Ascorbate Oxidation: UV Absorbance of Ascorbate and ESR Spectroscopy of the Ascorbyl Radical as Assays for Iron

Catalytic transition metals are an absolute requirement for the aerobic oxidation of ascorbate monoanion. Thus, for example, the concentration of iron can be determined by the metal-dependent rate of ascorbate oxidation in near-neutral solutions. The lower limit of detection of iron, as Fe(III)EDTA, by monitoring the decrease in absorbance at 265 nm of ascorbate is about 200 nM. However, by measuring the concentration of the ascorbyl radical by ESR spectroscopy the lower limit is about 10 nM. Using these assays, I have shown that the typical microliter laboratory syringe can introduce significant iron into solutions. Thus, for studies involving iron, these two tests can be used to determine the amount of contaminating iron in reagents as well as iron from other sources such as laboratory equipment.