Three-pulse ESEEM

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).

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.)... 

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.

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...

The main advantage of the three-pulse ESEEM experiment as compared to the two-pulse approach lies in the slow decay of the stimulated echo intensity determined by /, which is usually much longer than the phase memory time that limits the observation of the two-pulse ESE. 

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.

In a three-pulse ESEEM experiment the time T between the second and the third pulse is increased while the time x between the first and second pulse is kept constant. In contrast to the two-pulse ESEEM experiment, the three-pulse ESEEM spectra do not contain sum and difference frequencies as illustrated schematically in Fig. 2.21 for an S = Vi species with anisotropic hyperfine coupling due to a proton. Both spectra contain lines with nuclear frequencies and v expected for = /2. The combination lines at v v seen as satellites in the two-pulse spectrum do not appear in the corresponding 3-pulse spectrum. On the other hand lines can escape detection in the 3-pulse spectrum for certain values of the time x between the first and second pulse at so called blind spots. It is therefore customary to record several 3-pulse specfra with different values of x. 

The spectra can become quite complex when the nuclear quadrupole coupling is appreciable, as for nitric oxide (NO) introduced into Na-A zeolite discussed in Chapter 6. It is also known that NO tends to dimerize forming an 5 = 1 species under these conditions [39]. A triplet state complex is formed interacting with one or more Na nuclei in the zeolite [40]. The spectrum obtained after FT of the three-pulse ESEEM signal in Fig. 2.22 is difficult to analyze by visual inspection. Methods to obtain the hyperfine and quadrupole couplings by simulations are described in Chapter 3. 

Overlap of lines can make analysis difficult when several nuclei contribute in the one-dimensional (ID) two- and three-pulse ESEEM spectra. Eollowing the development in NMR, methods to simplify the analysis involving two-dimensional (2D) techniques have therefore been designed. The Hyperfine Sublevel Correlation Spectroscopy, or HYSCORE method proposed in 1986 [14] is at present the most commonly used 2D ESEEM technique. The HYSCORE experiment has been applied successfully to study single crystals, but is more often applied to orienta-tionally disordered systems. It is a four-pulse experiment (Fig. 2.23(a)) with a k pulse inserted between the second and the third k/2 pulse of the three-pulse stimulated echo sequence. This causes a mixing of the signals due to the two nuclear transitions with m.s = Vi of an 5 = Vi species. For a particular nucleus two lines appear at (v , V ) and (V ", v ) in the 2D spectrum as shown most clearly in the contour map (d) of Fig. 2.23. The lines of a nucleus with a nuclear Zeeman frequency... 

Figs. 3c and 3d display the three-pulse ESEEM patterns for the multiline EPR signal of untreated PSII membranes prepared in 2H2O buffer and illuminated at 195 K. The time and frequency domain patterns again show clear evidence of deuterons located on inner sphere molecules, presumably water... 

Figure 2 Section of a three-pulse ESEEM for a single crystal of [Ni acacen)]0.5H2O doped with [Co acacen)] at 4.2 K. (A) The ESEEM spectrum (B) the Fourier transform of (A) showing the transition frequencies of the nitrogens and some of the protons. (Reprinted from Schweiger A (1986) Moderne methodische ent-wicklungen in der Electronenspinresonanz-Spectroskopie. Chi-miaAO-. 111-123.)...

Figure 1 Experimental (---------) and simulated ( ) three-pulses ESEEM data for Cu in gallosilicate with...

The lack of combination frequencies in three-pulse ESEEM may sometimes be detrimental. If the isotropic and dipolar hyperfine couplings are much smaller than V/, the maximum of the sum combination frequency v+ is given below. ... 

The disadvantage of the fast echo decay in two-pulse ESEEM can be circumvented with the three-pulse ESEEM experiment shown in Figure 5b. In this pulse sequence the first two nil pulses create nuclear coherence that develops during the evolution time T and decays with the transverse nuclear relaxation time 72n which is usually much longer than the corresponding relaxation time 7m of the electrons. The third nJl pulse transfers the nuclear coherence back to observable electron coherence. The modulation of the stimulated echo is given by... 

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