Spin Relaxation Measurements Using First-Harmonic Out-of-Phase Absorption EPR Signals: Rotational Motion Effects

Abstract

A recent survey of nonlinear continuous-wave (CW) EPR methods revealed that the first-harmonic absorption EPR signal, detected 90 degrees out of phase with respect to the Zeeman modulation (V(1)(')-EPR), is the most appropriate for determining spin-lattice relaxation enhancements of spin labels (V. A. Livshits, T. Páli, and D. Marsh, 1998, J. Magn. Reson. 134, 113-123). The sensitivity of such V(1)(')-EPR spectra to molecular rotational motion is investigated here by spectral simulations for nitroxyl spin labels, over the entire range of rotational correlation times. Determination of the effective spin-lattice relaxation times is less dependent on rotational mobility than for other nonlinear CW EPR methods, especially at a Zeeman modulation frequency of 25 kHz which is particularly appropriate for spin labels. This relative insensitivity to molecular motion further enhances the usefulness of the V(1)(')-EPR method. Calibrations of the out-of-phase to in-phase spectral intensity (and amplitude) ratios are given as a function of spin-lattice relaxation time, for the full range of spin-label rotational correlation times. Experimental measurements on spin labels in the slow, intermediate, and fast motional regimes of molecular rotation are used to test and validate the method.

Application of the out-of-phase absorption mode to separating overlapping EPR signals with different T1 values

“…These apply to experimental conditions that are likely to optimize resolution in the V 0 1 -EPR spectra (see Section 2). Previous calibrations apply solely to high H 1 -values that are designed to optimize T 1 -determinations from single-component spectra [1,2].…”

“…At a modulation frequency of 100 kHz and moderately strong saturation, the out-of-phase and in-phase signals can become of comparable intensity. Even under conditions giving rise to rather low out-of-phase/in-phase ratios in the non-linear regime, useable signal strength can be obtained in the out-ofphase V 0 1 -EPR spectrum because the latter is routinely recorded at a much higher microwave power than are conventional spin-label EPR spectra [1,2]. For the example given above, it is predicted from Eqs.…”

“…However, the strength of the out-of-phase V 0 1;1 -signal increases with increasing modulation frequency (see Eqs. (2) and (12)). As a compromise between efficiency of signal separation and concentration sensitivity, modulation frequencies of 20-30 kHz are optimal.…”

“…We note that for T 1 @ 10 À6 s, which is typical for nitroxide spin labels, the inequality 2c 2 e H 2 1 T 2 1 ) 3 holds for microwave field strengths H 1 > 0.07 G. The latter value is well below the optimal H 1 normally used for V 0 1;1 measurements, which routinely is in the range 0.3-0.5 G [1,2]. Therefore, the dominant term in the numerator on the right of Eq.…”

“…Earlier, first-harmonic EPR signals detected in phase-quadrature with the Zeeman modulation, under conditions of partial microwave saturation, were used to determine spin-lattice relaxation times [1]. Sensitivity of the V 0 1 -EPR spectra also to molecular motion was investigated by spectral simulations for nitroxide spin labels, over the entire range of rotational correlation times [2]. It was shown that determination of the effective spin-lattice relaxation times from V 0 1 -EPR spectra is less dependent on rotational mobility than for other non-linear CW-EPR methods (see also [3]), especially at lower Zeeman modulation frequencies.…”