Raman microscopy in capillaries and at surfaces.

Abstract

Raman microscopy provides vibrational information with spatial resolution on the order of 1-5 $\mu$m. Recent instrumental developments, including the charge coupled device and volume holographic optical element prefilters, have led to significant improvements in detector efficiency and optical system throughput. The studies outlined in this thesis make use of these advancements to further develop Raman microscopy as an analytical technique. Experimental difficulties encountered in capillary electrophoresis (CE) are examined the further development of surface enhanced Raman spectroscopy (SERS) as a probe for neurotransmitter analysis is reported. In CE, the Raman microprobe reveals the existence of non-uniform intracapillary temperatures. These thermal variations are not predicted by current CE theory and are invisible to conductimetric or other thermometries which provide only global average intracapillary temperatures. The implications of thermal non-uniformities in CE separations are discussed. The Raman microprobe is also shown to be a useful tool for studying the kinetics of polymerization and the structure of polyacrylamide gels inside electrophoresis capillaries. Significant differences in reaction rate between bulk and capillary gels are reported. These differences can be attributed to inefficient heat dissipation in the bulk system. Histamine SERS potential and pH dependencies are reported. Spectral changes are discussed with respect to the orientation of the adsorbate on electrochemically roughened Ag electrodes. SERS has adequate sensitivity to measure histamine concentrations at release levels and can distinguish between the neurotransmitter and its primary mammalian brain metabolite 1-methylhistamine. Electrode preparation and protection from fouling are concerns in neurotransmitter SERS. The presence of lipid does not result in the degradation of SERS signal intensities. Further evidence is provided to illustrate that cellulose acetate coatings protect SERS electrodes from protein adsorption. Attention to surface preparation is important in SERS studies. Irreproducible SERS intensities arising from inadvertent loading of the SERS substrate with abrasive are reported.