Biological macromolecules are becoming increasingly important as a target for pharmaceutical research. Structural examination of this class of compounds is vital, both in terms of understanding the three-dimensional structures these molecules form as well as for quality control of bio-pharmaceutical products. UV polarised spectroscopy, such as circular dichroism (CD) is a powerful tool for this form of analysis, rapidly providing the user with structural information on the sample. The ease of data analysis combined with its non-destructive nature make UV polarised spectroscopy an ideal tool for this purpose. However, limitations in the current instrumentation, especially in terms of sample handling have placed significant barriers in the way of fully realising the potential of these techniques. To address the weaknesses of the currently available UV polarised spectroscopy accessories, new devices have been designed and tested to increase the utility of such techniques. A low volume capillary sample holder has been developed which significantly reduces the sample requirements for circular dichroism without loss of signal quality. This advance has been coupled with an HPLC auto-sampler to create a device that can process 96-well and in some cases 384- well plates. This device opens up a host of new applications for polarised UV spectroscopy, including refolding screening as well as freeing up user time. Additionally, a high precision demountable micro scale cuvette has been developed that reproducibly assembles to the same path length every time. The percentage error of path-length for this new cuvette is of the same order as currently commercially available 1 mm cuvettes. This new accessory should prove highly advantageous for the bio-pharmaceutical industry, as it allows accurate path-length CD analysis for high concentration samples. Additionally, the first confirmed linear dichroism (LD) spectrum of a bacterium and flagella are reported, opening up the potential for real time UV spectroscopic analysis of living bacteria. By using the work contained within this thesis, it should be possible to construct a new form of high path-length accuracy, low volume, multiple-sample UV polarised spectroscopy accessory that could be used for both CD and LD analysis.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:560292 |
| Date | January 2011 |
| Creators | Waldron, Daniel E. |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/51549/ |
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