Detection of radical adducts of 5,5-dimethyl-1-pyrroline n-oxide by the combined use of high-performance liquid chromatography with electrochemical detection and electron spin resonance.
Although high-performance liquid chromatography with electrochemical detection (HPLC-EC) has been previously used to analyze radicals trapped by 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), these techniques omitted the critical use of an internal standard. To improve reliability we have incorporated 3-carbamoyl-2,2,5,5-tetramethylpyrrolidin-1-yloxy free radical (CTPO) as a stable, oxidizable, and paramagnetic internal standard. Radicals generated during the uv photolysis of hydrogen peroxide were trapped by DMPO, analyzed by HPLC-EC, and quantified by relative comparison of peak areas to those of CTPO. DMPO adducts of hydroxyethyl, hydroxymethyl, and carbon dioxide anion radicals have been further characterized by ESR analysis of their 13C isotopes sampled from the HPLC eluant. Studies of the voltage dependence of the electrochemical signal demonstrate how this can be used to further confirm the identity of artifactual HPLC-EC peaks with similar retention times.
Towell
J
J
Department of Chemistry, Carroll College, Waukesha, Wisconsin.
Kalyanaraman
B
B
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003497:Cyclic N-Oxides; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D005609:Free Radicals; D013113:Spin Labels
D003497:Cyclic N-Oxides; D005609:Free Radicals; D013113:Spin Labels; C017245:5,5-dimethyl-1-pyrroline-1-oxide