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The analysis of rubber accelerators particularly zinc dithiocarbamates (ZDTCs) was carried out by high performance liquid chromatography (HPLC) to develop an improved analytical method for the determination of rubber accelerators. Further understanding of their chemical and chromatographic behaviour has been established by investigating a few conventional as well as the latest reported methods, which include the pre-column derivatisation of ZDTCs to cobalt (III) complexes, direct determination by the addition of other protective agent and pre-column derivatisation of ZDTCs to copper (III) complexes.

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This thesis describes the design and development of novel micro analytical devices for application in on-line process analytics. The work describes the design, development, numerical simulation and application of these devices for two specific cases: (i) electrochemical detection of bio(chemical) species at micro-scale and (ii) separation and purification of biological reagents using immobilised metal affinity chromatography at micro-litre scale. Chapter 1 provides a general overview and background to the field of process analytics, microreactors and theory related to the mass transfer inside the electrochemical microfluidic devices and meso-chromatography columns. Chapter 2 provides an overview of microfabrication methods and the numerical simulations employed for the development of micro analytical devices used in this thesis. Chapter 3 describes an experimental voltammetric study of enzyme cofactors in batch and hydrodynamic systems and also provides a numerical investigation of mass transfer over electrodes inside microreactors. Chapter 4 investigates the effect of hydrodynamic focusing within a microfluidic device in detail, using experimental and numerical techniques. The quantification of the results was carried out using a pseudo two-dimensional, steady state backward implicit finite difference model. A series of studies, interrogating the effects of volumetric flow rate, volume ratio and lead-in length, were carried out to quantitatively investigate hydrodynamic focusing. Chapter 5 details the development and fabrication of patterned photopolymerised and electrochemically polymerised (conducting) monoliths with dimensions in the range of 100-1000μm. The photopolymerised monoliths were characterised using hydrodynamic methods in order to study the flow profile. Electrochemical techniques were used to characterise the conducting monoliths and its composites, using N,N,N’,N’-tetramethyl-p-phenylenediamine. Chapter 6 describes an application of the photopatterned monoliths. A meso-chromatography column was fabricated and immobilised metal affinity chromatography at meso and micro-litre scale was studied inside these columns. Proteins with polyhistidine tags were shown to be successfully separated, purified and quantified under batch and hydrodynamic conditions.

This thesis examines new approaches to evaluate the effects of fouling on process chromatography. Fouling can have a serious, negative impact on the performance of chromatography and considerable effort is normally spent to prevent fouling species reaching the column, or in developing clean-in-place (CIP) protocols of ever increasing complexity to mitigate their effects. Despite this, the knowledge of chromatographic fouling often seems anecdotal, with only a few systematic investigations currently reported in literature. Furthermore, conventional approaches to investigate chromatographic fouling only provide an overall indication of their state. New approaches to investigate fouling at increasingly fine detail are studied in this thesis and provide valuable insights to the mechanism of fouling. At the whole-column level, the method of frontal analysis was used to determine the effects of fouling a packed bed column (DEAE Sepharose FF) with yeast homogenate. The shape and position of breakthrough curves generated by frontal analysis were used to quantitatively assess the impact of fouling on binding capacity and to qualitatively infer the overall changes in mass transfer properties. In particular, the effects of solids particulate and different modes of applying the fouling stream to the column were examined. Breakthrough curve analysis was also used to investigate the effectiveness of a rigorous CIP procedure in restoring the characteristics of a fouled column. An extended reverse-flow technique using an acetone tracer was then developed to quantify the dispersive effects of fouling on defined axial sections within a packed bed column, giving more than an overall indication of the fouling condition. The influence of column diameter, bed length and two different header designs on the extent of fouling were examined. The technique allows the band broadening effects due to reversible macroscopic factors, such as flow maldistribution in the flow distributor and inside the packed bed caused by packing heterogeneity, to be separated from irreversible microscopic factors, such as intraparticle diffusion, external fluid film mass transfer and interparticle axial dispersion. It was shown to be a simple, nondestructive method for investigating chromatographic fouling at an intra-column level. Finally, confocal scanning laser microscopy (CSLM) was proven to be a powerful technique to directly visualise fouling at a single-bead level. A particularly aggressive fouling stream of partially clarified E. coli homogenate was used to challenge an anion exchange resin (Q Sepharose FF) in a finite bath, and subsequently in a packed structure under flow conditions. The fouling caused by the material was visualised by fluorescently labelling DNA and host cell proteins in the fouling stream and by measuring the binding capacity and uptake rate for a fluorescently-labelled test protein, BSA. The use of CSLM also allowed the applications of various CIP procedures to be visually followed. The competitive adsorption of whole cells or cell debris and DNA to Q Sepharose FF has also been visualised. Confocal images obtained provide insights to the spatial distribution of key foulant types within a single bead. This thesis concludes with recommendations for future work which will seek to extend the analysis to situations where fouling occurs in a flow situation by the design of appropriate flow cells and methods of analysis.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been implemented as a means to separate and detect tetraether lipid cores derived from the complex lipids of Archaea. Distinct dissociation pathways during tandem mass spectrometry were noted for the lipid cores, providing information regarding their structure on-line. Analysis of cellular material from species of Methanothermobacter and Sulfolobus revealed tetraether lipid cores which contain up to four cyclopentyl rings per etherified alkyl chain, including structures identified previously in each genera. Identical structures were similarly identified in novel isolates from New Zealand hot springs. Product ions in the MS/MS spectra of the lipid cores include those formed from individual losses of both ring-containing C40 alkyl chains, allowing the reported structures to be verified with respect to the distribution of the rings within the two chains. A number of additional, hitherto unreported isomers and higher homologues of the ring-containing structures were resolved, both chromatographically and/or by characteristic product ions in MS/MS. Structures in which the two chains appear to be conjoined by a covalent link were also identified in Ignisphaera aggregans, the first such identifications in a Euryarchaeote. The array of structures revealed highlights both the complexity of the archaeal lipidome, which is more extensive than has been attributed previously, and the potential of LC-MS/MS as a powerful tool for probing tetraether lipid core structure. Ether lipid cores extracted from ancient aquatic sediments and contemporary soil were used to investigate the scope of LC-MS/MS for profiling of extremely complex distributions sourced from ecological communities as opposed to single organisms. Over 100 ether lipid components in total were identified during the studies, the vast majority of which represent novel structures. These include isoprenoid lipid cores of known archaeal origin and structures which may represent their transformation products; triolic structures in which one of the two capping glycerol moieties has been lost and chain or glycerol methylated higher homologues. A wealth of non-isoprenoid lipid cores were similarly identified, with inferred structures suggestive of a eubacterial or mixed eubacterial/archaeal origin. The components, once constrained to more specific origins, may be of chemotaxonomic value for use in modern environmental profiling or in palaeoecological reconstructions made using fossilised lipid cores.

The memory colour reproduction is an important factor in judging image quality of photographic images of real life scenes. As the most important memory colour category, skin tone was extensively studied for preferred colour reproduction in this research. The methodology to study skin colour preference was then applied to study the colour preference of two other important colour categories: green foliage and blue sky. There are three essential parts for preferred skin colour enhancement: 1) building a skin colour model to detect skin colours or skin pixels; 2) finding a preferred skin colour region or a preferred skin colour centre; and 3) developing an algorithm to morph skin colours toward the preferred skin colour region. This study for skin colour enhancement started with the mathematical modelling of the skin colour region for skin colour detection. The modelling of skin colours was then applied to adjust skin colours of test images for psychophysical experiments that were to determine a preferred skin colour region. Finally, the skin colour modelling and the preferred skin colour centres were applied to adjust skin colours of digital photographic images for preferred colour reproduction. Two approaches were developed to model the skin colour distribution for skin colour detection. The first approach was to model a local colour region for general applications. A convex hull is constructed to fit the geometrical shape of a local region, and then the convex hull is approximated with mathematical formulae. The formulations and data fitting are adjusted with interactive 3-D visualization. The approach is flexible for fitting data gamut with various mathematical forms for different purposes. The other approach was to model skin colours with elliptical shapes. Three elliptical skin colour models were developed for skin colour detection. The first one is to model the skin colour cluster using a single ellipse ignoring the lightness (or luminance) dependency. It is simple and efficient, and the skin colour detection accuracy may be adequate for many applications. In the second model, the skin colour ellipse is adapted to different lightness so that the shape of the ellipse fits the skin colour cluster more accurately. The model is more complex to train and is less efficient in computation, but it is more accurate in skin colour detection. In the third method, an ellipsoid is trained to fit the skin colour cluster. It is almost as simple to train as the first model, but the skin colour detection accuracy is improved. Finally, these models were applied to train mixed skin colours, African skin colours, Caucasian skin colours, and Asian skin colours.

This research focuses on the development of whole column detection (WCD) for liquid chromatography (LC). The WCD uses electrochemical techniques for detecting the analytes passing through the separation column. Electrode array for in-column electrochemical detection (ICED) is fabricated along the separation column to enable whole column separation monitoring and allow better understanding on the affinity of particular analyte to the stationary and mobile phases. Numerical models were built to understand the feasibility and differences of electrochemical detection within an unpacked and packed column. From the simulated results, the surface area of the electrode was not hindered by the presence of the particles in flow condition. An electrochemical microfluidic device has been successfully fabricated on PET (polyethylene terephthalate) substrate using the reverse imprinting technique. The photolithographically produced gold metal electrode lines were imprinted into the PET substrate using a blank mould and produced an inlaid electrode array with overall step residue within 40 nm. The semi-cured thermoset polyester channel was irreversibly thermal bonded on the PET substrate. The devices were able to tolerate pressure in excess of 90 bars. The PET column was packed with 5 μm C18 silica beads to perform reverse phase chromatography separation. The array was electrochemically characterised using standard redox probes in both stagnant conditions and under flow. Both numerical modelling and experimental data show improved sensitivity under flow and a limiting current which scaled linearly with cubic root of volume flow rate. Isocratic and gradient mode chromatographic separations of neurotransmitters and metabolites: serotonin, dopamine, adrenaline, 5- HIAA and DOPAC were conducted in the fabricated device. Separation progress was electrochemically detected at multiple locations along the column. Whole column assessment on separation efficiency and column packing efficiency monitoring were conducted using the ICED.

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