Contributions to the Gaussian line broadening of the proxyl spin probe EPR spectrum due to magnetic-field modulation and unresolved proton hyperfine structure.

A simple expression is derived to compute the total Gaussian linewidth of a Voigt line that is broadened by sinusoidal magnetic-field modulation as follows: delta HPPG(Hm)2 = delta HPPG(0)2 + kappa 2Hm2, where delta HPPG(Hm) is the Gaussian linewidth observed with an modulation amplitude Hm/2 and delta HPPG(0) is the Gaussian linewidth in the limit of zero modulation. The field modulation contributes an additional Gaussian broadening of kappa Hm, where kappa is a constant, which adds in quadrature to delta HPPG(0) to give the total Gaussian linewidth. Denoting the overall linewidth of the Voigt line in the absence of modulation broadening by delta HPP0(0), it is shown, both by analytical means and by spectral simulation, that the constant kappa is equal to 1/2 in the limit of Hm < = delta HPP0(0); however, using values of Hm as large as delta HPP0(0) leads to only minor departures from kappa = 1/2. The formulation is valid both for Lorentzian and Voigt lines and is tested for 2,2,5,5-tetramethylpyrrolidin-1-oxyl-3-carboxylic acid (3-carboxy proxyl) in CCl4 and in aqueous buffer. This spin probe was studied because the proxyl group is the only major spin-probe moiety whose Gaussian linewidth had not been characterized in the literature. For 3-carboxy proxyl, it is found that delta HPPG(0) = 1.04 +/- 0.01 G independent of solvent polarity. Precision values of the 14N hyperfine coupling constant for 3-carboxy proxyl at 9.5 degrees C are as follows: 14.128 +/- 0.001 G in CCl4 and 16.230 +/- 0.002 G in aqueous buffer. The temperature dependence of delta HPPG(0) and the 14N hyperfine coupling constant are reported as empirical equations. Results of the present work taken together with previously published data permits accurate correction for the effects of inhomogeneous broadening due to unresolved hyperfine structure and modulation broadening for the majority of spin probes in common use.