The aim of this study was to investigate hydrophilic interaction chromatography (HILIC) and its application to metabolic profiling. The effect of different mobile phase modifiers on the retention behaviour of acidic and neutral test probes was studied on silica gel based columns which confirmed the hypothesis that HILIC is due to a combination of hydrophilic interactions and electrostatic interactions. Among the acidic probes tested, the more negatively charged maleic acid was not strongly retained until the strength of the ammonium ion in the mobile phase was high enough to shield the acid from repulsion by charged silanol groups on the silica gel surface. The neutral test probes were increasingly retained with increasing ionic strength of the mobile phase suggesting that the thickness of the water layer on the surface of the silica gel increased with the strength of the ionic modifier. The utility of three silica hydride columns, Cogent silica C, Cogent Phenyl Hydride and Cogent UDC cholesterol, for separating mixture of metabolites was successfully assessed with an alternating gradient from high organic to high aqueous and then from high aqueous to high organic, thus permitting two orthogonal selectivities to be obtained on the same column. A method for the reductive amination was developed for the analysis of sugars and sugar phosphates. This method was very successful with deuterated aniline as a tag which led to the separation of the three common hexoses glucose, galactose and mannose. The method was applied to the profiling of sugars in milk and in brain tissue. An extensive study of seal milk in comparison with other mammalian milks was also conducted which found that unlike the milks of terrestrial mammals, seal milk had very little lactose. Thus seal pups appear to rely on fat metabolism to produce energy.A notable feature of the milk was that it contained high levels of nicotinamide, an essential precursor for the production of the cofactor NAD+ which is required for the β-oxidation of fats.
|Date of Award||19 Sep 2016|
- University Of Strathclyde
|Supervisor||David Watson (Supervisor) & Darren Edwards (Supervisor)|