Biomedical EPR Part-B Methodology Instrumentation and Dynamics - Sandra R. Eaton

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not attained a popularity comparable to EPR, it is now used by an increased number of research groups, especially since ENDOR accessories have become commercially available from the Varian Associates (Hyde, 1998) in the seventies and from the Bruker GmbH (Schmalbein, 1998) in the eighties. The ENDOR technique has been briefly dealt with in several early monographs on EPR spectroscopy (Ayscough, 1967; Carrington and McLachlan, 1967; Scheffler and Stegmann, 1970; Wertz and Bolton, 1972; Atherton, 1973) and, in some length, in a few books specialized in multiresonance (Kevan and Kispert, 1976; Dorio and Freed, 1979). An excellent introduction into the ENDOR technique, as used for organic radicals in solution, is to be found in a review article (Kurreck et al., 1984) and, in more detail, in a book by same authors (Kurreck et al., 1988). The latter also contains a comprehensive account of the pertinent ENDOR studies up to 1988. The physical fundamentals underlying this doubleresonance technique can be grasped by considering the so called transientENDOR effect in the way presented by Kurreck et al. and adopted in a recent monograph on EPR spectroscopy (Gerson and Huber, 2003). The following treatment is a condensed version of a section in this monograph.

1.2Physical Fundamentals

Figure 1. Schemes relevant to the transient-ENDOR effect for a paramagnetic system consisting of one unpaired electron and one magnetic nucleus with I = 1/2 and (a)

Energy levels in absence of the saturation. (b) Effect of saturation of the ESR transition on

the populations. (c) and (d) Effect of the saturation of the NMR transitions and respectively, on the populations. Reproduced partly from (Kurreck et al., 1988) and (Gerson and Huber, 2003) by permission of VCH Publishers and Wiley-VCH, respectively.

The relevant schemes are shown in Figure 1. They depict four Zeemanenergy levels which, at a given field strength, B, of the magnetic field are characteristic of a paramagnetic system consisting of one unpaired electron and one magnetic nucleus X, such as proton, with the spin-quantum number

value in MHz into the coupling constant in mT, the unit of B. According

In an ENDOR experiment, one EPR transition is selected for further

(MW) irradiation. Consequently, as indicated in scheme (b), the populations

strongly reduced. In the next step, the system is subjected to an intense irradiation with radiofrequency (RF), which is scanned from 0 to higher values. At two frequencies, the NMR transitions become saturated, first at and, subsequently, at As a result, the populations in the pairs of the affected levels are equalized, as shown in schemes (c) and

than in so that in either case the EPR transition is desaturated, and the intensity of the EPR signal exhibits an increase, the so called ENDOR enhancement. Such an enhancement is, however, not directly verified, but its occurrence is confirmed by the NMR absorptions observed for the transitions and

1.3Spectra

Figure 2. Schematic presentation of an ENDOR spectrum arising from one nucleus X or a set of equivalent nuclei X with the coupling constant Reproduced from (Gerson and Huber, 2003) by permission of Wiley-VCH.

The ENDOR signals which arise from the NMR transitions and while scanning the RF are schematically shown in Figure 2. Contrary to the NMR experiment, their intensity is due to a population excess, of electron spins, which is by several orders of magnitude larger than the analogous excess number of nuclear spins. Thus, the sensitivity of ENDOR is much higher than that of NMR, although it is lower than that of EPR. It can readily be verified that any magnetic nucleus X or a set of such equivalent nuclei with the coupling constant gives rise to a single pair of ENDOR signals, irrespective of the nuclear spin-quantum number I and the nuclear factor This pair of signals generally appears in a separate NMR frequency range characteristic of X and For which holds for the ENDOR spectra reproduced in this chapter, the two signals appear at they are centered on the frequency, of the “free” nucleus X and separated by the coupling constant (Figure 2, top). On the other hand, for the two signals occur at they are centered on and separated by (Figure 2, bottom). The ENDOR signals can be

recorded as absorption A or as the first derivative as function of depending on whether modulation is applied to the magnetic field or to the frequency. The latter procedure was used to record the ENDOR spectra in Figures 3 – 7 shown in the following sections.

Although ENDOR is less sensitive than EPR, this deficiency is amply made good by the enormous increase in spectral resolution. As the width, of ENDOR signals (line-width) of ca. 0.3 MHz is comparable to of ca. 0.01 mT, generally achieved for EPR lines in a well-resolved spectrum of an organic radical in fluid solution, the increase in resolution by ENDOR relative to EPR spectroscopy is due to a drastic decrease in the number of lines. With each further set of equivalent nuclei X giving rise to pairs of ENDOR signals, the number of lines grows additively, and not multiplicatively as in EPR spectra. Irrespective of the number of nuclei in each set (1, 2, ... k) with the total number of ENDOR lines for k sets is thus 2k and not

A disadvantage of ENDOR spectroscopy is, that, unlike NMR, the intensity of a signal is not a reliable measure for the number of interacting nuclei giving rise to it. This is because the ENDOR enhancement and, therewith, the intensity of the ENDOR signals depends on whether the nuclear-spin relaxation responsible for the saturation of the NMR transitions

and can compete with the electron-spin relaxation effective in

relaxation, which takes care of inversions of electron spins is much more efficient than the nuclear-spin relaxation which causes inversions of nuclear spins so that the former must be slowed down by appropriate experimental conditions. When cross-relaxation processes with can be neglected, as is often the case with protons in organic and bioorganic radicals, this slowing down is achieved by using viscous solvents and/or low temperatures. The ENDOR experiment is impeded by an enhanced electron-spin relaxation, e.g. in the presence of heavy nuclei (which are often contained in transition metals of bioorganic molecules) or by the dynamic Jahn-Teller effect relevant to radical with an axial symmetry (rotational axis with n equal or larger than 3) in a degenerate ground state. An enhanced electron-spin relaxation is mostly conspicuous in the corresponding EPR spectrum, as hyperfine lines are broadened and difficult to saturate.

The ENDOR technique proved to be particularly useful for radicals of low symmetry with a large number of overlapping and/or incompletely resolved EPR lines (Gerson et al., 1975). Because of its lower sensitivity, it requires somewhat larger radical concentration than EPR spectroscopy, and its application to transient radicals is, therefore, more problematic. In order to increase the signal-to-noise ratio, the ENDOR spectra are usually accumulated by repeated recording and addition. Radical ions, which are