Four-pulse ESEEM

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. 

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). 

Fig. 11. Pulse sequence of the sum-combination (four-pulse ESEEM) and HYSCORE experiment. In the ID four-pulse ESEEM experiment, t = t2 = 772. In HYSCORE, tj and t2 are incremented independently to provide a 2D data set.

Figure 6. Examples of ID four-pulse ESEEM experiments, (a) Con arison of three-pulse ESEEM (i) and DEFENCE (ii) experiments of bis(r -benzene)vanadium(0), V(C6H6)2, diluted into polycrystalline ferrocene observer position, gi. (b) Combination-peak spectra of [Cu(H20)6] centers in a frozen water/glycerol solution measured at three different observer positions. The dashed lines mark the frequency 2 Hi. Modified with permission from [24] and [26]. Copyright 1995, American Institute of Physics.

Figure 1. Scheme of the pulse EPR sequences mentioned in this chapter, (a) Two-pulse ESEEM. (b) Three-pulse ESEEM. (c) Four-pulse ESEEM. When times fi and ti are stepped under the constraint of ti= ti= T, combination-peak experiment is performed. Two-dimensional HYSCORE spectroscopy is done using the same sequence, whereby t and are stepped independently. The second and third nil pulse are replaced by high-tuming-angle (HTA) pulses in a matched HYSCORE experiment, (d) SMART-HYSCORE. The first and third pulses are HTA pulses, (e) Davies ENDOR. (f) Mims ENDOR. (g) ELDOR-detected NMR. 

Goldfarb D, Bernardo M, Thomann H, Kroneck PMH, Ullrich V. 1996. Study of water binding to low-spin Fe(Ill) in cytochrome P450 by pulsed ENDOR and four-pulse ESEEM spectroscopies. J Am Chem Soc 118(11) 2686-2693. 

Astashkin AV, Raitsimring AM, Walker FA. 1999. Two- and four-pulse ESEEM studies of the heme binding center of a low-spin ferriheme protein the importance of a multi-frequency approach. Chem Phys Lett 306( l-2) 9-l 7. 

Martinez and co-workers performed three- and four-pulse ESEEM and HY-SCORE on this species. Hfcs to N1 = 1.9 MHz), N3 = 0.7 MHz) andN10(Tiso = 11.7 MHz with = 2.7 MHz and Tj = 29.7 MHz) could also be determined. The authors did not attempt to say which was N1 or N3, giving N(A) and N(B) we have taken the liberty to assign them tentatively by comparison with our own DFT calculations. ... 

An alternative 2D ESEEM experiment based on the four-pulse sequence depicted in figure B 1.15.12(B) has been proposed by Meliring and coworkers [40]. In the hyperfme sublevel correlation (HYSCORE) experiment, the decay of the echo intensity as a fimction of is governed by -relaxation, whereas the echo decay along the t2-axis is... 

ESR spectra of La Cs2/ Y Cs2, Ho Cs2, and Tm C82 taken from the solid soot extract were reported by Bartl et al. (1994, 1995a,b, 1996) and showed low resolved but split hyperfine structure, indicating that the metal atoms exist in ionic form in the fullerene cage also in the solid state. The research group also reported (Knorr et al., 1998) the principal values of the hyperfine tensor A and the relative orientation of g and A tensors of M C82 (M = Sc, Y, La) applying three- and four-pulse electron spin-echo envelope modulation techniques (ESEEM). 

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... 

Figure 5. Pulse sequences making use of the ESEEM effect, (a) Two-pulse sequence and the primary echo, (b) Three-pulse sequence and the stimulated echo, (c) Four-pulse sequence for the HYSCORE experiment.

The use of a remote-echo detector allows r values shorter flian the spectrometer deadtime to be employed [55]. This is important in two-pulse ESEEM experiments where the deadtime prevents the signal for times r < from being recorded. Also in the deadtime-free four-pulse experiments described in 3.3, a small T value is often needed to avoid blind spots. Bhnd spots are a particular concern for flie measurement of proton spectra at X-band, where flie signals typically extend from 5 to 25 MHz, and with a r = 100 ns blind spots occur at nh = 0, 10, 20,... MHz. 

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 electron-spm echo envelope modulation (ESEEM) phenomenon [37, 38] is of primary interest in pulsed EPR of solids, where anisotropic hyperfme and nuclear quadnipole interactions persist. The effect can be observed as modulations of the echo intensity in two-pulse and three-pulse experiments in which x or J is varied. In liquids the modulations are averaged to zero by rapid molecular tumbling. The physical origin of ESEEM can be understood in tenns of the four-level spin energy diagram for the S = I = model system... 

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