Nuclear magnetic resonance spectroscopy , or NMR , is the most valuable of the numerous spectroscopic techniques used for structure determination. Although we focused in this chapter on NMR applications with small molecules, more advanced NMR techniques are also used in biological chemistry to study protein structure and folding. When magnetic nuclei, such as 1 H and 13 C, are placed in a strong magnetic field, their spins orient either with or against the field. On irradiation with radiofrequency (rf) waves, energy is absorbed and the nuclei “spin-flip” from the lower energy state to the higher energy state. This absorption of rf energy is detected, amplified, and displayed as an NMR spectrum. Each electronically distinct 1 H or 13 C nucleus in a molecule comes into resonance at a slightly different value of the applied field, thereby producing a unique absorption signal. The exact position of each peak is called the chemical shift . Chemical shifts are caused by electrons setting up tiny local magnetic fields that shield a nearby nucleus from the applied field. The NMR chart is calibrated in delta units ( δ ) , where 1 δ = 1 ppm of spectrometer frequency. Tetramethylsilane (TMS) is used as a reference point because it shows both 1 H and 13 C absorptions at unusually high values of applied magnetic field. The TMS absorption occurs on the right-hand ( upfield ) side of the chart and is arbitrarily assigned a value of 0 δ . 13 C spectra are run on Fourier-transform NMR ( FT–NMR ) spectrometers using broadband decoupling of proton spins so that each chemically distinct carbon shows a single unsplit resonance line. As with 1 H NMR, the chemical shift of each 13 C signal provides information about a carbon’s chemical environment in the sample. In addition, the number of protons attached to each carbon can be determined using the DEPT–NMR technique. In 1 H NMR spectra, the area under each absorption peak can be electronically integrated to determine the relative number of hydrogens responsible for each peak. In addition, neighboring nuclear spins can couple , causing the spin–spin splitting of NMR peaks into multiplets . The NMR signal of a hydrogen neighbored by n equivalent adjacent hydrogens splits into n + 1 peaks (the n + 1 rule ) with coupling constant J .