Indirect photometric detection of anions in capillary electrophoresis using dyes as probes and electrolytes buffered with an isoelectric ampholyte
Johns, CA and Macka, M and Haddad, PR, Indirect photometric detection of anions in capillary electrophoresis using dyes as probes and electrolytes buffered with an isoelectric ampholyte, Electrophoresis, 21, (7) pp. 1312-1319. ISSN 0173-0835 (2000) [Refereed Article]
The use of highly absorbing anionic dyes as probes and isoelectric ampholytes as buffers in background electrolytes (BGEs) combined with the use of a light emitting diode (LED) as a light source has been studied for ultrasensitive indirect photometric detection in capillary electrophoresis (CE). Potential dyes and buffers were evaluated based on characteristics relevant to indirect photometric detection principles, such as the electrophoretic mobility of the probe dye, its solubility and adsorption behaviour, and the isoelectric point and buffering capacity of the ampholytic buffer. Two dyes, tartrazine and naphthol yellow S, and histidine as the ampholytic buffer, were selected for detailed investigation. Purification of the probes was vital to avoid anionic impurities interfering with the detection. For the electrolytes containing a purified probe (0.5 mM) and histidine as the isoelectric buffer (pI 7.7), hydroxypropylmethyl cellulose (0.05%) was effective in suppression of the electroosmotic flow (EOF). Analytical method performance characteristics were determined. For both probes, experimentally determined mobilities were generally close to literature values, excellent peak shapes and separation efficiencies of up to 298 000 theoretical plates were obtained, and detection limits were generally at the sub-M level. For the naphthol yellow S-histidine BGE, linearity and reproducibility were also evaluated, with excellent linearity being observed over a range of 5 - 500 M, and reproducibility (relative standard deviation, RSD) less than 1% for migration times and 2 - 8% for normalised peak areas. The approach developed was applied successfully to several real samples including tap water, mineral waters, and beer.