-42, 1995 Liu H S., Y C Wang, W Y Chen, The Sorption of Lysozyme and Ribonuclease onto Ferromagnetic Nickel Powder Adsorption of Single Components, Colloid Surface B, 5: pp.25-34, 1995 Macasek F., J D Navratil, S Dulanska, Magnetic sorbent for soil remediation - A waste for waste treatment, Separ Sci Technol., 37: pp.3673-3692, 2002 MacRae I C., Removal of pesticides in water by microbial cells adsorbed to magnetite, Water Res., 19: pp.825-830, 1985 140 References MacRae I C., Removal of chlorinated hydrocarbons from water and wastewater by bacterial cells adsorbed to magnetite, Water Res., 20: pp.1149-1152, 1986 Matsunaga T., S Kamiya, Use of magnetic particles isolated from magnetotactic bacteria for enzyme immobilization, Appl Microbiol Biotechnol., 26: pp.328-332, 1987 Mehta R V., R V Upadhyay, S W Charles, C N Ramchand, Direct binding of protein to magnetic particles, Biotechnol Tech., 11: pp.493-496, 1997 Moeser G D., K A Roach, W H Green, P E Laibinis, T A Hatton, Water-based magnetic fluids as extractants for synthetic organic compounds, Ind Eng Chem Res., 41: pp.4739-4749, 2002 Moffat G., R A Williams, C Webb, R Stirling, Selective Separations in Environmental and Industrial-Processes Using Magnetic Carrier Technology, Miner Eng, 7: pp.1039-1056, 1994 Molday R S., L L Molday, Separation of cells labeled with immunospecific iron dextran microspheres using high gradient magnetic chromatography, FEBS Lett., 170: pp.232-238, 1984 Moreno E M., M Zayat, M P Morales, C J Serna, A Roig, D Levy, Preparation of narrow size distribution superparamagnetic gamma-Fe2O3 nanoparticles in a sol-gel transparent SiO2 matrix, Langmuir, 18: pp.4972-4978, 2002 141 References Mosbach K., L Andersson, Magnetic ferrofluids for preparation of magnetic polymers and their application in affinity chromatography, Nature, 270: pp.259-261, 1977 Mosbach K., U Schroder, Preparation and application of magnetic polymers for targeting of drugs, FEBS Lett., 102: pp.112-116, 1979 Munro P A., P Dunnill, M D Lilly, Casein hydrolysis in stirred tank reactors using chymotrypsin immobilized on magnetic supports, Biotechnol Bioeng., 1981 Navratil J D., M T S Tsair, Magnetic separation of iron and heavy metals from water, Water Sci Technol., 47: pp.29-32, 2003 Norde W., A C I Anusiem, Adsorption, Desorption and Readsorption of Proteins on Solid-Surfaces, Colloid Surface., 66: pp.73-80, 1992 Norde W., J P Favier, Structure of Adsorbed and Desorbed Proteins, Colloid Surface., 64: pp.87-93, 1992 Norde W., C E Giacomelli, BSA structural changes during homomolecular exchange between the adsorbed and the dissolved states, J Biotechnol., 79: pp.259-268, 2000 Nunez L., B A Buchholz, M Kaminski, S B Aase, N R Brown, G F Vandegrift, Actinide separation of high-level waste using solvent extractants on magnetic microparticles, Separ Sci Technol., 31: pp.1393-1407, 1996 142 References O'Brien S M., R P Sloane, O R T Thomas, P Dunnill, Characterisation of nonporous magnetic chelator supports and their use to recover polyhistidine-tailed T4 lysozyme from a crude E-coli extract, J Biotechnol., 54: pp.53-67, 1997 O'Brien S M., O R T Thomas, P Dunnill, Non-porous magnetic chelator supports for protein recovery by immobilised metal affinity adsorption, J Biotechnol., 50: pp.13-25, 1996 Ozaki H., Z Liu, Y Terashima, Utilization of Microorganisms Immobilized with Magnetic Particles for Sewage and Waste-Water Treatment, Water Sci Technol., 23: pp.1125-1136, 1991 Park J W., P R Jaffe, Phenanthrene Removal from Soil Slurries with SurfactantTreated Oxides, J Environ Eng.-Asce, 121: pp.430-437, 1995 Perez C., K Griebenow, Fourier-transform infrared spectroscopic investigation of the thermal denaturation of hen egg-white lysozyme dissolved in aqueous buffer and glycerol, Biotechnol Lett., 22: pp.1899-1905, 2000 Peters Jr T., Serum albumin, Adv Protein Chem., 37: pp.161-245, 1985 Peters T J., Serum albumin, Adv Protein Chem., 37: pp.161-245, 1985 Pieters B R., G Bardeletti, Enzyme Immobilization on a Low-Cost Magnetic Support - Kinetic-Studies on Immobilized and Coimmobilized Glucose-Oxidase and Glucoamylase, Enzyme Microb Tech., 14: pp.361-370, 1992 143 References Roath S., Biological and Biomedical Aspects of Magnetic Fluid Technology, J Magn Magn Mater., 122: pp.329-334, 1993 Rocha F M., S C de Pinho, R L Zollner, M H A Santana, Preparation and characterization of affinity magnetoliposomes useful for the detection of antiphospholipid antibodies, J Magn Magn Mater., 225: pp.101-108, 2001 Roger J., J N Pons, R Massart, A Halbreich, J C Bacri, Some biomedical applications of ferrofluids, Eur Phys J.-Appl Phys., 5: pp.321-325, 1999 Rorrer G L., T Y Hsien, J D Way, Synthesis of Porous-Magnetic Chitosan Beads for Removal of Cadmium Ions from Waste-Water, Ind Eng Chem Res., 32: pp.21702178, 1993 Rosensweig R E., 1985 Ferrohydrodynamics Cambridge; New York: Cambridge University Press, xv, 344 p Rudge S R., T L Kurtz, C R Vessely, L G Catterall, D L Williamson, Preparation, characterization, and performance of magnetic iron-carbon composite microparticles for chemotherapy, Biomaterials, 21: pp.1411-1420, 2000 Rusetski A N., E K Ruuge, Magnetic Fluid as a Possible Drug Carrier for Thrombosis Treatment, J Magn Magn Mater., 85: pp.299-302, 1990 Ruuge E K., A N Rusetski, Magnetic Fluids as Drug Carriers - Targeted Transport of Drugs by a Magnetic-Field, J Magn Magn Mater., 122: pp.335-339, 1993 144 References Safarik I., Removal of Organic Polycyclic Compounds from Water Solutions with a Magnetic Chitosan Based Sorbent Bearing Copper Phthalocyanine Dye, Water Res., 29: pp.101-105, 1995 Safarik I., K Nymburska, M Safarikova, Adsorption of water-soluble organic dyes on magnetic charcoal, J Chem Technol Biotechnol., 69: pp.1-4, 1997 Safarik I., M Safarikova, Batch Isolation of Hen Egg-White Lysozyme with Magnetic Chitin, J Biochem Bioph Meth., 27: pp.327-330, 1993 Safarik I., M Safarikova, Isolation and removal of proteolytic enzymes with magnetic cross-linked erythrocytes, J Magn Magn Mater., 225: pp.169-174, 2001 Safarik I., M Safarikova, Magnetic nanoparticles and biosciences, Monatsh Chem., 133: pp.737-759, 2002 Safarikova M., I Safarik, The use of copper phthalocyannine dye immobilized on magnetic carriers for the isolation of planar organic compounds, Ecolog Chem., 5: pp.205-209, 1996 Safarikova M., I Safarik, Magnetic solid-phase extraction, J Magn Magn Mater., 194: pp.108-112, 1999 Safarikova M., I Safarik, One-step partial purification of Solanum tuberosum tuber lectin using magnetic chitosan particles, Biotechnol Lett., 22: pp.941-945, 2000 145 References Schuster M., E Wasserbauer, C Ortner, K Graumann, A Jungbauer, F Hammerschmid, G Werner, Short cut of protein purification by integration of celldisrupture and affinity extraction, Bioseparation, 9: pp.59-67, 2000 Scopes R K., 1987 Protein purification: principles and practice New York: SpringerVerlag, xv, 329 p Setchell C H., Magnetic separation in biotechnology - A review, J Chem Technol Biotechnol., 35B: pp.175-182, 1985 Shen L F., P E Laibinis, T A Hatton, Bilayer surfactant stabilized magnetic fluids: Synthesis and interactions at interfaces, Langmuir, 15: pp.447-453, 1999 Shimoiizaka J (1978) USA Patent Shinkai M., Functional magnetic particles for medical application, J Biosci Bioeng., 94: pp.606-613, 2002 Shinkai M., H Honda, T Kobayashi, Preparation of Fine Magnetic Particles and Application for Enzyme Immobilization, Biocatalysis, 5: pp.61-69, 1991 Suzuki M., M Kamihira, T Shiraishi, H Takeuchi, T Kobayashi, Affinity Partitioning of Protein-a Using a Magnetic Aqueous 2-Phase System, J Ferment Bioeng., 80: pp.78-84, 1995a 146 References Suzuki M., M Shinkai, M Kamihira, T Kobayashi, Preparation and Characteristics of Magnetite-Labeled Antibody with the Use of Poly(Ethylene Glycol) Derivatives, Biotechnol Appl Bioc., 21: pp.335-345, 1995b Takayasu M., D R Kelland, J V Minervini, Continuous magnetic separation of blood components from whole blood, IEEE T Appl Supercon., 10: pp.927-930, 2000 Tamaura Y., T Katsura, S Rojarayanont, T Yoshida, H Abe, Ferrite Process Heavy-Metal Ions Treatment System, Water Sci Technol., 23: pp.1893-1900, 1991 Taniguchi T., D Duracher, T Delair, A Elaissari, C Pichot, Adsorption/desorption behavior and covalent grafting of an antibody onto cationic amino-functionalized poly(styrene-N-isopropylacrylamide) core-shell latex particles, Colloid Surface B, 29: pp.53-65, 2003 Tanyolac D., A R Ozdural, BSA adsorption onto magnetic polyvinylbutyral microbeads, J Appl Polym Sci., 80: pp.707-715, 2001 Tatsumi K., S Wada, H Ichikawa, Removal of chlorophenols from wastewater by immobilized horseradish peroxidase, Biotechnol Bioeng., 51: pp.126-130, 1996 Tong X D., Y Sun, Agar-based magnetic affinity support for protein adsorption, Biotechnol Progr., 17: pp.738-743, 2001 Tong X D., B Xue, Y Sun, A novel magnetic affinity support for protein adsorption and purification, Biotechnol Progr., 17: pp.134-139, 2001 147 References Tremsina Y S., V I Sevastianov, S Petrash, W Dando, M D Foster, Competitive adsorption of human serum albumin and gamma-globulin from a binary protein mixture onto hexadecyltrichlorosilane coated glass, J Biomat Sci.-Polym E., 9: pp.151-161, 1998 Tyagi R., M N Gupta, Purification and Immobilization of Aspergillus-Niger Pectinase on Magnetic Latex Beads, Biocatal Biotransfor., 12: pp.293-298, 1995 Uhlen M., Magnetic Separation of DNA, Nature, 340: pp.733-734, 1989 van Leemputten E., M Horisberger, Immobilization of enzymes on magnetic particles, Biotechnol Bioeng., 16: pp.385-396, 1974 Varlan A R., W Sansen, A VanLoey, M Hendrickx, Covalent enzyme immobilization on paramagnetic polyacrolein beads, Biosens Bioelectron., 11: pp.443-448, 1996 Vermonden T., C E Giacomelli, W Norde, Reversibility of structural rearrangements in bovine serum albumin during homomolecular exchange from AgI particles, Langmuir, 17: pp.3734-3740, 2001 Wada S., H Ichikawa, K Tatsumi, Removal of Phenols with Tyrosinase Immobilized on Magnetite, Water Sci Technol., 26: pp.2057-2059, 1992 148 References Wada S., H Ichikawa, K Tatsumi, Removal of Phenols and Aromatic-Amines from Waste-Water by a Combination Treatment with Tyrosinase and a Coagulant, Biotechnol Bioeng., 45: pp.304-309, 1995 Wang W., Instability, stabilization, and formulation of liquid protein pharmaceuticals, Int J Pharm., 185: pp.129-188, 1999 Wassell D T H., G Embery, Adsorption of bovine serum albumin on to titanium powder, Biomaterials, 17: pp.859-864, 1996 Watson J H P., D C Ellwood, Biomagnetic Separation and Extraction Process for Heavy-Metals from Solution, Miner Eng., 7: pp.1017-1028, 1994 White D A., G Athanasiou, Removal of heavy metals using magnetic flocs, Process Saf Environ., 78: pp.149-152, 2000 Whitesides G M., R J Kazlauskas, L Josephson, Magnetic separations in biotechnology, Trends Biotechnol., 1: pp.144-148, 1983 Wooding A., M Kilner, D B Lambrick, Studies of the Double Surfactant Layer Stabilization of Water-Based Magnetic Fluids, J Colloid Interf Sci., 144: pp.236-242, 1991 Xue B., Y Sun, Protein adsorption equilibria and kinetics to a poly(vinyl alcohol)based magnetic affinity support, J Chromatogr A, 921: pp.109-119, 2001 149 References Xue B., Y Sun, Fabrication and characterization of a rigid magnetic matrix for protein adsorption, J Chromatogr A, 947: pp.185-193, 2002 Yoon J Y., J H Kim, W S Kim, Interpretation of protein adsorption phenomena onto functional microspheres, Colloid Surface B, 12: pp.15-22, 1998a Yoon J Y., J H Lee, J H Kim, W S Kim, Separation of serum proteins with uncoupled microsphere particles in a stirred cell, Colloid Surface B, 10: pp.365-377, 1998b Yoshida H., T Kataoka, Adsorption of Bsa on Cross-Linked Chitosan - the Equilibrium Isotherm, Chem Eng J Bioch Eng., 41: pp.B11-B15, 1989 Zaitsev V S., D S Filimonov, I A Presnyakov, R J Gambino, B Chu, 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