Study on spinel iron oxide nanoparticles

Synthesis, Characterisation and Applications of Iron Oxide Nanoparticles

GERMAN SALAZAR-ALVAREZ

Doctoral Thesis Stockholm, Sweden 2004

ISRN KTH/MSE--04/59--SE+CHEM/AVH ISBN 91-7283-884-1

KTH Materialvetenskap SE-100 44 Stockholm SWEDEN

Akademisk avhandling som med tillst˚ and av Kungl Tekniska h¨ogskolan framl¨agges till offentlig granskning f¨or avl¨aggande av filosofie doktorsexamen fredagen den 17 december 2004 kl 10.00 i D3, Huvudbyggnaden, Kungliga Tekniska h¨ogskolan, Lindstedtsv¨agen 5, Stockholm. c German Salazar-Alvarez, december 2004

Tryck: Universitetsservice US AB

iii Abstract This thesis deals with the synthesis, characterisation, and some applications of ferrimagnetic iron oxide nanoparticles. The iron oxide systems presented in this work are Fe3 O4 , γ-Fe2 O3 , Nix Fe3−x O4 , Cox Fe3−x O4 . Iron oxide nanoparticles were prepared using chemical synthesis methods. Nanoparticles with a narrow size distribution were synthesised using zone confinement methods such as nanoemulsions and the novel flow injection synthesis. The flow injection method consisted of the precipitation of iron oxide nanoparticles in a continuous or segmented flow in a capillary reactor under laminar flow. Also, the preparation of particles in a ∼ 40 g/batch scale was carried out in a computer controlled stirred reactor which allowed reproducible synthesis conditions such a ∆pH ∼ 0.1 and ∆T ∼ 1 ◦ C. Several chemical and hydrodynamic parameters were optimised to achieve high phase purity and magnetisation. These methods allowed the preparation of particles with tailored mean particle size from 3 up to 12 nm. Spinel iron oxide nanoparticles were also doped with Ni and Co. The influence of the synthesis parameters on the morphological and magnetic properties of nickel- and cobalt-doped magnetite (Nix Fe3−x O4 and Cox Fe3−x O4 ) was studied. Particle size, morphology, and composition of the materials are strongly dependent on reaction conditions, e.g., metal to hydroxide ratio and temperature. Cubic, spherical, and particles of Cox Fe3−x O4 with varying morphology could be obtained by varying the metal to hydroxide ratio. M¨ossbauer studies indicated a disordered structure with a relatively low degree of inversion in the spinel structure. The coercivity of the particles increased with the cobalt content. High resolution microscopy and electron diffraction studies showed that cubic particles are monocrystalline. Iron oxide nanoparticles (Fe3 O4 and γ-Fe2 O3 ) with a particle size of about 10 nm were coated with layers of inorganic materials such as silica and gold to render the particles higher functionality and vary the surface properties. Iron oxide nanoparticles were coated with silica in a controlled manner producing either single coated particles, with a mean particle size varying from 12 to 60 nm, or multiparticle-beads with mean particle size in the range of 50-250 nm. The use of iron oxide nanoparticles in two applications was studied, i.e., for DNA purification and as optical power limiting agents. Silica-coated iron oxide particles were investigated for use in magnetic purification of plasmid DNA of the size range 0.1–1.7 kbp. The prepared nanoparticles have shown higher DNA recovery yields when compared to commercial particles. Single silica-coated nanoparticles have DNA recovery yield of about 70%, while the beads of multiple particles showed a recovery of approximately 50%. The optical power limiting application of coated and non-coated iron oxide nanoparticles was investigated. Both aqueous suspensions of pristine iron oxide nanoparticles consisting mainly of γ-Fe2 O3 and silica-coated iron oxide nanoparticles had a clamping level of about 3 µJ. The photoinduced nonlinear light scattering is the dominating nonlinear mechanism. Silica-coated nanoparticles have shown a better performance as a result of their higher colloidal stability.

iv Sammanfattning I denna avhandlingen diskuteras syntes, karakterisering och till¨ampningar f¨ or nanopartiklar av j¨ arnoxid. De olika j¨arnoxidsystem som presenteras i arbetet best˚ ar av Fe3 O4 , γ-Fe2 O3 , Nix Fe3−x O4 , Cox Fe3−x O4 . Nanopartiklarna av j¨ arnoxid har framst¨allts via kemiska syntesmetoder. Nanopartiklar med liten varians i storleken (σ < 20%) och en storleksdistribution 3-8 mm framst¨ alldes i den zon isolering fr˚ an inverterade miceller och i ett nyutvecklat metod som bygger p˚ a fl¨odesinjektion (flow injection synthesis). ˚ A andra sida, f¨ or att f˚ a reproducerbar betingelser vid syntes av nanopartiklar i 40 grams skala har en automatiserad reaktor anv¨ants (∆pH ∼ 0.1 och ∆T ∼ 1 ◦ C). Flera kemiska och hydrodynamiska parametrar optimiserades f¨ or att h¨ ogre fasrenhet och b¨ attre magnetiska egenskaper skulle erh˚ allas. Dessa reaktionsbetingelser till˚ ater framst¨allning av nanopartikar i relativt stora m¨ angder i storleksomr˚ adet 3-12 nm. J¨ arnoxid nanopartiklar med spinell struktur framst¨alldes via doping med nickel och kobolt. Effekterna fr˚ an syntesbetingelserna p˚ a morfologin och magnetiska egenskaper hos partiklarna har studerats hos nickel och koboltdopad magnetit (Nix Fe3−x O4 respektive Cox Fe3−x O4 ) . Partiklarnas storlek, morfologi och kemiska sammans¨ attning var beroende av koncentrationsf¨orh˚ allandet mellan metall-/ hydroxidjon. Via ¨andringar i detta f¨orh˚ allande kunde Cox Fe3−x O4 partiklarnas utseende styras s˚ a att kubiska, sf¨ariska eller till och med odefinierade partiklar bildades. M¨ ossbauersspektroskopi har pekat p˚ a en oregelbunden katjondistribution i strukturen. Partiklarnas koercivitet ¨okade med kobolthalten. H¨ oguppl¨ osande transmissionselektronmikroskopi och elektrondiffraktion visade att de kubiska partiklarna var monokristaller. F¨ or att kunna p˚ averka partiklarnas funktionalitet och ytkemiska egenskaper har nanopartiklar av j¨ arnoxid med en partikelstorlek p˚ a 10 nm framst¨allts med ett ytskikt av oorganiskt material, s˚ asom kiseloxid och guld. Kiseloxid och guld anv¨ andes f¨ or att de har v¨alk¨anda kemiska egenskaper och har anv¨ants under en l¨ angre tid inom biologiska applikationer och i supraledareindustrin. Ett ytlager av kiseloxid har belagts b˚ ade p˚ a ytan av enskilda nanopartiklarna i storleksordningen 12-60 nm och p˚ a partikelaggregat, s k beads i storleksordningen 50-250 nm. Kiselbelagda nanopartiklar av j¨arnoxid har studerats f¨or anv¨andning inom till¨ ampningar f¨ or reng¨ orning av plasmid DNA i storleksspannet 0,1-1,7 kbp, och som optiska limiter. ˚ Atervinning av DNA med enskilda partiklar ¨ar upp till 70%, medan beads visat ˚ atervinning av ca 50%. Optiska begr¨ ansning studerades med suspensioner av coatade och uncoatade j¨ arnoxid nanopartiklar. B˚ ada suspensionerna har en kl¨amniv˚ a av ca 3 µJ. Ickelinj¨ ara spridning av laserljuset ¨ar den dominerande mekanism.

v

Ïðîãóëêà â ìèëþ ñ ëþáèìîé æåíùèíîé ñîñòàâëÿåò ëèøü ñòî øàãîâ.

Äàøåíüêå

Preface My graduate studies were originally related to studies on the recovery of heavy metals from aqueous media by polymeric membranes. This was an extension of the studies I carried out in Mexico and for which I received my Bachelor in Science in 1999. The work was completed by presenting the Licentiate in Engineering in 2001. During the period 1999-2001, most of my colleagues were working with the synthesis of nanoparticles for various applications ranging from electronic to biomedical. It was this interdisciplinary environment what caught my interest. After a discussion with my supervisor I started to perform some experiments and read more about the nanoworld immediately after presenting my Licentiate. Within half a year I joined the European project magnanomed, which was oriented towards some biomedical applications of iron oxide-based nanoparticles. During the time of the project we produced nanomaterials to be used for DNA separation and purification, work which we carried out in collaboration with the Max Planck Institute for Molecular Genetics in Berlin-Dahlem. The initial magnetic characterisation of the materials was undertaken by Liquids Research Ltd. at Bangor, Wales. Shortly after the end of the magnanomed project, in 2003, discussion were held with people from the Department of Polymer Technology at KTH to produce nanoparticles of substituted ferrites to be incorporated into polymeric composite structures. The discussions were extended a bit and they turned into two manuscripts -so far. Concomitantly with the main research efforts, during the period 2001-2004 small projects were carried out in collaboration with FOI to investigate the applicability of nanomaterials as optical limiting agents against laser radiation. During this last stage, collaborations were also established with the Department of Physics at Universitat Aut`onoma de Barcelona and the Department of Physicochemical, Inorganic and Structural Chemistry at Stockholm University. Regarding the present work, I would like to point out certain things. As far as it was possible, SI units and terminology∗ were used throughout the thesis and the manuscripts. The values for various physical and chemical constants were obtained from the Compendium of Chemical Terminology edited by the IUPAC.† Also, ∗

The definition of several terms can be found in http://wikipedia.org/ For more information about the chemical symbols, values of universal constants, etc., please refer to http://www.iupac.org/ †

vii

viii

PREFACE

in addition to the regular bibliographic information provided by the references to scientific articles, the document object identifier, DOI, was added whenever it was available.∗ G.S.A.

∗

For more information about the DOI please refer to http://www.doi.org/.

LIST OF PUBLICATIONS

ix

List of publications I G. Salazar-Alvarez, A.A. Zagorodni and M. Muhammed, Magnetite core-inorganic shell nanoparticles for biomedical applications by novel confined-zone synthesis, Proceedings of the 2nd IEEE Conference on Nanotechnology 2002, 75-78. II G. Salazar-Alvarez, M. Muhammed and A.A. Zagorodni, Novel flow injection synthesis of iron oxide nanoparticles with narrow size distribution, Submitted to Chemistry of Materials. III R.T. Olsson, G. Salazar-Alvarez, M.S. Hedenqvist, U.W. Gedde, F. Lindberg and S.J. Savage, Controlled synthesis of near-stoichiometric cobalt ferrite nanoparticles, To be submitted to Chemistry of Materials. IV R.T. Olsson, M.S. Hedenqvist, U.W. Gedde, G. Salazar-Alvarez, M. Muhammed, and S.J. Savage, Synthesis and characterisation of cubic cobalt ferrite nanoparticles, J. Am. Ceram. Soc., in press. V G. Salazar-Alvarez, R. Reinhardt, and M. Muhammed, Silica-coated superparamagnetic iron oxide nanoparticles for the purification of plasmid DNA, Submitted to Chemistry of Materials. VI G. Salazar-Alvarez, E. Bj¨orkman, C. Lopes, A. Eriksson, S. Svensson and M. Muhammed, Synthesis and nonlinear light scattering of nanoemulsions and nanoparticle suspensions, Submitted to Journal of Materials Chemistry.

x

PREFACE

Contributions of the author I Performing experiments, characterisation of the samples, evaluation of the results, and writing. II Planning of experiments, performing experiments, characterisation of the samples, evaluation of the results, and writing. III Planning of experiments, characterisation of the samples, evaluation of results, and writing. HRTEM analysis done by Stockholms University. IV Design of experiments, characterisation of the samples, evaluation of results, and writing. V Planning of experiments, characterisation of the samples, analysis of results, and writing. DNA purification carried out by Max Planck Institute for Molecular Genetics. VI Planning of experiments, characterisation of the samples, analysis of results, and writing of the manuscript. Optical measurements by the Swedish Defence Research Agency.

Other work not included 1 G. Salazar-Alvarez, A.N. Bautista-Flores, E. Rodr´ıguez de San Miguel, M. Muhammed and J. de Gyves, Transport characterisation of a PIM system used for the extraction of Pb(II) using D2EHPA as carrier. Journal of Membrane Science, in press. 2 A. Uheida, G. Salazar-Alvarez, E. Bj¨orkman and M. Muhammed, The removal of Co2+ from aqueous solution by γ-Fe2 O3 and Fe3 O4 nanoparticles. To be submitted to Separation Science and Technology. 3 G. Salazar-Alvarez, J. Nogu´es, F. Lindberg, R.T. Olsson, U.W. Gedde, M. Muhammed, On the structure and magnetic properties of disordered Cox Fe3−x O4−δ nanoparticles. In preparation 4 E. Bj¨orkman, G. Salazar-Alvarez and Mamoun Muhammed, Measurement of the H2 sorption properties of UO2(s) and fuel alloy particles Project report to Swedish Nuclear Fuel and Waste Management, SKB

Symbols and acronyms Latin A B Bhkl C CZ D E E g H HC J k k K m M n R R1 R2 Re S T1 T2 T V w Z

Hamaker constant (N·m) magnetic flux density (A/m, T, or Oe) full width at half maximum (rad) Curie’s constant molar concentration of species Z(m=mol/dm3 ) particle size (nm) energy (J) electrode potential (V) gravitational acceleration constant (9.81 m/s2 ) magnetic field strength (A/m, T or Oe) coercive field strength (A/m, T or Oe) exchange energy (J) Boltzmann’s constant (1.38 × 10−23 J/K) kinetic formation constant anisotropy constant (J · m−3 ) magnetic moment (J/T) magnetisation (A · m−1 · kg−1 or emu·cm−3 ) a number (dimensionless) ratio of occupied octahedral and tetrahedral sites spin-lattice relaxation rate (s−1 ) spin-spin relaxation rate (s−1 ) Reynold’s number (dimensionless) surface area (m2 ) spin-lattice relaxation time (s) spin-spin relaxation time (s) temperature (K) particle volume (m3 ) size polydispersity (dimensionless) formula units per cell

xi

xii

SYMBOLS AND ACRONYMS

Greeks ζ

θ λ µB ρ σ τ φ χ

electrokinetic surface potential or zeta-potential(mV) crystallographic measuring angle (deg or ◦ ) wavelength (nm) Bohr magneton (9.274015 × 10−24 J/T) density (g/cm3 ) standard deviation relaxation time (s) phase magnetic susceptibility (dimensionless or emu/Oe·g)

Subscripts av b bk B B C C fw H i in out M N P R s S SA T v X

average bulk backward reaction Brown blocking coercive Curie forward reaction hydrodynamic initial input output magnetic N´eel physical remanence sample saturation surface area thermal volume crystallite

Superscripts ◦

measured at standard room temperature and pressure

xiii

Acronyms AFF CFF DSC ED fcc fis HRTEM IMS IUPAC JCPDS MRI OPL SAED SI SPIO SQUID SFTR STP TEM TEOS THPC TMAOH USPIO XRD

anionic ferrofluid cationic ferrofluid differential scanning calorimetry electron diffraction face-centred cubic flow-injection synthesis high resolution transmission electron microscopy or micrograph immumomagnetic separation International Union of Pure and Applied Chemistry Joint Committee on Powder Diffraction Standards magnetic resonance imaging optical power limiting selected area electron diffraction Syst`eme International d’Unit´es small particles of iron oxides superconductive quantum interference device magnetometry segmented flow tubular reactor standard temperature and pressure (298 K and 0.101 325 MPa) transmission electron microscopy or micrograph tetraethyl orthosilicate tetrakis(hydroxymethyl)phosphonium chloride tetramethylammonium hydroxide ultra-small particles of iron oxides X-ray diffraction or diffractogram

xiv

SYMBOLS AND ACRONYMS

Contents Preface vii List of publications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix Symbols and acronyms

xi

Contents

xv

1 Background 1.1 Crystal structure . . . . . 1.2 Magnetic properties . . . 1.3 Applications of iron oxides 1.4 Scope of this work . . . .

. . . .

1 1 4 8 12

2 Fabrication of iron oxide nanoparticles 2.1 Chemical methods . . . . . . . . . . . . . . . . . . . . . . . . . . . .

15 15

3 Materials and methods 3.1 Synthesis of nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Characterisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 DNA assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

21 21 25 29

4 Results and discussion 4.1 Synthesis of various iron oxides . . . . . . . . . . . . . . . . . . . . . 4.2 Magnetic characterisation . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Applications of magnetic nanoparticles . . . . . . . . . . . . . . . . .

31 31 43 45

5 Conclusions

51

Bibliography

57

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One

Background The iron oxides (generic name for iron oxides, hydroxides, oxyhydroxides, and other related compounds) have been known for millennia. Such minerals were used originally as pigments for paints during the Palaeolithic. Much later, they were used in the magnetic compass when it was invented in China. This was the first application of magnetic iron oxides, also known as lodestones, used by early navigators to locate the magnetic ’north’. The most plentiful deposits of these stones were found in the district of Magnesia in Asia Minor, hence the mineral’s name became magnetite. Magnetite can be found naturally occurring in molluscs, bees, pigeons, magnetotactic bacteria, and algae. The biogenic formation of magnetite crystals in Magnetobactericum sp. has received a great deal of attention[Sch99] due the narrow-size distribution of the crystals (magnetosomes) and their magnetic properties.[PM02] Synthetically, magnetite can be formed by precipitation in alkaline aqueous media of a mixture of Fe3+ and Fe2+ , by oxidation of Fe(II) solutions or Fe(OH)2 , by interaction of Fe2+ with ferrihydrite, or decomposition of organic precursors, etc.[CS03, WKC+ 04]

1.1

Crystal structure

Several iron oxides share crystal structure with other minerals. For instance, goethite is isostructural with diaspore (α-AlOOH), haematite with corundum (Al2 O3 ), and magnetite with spinel (Mg Al2 O4 ). Thus, the denomination spinel ferrites∗ for the latter case. The structure of iron oxides is dominated by the arrangement of the oxygen or hydroxide anions. The cations occupy different positions relative to these layers of anions. Table 1.1 lists crystallographic information regarding some of the iron oxides. In the case of spinel ferrites, the oxygens form a fcc sublattice with the ∗

Ferrites are defined as a material composed of Fe3+ ions as the main cationic component. There are three main families of ferrites: spinel ferrites, garnet ferrites, and hexaferrites. In metallurgy, the term ferrite has a different meaning.

1

2

CHAPTER 1. BACKGROUND

c

a

b b c

a

Fe+3

Fe O

Fe+2.5 O-2

(a)

(b)

Figure 1.1: Representation of the inverse spinel structure of (a) magnetite and (b) maghemite.

cations occupying 16 octahedral (B-sites) and 8 tetrahedral positions (A-sites). Figure 1.1 shows the unit cell corresponding to magnetite and maghemite, typical inverse spinel ferrites. Maghemite has basically the same crystal as magnetite. However maghemite can III be considered as an Fe(II)-deficient magnetite with formula (FeIII 8 )A [Fe40/3 8/3 ]B O32 , where  represents a vacancy, A indicates tetrahedral positioning and B octahedral.

W¨ ustite

Haematite Magnetite Maghemite

Feroxyhyte High pressure Ferrihydrite Bernalite

Goethite Lepidocrocite Akagan´eite Schwertmannite

Mineral

Chemical Composition α-Fe O OH γ-Fe O OH β-Fe O OH Fe16 O16 (OH)y (SO4 )z · nH2 O δ-Fe O OH δ 0 -Fe O OH Fe OOH Fe5 O8 · 4H2 O Fe OH3 Fe OH2 α-Fe2 O3 Fe3 O4 γ-Fe2 O3 -Fe2 O3 β-Fe2 O3 Fe O Cubic

Crystallographic system Orthorhombic Orthorhombic Monoclinic Tetragonal Hexagonal Hexagonal Orthorhombic Hexagonal Orthorhombic Hexagonal Hexagonal Cubic Cubic Orthorhombic Fm3m

Space group Pnma Bbmm I2/m P4/m P3ml P3ml Pn21 m P31c; P3 Immm P3ml R¯3c Fd3m P43 32 Pna21 0.4302

Unit cell dimensions (nm) a b c β◦ 0.9956 0.30215 0.4608 0.3071 1.2520 0.3873 1.0560 0.3031 1.0483 90.63 1.066 0.604 0.293 0.449 0.293 0.456 0.4932 0.4432 0.2994 0.2955 0.937 0.7544 0.7560 0.7558 0.3262 0.4596 0.5034 1.3752 0.8396 0.83474 0.5095 0.879 0.9437

1 2 2 4 8 1 6 8 8 8

4 4 8

Z

Table 1.1: Crystallographic information for various iron oxides, from [CS03]. A = Cl− ; 1/2 SO2− 4 , Z is the number of formula units in the unit cell.

1.1. CRYSTAL STRUCTURE 3

4

CHAPTER 1. BACKGROUND

(a)

(b)

(c)

(d)

Figure 1.2: Different orientations of magnetic dipoles: (a) paramagnetic, (b) ferromagnetic, (c) antiferromagnetic, and (d) ferrimagnetic.

1.2

Magnetic properties

There are various forms of magnetism that arise depending on how the dipoles interact with each other. Figure 1.2 shows a schematic representation of the different types of arrangements of magnetic dipoles.

1.2.1

Paramagnetism

Due to the magnetic field generated by unpaired electrons, atoms may behave as small magnets under the influence of an external magnetic field. However, when the applied magnetic field is removed the thermal fluctuations would make the magnetic moment of the paramagnetic material to move randomly. Under relatively low magnetic field saturation this effect can be described by Curie’s law: B (1.1) T where M is the resulting magnetisation, B is the magnetic flux density of the applied field, T is the absolute temperature, and C is a material-specific Curie constant. The expected behaviour is that paramagnetic materials would increase their magnetisation with the applied magnetic field but decrease with the temperature. In general paramagnetic effects are small and the response of the material to the magnetic field, i.e., the magnetic susceptibility, χ, is in the order of 10−3 to 10−5 , which is several orders of magnitude smaller than that of ferromagnetic materials. M =C

Saturation magnetisation,MS , is the point at which all the spins are aligned with the magnetic field; the coercive field, HC , is the internal magnetic field of the material; and the remanent magnetisation, MR , is the magnetisation retained by the material. Figure 1.3 depicts the position of the aforementioned points in a diagram so-called ’hysteresis loop’.

1.2. MAGNETIC PROPERTIES

5

M

MS Mr

χi HC

H

Figure 1.3: Magnetisation (M ) vs. applied field (H) for ferromagnetic (solid line), paramagnetic (broken line), and diamagnetic materials (dotted line). HC represents the coercive field of the material, MS the saturation magnetisation, Mr the remanent magnetisation, and χi the initial susceptibility.

1.2.2

Ferrimagnetism

In many ionic crystals the exchange energy,∗ J, between the spins of two neighbouring atoms is negative. This occurs due to the fact that the spin of the shared electron from the metal ions with the oxygen follows Hund’s rule and Pauli’s principle. Thus, if the metal ion have an outer shell less than half-occupied all the spins will be aligned parallel inside the shell and antiparallel with respect to the other metal ion. This means that antiparallel arrangement is the one that provides with the lowest energy. As mentioned earlier, in the case of spinel ferrites, the metal ions in the unit cell exist in 8 tetrahedral and 16 octahedral positions, where the metal ions are arranged into two sublattices A and B corresponding to tetrahedral and octahedral places. Neutron diffraction studies showed that there are two types of spinel fer∗

The difference in energy of two electrons in a system with antiparallel and parallel spins is called the exchange energy.

6

CHAPTER 1. BACKGROUND

E1

E0 M1

O

Jeff

M2

Figure 1.4: Ground state and excited state of two metal ions, M1 and M2 . The spin configurations as drawn become intermingled, causing an extra reduction of ground state energy; this does not occur if M1 and M2 have parallel spins. Adapted from [SW59].

rites: normal and inverse. Studies demonstrated that the normal spinel ferrites are paramagnetic whereas the inverse spinel ferrites behaved as ferromagnetic. In the case of normal spinel ferrites, the divalent metal ions are located in the sublattice A and the iron(III) ions are located in the sublattice B; whereas in the case of inverse spinel ferrites one half of the iron(III) ions occupy the sublattice A with the sublattice B occupied by the other half of iron(III) ions and the divalent metal ions. In spinel iron oxides, the metallic ions are separated by an oxygen(II) ion and since the distance between the metallic ions is too large, it was proposed that there is a superexchange between the metallic ions through the oxygen ion.[N´ee48, N´ee52] Figure 1.4 shows a schematic representation of the superexchange interactions between two metal ions through an oxygen. Half-century ago Louis N´eel explained why in the case of inverse spinel ferrites the metal ions in the sublattice A are antiparallel with respect to the metal ions in the sublattice B.[N´ee49] The net magnetic moment of the material is the difference in magnetic moments of sublattices A and B, which explained why the magnetic moment per formula unit was lower than the otherwise expected value. Thus, the higher the magnetic moment of the divalent cation, the higher the magnetisation.∗ For instance, manganese ferrite having Mn(II) with 5 unpaired electrons, i.e., 5 µB has a larger saturation magnetisation than that of nickel ferrite which is 2 µB .

∗

ions.

This rule does not apply for compositions containing both paramagnetic ions and diamagnetic

1.2. MAGNETIC PROPERTIES

1.2.3

7

Superparamagnetism

Superparamagnetic relaxation[BL59] can be explained using spherical particles – with uniaxial anisotropy– as a model system. If we consider spherical particles, the magnetic anisotropy can be approximated to be proportional to the particle volume, V . The energy barrier that separates easy magnetisation axes is the magnetic anisotropy energy, Kv V , with Kv as the volume anisotropy constant. For a small particle, the particle volume (size) is small and the magnetic energy Kv V may become comparable to (or lower than) the thermal energy (see Eq. (1.2)). Thus, the magnetic moment of the particle may fluctuate behaving as a paramagnetic atom but with a total magnetic moment that can be up to 1000 times larger than that of a single atom. Hence the term superparamagnetism. Kv V ≤ 25kT

(1.2)

Nevertheless, the moments of the particles follow a Boltzmann distribution when subjected to external applied magnetic field and thermal equilibrium. The orientation of the magnetic moment of the (non-interacting) paramagnetic particles can then be described by the Langevin function given by Eq. (1.3).[AK22]   1 mH mav = m coth α − ; α= α kT

(1.3)

where H is the applied magnetic field, k is Boltzmann constant, T is the absolute temperature, and m is the magnetic moment. Beside the dependence of the magnetisation on the particle size and composition, the magnetic properties depend greatly on the temperature. From Eq. (1.2) it is possible to obtain a critical temperature defined as the blocking temperature, TB , above which ferro- or ferrimagnetic particle will behave as superparamagnetic (Eq. (1.4)). TB =

1.2.3.1

Kv V 25k

(1.4)

Magnetic relaxation

Considering particles under the influence of an external magnetic field, it can be shown that when the applied magnetic field is removed the magnetisation of the particles is reversed. There are two main mechanisms for the magnetisation reversal: spin rotation or particle rotation, which describe whether the change in direction of the magnetic moment of the particle is due to the reversal of the magnetic spin of the particle or the actual physical rotation of the particle. The time required for the reversal of the magnetic moment of the particle (spin rotation) is related to the magnetic anisotropy of the material, N´eel relaxation time, τN . The mathematical expression of N´eel relaxation time is shown by Eq. (1.5).

8

CHAPTER 1. BACKGROUND

 τN = τ0 exp

Kv VM kT



; τ0 ∼ 10−9 s

(1.5)

The characteristic time for the randomisation of the magnetisation due mainly to the thermal motion of the particle is known as Brown relaxation time, τB , expressed by Eq. (1.6). τB =

3VH η kT

(1.6)

For a superparamagnetic material τN  τB , which means that the magnetisation reversal occurs by rotation of the magnetic axis. Alternatively, when the magnetic moment of the particle is locked to a given crystallographic direction (easy axis), the particle is thermally blocked and τB  τN . Previously it was mentioned that, under certain circumstances of particle size and temperature, ferro- or ferrimagnetic particles may be considered superparamagnetic (see Eq. (1.2) and (1.4)). The criterion of superparamagetism is regularly assumed considering particles with a relaxation time lower than 100 s. The experimental techniques used for the measurement of magnetic properties have different measuring times and thus a particle may appear as superparamagnetic using a slow technique such as SQUID or VSM (characteristic measuring time tens of seconds) whereas when measured with a fast technique such as M¨ossbauer spectroscopy (characteristic measuring time 10−7 s), it may appear as ferri- or ferromagnetic. In the case of Fe3 O4 with an anisotropy constant, Kv = 4.4 · 104 J · m−3 , the critical superparamagnetic particle size at room temperature, T = 290 K, is between 9 and 17 nm.

1.3

Applications of iron oxides

In the recent years there has been an increased interest in the application of nanotechnology to biotechnology.[WH00] Thus, a number of different uses of nanoobjects has been investigated. In the case of iron oxide-based materials, the focus has been on separations and diagnostics,[WKJ83, Shi02] DNA analysis,[Uhl89] and recently, on the use of iron oxides as contrast enhancers for magnetic resonance imaging (MRI)[JLJ+ 88, SWE+ 88], among others.

1.3.1 1.3.1.1

Biomedical applications Cell separations

Micrometre-sized magnetic particles have been used as commercial applications in biology, biomedicine and biotechnology for the separation of cells. The separation of cells, or immumomagnetic separation (IMS), is achieved by covalently binding

1.3. APPLICATIONS OF IRON OXIDES

9

magnetic nanoparticles –or magnetised beads∗ – to the cells. The bead carrying a specific antibody -usually monoclonal antibodies- on the surface binds selectively the target cell which can then be removed from the suspension with the help of a magnet. The IMS has been used for the purging and isolation of cancer cells, studies of HIV and AIDS, isolation of granulocytes, isolation of cells from various tissues, stem cells, etc.[PBMPS97] 1.3.1.2

DNA analysis

The separation and purification of nucleic acids has become more and more important for the accurate mapping of the genome. Nucleic acids are usually extracted from other cell components after cytolysis and repetitive washing procedures. The use of superparamagnetic particles for the analysis of plasmids and nucleic acids was introduced nearly 20 years ago by Uhl´en et al.[SHO+ 88] Separation and purification of DNA with magnetisable microspheres (with a typical size range of 200–2000 nm) has been reported by numerous groups.[SHO+ 88, Uhl89, HOMRS94, DTSB98] The DNA molecule is able to wrap around nanoobjects in a similar way it does around the histone core in the nucleosomes.[LMR+ 97] This finding indicates that DNA purification can be achieved even with simple nonfunctionalised nanoparticles such as silica nanoparticles. However, to improve the efficiency and selectivity of the process the surface of the particles is functionalised with specific molecules, e.g. streptavidin, which can bind non-covalently to biotinylated-DNA.[SHO+ 88] 1.3.1.3

MRI contrast agents

Magnetic resonance imaging (MRI) is based on the nuclear magnetic resonance of protons in the molecules, mainly water, that exist in a given tissue. Since the local environment of a given tissue varies depends on its position in the body it is possible to use MRI to identify various types of tissues. The characteristic measure in MRI is the proton relaxation rate or its inverse, the proton relaxation time, R1 ≡ 1/T1 and R2 ≡ 1/T2 . In this technique there are two different types of proton relaxation times: the longitudinal relaxation time, T1 , (spin-lattice relaxation) and the transverse relaxation time, T2 (spin-spin relaxation). Hence, there are two types of contrast agents: positive agents, which act on T1 providing a positive enhancement of the signal, appearing bright on the MRI scan; and the negative agents, which reduce the signal and give a negative enhancement, appearing as dark spots in the scan.[Pro22] Typical positive contrast agents are paramagnetic compounds based on rare earth ions, typically gadolinium-containing chelates. Nevertheless, other alterna∗

Beads is the term hereby used for particles or group of particles dispersed in an inorganic or organic matrix in a second step after the synthesis of the particle. The beads usually have a size from 100 nm to several micrometres and spheroidal shape.

10

CHAPTER 1. BACKGROUND

tives are sought after due to the high toxicity of the heavy metals and the low contrast enhancement. However, negative contrast enhancers (T2 agents) as iron oxide nanoparticles have been used due to a much higher magnetisation per concentration unit,[JLJ+ 88, SWE+ 88] reported to be at least one order of magnitude higher than gadolinium-containing molecules.[Bjø02] For a more complete survey of the relaxation effects induced by superparamagnetic particles refer to Roch et al.[RMG99]

Passive targeting Insofar there have been two different types of superparamagnetic contrast agents based on iron oxides that are clinically approved: the small particles of iron oxides (SPIO, 40 < DH < 200 nm) and the ultra-small particles or iron oxide (USPIO, DH < 40 nm). Due to their large size, the SPIO agents are rapidly cleared (several minutes) from blood by the reticuloendothelial system organs such as liver and spleen, thus enabling the imaging of lesions in such organs. On the other hand, the smaller USPIO agents have longer residence times (hours), allowing for imaging of the lymphatic system.[MVGD04] Furthermore, it has been reported that the use of USPIO permits imaging for more than 24 h, and sometimes as long as five days.[NVB+ 04] USPIO have lower T1 /T2 ratio than SPIO, which leads to a higher contrast on T2 -weighted images and enables the use of relatively weak magnetic fields (< 0.5 T).[KFG+ 99]

Active targeting The surface of USPIO and SPIO agents is functionalised with specific biomolecules to actively target certain tissues to be visualised by MRI technique. Several attempts have been performed to improve their surface properties, e.g. coating with different types of biocompatible materials, such as protein, polysaccharide, DNA fragments, etc.[MKCB+ 04, PST+ 04, ICB+ 01] A schematic representation of the degree of functionalisation required for such particles is given by Fig. 1.5. The visualisation of certain organs needs special functional properties of coating layer.[AMOB03] Thus, for instance, Kim et al. reported the use of polysaccharide-coated superparamagnetic iron oxide nanoparticles (SPION) for the MR imaging of brain tissue.[KZK+ 01, Kim02]. Also, in case of lesions detection in the brain, e.g., multiple sclerosis, Alzheimer’s disease, etc.; The MRI contrast agent has to pass across the blood brain barrier (BBB).[RPB+ 03, PWC+ 02] However, there are limited reports on the penetration of the BBB by nanoparticles.[NH02] Moreover, after passing the BBB those agents have to be recognised and up-taken by cells. Receptor mediated endocytosis has been proposed as possible way to introduce contrast agent in vivo and several studies have been performed where various types of cells were labelled by SPIO and further visualised by MRI.[AFHH+ 03]

1.3. APPLICATIONS OF IRON OXIDES

11

Figure 1.5: Representation of functional nanoparticles with a protective coating layer enhanced with specific ligands.

1.3.2 1.3.2.1

Other technological applications Optical power limiting agents

Advances in the development new communication methods and new sources of radiation, particularly optical power sources such as lasers, had resulted in research oriented towards the protection from exposure to such sources. In this context, the development of optical power limiting (OPL) has been focused on the design of novel materials having nonlinear optical phenomena upon irradiation with high energy sources.[Hol99] Figure 1.6 shows linear and nonlinear optical phenomena as well as the clamping level that can be achieved with a nonlinear mechanism. The clamping level demonstrates that a very pronounced optical limiting can be achieved even at high energy fluences. Among the processes that produce optical limiting are two-photon absorption, reverse saturable absorption, nonlinear scattering, nonlinear beam fanning, beam steering, photochroism, etc.[PHJ+ 99] Several types of laser blocking compounds and being extensively studied for protection from laser radiation, including liquid crystals,[UCS86, THD+ 97, KWG+ 98] dyes,[WHS+ 92, MRL98] and suspensions of carbon nanotubes,[VAR+ 99, LZQ+ 03] fullerenes[NW95, BS02] and different nanoparticles.[MSS92, JBHF98] 1.3.2.2

Ferrofluids

Ferrofluids are made of ferro- or ferrimagnetic single-domain particles suspended in a continuous medium with no long-range order between the particles. Some application areas of ferrofluids are: sealing, vibration damping, heat transfer, bearing, sensing/detection, etc.[RM90]

12

CHAPTER 1. BACKGROUND

E out linear absorption clamping level nonlinear absorption E in

Figure 1.6: OPL curves: the solid line represents the linear limiting mechanism whereas the broken line shows the nonlinear mechanism. The dotted line shows the possible clamping level that can be achieved.

There are certain stability requirements for the preparation of ferrofluids.[Ros85] These are influence by the thermal energy ET = kT , the magnetic energy EM = µ0 M HV , and the gravitational energy EG = δρV gDP . It is then possible to establish dimensionless relationships that provide information on the stability of a ferrofluid under the influence of a particular field. The maximum particle size that would be stable against settling under the influence of a small hand-held magnet can be determined by Eq. (1.7) ET kT = ≥1 EM µ0 M HV

(1.7)

Rearrangin Eq. (1.7) for the particle diameter DP = (6V /π)1/3 and assuming that H = 8 × 10−4 A/m, M = 4.46 × 105 A/m, and T = 298 K, gives a DP ∼ 8 nm. At room temperature the normal gravitational field have little influence on the stability of a ferrofluid. Also, the attractive van der Waals forces must be taken into consideration. These fluctuating dipole-dipole interactions are always present and are of great importance in neutral particles.

1.4

Scope of this work

This work is concerned with the synthesis of iron oxide nanoparticles with inverse spinel structure using different chemical methods with controlled particle size, particle size distribution, composition, and morphology. The surface modification of the particles was undertaken with different inorganic materials, namely silica and gold. Also, the particles were investigated to evaluate their application for the purification of DNA, as a contrast agent for magnetic resonance imaging, and as novel optical power limiting agents.

1.4. SCOPE OF THIS WORK

13

Papers I and II deal with the use of a novel method for the synthesis of these particles by flow-injection technique for the manufacturing of iron oxide nanoparticles in the size range of 1-10 nm with a narrow size distribution. In this study we investigate the hydrodynamic effects of different mixing schemes on the preparation of magnetite nanoparticles, as well as the factors influencing the size distribution of the synthesised particles. Papers III and IV are related to studies on the synthesis of cobalt ferrite particles. These papers present a detailed study of the effect of the chemical synthesis parameters (concentration and concentration ratios, temperature, digestion time and addition rate) on the particle characteristics: composition, particle sizes, particle size distribution, crystal structure, degree of crystallinity and morphology. The studies on the application of iron oxide nanoparticles are presented in papers V and VI. In paper V, the work is focused on the preparation of magnetic nanoparticles of magnetite (Fe3 O4 ) and maghemite (γ-Fe2 O3 ) with high saturation magnetisation, their coating with silica and their application in the magnetic purification of DNA. The suspensions of particles were characterised with traditional vibrating sample magnetometry and the novel nuclear magnetic relaxation. Finally, paper VI is concerned with studies on the effectiveness of nanoemulsions and suspensions of pristine and silica-coated iron oxide nanoparticles as novel optical power limiting agents against laser radiation.

Two

Fabrication of iron oxide nanoparticles 2.1

Chemical methods

In the last decade, the chemical methods of preparation of nanoparticles have been used for the fabrication of several types of nanoparticles where a greater control on the size, composition, etc., could be achieved. Iron oxide nanoparticles have been prepared by a variety of methods[WKC+ 04] including sonochemical reactions,[SKG+ 97] mechanochemical synthesis,[JZRM95] hydrolysis[KKA96, SSI98] and thermolysis[HCP+ 02] of precursors, polymeric matrices,[CGLQRR97, HY02] and co-precipitation in bulk,[TM82, PK89] flow injection synthesis,[SAMZ04] and in the confined zones of microemulsions.[MP96] The previous methods were used to prepare particles with homogeneous composition and narrow size distribution. However, the co-precipitation method in bulk has shown to be an economic and versatile technique used to synthesise large amounts of materials with different compositions and particle sizes.[Mat98] It has been found that the particles obtained in batch operation of the co-precipitation method have a broad size distribution and therefore a higher control of the synthesis conditions are required.

2.1.1

Synthesis of magnetite and maghemite

Magnetite can be formed by the addition of alkali solution to an aqueous solution containing Fe3+ and Fe2+ in a molar ratio of 2, which leads to the formation of green rust complexes and thence to a dark red complex with formula 2+ FeII FeIII 2 Ox (OH)2(3−x) · xH2 O from which magnetite particles precipitates.[CS03] Also, it has been suggested that the formation of magnetite involves the interaction of Fe2+ with precipitated ferrihydrite. In all cases the solubility product of magnetite has to be exceeded to be able to precipitate. The hydrolysis of iron(III) species proceeds through the formation of low molecular weight species (Eq. (2.1)) and that above OH/Fe ∼ 1 these species interact to produce polynuclear species (Eq. (2.2)). 15

16

CHAPTER 2. FABRICATION OF IRON OXIDE NANOPARTICLES

Fe3+ + H2 O Fe OH2+ + H+ 2+

Fe OH

+ H2 O

Fe(OH)+ 2

+H

(2.1a)

+

(2.1b)

kf w

−− * 2Fe OH2+ ) − − Fe2 (OH)4+ 2

(2.2)

kbk

The formation of the dimer is a fast reaction with a kf w = 630 m/s whereas the dissolution is very slow in the absence of protons, which suggest that further polymerisation may be very fast.[CS03] In the particular case of the spinel ferrites, the formation of magnetite, for instance, can be considered to proceed according to the overall reaction expressed by Eq. (2.3). Fe2+ + 2Fe3+ + 8OH− → Fe3 O4 + 4H2 O

(2.3)

whereas maghemite is formed primarily by the topotactic∗ oxidation of magnetite (Eq. (2.4)). The oxidation of synthetic magnetite to maghemite occurs via the formation of a solid solution of γ-Fe2 O3 in Fe3 O4 [CGLS65, GFM68] with the cations migrating to the surface and layers of oxygen being added to the structure to preserve the crystal structure.[GFM68]      2H2 O2   2H2 O  2H2 S O4 2H2 SO3 4Fe3 O4 + → 6γ − Fe2 O3 + (2.4)     O2 − From Eq. (2.3) it is clearly seen that in order to obtain Fe3 O4 the spontaneous oxidation of Fe2+ (E◦Fe3+ /Fe2+ = 0.77 V) by O2 (E◦O2 /H2 O = 1.23 V) has to be prevented. Otherwise, the most common by-products of the hydrolysis of Fe3+ , i.e., goethite and haematite, are formed. Fe3+ + 3OH− → α − Fe OOH + H2 O(at around 25 ◦ C) 2Fe

3+

−

+ 6OH → α − Fe2 O3 + 3H2 O(at around 100

◦ C)

(2.5a) (2.5b)

It has been reported that the end product of the oxidation (STP and below 600 of small crystals (< 3000 ˚ A) of pure magnetite is γ-Fe2 O3 , whereas the end product of the oxidation of large crystals (> 3000 ˚ A) is α-Fe2 O3 .[FG70] Nevertheless, it has been also reported that samples of magnetite containing impurities of α-Fe2 O3 transforms completely into haematite via epitaxial growth† on the [111]

◦ C)

∗ A topotactic transformation is characterised by internal atomic displacements, which may include either loss or gain of material, so that the initial and final lattices are in coherence. † Epitaxial growth is the growth of crystals of one mineral on the crystal face of another mineral, such that the crystalline substrates of both minerals have the same structural orientation.

2.1. CHEMICAL METHODS

17

Figure 2.1: Schematic representations of reverse micelles and nanoemulsions (a) o/w nanoemulsion, (b) w/o nanoemulsion, (c) bi-continuous dispersion, (d) isolated and aggregated o/w dispersion, and (e) isolated and aggregated w/o dispersion.

faces of the spinel crystal whereas large crystals form nuclei of α-Fe2 O3 , and that it is the formation of nuclei what determines the behaviour of the particles when exposed to oxygen.[CFG+ 68]

2.1.2

Confined-zone processing

The fabrication of nanoparticles with a given composition and structure requires a high degree of control on the reproducibility of the synthetic procedures. Some of the known problems are an uncontrolled mixing, low production (small units), lack of control of the residence time, etc. Zone confinement has been used to produce monodispersed particles, i.e., particles with very narrow size distributions, and to tailor the particle’s morphology. One of the techniques applied recently, is the confinement of the reaction to cavities with pre-defined size and shape, so-called templating, e.g., synthesis of particles in micro-emulsions,[OKC+ 01, MP96, PP97] sol-gel porous matrices,[MZM+ 02] aerogels,[CRM+ 02] ion exchange resins,[SFIB01, ZGW+ 92] and in cellulose.[SFIB01] 2.1.2.1

Synthesis in nanoemulsions

Emulsions consist of two (or more) immiscible liquids, such as oil (organic solvent) and water (aqueous solution), in which a discontinuous phase is formed as fine droplets dispersed in a continuous phase. The dispersion is achieved by mechanical mixing to produce turbid, thermodynamically unstable solution, where the droplet size of the dispersed phase is in the order of 1 – 50 µm.

18

CHAPTER 2. FABRICATION OF IRON OXIDE NANOPARTICLES

The addition of a surfactant with a suitable concentration stabilises the emulsion as it reduces the interfacial tension thus producing thermodynamically stable and transparent solutions, known as microemulsions. The droplet size in microemulsion systems is generally below 150 nm and therefore transparent to visible light. Nanoemulsion is actually an updated term of microemulsion as its dimension of the droplets is limited to the nanometre scale. The formation of the dispersed phase in an oil-surfactant-water (o/s/w) nanoemulsion system can be controlled at will; i.e. to form oil-in-water (o/w) or water-in-oil (w/o) nanoemulsions (see Figure 2.1). The oil and water phases often contain several dissolved components and therefore the selection of the surfactant (and co-surfactant) employed depends on the physicochemical characteristics of the system. Thus, a nanoemulsion requires to be designed for specific solutes in either the organic or aqueous phases by selecting an appropriate polar head (hydrophiliclipophilic balance). Several types of surfactants as cationic, anionic, or non-ionic can be used. The specific composition of a nanoemulsion can be determined according to a ternary phase diagram of the o/s/w system. Therefore, it is essential to determine such phase diagrams for different organic solvents (oils) and surfactants, which determine the structures and properties of a nanoemulsion to be concerned. 2.1.2.2

Concept of flowing confined-zone synthesis

The flow-injection synthesis (fis) technique consists of continuous or periodic microinjection of reactants into a carrier solution. The fis technique takes advantage of the flow-injection systems introduced by R˚ uˇziˇcka and Hansen[RH82, RH00] and have such characteristics[VdC84]: – Injection of reactant directly into the carrier stream, achieving always conditions of laminar flow (Reynolds number, Re  2000) which provides with high mixing homogeneity. – Possibility of segmenting the flow with inert fluids, thus providing the system with pool zones confined to volumes of few microlitres. – Control over the dispersion of the injected reactants by manipulation over the geometric and hydrodynamic conditions of the system. – High degree of automation by including a computerised detector in the manifold array. – High reproducibility in injection volumes and residence times. The fis integrates several components, such as the injection system, the propulsion unit, the capillary reactor, and the detector or container. The injection system consists, generally, of a T-shaped injector where the reactants mix head-on with high precision in time and volume. The injector can be replaced by a computercontrolled manifold that enables the system with segmentation capabilities. The injection system can be tailored so as to provide the chemical reaction with the

2.1. CHEMICAL METHODS

19

required limitations such as inert atmosphere, temperature control, etc. The propulsion unit comprehends the reactant, carrier, inert media solutions, and a mean for the transportation of sample such as a peristaltic pump. The capillary reactor consists of a capillary system where the solutions are injected and where the reaction takes place. The residence time of the reactants and products in the reactor which is the extent of the reaction, is controlled by the length of the capillary and the pumping rate of the solutions. The fis capillary reactor is constructed of Teflon tubing and has an internal diameter of 0.1 to 1 mm and a varied length so as to provide reaction pools of few microlitres without the need of using microsystems technology. One of the main characteristics of the system, along with the reduced volumes is that the reaction point can be adjusted. For a continuous injection where the carrier participates as reactant, the reaction takes place at the point of mixing, i.e, the injection point, which as opposite to the earlier reported[JDB+ 03] segmentedflow tubular reactor (SFTR) where the reaction takes place in a micromixer outside of the capillary system. In both cases the reaction evolves under laminar flow conditions, whose advantages in mixing have been recently reported.[HBBR03] The fis methodology can be used for the production of nanosized materials, whereas the SFTR has been applied to the synthesis of micron-sized ceramic powders.

Three

Materials and methods 3.1

Synthesis of nanoparticles

Two different approaches were used for the synthesis of the magnetic nanoparticles: bulk solution and confined-zone processing. The bulk solution processing was carried out in a stirred batch reactor with volumes of 100 to 1000 cm3 . The pH, temperature and stirring sped were carefully adjusted to minimise variations throughout the reaction time. In the second method, the reaction was carried out in confined zones provided by nanoemulsions and our earlier developed method of flow injection synthesis with constant hydrodynamic conditions.[WM99]

3.1.1 3.1.1.1

Batch synthesis in stirred reactor Synthesis of Fe3 O4 and γ − Fe2 O3

Iron(II) and iron(III) chlorides (CFe(III) /CFe(II) = 2) were dissolved under argon atmosphere in a deoxygenated aqueous solution of HCl 0.2 m to a total iron concentration of 1.5 m(iron source solution). The iron source solution was added to a deoxygenated aqueous solution of N H4 OH at 70 ◦ C under mechanical stirring. The reaction was allowed to proceed for 45 min under an atmosphere of argon followed by decantation using a magnet to retain the black gel containing the magnetite nanoparticles. Magnetite particles were obtained by washing the gel several times with deoxygenated water, adjusting the pH to neutral, and drying it under vacuum. Maghemite particles were obtained by washing the gel several times with an aqueous solution of H N O3 0.2 m and finally drying the particles in air. 3.1.1.2

Synthesis of Nix Fe3−x O4

An aqueous solution of nickel(II) chloride was mixed with a freshly prepared aqueous solution of iron(II) chloride. The metal solution was then added to an ammonium hydroxide solution under vigorous mechanical stirring. The pH of the reaction was 21

22

CHAPTER 3. MATERIALS AND METHODS

kept at 11.5-12.5 with additions of ammonium hydroxide solution and the reaction was allowed to proceed 20 min after a dark colouration appeared. The gel was then washed several times with water and dried. 3.1.1.3

Synthesis of Cox Fe3−x O4

An aqueous solution containing iron(II) sulfate and cobalt(II) chloride (CFe2+ /CCo2+ = 2) was heated up to 90 ◦ C and added to an aqueous solution of sodium hydroxide and potassium nitrate under mechanical stirring. The total mass balance in the finally mixed system was constant. The potassium nitrate was used as a mild oxidation agent in order to oxidise ferrous ions to ferric ions.[TM82] During all experiments the concentration ratio of nitrates to iron(II) was kept constant and equal to 0,66. A black precipitate was formed after mixing of the two solutions. The reaction mixture was allowed to proceed for a given period of time, after which the supernatant was decanted by sedimenting the particles using a magnet. The particles were washed with water, dried, and milled manually. 3.1.1.4

Controlled stirred batch reactor

In order to exert higher degree of control over the synthesis conditions, a fullyautomated computer-controlled batch reactor was constructed. The reactor has a production capacity of ca. 40 g per batch of iron oxide. Among the controllable conditions are pH, temperature, stirring speed, and rate of addition of reactants. As schematically depicted in Figure 3.1, the reactor consists of iron source solutions that are pumped to the reaction vessel by peristaltic pumps, whose speed is controlled by the main computer. With a computerised pH meter in the reaction vessel the addition of acidic or basic solutions are regulated as to keep a constant pH throughout the synthesis with a σ = 0.1 pH units. The feedback obtained from the temperature probe provides information used to regulate the synthesis temperature within 1 ◦ C variation.

3.1.2 3.1.2.1

Synthesis in confined zones Synthesis of iron oxides by Flow Injection

The design of the flow injection system is described elsewhere.[WM99] The homebuilt system included a multi-channel peristaltic pump (Gilson Minipuls 2, France), 0.5 mm Teflon tubing lines, and a T-shaped connector or a manifold injector. The valve of a Flow Injection Analysis apparatus Fia05 (Biofak, Sweden) was used as the manifold valve gave periodic and reproducible microinjections when controlled by a computer system. The Reynolds number was calculated for the different manifold configurations and flow rates and it was always below 200, indicating that conditions of laminar flow were always attained. Typically, an acidic solution of iron with an Fe3+ : Fe2+ ratio of 2 was mixed with a stream of Na OH . The synthesis temperature was controlled with a thermostat

3.1. SYNTHESIS OF NANOPARTICLES

23

Figure 3.1: Schematic representation of the automated batch reactor and its peripheral components.

and set to 80 ◦ C. The particles were then collected into a separation vessel where an external magnet was used to collect the product that was later washed thoroughly. Samples were dried in air and under vacuum.

3.1.2.2

Synthesis of iron oxides in nanoemulsions

Nanoemulsions composed of reverse micelles were used for the synthesis of iron oxide nanoparticles. The nanoemulsion system consisted of AOT-BuOH/cHex/H2 O with a molar ratio of surfactant to water of 2,85 and a surfactant to co-surfactant molar ratio of 1. A sequential synthetic procedure was used in order to prepare the nanoparticles. One nanoemulsion containing the iron source and another containing a solution of sodium hydroxide, were mixed to form the magnetite nanoparticles. The nanoemulsion was lysed with acetone to remove the particles from the surfactant and washed several times with ethanol.

24

CHAPTER 3. MATERIALS AND METHODS

3.1.3 3.1.3.1

Surface modification of nanoparticles Preparation of ferrofluids

Cationic and anionic ferrofluids were prepared modifying the procedures described by Massart.[Mas81] Briefly, the black gel obtained from the synthesis of the particles was peptised with nitric acid solution at pH=2, followed by rinsing with degassed water, the last step was repeated four times. A cationic ferrofluid (CFF), consisting mainly of maghemite, was obtained by rinsing one more time the gel with HNO3 solution, whereas an anionic ferrofluid (AFF) consisting mainly of magnetite was prepared by rinsing the gel with a TMAOH solution. Finally, the ferrofluids were dialysed against the respective acidic or alkaline solution for 24 h refreshing the solutions every 8 h. This procedure would yield stable ferrofluids with a wide concentration range, from 1 mg/cm3 to 40 mg/cm3 . 3.1.3.2

Silica coating of particles

Materials Besides the reagents already reported for the preparation of iron oxide, other chemicals used are tetraethyl orthosilicate (TEOS, 99%, Aldrich) and sodium trisilicate (27% Si O2 in 14% Na OH, Aldrich). Procedure Iron oxide particles from solutions AFF and CFF were coated∗ with a dense silica layer. A solution of sodium trisilicate was passed through an cationic exchange and the pH of the eluate was kept at 9. The thickness of the silica shell was varied by mixing the acidified silicate solution with the AFF or CFF at different silica/iron oxide ratios ranging from 0.05 to 5.4. To further increase the thickness of the coating shell, the silica layer on the particles was grown using the St¨ober method. [SFB68] Ammonia and various amounts of TEOS were dissolved in pure ethanol, to which a suspension of particles was added, the vessel was capped and shaken for 24 h. The suspension was neutralised with a H Cl solution, dialysed against water and concentrated by evaporation. Multi-particle beads were prepared by varying the initial mass ratio of iron oxide to sodium trisilicate and growing the shell with the St¨ober method. To check the quality of the silica coating, the coated particles were added to a 0.1 m HCl solution and the iron concentration which could eventually be leached was measured after one day. 3.1.3.3

Gold coating of particles

Materials Besides the reagents already reported for the preparation of iron oxide, other chemicals used are hydroxylamine (NH2 OH, 98%, Aldrich), tetrakis(hydroxymethyl)phosphonium chloride (THPC, 80% in water, Aldrich) and gold pellets (99.9999%). ∗

A coated nanoparticle is defined as a fully surrounded particle by a well defined close layer of another material.

3.2. CHARACTERISATION

25

Procedure Iron oxide nanoparticles were prepared by dissolving iron(III) chloride and iron(II) sulfate with a molar ratio of CFe3+ /CFe2+ = 2 in a deoxygenated aqueous solution containing 0.1 m H Cl . The iron source solution was added to an aqueous solution of Na OH . The reaction was allowed to proceed for 1.5 h at 80 ◦ C under mechanical stirring. The gel containing the particles was rinsed with deoxygenated water several times and collected with a magnet. 2.5 cm3 of a suspension of the iron oxide nanoparticles with a concentration of about 20 mg/cm3 were diluted to 100 cm3 with water. To this, 50 cm3 of 1.87 mm N H2 OH ·H Cl were added under mechanical stirring. The pH after the addition of hydroxylamine was around 11. After 10 min 100 cm3 of a solution containing 0.44 µm Au(OH)3 was added.∗ The colour of the solution turned from dark brown to dark purple after 10-20 min and the reaction was allowed to proceed for 40 more min. After the last step the particles were rinsed with water several times.

3.2

Characterisation

3.2.1

Crystal structure

Phase identification of the precipitated powders was performed by comparing X-ray powder diffraction (XRD) data obtained with a PW 1830 diffractometer (Philips, λ = 0.154021 nm) against the JCPDS standards database. The obtained spectra were corrected for instrumental line broadening and refined with the program celref.[LB22]

3.2.2

Electron microscopy

Particle imaging was undertaken with a JEM-2000EX (JEOL) transmission electron microscope operating at an accelerating voltage of 200 kV. Specimens were dispersed in ethanol under sonication and deposited onto 200 mesh carbon-coated copper grids (Agar Scientific). The excess of solvent was blown-dried with a gentle stream of N2 . High resolution images and single-particle electron diffraction patterns were acquired with a JEM-3010 (JEOL) high resolution transmission electron microscope operating at an accelerating voltage of 400 kV using a camera distance of 1 m. Specimens were dispersed in butanol under sonication and deposited onto carbon-coated grids. The excess of solvent was evaporated with light irradiation.

3.2.3 3.2.3.1

Surface characterisation ζ-potential

The electrokinetic surface potential of the particles was determined by measuring the electrokinetic sonic amplitude (ESA) with a ESA-9800 (Matec Sciences). ∗

The Au(OH)3 solution was prepared by adding 2.49 g of K2 C O3 to 1 dm3 of an aqueous solution containing 0.44 µm Au3+

26 3.2.3.2

CHAPTER 3. MATERIALS AND METHODS Surface area

Specific surface area (SSA ) of the powders was determined by measuring the nitrogen desorption using the BET method in a Flowsorb II 2300 (Micromeritics).

3.2.4 3.2.4.1

Magnetic characterisation Vibrating Sample Magnetometry

The principle of VSM is the measurement of the electromotive force induced by a ferromagnetic sample when it is vibrating at a constant frequency, under the presence of a static and uniform magnetic field. Magnetic characterisation of the materials was undertaken by analysing approximately 40 mg of powder in fields up to 11 kOe at a vibration frequency of 67 Hz. The magnetic field error was estimated to be 2 Oe. The measurements were carried out at room temperature in an Oxford Instruments 1.2 VSM, at the Laboratori de Propietats Magn`etiques i t`ermiques of the Universitat Aut`onoma de Barcelona. The powders were weighted, pressed into a small quartz container and finally mounted at the end of a rigid ceramic rod. To avoid the movement of the powders inside the tube they were compacted using quartz wool. To get optimum signal the samples are previously centred in angle and height, applying a field of 2 kOe. Subsequently they are demagnetised using a sequence of alternating decreasing fields. The VSM was calibrated in magnetic moment and sample temperature. The magnetic moment calibration was carried out using a Ni standard (sphere) with known saturation magnetization (MS = 54.4 emu/g). The temperature calibration was performed using ferromagnetic alumel and Ni standards, with Curie temperatures of 438 and 627 K, respectively.

3.2.4.2

M¨ ossbauer spectroscopy

The M¨ossbauer effect is based on the absorption of γ-radiation by 57 Fe . The source are 57 Co atoms in a matrix of Rh. The 57 Co nuclei decay to 57 Fe in the excited state with spin 5/2 (half-life time approx 10−9 s) and these transform fast to the state 3/2 (half-life time approx 10−7 s) and then to the state 1/2. The radiation emitted during this last step is the one that allows for the excitation of the 57 Fe atoms present in the sample. The abundance of 57 Fe is 2.2 %, which permits an efficient and statistically large enough population of atoms for a quantitative measurement. M¨ossbauer spectra for different samples were recorded at room temperature using a source of 57 Co atoms in a matrix of Rh. The working speed range was from -8 mm/s to 8 mm/s. A thin foil of α − Fe was used as standard to determine the baseline of the speed.

3.2. CHARACTERISATION

a

27

b

DP DH

c

d

DM

DX

Figure 3.2: Schematic representation of particle sizes as determined with various techniques.

3.2.5

Thermal analysis

Thermochemical analysis of the produced particles was performed with a differential scanning calorimeter DSC 2920 (TA Instruments). Measurements of the heat flow in the temperature range of 25-600 ◦ C were recorded for few milligrams of sample placed in aluminium pans and heated at a rate of 10 ◦ C/min.

3.2.6

Particle size measurements

The developments in the preparation of particles have led to a wide number of particulate materials with different characterisation techniques providing different information on the materials. Thus, there are a number of ’particle sizes’ that arise depending on the measuring technique, as shown schematically in Figure 3.2. a) Physical size, DP : This refers to the “true” size of the particle and is usually obtained with electron microscopy. b) Hydrodynamic size, DH : The hydrodynamic size or diameter of a particle is usually determined using photon correlation spectroscopy (PCS). This length indicates the diameter of a particle including the fluid molecules around the electrostatic double layer. c) Magnetic size, DM : Means the size of the magnetic core of a particle. The size is usually determined from magnetic hysteresis loops. The magnetic size is the crystalline core and it does not include the non-stoichiometric surface layer. Therefore this is generally smaller than the physical size.

28

CHAPTER 3. MATERIALS AND METHODS

d) Crystallite size, DX : The size corresponds to the mean value of the crystalline domain size of the particles, calculated from Scherrer’s equation which uses the line broadening of X-ray diffraction measurements. e) Surface areal size, DS : Calculated from the gas adsorption isotherms assuming a specific shape of the particles. 3.2.6.1

Physical size measurements

The diameters of >500 particles were measured on digitised TEM micrographs with the image analysis program ImageJ.[Ras01] Mean diameters were thus obtained by fitting the experimental data with a lognormal distribution function, as suggested by O’Grady and Bradbury,[OB83] i.e.,  0 )2  ((ln DP ) − ln DP 1 f (DP ) = √ exp − 2 2wP 2πwP DP

(3.1)

0 exp(w 2 /2) and w as the standard deviation with mean diameter < DP >= DP P P 0 . The standard deviation of the mean diameter σ is around ln DP P 0 2 2 1/2 σP = DP [exp (2wP ) − exp (wP )]

3.2.6.2

(3.2)

Hydrodynamic size measurements

All samples were measured by dynamic light scattering (DLS) to quantify the degree of agglomeration. DLS measurements were carried out with a BI-90 particle sizer (Brookhaven Instruments Corp.). 3.2.6.3

Magnetic size measurements

Measurements were carried out at room temperature in particle suspensions. The size of the magnetic cores DM were determined by assuming a lognormal distribution of particle volumes for the Langevin function, i.e.,  DM =

18kT πMsb

r

χi 3Ms · H0

1/3 (3.3)

which has a standard deviation wVSM around the magnetic core of σM

1 = 3



3χi ln Ms · 1/H0

1/2 (3.4)

k represents Boltzmann’s constant, T is the temperature, Msb is the saturation magnetisation of the bulk material and Ms is that of the sample; χi is the initial susceptibility (χi = dM/dHH→0 ), and 1/H0 is the intercept on the 1/H for the high field extrapolation of the magnetisation versus 1/H.

3.3. DNA ASSAYS 3.2.6.4

29

Crystallite size measurements

The crystallite size DX was determined from XRD measurements using the Scherrer’s equation DX =

0.9λ Bhkl · cos θ

(3.5)

where Bhkl represents the full width at half maximum of the main peak expressed in radians and θ is the position of the peak. 3.2.6.5

Surface area measurements

Particle sizes (DSA ) were calculated from the SSA and the specific density of the particles measured assuming spherically shaped particles according to: DSA =

3.3

6000 ρ · SSA

(3.6)

DNA assays

Pristine (uncoated) iron oxide nanoparticles and silica-coated particles were used to purify plasmid DNA fragments of various sizes in the range 0,1–4 kbp (1 kb=1000 base pairs). Purifications were carried out both manually and using 96- and 384well experimental set-ups. Initial manual DNA purification with the magnetic beads was carried out by binding the DNA to the beads, removing excess of materials by washing the beads, and eluting the DNA from the particles.

Four

Results and discussion In this chapter are presented the results of the investigation of the different factors that influence the synthesis of superparamagnetic and ferrimagnetic iron oxide nanoparticles , i.e., iron, cobalt and nickel spinel ferrites with narrow size distribution and different morphologies. The incorporation of the materials into stable ferrofluids and the coating with inorganic compounds, such as silica and gold, as well as organic surfactants is also reported. In the last section of the chapter some of the investigated applications of iron spinel ferrites are presented.

4.1 4.1.1 4.1.1.1

Synthesis of various iron oxides Preparation of magnetite and maghemite Control of crystallographic phase

The synthesis of particles was accomplished using the information provided by chemical equilibrium diagrams. Several experiments were performed in order to investigate the effect of the synthesis temperature on the final crystallographic phase of the particles. Figure 4.1 shows the DSC analysis of the prepared particles, where it is possible to observe two transitions, one endothermic at ∼100 ◦ C, associated to the desorption of surface water; and an exothermic at ∼450 ◦ C, related to the oxidation of magnetite to maghemite, Fe3 O4 → γ-Fe2 O3 . Particles were then synthesised at temperatures higher than 60 ◦ C to minimise the presence of adsorbed water. Experiments were also performed using different iron(III) to iron (II) ratios to determine the boundaries at which spinel iron oxides are formed (Figure 4.2(a)). According to the results, it is possible to see that goethite is formed when there is no Fe(II) present and that even a small amount of iron(II), i.e., a high Fe(III) to Fe(II) ratio, is enough to induce the formation of the inverse spinel structure. However, due to the peak broadening associated with the small size of the particles, it is not possible to make a differentiation between magnetite and maghemite based solely on the XRD results. The particles were characterised by differential scanning 31

32

CHAPTER 4. RESULTS AND DISCUSSION

o

80 C o

Exo

70 o C 60 oC 50 C o

40 C

0

100

200

300

400

Temperature (oC)

500

600

Figure 4.1: Calorimetric analysis of the particles synthesised at different temperatures.

calorimetry (Figure 4.2(b)) showing an endothermic peak centred at ∼ 270 ◦ C, which was associated to the dehydroxylation of hydroxide species present in goethite, 2α-FeO OH → α-Fe2 O3 +H2 O . It is therefore important to avoid the excess of Fe2+ during the wet preparation of Fe3 O4 . Magnetite particles were synthesised and the digestion time was investigated. The size of the particles, as determined by XRD, did not change significantly. On the other hand, the most drastic change was found in the density of the materials. In order to eliminate the possibility of interference by water or incomplete dehydration, the as-synthesised powders were dried for 6 h at 120 ◦ C. Figure 4.3 shows the density variations with the digestion time, which is a result of the variation in the formation of different phases. In section 2.1.1 it was indicated that the formation of magnetite (or maghemite) proceeds through the formation of polynuclear hydroxide complexes. Hence, the gradual dehydroxylation of these complexes yields Fe3 O4 (or γ-Fe2 O3 ). The formation of different (hydr)oxide with the time result in large variations in the macroscopic density of the materials. Table 4.1 shows the density of different iron(II) and iron(III) (hydr)oxide compounds. However, the XRD diffractograms did not indicate the presence of such secondary phases, although their absence of such secondary phases may be due to the high noise-to-signal ratio arising from the low crystallinity of the samples.

4.1. SYNTHESIS OF VARIOUS IRON OXIDES

33

Ratio = 2

Ratio = 1

*

* Ratio = oo

* **

*

20

30

*

*

40

**

* *

50

* *

60

*

70

o

Diffraction angle ( 2θ)

Exo

(a)

0

100

200

300

400

500

600

o

Temperature ( C)

(b)

Figure 4.2: (a) XRD (b) and DSC analysis of particles synthesised with Fe(III):Fe(II) ratios of 1, 2, ∞ in a batch reactor. The symbols indicate peaks corresponding to (∗) α-FeOOH, to () inverse spinel, and to () α-Fe2 O3 .

34

CHAPTER 4. RESULTS AND DISCUSSION

5,0

Density (g·cm3)

4,8

4,6

4,4

4,2

10

100

1000

time (min) Figure 4.3: Variation in the density of iron oxide nanoparticles as a function of reaction time.

Table 4.1: List of properties of some iron (hydr)oxide compounds. Values obtained from [Lid04]. Compound Fe(OH)2 Fe(OH)3 Fe O OH α-Fe2 O3 Fe3 O4 Co Fe2 O4 a

Density (kg/m3 ) 3200 3120 4260 5250 5170 5240a

Theoretical value.

4.1. SYNTHESIS OF VARIOUS IRON OXIDES

(a) Flow-injection synthesis

35

(b) Batch synthesis

Figure 4.4: TEM micrograph and respective particle size distribution analysis for iron oxide nanoparticles prepared by fis (a) and batch methods (b).

4.1.1.2

Control of particle size and size distribution

Particle analysis by TEM revealed spheroidal particles with sizes in the nanometre scale. Figure 4.4 shows the TEM for particles obtained with the continuous injection scheme (Scheme A) under standard conditions and those obtained in a small batch reactor (∼ 50 cm3 ) in identical chemical conditions. From the respective particle size distribution analysis it can be seen that the particles synthesised using a fis-based method have a lower particle size and lower polydispersity than those obtained in a batch reactor. The values determined for particles prepared in a fis reactor were 3,3 nm with a polydispersity of 0,23 compared to 4,3 nm with polydispersity of 0,33 determined for particles prepared in a batch reactor. The differences in size and distribution width (polydispersity), provides indication that the mixing in the capillaries is much faster and homogeneous than in batch reactor. The Reynolds number was calculated for the fis conditions hereby studied and it was always close to zero, indicating that conditions of laminar flow were always attained.

4.1.2 4.1.2.1

Synthesis of nickel and cobalt-doped magnetite Synthesis of Nix Fe3−x O4

Spinel ferrite nanoparticles were initially prepared using only iron salts as starting material. However, the properties of the ferrites vary dramatically as their composition changes. The synthesis of nanoparticles with more complex composition was then studied. The synthesis of ferrite nanoparticles based on nickel with a chemical composition Nix Fe3−x O4 was carried out. The as-precipitated particles were characterised for the composition with XRD. The obtained diffractograms are shown in Figure 4.5.

36

CHAPTER 4. RESULTS AND DISCUSSION

*

(d)

* 20

30

(c)

*

* 40 o

* 50

o

*

* 60

70

o

o

20

30

40

50

60

70

20

30

40

50

60

70

20

30

40

50

60

70

(b)

(a)

o

Scattering angle ( 2θ)

Figure 4.5: XRD diffractograms of Nix Fe3−x O4 particles prepared with an initial nickel content of (a) 0, (b) 5, (c) 10, and(d) 30 at%. The symbols represent the identified peaks for spinel ferrite (∗) and haematite phases(◦ ).

The results shown in Figure 4.5 indicate the formation of the spinel ferrite phase in all samples. The presence of haematite was detected only in the samples prepared with low amount of nickel (5 and 10 at%). Chemical analysis of the powders revealed that the nickel content in the samples that developed haematite is nearly negligible, whereas the sample prepared with a nickel content (30 at%) close to the stoichiometric one had an actual nickel content of about 20 at%. The crystallite size, as determined with Scherrer’s equation, was about 25 nm for all samples. The latter suggests that the nickel has practically no influence on the particle formation and that it might incorporate into the spinel lattice at an intermediate stage. The development of haematite in Nix Fe3−x O4 and Fe3−x O4 nanoparticles has been observed whenever there was an excess of Fe2+ in solution. As discussed earlier, haematite can grow epitaxially on magnetite or maghemite and therefore the surface area influences the kinetics of transformation. 4.1.2.2

Synthesis of Cox Fe3−x O4

Figure 4.6 shows that the cobalt content in the analysed solids can be adjusted in a controlled manner by varying the metal ion to hydroxide ion concentration ratio

4.1. SYNTHESIS OF VARIOUS IRON OXIDES

37

5,2 5,1

Density (g·cm-3 )

5,0 4,9 4,8 4,7 4,6 4,5 0,80

0,85

0,90

0,95

1,00

1,05

1,10

1,15

1,20

x (Cox Fe3-x O4 )

Figure 4.6: Iron/cobalt molar ratio of nanoparticles as a function of the metal ionhydroxide ion molar ratio of the mixed suspension, [Me2+ ]M /[OH− ]M . The digestion time was 3 h.

in the mixed solution, [Me2+ ]M /[OH − ]M . The control of the particle composition can also be achieved by varying the initial iron to cobalt ratio.[TFM90, LK03] In the present study, particles with a cobalt composition corresponding to x values in the range 0,86 to 1,14 were synthesized. Although the initial iron to cobalt ratio prior to mixing was constant (stoichiometric ratio) the resulting phase has varying composition. Figure 4.7 shows the variation of density as a result of varying the metal to hydroxide ratio [Me2+ ]M /[OH− ]M . The pronounced decrease in density can be attributed to the gradual increase of secondary phases. Table 4.1 shows the density of various metal (hydro)oxides that may form during the precipitation of cobalt ferrite. The density values for magnetite, cobalt ferrite, and haematite are quite similar. However, the density of the hydroxide species is much lower. The redox potential in the system imposed by nitrate and iron(II) ions discard the existence of Fe(OH)2 . Hence, the secondary phases that may lower the density are Fe(OH)3 and Fe O OH . The presence of these phases was detected by XRD measurements carried out on particles withdrawn from the reaction liquors at regular time intervals and at high [Me2+ ]M /[OH− ]M . 4.1.2.3

Variation of particle morphology

Variation of the metal to hydroxide ratio [Me2+ ]/[OH− ] resulted in particles with different morphology. The particles were mainly cubic and spherical, although irregularly shaped particles were also observed. Fig. 4.8 shows electron micro-

38

CHAPTER 4. RESULTS AND DISCUSSION

5200 5100

Density (kg·m-3 )

5000 4900 4800 4700 4600 4500

0,30

0,35

0,40

0,45

0,50

0,55

0,60

-

[Me2+]M /[OH ]M

Figure 4.7: Variation of the density of the particles with the metal to hydroxide ratio. The amount of metal ions was varied while keeping a constant amount of sodium hydroxide, nOH− = 0, 528 mol. The volume of the suspension was 1,6 dm3 and all the samples were digested for 3 h.

graphs of particles observed under different conditions. Spherical and cubic particles were equal in amounts when the precipitation conditions were adjusted to generate the compositions close to the empirical stoichiometric value for cobalt ferrite, Cox Fe3−x O4 , x = 1. Several physical properties of particles are dependent on the surface composition. In this work we also studied the crystallinity and the presence of amorphous phases in the particles with HRTEM. Figure 4.9 shows the ED pattern and HRTEM of a cubic particle taken from a [111] zone axis. The measured lattice fringes correspond to the unit cell of inverse spinel ferrite. Also, the high crystallinity of the particles can be observed, for which we can assume that each cubic particle is a single crystal with no noticeable amorphous surface layers.

4.1.3

Surface modification

According to the double layer theory, most of the transition metal oxides have a point of zero charge (PZC) when suspended in aqueous media. At the PZC there is a minimum in the surface charge density and due to the positive Hamaker constant that exist between two surfaces of the same composition, there would be a strong attraction between the particles. Therefore, at neutral pH values iron oxide nanoparticles (PZC∼7) tend to aggregate into large clusters, causing the loss of properties related to superparamagnetic relaxation. The surface properties can be varied by coating the particles with a layer of organic surfactant.

4.1. SYNTHESIS OF VARIOUS IRON OXIDES

39

Figure 4.8: TEM of particles prepared with a [M2+ ]/[OH− ] ratio of (top) 0,2, (middle) 0,3, and (bottom) 0,5. Note the different scale bars used in the figures.

40

CHAPTER 4. RESULTS AND DISCUSSION

Figure 4.9: ED pattern and HRTEM of a cubic particle of cobalt ferrite taken from the zone axis [111]. The lattice fringes correspond to the unit cell of inverse spinel ferrite.

4.1.3.1

Coating of iron oxide nanoparticles by silica

In this work we studied the coating of iron oxide nanoparticles with a shell of silica since it has been previously reported that silica does not affect the magnetic properties of the coated magnetic particles.[dGBMP99] The thickness of the coating layer was further increased by growing the silica shell via slow hydrolysis of tetraethoxy silane. It was observed that the thickness of the shell could be controlled by adjusting the total amount of added silane. From the graph, it can be seen that the thickness of the shell increases with the TEOS added and has a maximum at 0.15 cm3 of TEOS, then it decreases abruptly and increases at a slow rate. From the electron micrographs (4.10) it was observed that at this concentration of TEOS there is a homogeneous nucleation of silica which increases steadily with the TEOS added to the suspension.The reason why the nuclei do not increase in size is due to the fact that the number of nuclei increases instead. 4.1.3.2

Coating of iron oxide nanoparticles with gold

Experiments were carried out in order to coat iron oxide nanoparticles with a layer of gold. The first test were oriented towards the preparation and characterisation of gold nanoparticles. The citrate reduction method following the protocol reported by Kooij et al.[KBWP02] which yields gold particles with an average size of about 20 nm. However, the gold particles prepared by this route are too large and other systems were investigated. The method developed by Duff et al. was used to have a better size control.[DB93] Gold seeds with a particle size below 1 nm (below detection limit of JEOL-2000FX) were grown with the seed-mediation method. Figure 4.11 shows the TEM of gold nanoparticles grown with a single iteration of the growth process. The size of the particles could be tailored up to 20-30 nm adjusting the number of iteration steps in the growth process.

4.1. SYNTHESIS OF VARIOUS IRON OXIDES

41

Figure 4.10: Electron micrographs showing the coating of iron oxide nanoparticles with silica layers of different thickness. The cores consisted of iron oxide nanoparticles with a size of about 10 nm.

42

CHAPTER 4. RESULTS AND DISCUSSION

Figure 4.11: TEM of gold particles grown by the seed-mediation method.

Following this, several alternatives were tested in order to obtain iron oxide nanoparticles with a gold shell. Among the investigated alternatives were the sequential synthesis in batch, sequential synthesis in nanoemulsions, surface reduction, etc. The only method that proved to be effective is the adsorption of hydroxylamine on the surface of the nanoparticles with the further reduction of gold(III) ions on such surfaces. The size of the gold clusters that form on the surface of the nanoparticles was increased by a seed-growth method. Figure 4.12 shows iron oxide nanoparticles with small gold clusters of about 1 nm on their surface. The gold coverage of the particles was measured to be about 40 %. The reduction of gold by hydroxylamine is a heterogenous nucleation process that required an existing surface to nucleate upon. Experiments performed without iron oxide nanoparticles, this is, in the absence of seeds, did not show the formation of gold crystals. Lyon et al.[LFS+ 04] have recently shown that the coating of iron oxide nanoparticles with a particle size of about 9 nm with a shell of gold with a thickness of about 50 nm using a similar methodology. The findings in this work and in that of Lyon et al. demonstrate that it is possible to attach gold particles to the surface of iron oxide nanoparticles. The full covering of the iron oxide nanoparticles can be then achieved with the seed-mediated growth of the gold clusters, as earlier reported.[Hay89]

4.2. MAGNETIC CHARACTERISATION

43

Figure 4.12: TEM of iron oxide nanoparticles coated by smaller gold nanoparticles grown by the seed-mediation method. The image was focused on the small gold particles. The arrow indicates an isolated iron oxide nanoparticle for clarity.

4.2 4.2.1

Magnetic characterisation Silica-coated iron oxide nanoparticles

Measurements of the magnetisation versus applied magnetic field undertaken on the starting ferrofluids as well as on suspensions of the silica-coated materials are displayed in Figure 4.13. Bulk saturation magnetisation values were calculated from the saturation magnetisation values of the ferrofluids and the total iron concentration of each sample assuming that all iron was in the form of maghemite. None of the samples exhibited magnetic hysteresis at room temperature and the saturation magnetisation value for particles with silica monolayer was found to be 74 emu/g, whereas the value found for pristine particles was 79 emu/g. It is important to highlight that the saturation magnetisation value for the pristine particles is about 90% of the theoretical value for maghemite.[PS95] From a plot of the reduced saturation magnetisation (M/MS ) it was seen that all the curves converge into a master curve. Therefore, it is possible to assume that at this level of magnetic volume fraction ( 7-10 mg/cm3 ) there are no particle-particle interactions and thus, the magnetisation of the suspension is proportional to the volume fraction of the magnetic content, i.e., M (H, φ) = M (H)(φmag ).

44

CHAPTER 4. RESULTS AND DISCUSSION

1.2

Reduced magnetisation

1.0 0.8 0.6 0.4 0.2 0.0 -0.2

0

2000

4000

6000

8000

10000

Applied field (Oe)

Figure 4.13: Magnetisation curves for pristine iron oxide nanoparticles (open squares) and particles with a silica monolayer (open circles). Table 4.2: List of samples analysed with M¨ossbauer spectroscopy. Sample ID Co19 Co21 Co22 Co23 Co25

4.2.2

Density (g/cm3 ) 4,44 4,56 5,01 5,02 4,84

DS (nm) 91 95 80 90 92

Cobalt ferrite nanoparticles

Analysis of the particles carried out below the blocking temperature of cobalt ferrites showed almost identical M¨ossbauer spectra. Figure 4.14 presents the results of the measurements carried out at 90 K. It can be seen that the peaks of the samples are superimposed, which does not allow for an unambiguous determination of the the degree of inversion of the sample, i.e., [B]/(A) ratio. The spectra taken at room temperature were fitted with four sub-spectra (1 for A sites, 3 for B site). The fits of the spectra taken at room temperature for samples Co19, Co21, Co22, Co23, and Co25 shown in Figure 4.15 result in a ratio R = [B]/(A) between 1.66 and 2.0. The numbers for the distribution of the iron ions in the different sites depend on the precise information about the total Fe/Co ratio as determined from chemical analysis. For instance, for sample Co25 with a [B]/(A) = 1, 68, (Co0,25 Fe0,75 )[F e1,25 Co0,75 ]O4 for a Fe/Co = 2,0. On the other hand, we obtain (Co0,30 Fe0,70 )[F e1,28 Co0,72 ]O4 for

4.3. APPLICATIONS OF MAGNETIC NANOPARTICLES

45

Figure 4.14: M¨ossbauer spectra taken at 90 K for samples Co19, Co21, Co22, Co23, and Co25. Note that only sample Co19 shows a minor component in the central part of the spectrum.

Co21 with a [B]/(A) = 1,77 if Fe /Co = 2, 0. However from chemical analysis, we have that Fe /Co = 2, 42, thus the composition of the material can be written as (Co0,24 Fe0,76 )[F e1,35 Co0,65 ]O4 . From the measurements at 90 K and room temperature it can be observed that all samples have similar pattterns except Co19 which showed a small fraction of superparamagnetic components. Analysis of the results showed a disordered inverse spinel structure, which normally has the cobalt ions occupying only [B]-sites. The fits shown here are approximate since for an unambiguous determination of the distribution of the iron and cobalt ions in the (A) and [B] sites, it is required to obtain M¨ossbauer spectra under an external magnetic field.

4.3 4.3.1

Applications of magnetic nanoparticles DNA separation

Different types of iron oxide nanoparticles were used for the purification of DNA: pristine nanoparticles , single-core iron oxide nanoparticles coated with a silica monolayer and with a layer of 1-2 nm, and also silica beads containing several iron oxide cores with a bead size of 100-250 nm . The results presented in Table 4.3.1 show that the recovery capacity of the silicacoated particles with a thin layer (b) is the highest among the different particles tested. To evaluate the origin of such a difference the hydrodynamic radius was

46

CHAPTER 4. RESULTS AND DISCUSSION

Figure 4.15: M¨ossbauer spectra taken at room temperature for samples (a) Co19, (b) Co21, (c) Co22, (d) Co23, and (e) Co25. The dots represent the measured points and the continuous lines correspond to the fitting.

4.3. APPLICATIONS OF MAGNETIC NANOPARTICLES

47

Table 4.3: Recovery of DNA using iron oxide nanoparticles with and without silica coating using a 384-well plate. (a) corresponds to pristine nanoparticles, (b) to single-core iron oxide nanoparticles coated with a very thin layer of silica, a layer of 1-2 nm (c), (d) to silica beads containing several iron oxide nanoparticles, and (e) Nanomag-D beads (Micromod GmbH, Germany) with particle size of 130 nm. Sample (a) (b)

(c) (d) (e)

DNA Plasmid size (kbp) 1,7 1,7 1 0,5 0,256 0.1 1,7 1,7 1,7

Recovery yields for different concentrations (%) 15 µg/rxa 7,5 µg/rx 3,75 µg/rx 1,875 µg/rx 26 45 37 6 67 63 55 51 57 61 – – 44 55 – – 57 53 – – 8 22 – – 25 20 18 19 29 47 55 – 44 – – –

a

15 µg of total iron per reaction correspond approximately to 100 µg of particles per reaction.

measured. It was found that the particles in (a) had a hydrodynamic particle size of about 30 nm, considerably smaller than that of the other samples. The values obtained with the beads (d) are higher than those obtained with the aggregates particles (b). This may be explained by the fact that the recovery yields are measured per concentration unit of iron. Hence, the beads have a larger amount of silica per particle which results in a higher surface area per iron oxide particle. Nevertheless, the recovery of the beads is significantly lower than that obtained with the particles in (a). Mornet et al.[MVB+ 00] suggested that there are two possible places in the DNA double strand that may accommodate the nanoparticles in a manner that resembles the wrapping of the DNA around the histone core in the cellular nucleus. However, the suggested sites have a size of few nanometres, hence the beads may be too large (with a particle size larger than 150 nm) to be accommodated in the same position as the smaller particles. This also explains why increasing the concentration of beads does not increase the recovery rates whereas in the case of (b) an increase of the concentration of the particles increases the recovery to nearly 70%. The recovery of DNA with the studied particles is comparable to that reported by Yoza et al.[YMM02] who used amine-functionalised particles in the size range 50–5000 nm. The positively charged amino group (around neutral pH) binds electrostatically to the negatively charged DNA. Although the particles used in this work are non-functionalised, the recoveries are in the same order of magnitude and in some cases higher that those reported for amine-functionalised materials. A great advantage of using silica-coated particles resides in the fact that these particles can be readily regenerated by ’salting out’ the DNA form their surface and re-use them several times.[HOMRS94]

48

CHAPTER 4. RESULTS AND DISCUSSION

100

Eout (µJ)

10

1

0,1 0,1

1

10

100

1000

Ein (µJ)

Figure 4.16: Optical power limiting of iron oxide nanoparticles coated with a silica monolayer.

4.3.2

Optical limiting

Suspensions of pristine iron oxide nanoparticles were used to study their optical limiting properties. The particles studied had a spheroidal morphology with a diameter of about 10 nm and a standard deviation of 25%. The secondary particle size, as determined by DLS (DH ), was about 60 nm, which implies that agglomerates consisting of tens of nanoparticles were formed. Also, iron oxide nanoparticles with a silica monolayer were studied. The silica layer modifies the surface properties of the particles by lowering the point of zero charge from about 7 to around 3 (results not shown). At pH values close to the point of zero charge the surface charge density is very low and the electrostatic repulsion can no longer prevent the particle aggregation. The secondary particle size of iron oxide nanoparticles with a silica coating was 25 nm, i.e., the agglomerates consisted only of few particles, which increased the concentration range that can be used and improves the hydrodynamic stability of the suspension. Furthermore, the silica coating on the particles improves not only the colloidal stability of the suspension but also serves as a protective layer to avoid leaching of Fe3+ ions from the surface. The transmission spectra of pristine iron oxide nanoparticles and silica-coated iron oxide nanoparticles was measured at 532 nm. The transmittances were quite similar and in excess of 60%. The transmittance of both iron oxide suspensions was dependent of the wavelength and a maximum of about 84% transmittance was measured for both samples at around 750 nm. Figure 4.16 shows the results of the optical power limiting measurements for silica-coated iron oxide nanoparticles. The clamping level was determined to be around 3 µJ for both suspensions

4.3. APPLICATIONS OF MAGNETIC NANOPARTICLES

49

of pristine and silica-coated iron oxide nanoparticles. It has been suggested that carbon nanoparticles present a strong nonlinear scattering associated to a laserinduced plasma formation and generation of bubbles.[MSS92] Considering that the iron oxide nanoparticles are also inorganic materials it could be assumed that a similar mechanism is responsible for the non-linear behaviour. Yu et al. reported that colloidal suspensions of Fe2 O3 nanoparticles show a very large nonlinear refractive index.[YZG+ 97, YZG00] However, it is not clear in the report which crystallographic phase with the composition Fe2 O3 was studied. In addition, it has been reported that the nonlinear mechanism might be associated with the excitation of the magnetically-coupled Fe3+ ions as well other electronic resonances.[HYY96] Hence, there are clear differences in the electronic properties of the materials associated with the crystallographic structures.

Five

Conclusions In the present study the synthesis of iron oxides nanoparticles and their use in two different applications is reported. Several types of iron oxide nanoparticles with various composition, i.e., Fe3 O4 , γ-Fe2 O3 , Cox Fe3−x O4 , and Nix Fe3−x O4 ; all with the inverse spinel structure were prepared. Iron oxide nanoparticles consisting mainly of γ-Fe2 O3 with an average particle size of about 10 nm have have shown higher saturation magnetisation and proton relaxivity rates than most of the reports in the literature. The increase in magnetisation of the particles was attributed to a higher crystallinity. Also, a new method for the synthesis of iron oxide nanoparticles with a narrow size distribution by the flow-injection technique was developed. The particles thus synthesised have smaller size and narrower particles size distribution than those obtained with a stirred batch reactor. It was found that segmentation of the flow is not required for the synthesis of iron oxides and that continuous flow can be used without altering the particles’ morphology or quality. The flow-injection synthesis of particles can be performed in aqueous or bi-phasic media reducing considerably the volumes of organic phase needed in microemulsion-based synthesis. Particles with a composition of Cox Fe3−x O4 and Nix Fe3−x O4 have been prepared and showed the spinel structure and the metal doping, x was was varied in the range 0, 8 ≤ x ≤ 1, 2. The composition of the iron oxide particles was tailored by co-precipitating a mixed metal solution with hydroxide ions. The degree of doping was found to be dependent on the molar ratio of the metal solution to hydroxide solution, this is, on the presence of excess of metal ions or hydroxide ions. The purity of the samples was initially characterised by large changes in the density of the material and confirmed by X-ray diffraction measurements. Stable aqueous ferrofluids were prepared by oxidising the surface of magnetite nanoparticles, which were later used to coat the particles with a silica layer. The silica shell thickness was adjusted by varying the mass ratio of silica to iron oxide, whereas sonication was used to decrease the number of particles with multiple core without requiring the centrifugation of the material. In the present study, iron oxide nanoparticles have been evaluated for their use 51

52

CHAPTER 5. CONCLUSIONS

in biomedical applications and for the magnetic separation of DNA. The particles present a very large contrast which is related to the relatively large magnetic moment of the nanoparticles. The particles presented a large R2 /R1 ratio, which indicates that they can be used for in-vitro diagnostic applications. The magnetic separation of DNA by silica-coated magnetic nanoparticles was readily achieved. The recovery rates of the particles were similar to commercial materials and particles containing surface functional groups with the advantage of having non-specific interactions between the particles and the nucleic acids. The recovery rates were greatly affected by the hydrodynamic particle size. The presence of two different mechanisms for the binding of DNA depending on the particle size is suggested by the differences in recovery rates for small particles (below 50 nm) and large particles (above 150 nm). The high magnetisation of the particles combined with a large surface area allows a faster and more efficient purification methods to be achieved. The optical power limiting application of coated and non-coated iron oxide nanoparticles was investigated. Both aqueous suspensions of pristine iron oxide nanoparticles consisting mainly of γ-Fe2 O3 and silica-coated iron oxide nanoparticles were yellowish and had a clamping level of about 3 µJ. The photoinduced nonlinear light scattering proved to be the dominating nonlinear mechanism. The slight better performance of the silica-coated particles was attributed to their higher colloidal stability.

Suggested future work The synthesis of iron oxide nanoparticles with few different compositions was studied. Nevertheless, there are a number of applications that require materials with more complex stoichiometry and metal ions with different aqueous behaviour. Hence, the investigation of parameters found to have a large impact on the composition of the nanoparticles, such as the metal to hydroxide ratio, is required. Once the chemical parameters are fixed, the flow-injection synthesis method can be used to prepare materials with a smaller particle size and narrower particle size distribution than those obtained with stirred reactors. However, the yield is still very low and it requires further studies to scale it up. Also, materials with compositions that need careful mixing conditions could be investigated. Regarding the surface modification of iron oxide nanoparticles, the silica-coating proved to be advantageous for the preparation of dispersed suspensions. Nonetheless, the surface properties can be greatly modified to enhance their functionality. For instance, it has been shown that folate-functionalised particles can be used for the labelling of certain types of cancerous cell. Tissues with a high concentration of these particles can later be imaged with magnetic resonance imaging. The magnetic and structural properties of the prepared materials were studied by different techniques. Nevertheless, it is not clear the role of the secondary phases in the case of doped-magnetite. Furthermore, magnetometry and XRD investiga-

53 tions carried out in the present studies are insufficient for the detailed characterisation of the particles. The magnetic properties of the particles are dependent on their structure, composition, and morphology. Hence, a more detailed study on the structure-properties relationship may be needed. A clear understanding of this relationship is required to understand magnetic phenomena related to, e.g., exchange bias mechanisms, microwave absorption, magnetic relaxation, etc.

Acknowledgements First of all I am indebted to my supervisor, Prof. Mamoun Muhammed, for the efforts put during the course of the work, his confidence in me and all the support through the years. I also appreciate the efforts from Eva Bj¨orkman, Maria Mikhaylova, and Muhammet Toprak for their help, valuable scientific and non-scientific advices, proofreading this thesis and standing by me in tough times. I would like to acknowledge the fruitful collaboration and discussions with Richard T. Olsson, Ulf W. Gedde, and Mikael S. Hedenqvist at the Department of Polymer Technology; Cesar Lopes, S¨oren Svensson, and Steven J. Savage at the Departmenf of Functional Materials of FOI; Fredrik Lindberg at the Department of Structural, Physical and Inorganic Chemistry of the Stockholm University; Josep Nogu´es, at the Department of Physics of the Universitat Aut`onoma de Barcelona; Sophie Laurent and Robert Muller at the Department of Organic Chemistry of the University of Mons-Hainaut in Belgium. I thank the local collaborators for without their discussions/chat/àíåêäîòû this work would have been much harder: Andrei Z, Erik L, Uheida, Othonas, Zongyin, Pascale, Andy, Michel, Y Zhang, Kim, Shanghua, Jian, Jackie call-me-Bill, Raph, Tib˚ a. Also, I thank the people at the innebandy club: Hasse, Wolfgang, Dilip, ¨ Johan E, Sylwester; and people from other divisions in MSE: David, Johan O, Lyuba, Amita, Fred, Nabil, Ulf, K2 , Athula, Bengt, Martin Schwind, Patrik F, Anders T, Thobias S, Tosse, Rosa, Ricardo, Era, and Tripathi. Parts of this work have been supported through grant from the EU-Fifth Framework programme Contract GRD5-CT2000-00375; Swedish Defence Research Agency, FOI; Swedish Nuclear Fuel and Waste Management, SKB; and KTH Rektors g˚ avomedel.

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D.D. Stark, R. Weissleder, G. Elizondo, P.F. Hahn, S. Saini, L.E. Todd, J. Wittenberg, and J.T. Ferrucci. Superparamagnetic iron oxide: clinical application as a contrast agent for mr imaging of the liver. Radiology 168 (1988) 297–301. URL http://tinyurl.com/5e79f.

[TFM90]

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[THD+ 97]

X. Tiejun, D.J. Hagan, A. Dogariu, A.A. Said, and E.W. van Stryland. Optimization of optical limiting devices based on excited-state absorption. Appl. Opt. 36 (1997) 4110–4122.

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[UCS86]

C. Umeton, G. Cipparrone, and F. Simoni. Power limiting and optical switching with nematic liquid-crystal films. Opt. Quantum Electron 18 (1986) 312–314.

[Uhl89]

M. Uhlen. Magnetic separation of DNA. Nature 340 (1989) 733– 734.

[VAR+ 99]

L. Vivien, E. Anglaret, D. Riehl, F. Bacou, C. Journet, C. Goze, M. Andrieux, M. Brunet, L. Lafonta, P. Bernier, and F. Hache. Single-wall carbon nanotubes for optical limiting. Chem. Phys. Letters 307 (1999) 317–319. http://dx.doi.org/10. 1016/S0009-2614(99)00528-X. `res, C. Chane ´ac, E. Tronc, and J.P. Jolivet. Size L. Vayssie tailoring of magnetite particles formed by aqueous precipitation: An example of thermodynamic stability of nanometric oxide particles. J. Colloid Interface Sci. 205 (1998) 205–212. http://dx.doi.org/ 10.1006/jcis.1998.5614. ´rcel Cases and M.D. Luque de Castro. An´ M. Valca alisis por Inyecci´ on en Flujo. Departamento de Qu´ımica Anal´ıtica-Universidad de C´ordoba, C´ordoba, 1984. In Spanish.

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[WH00]

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[WHS+ 92]

T.H. Wei, D.J. Hagan, M.J. Sence, E.W. Van Stryland, J.W. Perry, and D.R. Coulter. Direct measurements of nonlinear absorption and refraction in solutions of phthalocyanines. Appl. Phys. B 54 (1992) 46–51.

[WKC+ 04]

M.A. Willard, L.K. Kurihara, E.E. Carpenter, S. Calvin, and V.G. Harris. Chemically prepared magnetic nanoparticles. Int. Mater. Rev. 49 (2004) 125–170. http://dx.doi.org/10.1179/ 095066004225021882.

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[WM99]

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[YMM02]

B. Yoza, M. Matsumoto, and T. Matsunaga. DNA extraction using modified bacterial magnetic particles in the presence of amino silane compound. J. Biotechnol. 94 (2002) 217–224. http://dx. doi.org/10.1016/S0168-1656(01)00427-8.

66

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[YZG+ 97]

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[YZG00]

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[ZGW+ 92]

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67

“It goes like this. Let’s see now: ’Protect me from knowing what I don’t need to know. Protect me from even knowing that there are things to know that I don’t know. Protect me from knowing that I decided not to know about the things that I decided not to know about. Amen’ . . . Oh yes, add ’Lord lord lord’, it makes it more profound: ’Lord lord lord. Protect me from the consequences of the above . . . ’ D. Adams, The ultimate hitchhiker’s guide to the galaxy Del Rey Books, New York, 2002.