Abstract
Methods to detect intermodulation sidebands produced upon irradiation of an EPR spin system by two closely spaced microwave frequencies (separation ω 1−ω 2) were described by Sczaniecki et al. ( J. Chem. Phys. 94, 5907, 1991). The first pair of sidebands correspond to absorption of two quanta from one source and emission of one quantum from the other source and are therefore designated as three-quantum transitions. Similarly, the second pair of sidebands are five-quantum transitions. At low incident power, it is shown that the signal intensities of three-quantum spectra of spin labels are proportional to the spin-lattice relaxation time T 1, using [O 2] as a convenient T 1 modulator. The ratio of three-to five-quantum signal intensities also is proportional to T 1. Signal intensities were studied as a function of the separation of the two irradiating frequencies. The three-quantum intensity drops to 1 2 at (ω 1 − ω 2) T 1 ≃ 1, which was confirmed by pulse experiments and also by varying the frequency difference and [O 2]. Hydrophobic and hydrophilic components of the spin label Tempo in equilibrium with a lipid bilayer were separated by subtraction of three-quantum spectra obtained in the presence and absence of O 2. Multiple-quantum EPR is found to be a useful tool in spin-label oximetry, in discrimination of overlapping spectra with differing T 1 values, and in estimating numerical values of T 1.
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