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. Author manuscript; available in PMC: 2009 Aug 13.
Published in final edited form as: J Am Chem Soc. 2008 Jul 18;130(32):10780–10787. doi: 10.1021/ja803083z

Figure 6.

Figure 6

High-field components of the EPR spectra of aTAM4 measured in 1.5 mM pyrophosphate buffer, pH 9.9 (a), pH 5.8 (b), and pH 7.8 (c), at various oxygen partial pressures: 0% (black), 10% (red), 20.7% (blue). Spectral parameters: microwave power, 0.63 mW; time constant, 40.96 ms; conversion time, 160 ms; sweep time, 327.68 s; modulation amplitude, 0.1 G; sweep width, 8 G; number of points, 2048. Experimentally measured peak-to-peak line widths are ΔHpp = 160 mG (a) and ΔHpp = 280 mG (b). Fitting the calculated spectra to the experimental spectra a and b at 0% oxygen using hfs given in the caption to Figure 2 and describing individual line shape by convolution of Lorentz and Gauss profiles22 yields the following peak-to-peak line widths: (a) ΔHL = 96 mG, ΔHG = 100 mG, and (b) ΔHL = 97 mG, ΔHG = 94 mG. Note that the values of the obtained parameters for the nonprotonated radical (a) are in agreement with the previously reported equation for convolution of Lorentz and Gauss profiles (ΔHL/ΔHPP+ΔHG2/ΔHPP2=1).22 This is not valid for the protonated form of the radical (b) due to the fact that ΔHpp represents peak-to-peak line width of partially unresolved doublet aroused from additional proton hfs, aH(NH+Et2) = 0.15 G (Figure 2). Fitting the calculated spectra of R and RH+ forms to the experimental spectra a and b, respectively, measured at various concentration of oxygen yield the values of ΔHL shown in Figure 7. Fitting the calculated spectra to the experimental spectra c measured at intermediate pH and various concentrations of oxygen yields the values of peak-to-peak line width, ΔHL, and fraction of protonated form, fRH+, allowing simultaneous [O2] and pH determination using corresponding calibration curves (Figure 7 and Figure 5a, respectively), namely: (black) ΔHL = 105 mG (1.3%), fRH+ = 0.58 (pH 7.85); (red) ΔHL = 166 mG (11.3%), fRH+ = 0.57 (pH 7.88); (blue) ΔHL = 227 mG (21.5%), fRH+ = 0.62 (pH 7.8). The latter examples demonstrate an accuracy in pH (±0.1) and oxygen (±2%) determination using aTAM4 probe in the range of pH around its pKa.