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Spectroscopy is a powerful tool for the investigation of chemical phenomena, but often is hindered in the analysis of species in chemical mixtures—in which chemical interferences can convolute the attainable spectroscopic information. Separation techniques such as liquid chromatography and capillary electrophoresis provide a means of deconvoluting such information but typically are performed over timescales much longer than the life span of highly unstable compounds. This dissertation describes the development of the world’s fastest separation technique, microsecond electrophoresis, and application of this technique to the study of transient species. Chapter 1 provides a discussion of issues involved in the performance of high speed electrophoretic separations. Chapter 2 describes development of multiphoton excited (MPE) optical injection and detection strategies which allow transient species to be analyzed using electrophoresis over low micron separation distances. Chapter 3 presents a method by which separation capillaries are modified such that electric field strengths exceeding 0.1 MV/cm can be used to perform separations (resulting in a ~ 100-fold reduction in analysis time over previous high speed separation techniques). Chapter 4 presents further instrumental developments which allow differently-charged species to be detected at different locations within the separation channel (allowing “infinite resolution’ microsecond separations to be performed). Chapter 5 details work towards application of low-timescale electrophoretic separations to the study of folding protein and peptide species.