Hyperfine from ENDOR spectrum

Figure 2A shows a pulsed ENDOR spectrum of an oxo-Cr(V) complex where the unpaired electron is interacting with a 1H nucleus with principal hyperfine values Hax = Hay = — 2 MHz and 1 a- 5 MHz. In this case, the isotropic hyperfine value is Haiso = (%+ % + V)/3 = 0.33 MHz ( 0.11x10 4 cm ), a value that is not resolved in the CW-EPR spectrum. The hyperfine contribution of this proton is, however, clearly resolved in the ENDOR spectrum. The ENDOR spectrum is centered around the proton Larmor frequency (vH), identifying the contribution as stemming from an interaction with a H nucleus. The principal values can be read directly from the spectrum, as indicated in Fig. 2A. 

The EPR lines encountered in photosynthetic material often consist of the envelope of many hyperfine lines (they are inhomogeneously broadened Gaussians) and contain little structural information. From their ENDOR spectrum, however, several hyperfine coupling constants could be determined. 

Thus, as a consequence of the selection of orientation subsets by choice of field value, the ENDOR spectrum changes as a function of the external field position or g value at which it is measured. A series of ENDOR spectra collected at fields across the EPR envelope samples different sets of molecular orientations. An example of this is shown in Fig. 6B. It can be seen that at the extreme g values (g, and g ) the ENDOR spectrum is the least complex, whereas at intervening g values the ENDOR spectrum shows multiple frequencies. The analysis of such spectra taken at several magnetic field positions gives the full tensor of the hyperfine interaction A, from which the isotropic and anisotropic components can be deduced. Procedures for this analysis have been described in detail elsewhere. 

The frequencies and the amplitude of the modulation measured in an ESEEM experiment can both be simulated to derive information about the hyperfine interaction. The ESEEM-derived spectrum in the frequency domain is related to, but not necessarily identical to, an ENDOR spectrum. Thorough reviews of this technique and its analysis are found elsewhere" here we give a brief comparison between the information derived from ESEEM and that directly determined by ENDOR. In an ESEEM experi-... 

Fig. 19. X-band Mims ENDOR spectrum showing resolved deuteron hyperfine couplings in nitrile hydratase. Solid trace represents sample in buffer, and dashed trace is sample in HjO buffer. The patterns are centered at the Larmor frequency of 1.97 MHz (vertical bar) and show first-order splittings from both the hyperfine and quadrupole interactions. Conditions 3000 G, 9.4 GHz, 16 nsec pulse lengths, t = 376 nsec, rf pulse length 60 /isec, repetition rate 5 Hz, temperature 2 K. (Adapted from Jin et al. )...

A flavin anion radical shows a markedly different proton ENDOR spectrum as compared to that of a neutral radical. The most pronounced differences are, of course, the absence of the signal from H5 and the larger splittings of the signal pairs arising from H8ot and H6 in the anion radical case. Hence, in addition to the g-tensor, the hyperfine pattern of a flavin radical allows for an unambiguous discrimination of the radical s protonation state [33, 34]. 

Fig. 2.5 ENDOR spectrum of the malonic acid radical, HC(COOH)2 in a single crystal of malonic acid. The vj lines corresponding to ms = /2 for the H nucleus differ in intensity due to hyperfine enhancement and relaxation factors. Additional hnes A, B and C are attributed to other species that are not apparent in the ESR spectrum, while the overlapping features 2- 5 represent weak couplings due to H at neighbour molecules based on the EIE spectra to the right. The line marked by a square is an instrumental artifact. The ENDOR spectrum was recorded at a magnetic field value marked by in the ESR spectrum. The figure is reproduced fl om [24] with permission from the American Chemical Society.

Even more complex ESR spectra can occur for nitrogen-containing aromatic radicals of the type in Fig. 3.4. A stickplot analysis is not easily performed. At this level of complexity a combination of ESR and ENDOR measurements, simulation of the observed spectra, and theoretical calculations of hyperfine coupling constants is often applied to obtain a reliable assignment. The procedure to obtain the coupling constants from the ENDOR spectrum of the Wurster blue cation is indicated in Exercise E3.3. ENDOR lines due to can be observed more easily than signals due to " N, a phenomenon that is quite typical in CW-ENDOR studies. The hyperfine couplings due to N nuclei may therefore have to be deduced from simulation of the ESR spectrum. 

E3.2 (a) Construct the ESR stickplot of two equivalent (/ = 1) by successively drawing the stickplot due to the first nucleus and the stickplot of the second on top of each line of the first. What is the intensity ratio of the five hyperfine lines of the two equivalent (b) The ESR spectrum and stickplot pattern due to the N2H4 radical in solution shown below is rather complex due to similar coupling constants of four equivalent (an = lEO G) and two equivalent (aN = 11.5 G). Predict the shape of the ENDOR spectrum at X-band, assuming that signals are obtained both from H and... 

From the ENDOR spectrum of I in frozen RC solution (100 K) at least 16 hyperfine splittings were obtained (see Fig. 2). Except for an effect on the overall ENDOR intensity, long (2 min) or brief... 

ENDOR spectroscopy offers enhanced resolution compared with conventional ESR for example, isotropic hyperfine interactions as small as 0.004 mT can be measured. This enhanced resolution is achieved partly because the technique lies between ESR and nuclear magnetic resonance (NMR) and also because redundant lines are eliminated from the spectrum essentially the ESR signal is monitored while sweeping through NMR frequencies. The simplification of the spectrum arises from the fact that each fl-value produces only two lines in the spectrum, irrespective of how many nuclei contribute to that hyperfine coupling constant. Other advantages of the method are ... 

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