Singlet oxygen detection in skim milk by electron spin resonance spectroscopy

2,2,6,6-Tetramethyl-4-piperidone (TMPD) can react with singlet oxygen to produce the corresponding nitroxide, 2,2,6,6-tetramethyl-4-piperidone-1-oxyl (TAN), which can be detected by electron spin resonance (ESR) spectroscopy. ESR detected the formation of TAN in 5 muM riboflavin phosphate buffer, or skim milk containing 20 mM TMPD,during illumination. The effects of illumination, riboflavin, and oxygen on the formation of TAN indicated that the presence of each was required for singlet oxygen formation. The photoreduced riboflavin might reduce TAN to the hydroxylamine of TAN, thus decreasing the paramagnetic TAN signal. Maximum concentration of TAN was obtained at an added 10 mM riboflavin in buffer solution or in skim milk after 15 min of illumination in the presence of oxygen.