RESEARCH ARTICLE Open Access Free radicals produced by the oxidation of gallic acid: An electron paramagnetic resonance study Angelique C Eslami 1 , Wanvimol Pasanphan 2 , Brett A Wagner 1 , Garry R Buettner 1* Abstract Background: Gallic acid (3,4,5-trihydroxybenzoic acid) is found in a wide variety of plants; it is extensively used in tanning, ink dyes, as well as in the manufacturing of paper. The gallate moiety is a key component of many functional phytochemicals. In this work electron paramagnetic spectroscopy (EPR) was used to detect the free radicals generated by the air-oxidation of gallic acid. Results: We found that gallic acid produces two different radicals as a function of pH. In the pH range between 7-10, the spectrum of the gallate free radical is a doublet of triplets (a H = 1.00 G, a H = 0.23 G, a H = 0.28 G). This is consistent with three hydrogens providing hyperfine splitting. However, in a more alkaline environment, pH >10, the hyperfine splitting pattern transforms into a 1:2:1 pattern (a H (2) = 1.07 G). Using D 2 O as a solvent, we demonstrate that the third hydrogen (i.e.a H = 0.28 G) at lower pH is a slowly exchanging hydron, participating in hydrogen bonding with two oxygens in ortho position on the gallate ring. The pK a of this proton has been determined to be 10. Conclusions: This simple and novel approach permitted the understanding of the prototropic equilibrium of the semiquinone radicals generated by gallic acid, a ubiquitous compound, allowing new insights into its oxidation and subsequent reactions. Background Gallic acid (3,4,5-trihydroxybenzoic acid), found in a variety of plants, is extensively used in tanning, ink dyes, as well as in the manufacturing of paper [1]. In addition, the gallate moiety is a key component of many foods and drinks, e.g. there are two gallate moieties in the important polyphenol, (-)- epi- gallocatechin-3-gallate (EGCG); this and related polyphenols are responsible for the antioxidant, anticarcinogenic, and antiviral proper- ties of some of the most widely consumed beverages in the world, such as green tea [2,3]. The three aromatic phenoxyl groups of gallic acid are prone to oxidation with the formation of hydrogen peroxide, quinones, and semiquinones [4]. We have observed the formation of two distinct semiquinones formed upon the oxidation of gallic acid. Here we have investigated the nature of these two different radicals. Experimental Gallic acid (3,4,5-trihydroxybenzoic acid, CAS No.: 149- 91-7) was obtained from Sigma, USA; sodium hydroxide was from Fisher. All experiments were carried out in 100 mM potassium phosphate. The final concentration of gallic acid was 1 mM, unless noted otherwise. All solutions above neutral pH (alkaline solutions) were pre- pared by adjusting with 1 M sodium hydroxide solution. EPR spectroscopy was done using a Bruker EMX spec- trometer equipped with a high-sensitivity cavity and an Aqua-X sample holder. Spectra were obtained at room temperature (24-27°C). Typical EPR parameters were as follows: 3510 G center field; 10 G sweep width; 9.852 GHz microwave frequency; 20 mW power; receiver gain varied as needed; modulation frequency of 100 kHz; modulation amplitude of 0.10 G; conversion time of 40.96 ms; and time constant of 20.48 ms with 20 X-scans for each 1024 point spectrum. Spectral simula- tions of EPR spectra were performed using the WinSim program developed at the NIEHS by Duling [5]. In the experiments to determine the second pK a of the gallate free radical 5.0 mL of 10 mM gallic acid (final * Correspondence: garry-buettner@uiowa.edu 1 Free Radical and Radiation Biology, Med Labs B180, The University of Iowa, Iowa City, IA 52242-1181, USA Full list of author information is available at the end of the article Eslami et al. Chemistry Central Journal 2010, 4:15 http://journal.chemistrycentral.com/content/4/1/15 © 2010 Eslami et al