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«By Yusuf Nur A thesis submitted to The University of Birmingham for the Degree of DOCTOR OF PHILOSOPHY School of Geography, Earth and Environmental ...»

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Here µ is the electrophoretic mobility, ε is the electric permitivity,  stands for the viscosity of the surrounding liquid,  is the zeta potential, with a simple algebraic manipulation,  can be made the subject of the formula and calculated from the electrophoretic mobility of the charged particles.

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3.1.6 Surface Plasmon Resonance (SPR) spectroscopy.

The free conductive valence electrons on the surface of the metals oscillate naturally due to the storing attraction effect of the positive charge of the atomic nuclei. This oscillation causes an electron density waves (surface Plasmon) with characteristic frequency on the surface of the metal. The nature of the force between electrons and nuclei is coulomb forces which is indirectly proportional to the charges separation distance and directly proportional to the strength of the charges. The separation distance of the charges (electrons and nuclei) in the metal is given by Equation 3-16 below.

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Here, K is constant, e stands for the charge of the electrons and S is the separation distance between charges.

The oscillation of the electrons around the positively charged nuclei can be treated like classical harmonic oscillator and below can be used for the energy of harmonic oscillator.

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2 ώp is the frequency of the harmonic oscillator. Using the last three equations and rearranging them for the frequency of oscillation the following equation can be derived which gives the frequency of the oscillating electron density ( surface Plasmon) waves.

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If the frequency of incident light photons matches the natural characteristic frequency (ώp) of the electron density waves of the metal, the energy of the photons is absorbed (Pockrand et al., 1978) and surface plasmon resonance (SPR) occurs (Gordon Ii and Ernst, 1980) for planar surface and localised surface plasmon resonance (LSPR) for nanoscale sized metals (see Figure 3-6 below).

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The wavelength of the maximum absorption depends on the type, shape, size and the environmental surrounding of the nanoparticles (Kreibig and Vollmer, 1995) In the most metal nanoparticles this maximum absorbance which causes the localised surface Plasmon resonance falls in the ultraviolet region of the electromagnetic spectrum. In the case of gold and silver nanoparticles, the resonance takes places in the visible region due to interband transitions. This absorption wave can be recorded with UV-Vis spectrometer to identify the type of metal in the sample or to study any changes in the surrounding media. To quantify the amount of light absorbed by sample, the concept absorbance A is used which is defined as.

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Where Io and I stand for respectively the intensity of the light at certain wavelength (λ) before and after it has passed through the sample under study. Absorbance is a fraction of two intensities so it has no unit but many times it is reported in absorbance unit (AU). Figure 3-7 gives a diagram representation of the typical dauble beam Uv_vis spectroscopy.

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Figure 3-7: Simplified Schematic representation of the main parts of Uv-vis spectrophotometer showing the light sources the monochromater, reference and sample holder and the detector.

3.1.7 Field Flow Fractionation (FFF) Field flow fractionation is a very versatile sizing and separation technique which has many applications in the different scientific areas including food chemistry, medicines, biology and environmental science (Wittgren and Wahlund, 1997, Petteys and Schimpf, 1998, Chmelik, 2007). The range of sizes that can be separated may vary from 0.001 to 100 µm (Dulog and Schauer, 1996) which is the whole range of colloidal, macromolecules (Ratanathanawongs Williams et al., 2006), nanoparticles (Lohrke et al., 2008, Baalousha et al., 2011) and particulates (Kirkland et al., 1990) in different carried solvents. What makes FFF so versatile to separate a wide range of sizes is because of the variety of different nature fields which can be used to achieve separation (Shendruk and Slater, 2012). The one main difference between the different separation types of the commercial FFF is the nature of the applied field which can be either electric field (Giddings et al., 1974), temperature gradient (Caldwell et al., 1972), sedimentation field (Giddings et al., 1975) or crossflow (Giddings et al., 1976). FFF instruments contains similar components (carrier, pump, injection port, separation channel, detector and computer see Figure 3-8 below) of the traditional liquid chromatography (Myers,

1997) though the basic separation principles are, as will be explained later, different.

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Figure 3-8: Schematic diagram of field flow fractioning with all main parts illustrated (FFF) A detailed theory of the relevant principles of the FFF has been given in a number of papers (Myers, 1997, Giddings, 1993a, Giddings et al., 1977, Giddings, 1966). Here, only a short introduction of the basics of cross-flow normal mode of the FFF is given, which is suitable for the NPs in this project. Of the number of assumptions applied to develop a theoretical concept of the FFF retention the main ones are: the uniformity of the field across the channel, and the lack of interaction between particles and between wall particles interaction (Messaud et al., 2009).

FFF separation is achieved due to the combined effect of two opposing transport processes (Giddings, 1966). An applied cross-flow which is perpendicular to the liquid flow direction pushes the particles in the samples toward the accumulation wall (Bos and Tijssen, 1995).

This is followed by continuous diffusion of the particles from the accumulation wall due to concentration gradient set up by the cross flow where the highest concentration are by the wall. The thickness of the cloud of particles L can be related to the above mentioned main processes through Equation 3-21.

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D is the diffusion coefficient and U is the side way velocity caused by the cross flow. If the thickness L is divided by the thickness of the channel (w) an alternative dimensionless parameter λ can be defined as follows.

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From the theory of FFF (Giddings, 1973, Giddings et al., 1976), λ is related to experimental parameter R ( void volume/ retained volume) through Equation 3-24 below.

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Experimentally, R can be obtained by the ratio of the void time t0 and the retention tr(Messaud et al., 2009). Then Equation 3-23 can be used to give λ. With the λ so obtained, Equation 3-21 is applied to calculate the value for the diffusion coefficient of the particles

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D stands for the diffusion coefficient, T is the absolute temperature in degrees Kelvin of the sample, KB is the distribution Boltzman constant,  is the viscosity of the solvent and A is the radius of the spherical particles.

There are two modes of operations for FFF techniques with different separation mechanisms determined by the size of the particles to be separated (Shendruk and Slater, 2012). In the normal Brownian dominated mode, submicron and nanosized particles are separated by the balance of the two transportation processes explained earlier in this section. While for bigger particles, the diffusion process is negligible and particles roll on the bed of the channel so a sterical interaction between the particles and the surface of the channel determines the separations and this mode is called steric mode. Since bigger particles roll faster than smaller ones the separation is opposite to the normal mode (Reschiglian et al., 2005) (see Figure 3-9 for further illustration). The shape of the flow in the channel is parabolic with maximum speed in the centre (Giddings, 1993b) see Figure 3-9 below. Since smaller particles diffuse faster, they will reach further away from the wall into the area of the higher laminar velocity and they are transported faster and detected earlier than the bigger particles which are situated near the wall where the laminar flow of the carrier fluid has the lowest velocity (Caldwell et al., 1979).

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3.1.8 Inductively Coupled Plasma Mass Spectrometry (ICP-MS) ICP-MS can be applied to measure traces of nearly most of the elements in the periodic table;

it is an extremely powerful analysis method for metal (Hirner, 2006) and non-metallic (Wuilloud and Altamirano, 2006) elemental measurements. The combinations of low detection limit, lower than part per trillion (Ray et al., 2004, Moldovan et al., 2004), and the short measurement time (Montaser, 1998, Nelms, 2005) make it suitable for many fields in both research and applied science (Ammann, 2007) and superior to other types of elemental analysis instruments such as atomic absorption spectrometer (AAS) and inductively coupled plasma –optical emission spectrometer (ICP-OES) (Thomas, 2004, Jarvis, 1988). Examples of the fields where ICP-MS has been applied include environmental(Butler et al., 2011), biomedical, forensic (Ulrich et al., 2004), food industry, life sciences (Bettmer et al., 2006) and many more. Samples used for ICP-MS can be any state liquid, solid or gas.

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followed by the first commercial ICP-MS introduced by Perkin Elmer SCIEX in 1983 (Rakhi et al., 2008). Generic components for most ICP-MS components which are similar to other mass spectrometers include: Sample introduction system, ICP torch and radio frequency (RF) coil, Interface, Vacuum system, electrical lenses (electrostatic analyser ESA), quadrupole mass filter and detector (PerkinElmer, 2011) (see Figure 3-10 below for illustration). In the ICP-MS, ions are generated in the induction system and introduced in the vacuum area of the mass spectrometer, and then they are accelerated by electrostatic lenses. The separation of ions on the basis of their mass/charge ratio is achieved through a mass analyser filter (quadrupole) and they are finally detected by an electron multiplier at the far end of the mass spectrometer system. To find the concentration of the element under study, its electrical signal from the detector is compared with a signal given by a certified reference material used to calibrate the system (Zeisler et al., 2006).

Figure 3-10: A diagram showing the cross section of the different components of modern quadrupole ICPMS(Wum, 2012) 3.1.9 Potentiometric Titrations Method Potentiometric titration is an analytical method where an electrochemical cell measures the

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potentiometric titration can be applied to different types of reaction such as redox, complexion, precipitation and many more the most popular reaction is the acid base titration.

Certain volume (V) of an acid with unknown concentration of H+ is titrated with volume v of base with known concentration of OH-. The potential difference between the two electrodes is then given in the form of pH due to the calibration that was conducted prior to the measurement. The change of the pH against the volume of titrant used can be recorded and plotted as a graph (see Figure 3-11 below). The inflection point of the graph is where maximum change occurs and corresponds to the end point of the reaction.

Figure 3-11: Titration of acid with strong alkali.(Floridauniversity, 2012) Another alternative way of finding the end point of the reaction is the applying of the Gran method which is explained in the following paragraphs. The section of the titration curve before the end point in the acidic region applies that the concentration of the hydrogen ions can be calculated using Equation 3-26 below (Gran, 1950).

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acid solution respectively, vb and [OH-] are the volume of the base added and the concentration of the OH- ions. Manipulating Equation 3-26 slightly provides Equation 3-27 below.

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If the part before the equal sign in Equation 3-27 is plotted against vb a linear equation with slope of [OH-]0 will be found. The intercept of this linear relationship is v0 *[H+]0 which is the number of moles of H+ ions in the unknown original sample.

When the unknown is [OH-], the alkaline region of the titration curve can provide solution. In this region following Equation 3-28 applies for the initial concentration of the unknown OHconcentration (Gran, 1950).

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Where Kw is the water constant and is equal to [H+] *[OH-] = 10-14. Similar manipulation as Equation 3-26 will provide a linear equation with gradient of [OH-]0/Kw (Gran, 1952).

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4.1 Introduction This chapter describes the sample preparation techniques and analytical methods used to synthesise and characterise gold nanoparticles (AuNPs) of different capping agents (citrate and Polyvinyl pyrrolidone ( PVP)) and sizes both as prepared and in bacterial growth media.

In order to understand the effect of NPs on planktonic bacteria the properties of nanoparticles need to be fully understood so that the effect property relationship can be studied. It is important to examine and understand for example the stability of NPs in the bacteria growth media prior to their exposure to the actual bacteria since any change in the physicochemical properties of the NPs may cause unpredicted and hard to interpret results. The more we know about the properties of the nanoparticles the easier it will be for us to identify the nanoparticles property and bacterial response relationship (Stone et al., 2010). After a short introduction to the different synthesis method used to synthesise NPs, this chapter will give detailed description of how characterisation techniques are applied practically in this project.

The last section of this unit will be devoted to microbiological analysis techniques including media preparation, sterilisation techniques, staining, fixation, sectioning and bacteria quantification methods.

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