3- Pulse ESEEM

Fig. 2.20 (a) 2-pulse ESEEM from CO2 radicals in an X-irradiated powder of lithium formate. The echo envelope shows modulations due to Li ions adjacent to the COf radicals. The smooth dashed curve shows an exponential decay with the phase memory time Tu (b) FT-tiansform of (a), (c) 2-pulse sequence employed in the experiment (Data provided by Dr. H. Gustafsson)... 

Fig. 3.31 Experimental and simulated 3-pulse ESEEM spectra for trapped solvated electrons in DOCH2CH2OD (left, above) and HOCD2CD2OH (right, above) frozen glass (above), and 2-pulse ESEEM for DOCH2CH2OD (below). The suggested solvation structure (right, below) has re h(01) = 0.27 and re h(02) = 0.32 nm. The figure is adapted from [M. Narayana et al. J. Chem. Phys. 81, 2297 (1984)] with permission from the American Institute of Physics...

Tissue samples were excised from animals following a four-day program of intravenous VOSO4 injections once per day. CW and 2-pulse ESEEM spectra were measured on the tissue samples directly. Reasonable SIN levels were observed in the CW spectra, allowing for orientation selection of flic paramagnetic species by measuring the ESEEM spectrum at the parallel and perpendicular features of die... 

The main advantage of tlie tln-ee-pulse ESEEM experiment as compared to the two-pulse approach lies m the slow decay of the stimulated echo intensity detemiined by T, which is usually much longer than the phase memory time Ty that limits the observation of the two-pulse ESE. 

More sophisticated pulse sequences have been developed to detect nuclear modulation effects. With a five-pulse sequence it is theoretically possible to obtain modulation amplitudes up to eight times greater than in a tlnee-pulse experunent, while at the same time the umnodulated component of the echo is kept close to zero. A four-pulse ESEEM experiment has been devised to greatly improve the resolution of sum-peak spectra. 

Muns ENDOR mvolves observation of the stimulated echo intensity as a fimction of the frequency of an RE Ti-pulse applied between tlie second and third MW pulse. In contrast to the Davies ENDOR experiment, the Mims-ENDOR sequence does not require selective MW pulses. For a detailed description of the polarization transfer in a Mims-type experiment the reader is referred to the literature [43]. Just as with three-pulse ESEEM, blind spots can occur in ENDOR spectra measured using Muns method. To avoid the possibility of missing lines it is therefore essential to repeat the experiment with different values of the pulse spacing Detection of the echo intensity as a fimction of the RE frequency and x yields a real two-dimensional experiment. An FT of the x-domain will yield cross-peaks in the 2D-FT-ENDOR spectrum which correlate different ENDOR transitions belonging to the same nucleus. One advantage of Mims ENDOR over Davies ENDOR is its larger echo intensity because more spins due to the nonselective excitation are involved in the fomiation of the echo. 

Fig. 16. Three-pulse ESEEM spectrum of the Rieske cluster in hovine heart submit-ochondrial particles at gy = 1.89 and 3.7 K. The pairs of trEmsitions belonging to the two nitrogen atoms are indicated. Conditions of measurement EU-e as stated in (87).

HYSCORE, is a 2D four-pulse ESEEM technique which provides correlation between nuclear frequencies originating from different electron manifolds. The sequence of four microwave pulses is tx/2—x—tx/2—/tx— t2-nl2-x-echo where the echo amplitude is measured as a function of tx and t2 at fixed x. The a-proton anisotropic couplings can be detected by this technique (Konovalova et al. 2001a, Focsan et al. 2008). 

Three-pulse ESEEM spectrum of perdeuterated P-carotene imbedded in Cu-MCM-41 exhibits an echo decay with an echo modulation due to deuterons. The three-pulse ESEEM is plotted as a function of time, and curves are drawn through the maximum and minima. From ratio analysis of these curves, a best nonlinear least-squares lit determines the number of interacting deuterons, the distance (3.3 0.2A), and the isotopic coupling (0.06 0.2MHz). This analysis made it possible to explain the observed reversible forward and backward electron transfer between the carotenoid and Cu2+ as the temperature was cycled (77-300 K). 

Carotenoid radical formation and stabilization on silica-alumina occurs as a result of the electron transfer between carotenoid molecule and the Al3+ electron acceptor site. Both the three-pulse ESEEM spectrum (Figure 9.3a) and the HYSCORE spectrum (Figure 9.3b) of the canthaxanthin/ A1C13 sample contain a peak at the 27A1 Larmor frequency (3.75 MHz). The existence of electron transfer interactions between Al3+ ions and carotenoids in A1C13 solution can serve as a good model for similar interactions between adsorbed carotenoids and Al3+ Lewis acid sites on silica-alumina. 

FIGURE 9.3 Spectra of the mixture of canthaxanthin (2mM) and A1C13 (2mM) in CH2C12 measured at 60 K at the field B0=3349G and microwave frequency 9.3757 GHz (a) superimposed plot of a set of three-pulse ESEEM spectra as the modulus Fourier transform and (b) HYSCORE spectrum measured with a x=152ns. (From Konovalova, T.A., J. Phys. Chem. B, 105, 8361, 2001. With permission.)... 

Analytical expressions for the primary echo modulation function of an B = 1 /2, / = 1/2 system were worked out for some of the earliest ESEEM studies that appeared in the literature. Perhaps the most general of these theoretical treatments is that of Mims where the two-pulse ESEEM function is given by... 

Figures 1 and 3 show that although the modulations of the three-pulse, or stimulated echo are less intense than those of its two-pulse counterpart, the resolution is much higher and the spectrum is simplified because combination peaks only enter into the data through the presence of multiple ESEEM-active nuclei. Equation (8) shows that for an S = 1 /2, 7 = 1/2 spin system, judicious selection of the r-value can control the ESEEM amplitudes of the hyperfine frequencies from a and electron spin manifolds allowing them to be optimized or suppressed. For weakly coupled protons, where the modulation frequencies from both electron spin manifolds are centered at the proton Larmor frequency, x can be set at an integer multiple of the proton Earmor frequency to suppress the contributions of this family of coupled nuclei from the three-pulse ESEEM spectrum. It is common for three-pulse ESEEM data to be collected at several r-values, including integer multiples of the proton Larmor period, to accentuate the other low frequency modulations present in the data and to make sure that ESEEM components were not missed because of T-suppression.

The discussion of the two-pulse ESEEM experiment given above might cause one to conclude that the measurement is of little use. There are a few reasons why these data should be collected as part of the overall routine of an ESEEM... 

Figure 8 Three-pulse ESEEM data (a) and (c) and corresponding ESEEM spectra (b) and (d) for Fe(II)NO-TauD treated with aKG and taurine. ESEEM data were collected under the following conditions microwave frequency, 9.723 GHz magnetic field strength, (a) 171.0 mT and (c) 346mT 90° — r — 90° — T — 90° sequence with 16ns pulses r value, 136ns T increment, 16ns repetition rate, 1 kHz events averaged/time point, 100 scans, 4 and sample temperamre, 4.2 K...

Fig. 8. Illustration of microwave pulse schemes for two-pulse and three-pulse ESEEM (adapted from Kevan and Bowman ). In the two-pulse experiment, the interpulse time t is varied and the amplitude modulation of the resulting electron spin echo is recorded. In the three-pulse experiment, t is fixed and the electron spin echo amplitude is recorded as a function of the interpulse time T.

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