Effective and reproducible capillary electrophoretic separation of thiols under conditions where exceptionally high current is generated

There is an escalating interest in the role of endogenous nitric oxide (NO) in biological systems and how this chemical regulates physiology in normal and disease states. In biological systems, the cellular concentration can be estimated, in the simplest form, by accounting for NO and its common metabolites, nitrate and nitrite. However, since NO is also known to interact with other chemical entities, such as thiols, it would be valuable to have a rapid qualitative assay that could account for thiol binding and S-N bond cleavage in the presence of different reducing agents. A separation buffer consisting of 10 mM phosphate, 10 mM HCl, and 250 mM KCl is shown to be adequate for the separation of glutathione, nitrosylated glutathione, and glutathione disulfide solubilized in 2 M HCl. The current observed under these separation conditions (249 μA at 11 kV) is extremely high by capillary electrophoresis (CE) standards, with a total power (current×voltage/capillary length) calculated to be in excess of 7 W/m. While this exceeds the approximately 1.0 W/m recommended by previous studies as a maximum for CE-based separations, we demonstrate that effective CE separation of thiols can, in fact, be accomplished under these conditions with acceptable reproducibility, provided that buffer depletion issues are addressed.