Determination of Ascorbate Concentration in a Raw Leaf with Electron Spin Resonance Spectroscopy

Abstract

The concentration of ascorbate in a raw leaf was determined by monitoring a nitroxyl radical reduction with X-band ESR. One piperidine nitroxyl derivative, 2,2,6,6-tetramethyl-4-hydroxypiperidine-1-oxyl (TEMPOL), was reduced according to first-order kinetics by more than 5-fold excess of ascorbate. The observed reduction rate was found to be proportional to ascorbate concentration in phosphate buffer (0.1 mol/dm³ , pH 7.4) at 25°C. The reduction of TEMPOL in leaf homogenate of sprouts of radish was measured; it was found to obey first-order kinetics. The ascorbate concentration in leaf was estimated from observed reduction rate and found to be virtually identical with that from a visible absorption spectrophotometric analysis. The features of our ESR method for sensitivity, procedure and measuring time were discussed with comparisons from other methods. The results indicated that the determination of ascorbate in a raw leaf by ESR spectroscopy was simple and effective.