| Capillary electrophoresis allows for exquisitely selective separations of analytes, given that the charge-to-friction ratio of each of the analytes in a given complex sample is different under the operational electrophoresis conditions. Typically the requisite mobility difference is obtained by conducting the separation in a bulk sieving media, such as a highly entangled polymer solution. However, the introduction of these viscous media into microscale separation channels can be challenging; thus it would be advantageous to conduct the separation in low-viscosity aqueous media. This research investigates a general separation modality, called End-Labeled Free-Solution Electrophoresis (ELFSE) and its complement Free-Solution Conjugate Electrophoresis (FSCE), which both enable high-resolution electrophoretic separations in aqueous buffer alone (i.e., in free solution).; For two important classes of analytes, namely highly charged polymers (e.g., DNA), and uncharged polymers (e.g., certain peptides, proteins, and polyethylene glycol (PEG)), electrophoresis in free solution does not provide the requisite difference in the charge-to-friction ratio, and high-resolution separations are not possible. ELFSE and FSCE are implemented through the end-on covalent derivatization of either of these analytes with a monodisperse polymer of the other class, which creates an analyte mixture with each species having a unique charge-to-friction ratio, enabling high-resolution separation of the analytes in free solution.; Specifically, the research shows ELFSE size-dependent separation of the DNA oligomers using monodisperse uncharged polymeric “drag-tags” covalently attached to charged DNA oligomers. Application of this system to a single-base extension biological assay allows multiplexed SNP genotyping analysis, which we have used to screen for common mutations in the p53 gene. Conversely, FSCE, using monodisperse charged DNA “engines” attached to uncharged polydisperse polymers (PEG and oligomeric peptide mimics), facilitates the separation of polydisperse uncharged synthetic polymers. In the latter separation class, theoretical treatment of the resultant data allows for precise calculation of the original uncharged polymers' degree of polymerization, and accurate molar mass characterization of the polydisperse polymer samples. The final chapter introduces an isotachophoretic method for increased detection sensitivity. |
