Fumio Moriya

Separation of free radicals by high-performance liquid chromatography with electron spin resonance detection

Abstract Studies on the separation of free radicals by high-performance liquid chromatography with electron spin resonance detection (HPLC-ESR) are described. Aqueous solutions of amino acids, dipeptides and tripeptides were γ-irradiated in the presence of a spin trap, 2-methyl-2-nitrosopropane. The stable aminoxyl radicals (spin adducts) produced were isolated by HPLC on cation-exchange columns. An ESR spectrometer was utilized as a detector for monitoring the eluted radicals. Various types of spin adducts were identified and characterized by ESR spectroscopy. The individual components of several pairs of diastereomeric radicals could be separated.

A Fundamental Study on Aqueous Solutions of 2-Methyl-2-Nitrosopropane as a Spin Trap

Several aspects of spin trapping with 2-methyl-2-nitrosopropane (MNP) were studied. Compounds produced from dimer MNP in aqueous solutions prepared for spin trapping were analyzed as t-butylnitrosohydroxylamine, t-butyl alcohol, and isobutene. The procedure for obtaining solutions containing the lowest concentrations of these products and the highest of monomer MNP with spin-trapping ability was established. The ESR signals of di-t-butyl-nitroxide in the light-irradiated aqueous MNP solution increased when the pH of the solution was raised. It was found that nitrous acid generated in the irradiated solution reacts with di-t-butylnitroxide and an unidentified compound, giving rise to a stable, positively charged diamagnetic intermediate. The production of the nitroxide from the intermediate caused by raising the pH might be relevant to the increase in ESR signals.

Application of the spin-trap HPLC-ESR method to radiation chemistry of amino acids in aqueous solutions

Our recent studies of the application of the newly developed spin-trap HPLC-ESR method to γ-radiolysis of aqueous solutions containing amino acids are reviewed. 2-Methyl-2-nitrosopropane (MNP) was used as a spin trap to convert generated unstable free radicals into relatively stable aminoxyl radicals, which were separated individually by HPLC with cation-exchange columns. Compounds derived from MNP during the preparation of aqueous MNP solutions were found to be t-butylnitrosohydroxylamine, t-butyl alcohol and isbutene. The preparation procedure of the solution in which these undesirable products are minimized is proposed. γ-Radiolysis of aqueous MNP solutions resulted in the formation of five aminoxyl radicals. The chromatographic retention times of the radicals were found to be different from those of the spin adducts from the amino acids studied here. Amino acids investigated in the present work were glycine, L-alanine, L-valine, L-isoleucine, L-leucine and DL-methionine. Twenty-five spin adducts from the amino acids were detected and identified by the method. The reactions by which short-lived radicals are produced in γ-irradiated aqueous solutions of the amino acids have been found to be H-abstraction by hydroxyl radicals and deamination by hydrated electrons.

Cautionary Note for Use of 2-Methyl-2-Nitrosopropane as Spin Trap

Abstract It could be found by means of optical absorption, NMR spectroscopy and high performance liquid chromatography that 2-methyl-2-nitrosopropane used as spin trap is converted slowly in aqueous solutions by heat to tert-butylnitrosohydroxylamine and tert-butyl alcohol, which do not spin trap.

Spin-Trap-Radical Chromatography of Spin Adducts Produced from L-Valine by γ-Irradiation

Abstract Diastereomeric spin adducts produced by reaction of 2-methyl-2-nitrosopropane with the short-lived radicals from L-valine by γ-irradiation could be separated and identified by means of high performance liquid chromatography and ESR spectroscopy.