Determining Organic Molecule Structures Using NMR Spectroscopy

NMR Spectroscopy Worksheet

Nmr spectroscopy is the most commonly used probe for determining the structure of organic molecules. It involves a spinning sample-holder inside of a strong magnet coupled with a radio-frequency emitter and receiver.

The results of a 1H NMR spectrum provide information on the number of signals (multiplets), their chemical shifts, and integrations. The spectra also show whether or not the protons are shielded or deshielded.

Splitting

The NMR spectrometer is a very powerful tool. It can tell you many things about your sample based on the magnetic properties of its nuclei and the electromagnetic influences on them from electrons and other nuclei nearby. The information derived from this is used to determine molecular structure, geometry and much more.

A key feature of NMR is the splitting patterns that can occur between different types of protons (nonequivalent). In the case of 1H NMR, this is known as multiplicity and it is calculated based on the n + 1 rule.

For example, proton peaks associated with hydrogens on the carbonyl group of ethanol appear between 8-10 ppm and those on an aromatic ring (sp2 or sp3) appear at 11-14 ppm. The integration table below shows the number of peaks, their shifts and the multiplicity as determined by the n + 1 rule. Each peak is also shown as a single peak or multiple peaks and their coupling to each other, denoted by J, is shown as well.

Shift

Using strong magnetic fields (usually 1 to 2 T) an nmr spectrometer generates a nucleus’s resonant frequency. When the resonant frequency of a given nucleus is compared with that of an internal standard, such as tetramethylsilane (TMS), it is possible to determine its chemical shift, or the location on the spectrum where the signal appears. Shifts are measured on a scale in parts per million.

Each unique H atom in a molecule will produce a signal on the NMR spectrum, with a peak for each corresponding to its chemical shift. For example, hydrogens attached to carbons of a methyl group will have very low chemical shifts, while those on electronegative carbons like aldehydes and ketones will have higher ones. Interpretation of these signals is important for NMR structure determination. For example, a protein’s backbone chemical shifts provide information on its secondary structure, sidechain conformations and dynamics. They can be combined with evolutionary distance restraints derived from homologous proteins in the PDB to enable fragment assembly techniques that reliably obtain high resolution structures [1-5]..

Signal

NMR uses an external magnetic field to excite nuclei in a sample, causing them to flip back and forth between a low energy state and a high energy state at a specific radio frequency. The signal produced by this process corresponds to the amplitude of these rotations and can be detected with sensitive radio receivers.

All atomic nuclei have a built in magnetic moment and angular momentum which gives them a property called spin, allowing them to have distinct resting energies. NMR can distinguish atoms with odd mass and atomic numbers, such as hydrogen (1H) and carbon (13C).

Due to symmetry, many compounds have multiple signals in their 1H NMR spectrum. Splitting of peaks reveals the influence of magnetic effects from neighboring nuclei. Hydrogen nuclei that are shielded by methyl groups or aliphatic molecules, have high chemical shift values and appear upfield in the NMR spectrum. Hydrogens attached to electronegative atoms or close to electronegative groups such as carboxylic acids, ketones and aldehydes are deshielded and have lower chemical shift values and appear downfield in the NMR spectrum.

Integration

NMR spectroscopy is the most powerful analytical technique available to determine three-dimensional structure of molecules from the atomic scale up. NMR spectroscopy provides information about covalent bonds, hydrogen-spin couplings and through-space interactions by observing resonances at specific frequencies.

NMR is an inexhaustible source of information about a sample and its constituents, even in the solid state. NMR experiments use a rotating sample-holder inside a strong magnet, a radio-frequency emitter and receiver, gradient coils (for diffusion measurements), and electronic controls.

The computer in the NMR instrument can be instructed to automatically integrate each peak in a spectrum. This tells you how many equivalent protons each signal represents. For example, if two signals in the methyl acetate spectrum have the same area ratio, each one corresponds to a set of three equivalent hydrogens. You can also calculate the number of hydrogens from the chemical shift of a peak, or from its multiplicity. (To calculate multiplicity, divide the area of a peak by its peak height.

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