La gélatine est un ingrédient utilisé dans de nombreuses industries et sa solubilité influence beaucoup ses propriétés fonctionnelles. Des défauts de solubilité sont parfois constatés, notamment suite à un stockage de la gélatine en grains à température élevée et humidité importante. Cette perte de solubilité pourrait être due à la présence de molécules de haut poids moléculaire. L'objectif de ce travail est d’apporter des éléments de compréhension sur la perte de solubilité observée dans les solutions de gélatine. L'utilisation d’une technique de Fractionnement par Flux-Force couplée à une diffusion de la lumière Multiangulaire, a mis en évidence la présence d’agrégats de haut poids moléculaires dans les solutions de gélatine (de 9.5 à 30.2.105 g.mol-1). Ces agrégats n’avaient jamais pu être identifiés par les techniques classiques d'exclusion stérique car elles sont souvent éliminées dans le volume d'exclusion des colonnes. La quantité d'agrégats formés ne cesse d'augmenter lors d'un traitement thermique à 75 °C, aboutissant à l'insolubilité de la gélatine. La compréhension du mécanisme à l’origine de cette perte de solubilité montre l'implication de la lysine disponible, dans l'apparition des agrégats. La lysine libre réagissant au cours du traitement thermique provoquerait la formation d'agrégats qui modifient le comportement de la gélatine en solution aqueuse. Les paramètres de caractérisation de l’AFlFFF-MALS permettent de discriminer partiellement des échantillons de gélatine dont les comportements en solubilité sont différents. Rajoutés aux paramètres classiques de caractérisation comme la viscosité à 6.67% et au dosage de la lysine disponible, la discrimination devient parfaite. / Gelatin is an important product for several industries and its solubility dramatically influences its functional properties. The lack of solubility observed in gelatine is supposed to be due to the occurrence of molecules with high molecular weights, especially after heat treatments. In order to be able to predict the gelatin behaviour, a new technique for its analysis has been developed with an Asymmetrical Flow Field-Flow Fractionation (AFlFFF-MALS) coupled to a multiangular light scattering. The AFlFFF-MALS analysis showed high molecular weight fractions in gelatin ranging from 9.5 to 30.2 105 g.mol-1 which has not been shown previously with alternative techniques such as size exclusion chromatography. After heat treatment of dry gelatine in an oven at 75°C, some huge aggregates appeared, of which size and density increased and led to partial insolubilisation of gelatin into water. The mechanism responsible for this phenomenon involved lysine residues which plays a very important role in gelatin properties. Quantification of available lysine in gelatin samples by LC-UV has been developed. Thermal treatment during 8 days led to a decrease of free lysine content whereas, at the same time, the molecular weight of gelatin fractions increased and α helixes formation in solution was strongly affected. Intermolecular cross-links led to high molar mass compounds and limited protein chain unfolding. From an industrial point of view, AFlFFF-MALS analysis can help to discriminate gelatine samples in regard to their solubility. If other parameters are added (6.67 % viscosity and free lysine) the discrimination was perfect.

The advent of microfluidic technology presents many difficulties but also many opportunities for separation science. Leveraging the potential of micro- and nanofluidic geometries is not only a matter of shrinking systems. Miniaturization can shift the relative importance of physical phenomena leading to separation. Theoretical and computational studies into the consequences of miniaturization are vital. Mesoscopic, multi-particle collision dynamics simulations are performed on polyelectrolytes and hard, colloidal solutes. Multiple variations of this simulation algorithm are implemented to achieve versatility for simulating non-equilibrium flows and dispersed solutes. The algorithm is extended to simulate the effects of finite Debye layers on the electro-hydrodynamics of electrophoresing macromolecules and used to study the electrophoresis of charged oligomers, polyelectrolytes and polyampholytes in both free-solution and confined geometries. Multi-particle collision dynamics simulations of hydrodynamic chromatography and field-flow fractionation are also performed to test the predictions of the derived unified, ideal retention theory. This unified, ideal retention predicts the transitions between multiple operational modes, including Faxén-mode FFF. Simulations and the theory show that increases in drag due to hydrodynamic interactions with microfluidic channel walls perturb the retention curves from the ideal predictions at large particle sizes. Further complications to field-flow fractionation including undesirable forces perpendicular to the flow direction, slip at channel walls and rectangular channel geometries are investigated. These theoretical studies lead to the proposal of several novel fractionation techniques, namely adverse-mode FFF, slip-mode FFF and polymer/depletant HC.

The advent of microfluidic technology presents many difficulties but also many opportunities for separation science. Leveraging the potential of micro- and nanofluidic geometries is not only a matter of shrinking systems. Miniaturization can shift the relative importance of physical phenomena leading to separation. Theoretical and computational studies into the consequences of miniaturization are vital. Mesoscopic, multi-particle collision dynamics simulations are performed on polyelectrolytes and hard, colloidal solutes. Multiple variations of this simulation algorithm are implemented to achieve versatility for simulating non-equilibrium flows and dispersed solutes. The algorithm is extended to simulate the effects of finite Debye layers on the electro-hydrodynamics of electrophoresing macromolecules and used to study the electrophoresis of charged oligomers, polyelectrolytes and polyampholytes in both free-solution and confined geometries. Multi-particle collision dynamics simulations of hydrodynamic chromatography and field-flow fractionation are also performed to test the predictions of the derived unified, ideal retention theory. This unified, ideal retention predicts the transitions between multiple operational modes, including Faxén-mode FFF. Simulations and the theory show that increases in drag due to hydrodynamic interactions with microfluidic channel walls perturb the retention curves from the ideal predictions at large particle sizes. Further complications to field-flow fractionation including undesirable forces perpendicular to the flow direction, slip at channel walls and rectangular channel geometries are investigated. These theoretical studies lead to the proposal of several novel fractionation techniques, namely adverse-mode FFF, slip-mode FFF and polymer/depletant HC.

Relationships between trace metals and nanoparticles were investigated using analytical transmission electron microscopy (aTEM) and asymmetric flow field flow fractionation (aFlFFF) coupled to both multi-angle laser light scattering (MALLS) and high resolution-inductively coupled plasma mass spectroscopy (HR-ICPMS). Riverbed sediment samples were taken from the Clark Fork River in Montana, USA where a large-scale dam removal project has released reservoir sediment contaminated with toxic trace metals (namely Pb, Zn, Cu and As) which accumulated from one and a half centuries of mining activities upstream. An aqueous extraction method was used to attempt to separate the nanoparticles from the bulk sediment. After analysis of initial results, it was found that low density clays were being selected for in this process and made up a major portion of the particles within the extracts. However, it was also realized that the metals of interest were associated almost exclusively with nano-sized Fe and Ti oxides. In order to more fully examine these relationships, a density separation method, using sodium polytungstate (2.8g/cm3), was developed to separate these higher density oxides from the lower density clays. The heavy fraction was then subjected to an aqueous extraction routine to extract the nanoparticulate fraction. FFF results indicated a smaller size distribution and more ideal fractionation with this method. The aFlFFF-HR-ICPMS profiles for Fe and Ti also matched strongly with the data for the trace metals. The majority of particles analyzed with the TEM were nano-sized Fe and Ti oxides (most commonly goethite, ferrihydrite and brookite), which typically had trace metals associated with them. In many cases, it was aggregates of these nano oxides that were found hosting trace metals. Nanoparticles and aggregates are known to behave differently than their bulk mineral phases or constituent particles, respectively. Nanoparticles are also capable of extended transport in the environment. For these reasons, it is important that their associations with toxic trace metals be extensively evaluated, as they will affect the bioavailability and toxicity of these metals with implications for any type of contaminant sediment relocation, dam removal or metal contaminated site. / Ph. D.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Polystyrene-block-poly(ethylene oxide) (PS-b-PEO) copolymers contain a hydrophilic PEO block and a hydrophobic PS block. PS and PEO have different affinities for most organic solvents and as a result, the PS-b-PEO copolymers are difficult to characterize in solution. In order to achieve a complete characterization of their molecular heterogeneity different techniques have been used. Recently FFF has become a cutting edge technology for polymer analysis because it possesses a number of advantages over conventional SEC and other liquid chromatographic techniques. The mild operating conditions allow the analysis of delicate and sensitive complex analytes such as complex polymer assemblies. The ability to analyze polymers with ultrahigh molar masses has also contributed to its significance in the characterization of polymers. In this study, the FFF behaviour of PS-b-PEO copolymers as well as PS and PEO homopolymers was investigated using Thermal FFF in different organic solvents and AF4. The aim of the study was the correlation of the thermodynamic quality of the solvents and the elution behaviour of the polymers. Unfortunately, PEO homopolymers have been found to interact with the membrane in AF4. Therefore, they were best characterized in organic solvents using Thermal FFF. In contrast to AF4 no specific interactions occurred due to the absence of a membrane. Results for Thermal FFF showed that in all utilized solvents, PS and PEO homopolymers were separated in the direction of increasing molar mass. For PS-b-PEO copolymers the retention in selective (good) solvents for PS was dependent on the molar mass of the PS block in the block copolymer. This was explained by the fact that in poor solvents PEO adopts a collapsed coil conformation while PS is present in extended random coil conformation. Results also showed that polymer retention was dependent on the temperature programme utilized. The fractionations by Thermal FFF indicated that some of the PS-b-PEO copolymer samples contained PS and PEO homopolymers as by-products. After semi-preparative fractionation these homopolymers were qualitatively identified using FTIR spectroscopy. / AFRIKAANSE OPSOMMING: Polistireen-blok-poli(etileenoksied) (PS-b-PEO) ko-polimere bevat 'n hidrofiliese politetileen oksied (PEO) blok en 'n hidrofobiese polistireen (PS) blok. PS en PEO het verskillende affiniteite vir die meeste organiese oplosmiddels, dit bemoeilik die karakterisering van PS-b-PEO ko-polimere in oplossing. Ten einde 'n volledige karakterisering van hul molekulêre heterogeniteit te bepaal moet ‘n verskeidenheid van tegnieke gebruik word. Onlangs het veldvloeifraksionering (FFF) baie grond gewen tov polimeer analise, aangesien dit verskeie voordele het bo tradisionele chromatografiese tegnieke soos grootte-uitsluitingschromatografie (SEC). Die ligte operasionele omstandighede laat die ontleding van ‘n verskeidenheid van polimere toe, enige iets van delikate polimeer komplekse tot ultra hoë molekulêre massa. In hierdie studie is die FFF gedrag van PS-b-PEO ko-polimere asook PS en PEO homopolimere ondersoek met behulp van Termiese FFF(ThFFF) in verskillende organiese oplosmiddels en onsimmetriese vloei-veldvloeifraksionering(AF4). Die doel van die studie was om die verband tussen die termodinamiese gehalte van die oplosmiddels en die eluering gedrag van die polimere te bepaal. Analise van PEO homopolimere was onsuksesvol aangesien daar interaksie was met die membraan. PEO is dus net geanaliseer in organise oplosmiddels met behulp van ThFFF, aangesien daar geen membraan is nie. Analise met ThFFF het gewys dat skeiding plaasvind volgens ‘n toename in molekulêre massa in organise oplosmiddels. Vir PS-b-PEO ko-polimere die retensie in selektiewe (goeie) oplosmiddels vir PS was afhanklik van die molekulêre massa van die PS blok in die ko-polimeer. ‘n Moontlike teorie is dat die PEO blok ‘n ineengestorte spoel struktuur vorm terwyl die PS blok ‘n uitgestrekte lukraake vorm aan neem. Resultate het ook getoon dat die polimeer retensie afhanklik was van die temperatuur program wat gebruik is. Die fraksionering deur ThFFF het aangedui dat sommige van die PS-b-PEO kopolimeer monsters bestaan het uit PS en PEO homopolimere as by-produkte. Hierdie is kwalitatief bewys deur analise van die fraksies na fraksionering van die ko-polimere met behulp van FTIR spektroskopie.

Abstract not available

<p>The work presented in this thesis describes the use of polystyrene nanoparticles as model surfaces for bioanalytical work. Nanoparticles constitute convenient platforms for the attachment of bioactive agents, and receptor coated particles offer high local concentration of binding sites for specific ligands with minimal steric hindrance. However, it is not only the amount of bound protein that matters, the proteins must also be immobilized at the surface in such ways that they fully retain their activity, while at the same time protecting the surface from unspecific uptake of undesired components. The present work relates to the controlled immobilization of multiple types of active biomolecules onto nanoparticle surfaces to make them multifunctional. The surface expansion offered by the nanoparticles, in combination with the closeness between the reactants co-immobilized on the same particle, enables coupled reactions to be carried at a higher rate than otherwise possible. Thus, particle-decorated surfaces of this kind are highly suitable for miniaturized bioanalytical systems. Sensitive microarray systems are under development, including lectin-coated nanoparticles for glycoprotein mapping and a diagnostic device for Point-of-Care testing with a nanoparticle-based detection system.</p><p>The full evaluation of protein attachment to nanoparticles requires precise analytical techniques for particle characterization, both in bare and coated form. The mass-sensitive SdFFF technique occupies a prominent position for particle characterization, as it offers both accurate determination of particle size and a quantification of adsorbed layers on small particles, whether of synthetic or biopolymeric nature. Here, this analytical technique is developed to precisely characterize nanoparticles that are sequentially coated with different layers, each rendering the particles a specific functionality. The thesis demonstrates how precise mass uptakes can be determined for each specific layer, and how control over the exact surface composition of the modified particles can be established for optimization of biological activity.</p>

The work presented in this thesis describes the use of polystyrene nanoparticles as model surfaces for bioanalytical work. Nanoparticles constitute convenient platforms for the attachment of bioactive agents, and receptor coated particles offer high local concentration of binding sites for specific ligands with minimal steric hindrance. However, it is not only the amount of bound protein that matters, the proteins must also be immobilized at the surface in such ways that they fully retain their activity, while at the same time protecting the surface from unspecific uptake of undesired components. The present work relates to the controlled immobilization of multiple types of active biomolecules onto nanoparticle surfaces to make them multifunctional. The surface expansion offered by the nanoparticles, in combination with the closeness between the reactants co-immobilized on the same particle, enables coupled reactions to be carried at a higher rate than otherwise possible. Thus, particle-decorated surfaces of this kind are highly suitable for miniaturized bioanalytical systems. Sensitive microarray systems are under development, including lectin-coated nanoparticles for glycoprotein mapping and a diagnostic device for Point-of-Care testing with a nanoparticle-based detection system. The full evaluation of protein attachment to nanoparticles requires precise analytical techniques for particle characterization, both in bare and coated form. The mass-sensitive SdFFF technique occupies a prominent position for particle characterization, as it offers both accurate determination of particle size and a quantification of adsorbed layers on small particles, whether of synthetic or biopolymeric nature. Here, this analytical technique is developed to precisely characterize nanoparticles that are sequentially coated with different layers, each rendering the particles a specific functionality. The thesis demonstrates how precise mass uptakes can be determined for each specific layer, and how control over the exact surface composition of the modified particles can be established for optimization of biological activity.

Gegenstand der Dissertation ist die größen- und eigenschaftsoptimierte Synthese und Charakterisierung von anorganischen Nanopartikeln in einer geeigneten Polyelektrolytmodifizierten Mikroemulsion. Das Hauptziel bildet dabei die Auswahl einer geeigneten Mikroemulsion, zur Synthese von kleinen, stabilen, reproduzierbaren Nanopartikeln mit besonderen Eigenschaften. Die vorliegende Arbeit wurde in zwei Haupteile gegliedert. Der erste Teil befasst sich mit der Einmischung von unterschiedlichen Polykationen (lineares Poly (diallyldimethylammoniumchlorid) (PDADMAC) und verzweigtes Poly (ethylenimin) (PEI)) in verschiedene, auf unterschiedlichen Tensiden (CTAB - kationisch, SDS - anionisch, SB - zwitterionisch) basierenden, Mikroemulsionssysteme. Dabei zeigt sich, dass das Einmischen der Polykationen in die Wassertröpfchen der Wasser-in-Öl (W/O) Mikroemulsion prinzipiell möglich ist. Der Einfluss der verschiedenen Polykationen auf das Phasenverhalten der W/O Mikroemulsion ist jedoch sehr unterschiedlich. In Gegenwart des kationischen Tensids führen die repulsiven Wechselwirkungen mit den Polykationen zu einer Destabilisierung des Systems, während die ausgeprägten Wechselwirkungen mit dem anionischen Tensid in einer deutlichen Stabilisierung des Systems resultieren. Für das zwitterionische Tensid führen die moderaten Wechselwirkungen mit den Polykationen zu einer partiellen Stabilisierung. Der zweite Teil der Arbeit beschäftigt sich mit dem Einsatz der unterschiedlichen, Polyelektrolyt- modifizierten Mikroemulsionen als Templatphase für die Herstellung verschiedener, anorganischer Nanopartikel. Die CTAB-basierte Mikroemulsion erweist sich dabei als ungeeignet für die Herstellung von CdS Nanopartikeln, da zum einen nur eine geringe Toleranz gegenüber den Reaktanden vorhanden ist (Destabilisierungseffekt) und zum anderen das Partikelwachstum durch den Polyelektrolyt-Tensid-Film nicht ausreichend begrenzt wird. Zudem zeigt sich, dass eine Abtrennung der Partikel aus der Mikroemulsion nicht möglich ist. Die SDS-basierten Mikroemulsionen, erweisen sich als geeignete Templatphase zur Synthese kleiner anorganischer Nanopartikel (3 – 20 nm). Sowohl CdS Quantum Dots, als auch Gold Nanopartikel konnten erfolgreich in der Mikroemulsion synthetisiert werden, wobei das verzweigte PEI einen interessanten Templat-Effekt in der Mikroemulsion hervorruft. Als deutlicher Nachteil der SDS-basierten Mikroemulsionen offenbaren sich die starken Wechselwirkungen zwischen dem Tensid und den Polyelektrolyten während der Aufarbeitung der Nanopartikel aus der Mikroemulsion. Dabei erweist sich die Polyelektrolyt-Tensid-Komplexbildung als hinderlich für die Redispergierung der CdS Quantum Dots in Wasser, so dass Partikelaggregation einsetzt. Die SB-basierten Mikroemulsionen erweisen sich als günstige Templatphase für die Bildung von größen- und eigenschaftenoptimierten Nanopartikeln (< 4 nm), wobei insbesondere eine Modifizierung mit PEI als ideal betrachtet werden kann. In Gegenwart des verzweigten PEI gelang es erstmals ultrakleine, fluoreszierende Gold Cluster (< 2 nm) in einer SB-basierten Mikroemulsion als Templatphase herzustellen. Als besonderer Vorteil der SB-basierten Mikroemulsion zeigen sich die moderaten Wechselwirkungen zwischen dem zwitterionischen Tensid und den Polyelektrolyten, welche eine anschließende Abtrennung der Partikel aus der Mikroemulsion unter Erhalt der Größe und ihrer optischen Eigenschaften ermöglichen. In der redispergierten wässrigen Lösung gelang somit eine Auftrennung der PEI-modifizierten Partikel mit Hilfe der asymmetrischer Fluss Feldflussfraktionierung (aF FFF). Die gebildeten Nanopartikel zeigen interessante optische Eigenschaften und können zum Beispiel erfolgreich zur Modifizierung von Biosensoren eingesetzt werden. / This work is focused on the formation, recovery and characterisation of inorganic nanoparticles in a tailor-made polycation-modified reverse microemulsion. The main aim is the choice of an adequate microemulsion for the synthesis of small, uniform, reproducible nanoparticles with specialn characteristics. The first part is focused on the incorporation of two different polycations, low molecular weight linear poly(diallyldimethylammonium chloride) (PDADMAC) and low molecular weight branched poly(ethyleneimine) (PEI) in different surfactant-based (CTAB – cationic, SDS – anionic, SB - zwitterionic) w/o microemulsions. In principle the incorporation of the polycations in the small water droplets is possible, but the influence of the polycations on the phase behaviour is different. Repulsive interactions induce a destabilisation of the w/o microemulsion for cationic surfactant CTAB, while the distinctive interactions between the anionic surfactant SDS and the polycations induce an extension of the phase range and a considerable stabilisation. In case of zwitterionic surfactant SB a partial destabilisation can be observed, according to the lower interactions with the polycations. The second part is focused on the formation of different, inorganic nanoparticles in these polyelectrolyte-modified reverse microemulsions as a template phase. The CTAB-based microemulsion is not adequate for the formation of CdS nanoparticles, according to the low tolerance towards the reactants (destabilisation effect). Furthermore the particle growth cannot be limited by the surfactant-polycation-film and a recovery of the nanoparticles from the microemulsion is not possible. The results show that the SDS-based quaternary template phase consisting of water, toluene-pentanol (1:1), and the anionic surfactant SDS in presence of PEI or PDADMAC can be successfully used for the synthesis of polymer capped inorganic nanoparticles (3 – 20 nm). CdS quantum dots, as well as gold nanoparticles are successfully synthesised in the microemulsion droplets. Especially PEI acts as a reducing and stabilizing agent and shows an additional, interesting template effect in the microemulsion. Unfortunately a recovery of the nanoparticles without a particle aggregation is not possible due to the strong surfactant polycation interactions, which lead to polycation-surfactant complexes. The SB-based microemulsion can be successfully used as a tailor-made polycation-modifiedreverse microemulsion for the formation of small, uniform nanoparticles (< 4 nm) with special characteristics. Especially a modification with PEI is optimal and for the first time small, fluorescent gold cluster (< 2 nm) can be synthesised in a SB-based microemulsion as template phase. The results show that the electrostatic interactions between the polycation and the surfactant are of high relevance especially in the solvent evaporation and redispersion process. That means only in the case of moderate polycation-surfactant interactions a redispersion of the polymer capped particles without problems of aggregation is possible and the size and characteristics are unchanged in the redispersed solution. By means of asymmetric flow field flow fractionation (af-fff) it becomes possible to separate the two cluster fractions from each other as well as from the nanoparticle fraction with diameter > 5 nm. This opens a way to use the nanoparticles with their interesting, optical characteristics in different new fields of application for example for modification of biosensors.

Recent advances in nanotechnology have led to the production of materials with nanoscale dimensions (nm) and properties distinctly different from their bulk (>100 nm) counterparts. With increased use, it is inevitable that nanomaterials will accumulate in the environment and there is concern that the novel properties of nanomaterials could result in detrimental environmental and human health effects. In particular, there has been concern recently regarding the use of silver (Ag) based nanomaterials as antimicrobial agents in consumer and medical products. Current regulations dealing with the discharge of metals into the environment are based on total concentrations with no consideration for the form (e.g., ionic, nanoparticle, colloid) which can largely determine toxicity. Methods for the identification and characterization of nanoparticulates within complex matrices are lacking and the development of robust methods for this purpose are considered a high priority research area. This research focuses on the development and application of a novel method for characterizing Ag manufactured nanoparticles (MNPs) within terrestrial environments, in particular in soil pore water, with applications relevant to other metal MNPs as well. The method was then applied to understand the dynamics and behavior of Ag MNPs in soil and soil amended with sewage sludge biosolids.