Four-dimensional spectroscopy techniques

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... 

Overlapping resonances in 2D NMR have limited protein-structure elucidation to fairly small proteins. However, three- and four-dimensional methods have been developed that enable NMR spectroscopy to be further extended to larger and larger protein structures. A third dimension can be added, for example, to spread apart a H- H two-dimensional spectrum on the basis of the chemical shift of another nucleus, such as N or - C. In most three-dimensional experiments, the most effective methods for large molecules are used. Thus, COSY is not often employed, but experiments like NOESY-TOeSY and TOCSY-HMQC are quite effective. In some cases, the three dimensions all represent different nuclei such as H- C- N. These are considered variants of the HETCOR experiment. Multidimensional NMR is now capable of providing complete solution-phase structures to complement crystal structures from X-ray crystallography. Hence. NMR spectroscopy is now an important technique for determining structure.s and orientation.s of complex molecules in solution. 

All chapters and appendices are written by Japanese researchers. This is simply because this book is based on a book written in Japanese and published in Japan in 2012. However, this book is not a translation of the Japanese book. In preparing it, four chapters in the original Japanese book were deleted and many paragraphs and sentences replaced, changed or deleted, and some new descriptions and explanations added, in order to make it more easily understandable, up to date, and practically useful. Appendix F, which did not exist in the Japanese book, was written by Isao Noda, originator of the two-dimensional correlation spectroscopy technique, and added to the present book. 

Recently two-dimensional solid-state heteronuclear chemical shift correlation experiments involving magic angle sample spinning (MASS) have been described. This technique results in an increase in the resolution of solid-state proton spectra by a factor of 5-10. Solid-state NMR spectroscopy has hitherto been confined to C-NMR spectra since the residual line widths in the spectra of solid samples are around 2 ppm, so that only four or five nonequivalent protons can be distinguished in the normal proton spectral region of 10-15 ppm. The increase in resolution attained by the 2D solid-state heteronuclear COSY experiment should promote further studies of the H-NMR spectra of solid samples. 

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