NMR spectroscopy
From Wikipedia, the free encyclopedia
Nuclear Magnetic Resonance Spectroscopy most commonly known as NMR Spectroscopy is the name given to the technique which exploits the magnetic properties of nuclei. This phenomenon and its origins are detailed in a separate section on Nuclear magnetic resonance or NMR. Two very important techniques are proton NMR and carbon-13 NMR, although some other nuclei can be measured as well.
Many areas of information can be obtained from this single phenomenon. In its simplest form NMR allows identification of individual atoms in a pure molecule. Much like using infrared spectroscopy to identify functional groups, analysis of a 1D NMR spectrum tells the scientist what atom environments (like a methyl proton), and in some cases how many atoms of each type, exist within the sample. NMR is based in quantum mechanical properties of nuclei, and as such is very reliable, predictable and reproducible. Since its advent, it has become the most important analytical tool available for organic chemists; it yields far more information than for example infrared spectroscopy.
The impact of NMR Spectroscopy on the natural sciences is substantial. It can be used to study mixtures of analytes; to understand dynamic effects such as change in temperature and reaction mechanisms; it can be used in the solution and solid state; and critically it is an invaluable tool in understanding protein and nucleic acid structure and function.
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[edit] Basic NMR Techniques
When placed in a magnetic field, NMR active nuclei (like 1H or 13C) resonate at a specific frequency, dependent on strength of the magnetic field. For example, in a 21 tesla magnetic field, protons resonate at 900 MHz. It is common to refer to a 21 T magnet as a 900 MHz magnet but it is important to note that different nuclei resonate at a different frequency at this field strength.
However, depending on the local chemical environment, different protons in a molecule each resonate at slightly different frequencies. Since this frequency is dependent on the strength of the magnetic field it is converted into a field-independent value known as the chemical shift. The chemical shift is reported as a relative measure from some reference resonance frequency (For the nuclei1H, 13C, and 29Si, TMS (tetramethylsilane) is commonly used as a reference.) This difference between the frequency of the signal and the frequency of the reference is divided by frequency of the magnetic field to give the chemical shift. The units of chemical shift are parts per million (ppm) because the difference in frequencies is usually in hertz while the frequency of the magnetic field is in megahertz. The formula for chemical shift is as follows:
By understanding different chemical environments, the chemical shift can be used to obtain some structural information about the molecule in question to assign signals to an atom or a group of atoms.
For example, in a proton spectrum for ethanol (CH3CH2OH) one would expect three specific signals at three specific chemical shifts. One for the CH3 group, one for the CH2 group and one for the OH. A typical CH3 group has a shift around 1 ppm, the CH2 attached to a OH has a shift of around 4 ppm and the OH has a shift around 2–3 ppm depending on the solvent used.
Because the molecular motion at room temperature makes each of the three methyl protons average out during the course of the NMR experiment (which typically takes a few ms), the protons become degenerate and form a peak at the same chemical shift.
Interestingly the shape and size of peaks are indicators of chemical structure too. In the example above—the proton spectrum of ethanol—the CH3 peak would be three times as large as the OH. Similarly the CH2 peak would be twice the size of the OH peak but only 2/3 the size of the CH3 peak.
Modern analysis software allows analysis of the size of peaks to understand how many protons give rise to the peak. This is known as integration—a mathematical process which calculates the area under a graph (essentially what a spectrum is). It is important to note that the analyst must integrate the peak and not measure its height because the peaks also have width—and thus its size is dependent on its area not its height. However, it should be mentioned that the number of protons, or any other observed nucleus, is only proportional to the intensity, or the integral, of the NMR signal, in the very simplest one-dimensional NMR experiments. In more elaborate experimets, for instance, experiments typically used to obtain carbon-13 NMR spectra, the integral of the signals depends on the relaxation rate of the nucleus, and its scalar and dipolar coupling constants. Very often these factors are poorly understood - therefore, the integral of the NMR signal is very difficult to interpret in more complicated NMR experiments.
| Multiplicity | Intensity Ratio |
|---|---|
| Singlet | 1 |
| Doublet | 1:1 |
| Triplet | 1:2:1 |
| Quartet | 1:3:3:1 |
| Quintet | 1:4:6:4:1 |
| Sextet | 1:5:10:10:5:1 |
| Septet | 1:6:15:20:15:6:1 |
The most reliable information for structure determination in a one dimensional NMR spectrum is the spin-spin coupling (also called J-coupling or scalar coupling) between NMR active nuclei. This coupling arises from the interaction of different spin states through the chemical bonds of a molecule, which results in splitting of signals into recognizeable patterns based on the pairing of spin states. As a result, this coupling can tell us information about the connectivity of atoms in a molecule. However, nuclei that are chemically equivalent (that is, have the same chemical shift) and nuclei that are distant (usually more than 3 bonds apart) do not show any splitting to each other. Therefore, for each neighboring chemically equivalent (spin ½) nuclei, the signal is split into a corresponding multiplet with intensity ratios following Pascal's triangle as described on the right.
For example, in the proton spectrum for ethanol described above, the CH3 group is split into a triplet with an intensity ratio of 1:2:1 by the two neighboring CH2 protons. Similarly, the CH2 is split into a quartet with an intensity ratio of 1:3:3:1 by the three neighboring CH3 protons. Under ideal conditions, the two CH2 protons would also be split again into a doublet to form a doublet of quartets by the hydroxyl proton, but intermolecular exchange of the acidic hydroxyl proton often results in a loss of coupling information.
Although this example uses protons for simplicity, any spin ½ nuclei such as phosphorus-31 or fluorine-19 couples in this fashion. For nuclei with spin greater than ½ such as deuterium (spin 1), because the spin quantum number has more than two possible values, the splitting patterns differ from the ones described above in Pascal's triangle. For NMR active nuclei coupled to deuterium (and other spin 1 nuclei), the deuterium splits the signal into a 1:1:1 triplet instead of a doublet as was the case for a spin ½ nuclei. Similarly, a spin 3/2 nuclei splits a signal into a 1:1:1:1 quartet and so on. Coupling combined with the chemical shift (and the integration for protons) tells us not only about the chemical environment of the nuclei, but also the number of neighboring NMR active nuclei within the molecule. In more complex spectra with multiple peaks at similar chemical shifts or in spectra of nuclei other than hydrogen, often coupling is the only way to distinguish different nuclei.
[edit] Correlation spectroscopy
- For more details on this topic, see 2D-NMR.
Correlation spectroscopy is one of several types of two-dimensional nuclear magnetic resonance (NMR) spectroscopy. This type of NMR experiment is best known by its acronym, COSY. Other types of two-dimensional NMR include J-spectroscopy, exchange spectroscopy (EXSY), Nuclear Overhauser effect spectroscopy (NOESY), total correlation spectroscopy (TOCSY) and heteronuclear correlation experiments, such as HSQC, HMQC, and HMBC. Two-dimensional NMR spectra provide more information about a molecule than one-dimensional NMR spectra and are especially useful in determining the structure of a molecule, particularly for molecules that are too complicated to work with using one-dimensional NMR. The first two-dimensional experiment, COSY, was proposed by Jean Jeener, a professor at Université Libre de Bruxelles, in 1971. This experiment was later implemented by Walter P. Aue, Enrico Bartholdi and Richard R. Ernst, who published their work in 1976.
[edit] Solid-State nuclear magnetic resonance
- For more details on this topic, see Solid-state NMR.
A variety of physical circumstances does not allow molecules to be studied in solution, and at the same time not by other spectroscopic techniques to an atomic level, either. In solid-phase media, such as crystals, microcrystalline powders, gels, anisotropic solutions, etc., it is in particular the dipolar coupling and chemical shift anisotropy that become dominant to the behaviour of the nuclear spin systems. In conventional solution-state NMR spectroscopy, these additional interactions would lead to a significant broadening of spectral lines. A variety of techniques allows to establish high-resolution conditions, that can, at least for 13C spectra, be comparable to solution-state NMR spectra.
Two important concepts for high-resolution solid-state NMR spectroscopy are the limitation of possible molecular orientation by sample orientation, and the reduction of anisotropic nuclear magnetic interactions by sample spinning. Of the latter approach, fast spinning around the magic angle is a very prominent method, when the system comprises spin 1/2 nuclei. A number of intermediate techniques, with samples of partial alignment or reduced mobility, is currently being used in NMR spectroscopy.
Applications in which solid-state NMR effects occur are often related to structure investigations on membrane proteins, protein fibrils or all kinds of polymers, and chemical analysis in inorganic chemistry, but also include "exotic" applications like the plant leaves and fuel cells.
[edit] NMR spectroscopy applied to proteins
Much of the recent innovation within NMR spectroscopy has been within the field of protein NMR, which has become a very important technique in structural biology. One common goal of these investigations is to obtain high resolution 3-dimensional structures of the protein, similar to what can be achieved by X-ray crystallography. In contrast to X-ray crystallography, NMR is primarily limited to relatively small proteins, usually smaller than 25 kDa, though technical advances allow ever larger structures to be solved. NMR spectroscopy is often the only way to obtain high resolution information on partially or wholly intrinsically unstructured proteins.
Proteins are orders of magnitude larger than the small organic molecules discussed earlier in this article, but the same NMR theory applies. Because of the increased number of each element present in the molecule, the basic 1D spectra become crowded with overlapping signals to an extent where analysis is impossible. Therefore, multidimensional (2, 3 or 4D) experiments have been devised to deal with this problem. To facilitate these experiments, it is desirable to isotopically label the protein with 13C and 15N because the predominant naturally-occurring isotope 12C is not NMR-active, whereas the nuclear quadrupole moment of the predominant naturally-occurring 14N isotope prevents high resolution information to be obtained from this nitrogen isotope. The most important method used for structure determination of proteins utilizes NOE experiments to measure distances between pairs of atoms within the molecule. Subsequently, the obtained distances are used to generate a 3D structure of the molecule using a computer program.
[edit] See also
- NMR tube - includes sample preparation
[edit] References
- ↑ Martin, G.E; Zekter, A.S., Two-Dimensional NMR Methods for Establishing Molecular Connectivity; VCH Publishers, Inc: New York, 1988 (p.59)
[edit] External links
- The Science of Spectroscopy - supported by NASA. Spectroscopy education wiki and films - introduction to light, its uses in NASA, space science, astronomy, medicine & health, environmental research, and consumer products.
- The Basics of NMR - A very detailed and technical overview of NMR theory, equipment, and techniques by Dr. Joseph Hornak, Professor of Chemistry at RIT
- NMR processing software
