When the 90° pulse exactly cancels out the signal, the delay corresponds to the time needed for 90° of relaxation. Spins that are chemically equivalent but are not indistinguishable (based on their coupling relationships) are termed magnetically inequivalent. In presence of external magnetic field, nuclei of atoms align themselves either in the direction of external magnetic field or in the opposite direction of the external magnetic field. [8], The TOCSY experiment is similar to the COSY experiment, in that cross peaks of coupled protons are observed. But the splitting patterns differ from those described above for nuclei with spin greater than ½ because the spin quantum number has more than two possible values. The most common types of NMR are proton and carbon-13 NMR spectroscopy, but it is applicable to any kind of sample that contains nuclei possessing spin. This makes it useful for identifying the larger interconnected networks of spin couplings. Early spectra at 60 MHz were more prone to distortion than spectra from later machines typically operating at frequencies at 200 MHz or above. Subsequently, the distances obtained are used to generate a 3D structure of the molecule by solving a distance geometry problem. 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If the ordered nuclei are now subjected to EM radiation of the proper frequency the nuclei aligned with the field will absorb energy and "spin-flip" to align themselves against the field, a higher energy state. In contrast to X-ray crystallography, NMR spectroscopy is usually limited to proteins smaller than 35 kDa, although larger structures have been solved. The first and most popular two-dimension NMR experiment is the homonuclear correlation spectroscopy (COSY) sequence, which is used to identify spins which are coupled to each other. or through-space (NOE) coupling, a response can also be detected on the frequency of the correlated nucleus. Note that we get the simplest ratio of H atom types (1:3, methyl : t-butyl) not the true ratio of 3:9. 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However, when a sample of these nuclei is place in an external magnetic field, the nuclear spins will adopt specific orientations much as a compass needle responses to the Earth’s magnetic field and aligns with it. In order to avoid detecting only signals from solvent hydrogen atoms, deuterated solvents are used where 99+% of the protons are replaced with deuterium (hydrogen-2).
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