Single HYSCORE

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... 

Fig. 2.23 Schematic HYSCORE spectrum showing (a) the HYSCORE sequence, (b) the 2D time-domain modulation signal, (c) the 2D HYSCORE spectrum and (d) the contour plot of a single crystal sample for an 5 = Vi species containing a H nucleus with an axitilly symmetric hyper-fine coupling. The magnetic field is at an angle 0 = 10° with the A axis. The nuclear Zeeman frequency vh 15 MHz is larger than the hyperflne coupling, i.e. Ai I < A < 2 vh...

Fig. 2.24 Contour plot of single crystal HYSCORE spectrum (a) and simulation (b) showing interaction between N of released ammonia and deaminated radical HsCCHCOO" in irradiated /-alanine. The figure is reproduced from [41] with permission from Elsevier...

HYSCORE has a higher sensitivity in the region of low nuclear frequencies than that of ENDOR spectroscopy. Single crystal measurements have therefore been applied to studies of nitrogen-containing paramagnetic species (nuclear Zeeman frequency v( " N) = 1 MHz at X-band) of interest in biochemical and fundamental applications. The local structure around the species may be obtained as discussed below for the radical H3CCHCOO in irradiated /-alanine. 

HYSCORE single crystal spectra of irradiated /-alanine in Fig. 2.24(a) show an interaction with nuclei (/ = 1) that is too weak to be resolved in CW-ESR and ENDOR spectra. As for I = Vi the nuclear transitions are identified most conveniently by a contour map of the type shown in Fig. 2.24. Software for the data processing to obtain the map is usually provided with commercial instruments. The interpretation is, however, complicated by the quadrupole interaction of the nucleus. The procedure to analyse the data employed in [41] was analogous to that applied for measurements of hfc and nqc tensors by ENDOR (Section 2.2.22). The analysis was supported by simulations (Fig. 2.24(b)) which also accounted for the lines observed in the left quadrant due to the low nuclear frequency of " N at X-band. The results were interpreted as due to a deaminated radical, H3CCHCOO , weakly interacting with the released ammonia group. 

Fig. 3.33 Schematic HYSCORE contour plot for an S = Vi single crystal species with an anisotropic hyperfine coupling due to two I = Vi nuclei. The spots symmetrically displaced from the diagonals correspond to nuclear frequencies with electron quantum number ms = 14.The correlation between the nuclear transitions for a particular nucleus is achieved by the Jt pulse inserted between the second and the third tt/2 pulse of the three-pulse stimulated echo sequence. The spots in the right quadrant are due to a nucleus with frequencies (Vd, vpi) for the two nuclear transitions corresponding to ms = 14. The frequencies to the right are small compared to the nuclear Zeeman frequency, while the spots in the left quadrant are for the opposite case with large frequencies (v, vpa)...

Figure 10 shows HYSCORE spectra from the Cu(II) N-confused tetra-phenylporphyrin (NCTPP) complex measured at X- and Q-band frequencies [42], The correlation peaks observed in the single-crystal like spectra, measured at g, are assigned to the remote nucleus of the inverted pyrrole. In the Q-band spectrum (Fig. 10b, weak-coupling case) the stronger peaks appear in the first quadrant. 

Figure 24. Sequences for chirp ENDOR experiments (a) Davies-type chirp ENDOR (b) Mims-type chirp ENDOR (c) Chirp-ENDOR-HYSCORE sequence and (d) Two-dimensional chirp ENDOR-HYSCORE spectram of a Cu(II)-doped glycine single-crystal. Cross-peaks in the first quadrant correspond to proton ENDOR lines, cross-peaks in the second quadrant to nitrogen ENDOR hnes. Modified with permission from [7]. Copyright 2001, Oxford University Press.

Currently the HYSCORE is monitored at a single point (detection gate delay 4)), rather than integrating over a detection gate, dx and dy define the time increments in the two dimensions and sx and sy correspond to the number of data (time) points calculated. Whilst the pulse sequence assumes dx and dy are identical, as is often the case experimentally, this condition is not strictly required... 

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