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. The two important techniques are proton NMR and carbon-13 NMR. Pacific Northwest National Laboratorys high magnetic field (800 MHz, 18. ... Pacific Northwest National Laboratorys high magnetic field (800 MHz, 18. ... Pacific Northwest National Laboratorys high magnetic field (800 MHz, 18. ...

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. IR spectrum of a thin film of liquid ethanol. ...

NMR Spectroscopy is much more powerful than this everyday usage. 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.

Basic NMR Techniques

The NMR sample is prepared in a thin walled glass tube.

When placed in a magnet, NMR active nuclei (like ¹H or ¹³C) resonate at a specific frequency. Frequency is dependent on the strength of the magnet. In a 21 tesla magnet protons resonate at 900 MHz. It is common to refer to a 21 T magnet as a 900 MHz magnet, but it is worth remembering that different nuclei resonate at a different frequency at this field strength. Image File history File linksMetadata Download high resolution version (1704x2272, 650 KB) Summary Picture taken by User:Kjaergaard. ... Image File history File linksMetadata Download high resolution version (1704x2272, 650 KB) Summary Picture taken by User:Kjaergaard. ... The tesla (symbol T) is the SI derived unit of magnetic flux density (or magnetic induction). ... Properties In physics, the proton (Greek proton = first) is a subatomic particle with an electric charge of one positive fundamental unit (1. ... MegaHertz (MHz) is the name given to one million (106) Hertz, a measure of frequency. ...

At 21 T, protons resonate at around 900 MHz. Different protons in a molecule each resonate at slightly different frequencies dependent on their local environment. 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. In nuclear magnetic resonance (NMR), the chemical shift describes the dependence of nuclear magnetic energy levels on the electronic environment in a molecule. ...

So nuclei in different environments have different chemical shifts. By understanding the different values of chemical shift we can assign each signal to an atom or group of atoms in the molecule under study.

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. Parts per million (ppm) is a measure of concentration that is used where low levels of concentration are significant. ...

It is because during the course of the NMR experiment (which typically takes a few ms) molecular motion makes each of the three methyl protons average out—they become "degenerate" which is a scientific way of implying identical. Mil-li-sec-ond One hundreth of a second ...

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 gives 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. In calculus, the integral of a function is a generalization of area, mass, volume and total. ...

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. Other types of two-dimensional NMR include J-spectroscopy, exchange spectroscopy (EXSY), and Nuclear Overhauser effect spectroscopy (NOESY.) 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.^[1] Correlation spectroscopy is one of several types of two-dimensional nuclear magnetic resonance (NMR) spectroscopy. ... It has been suggested that this article or section be merged with Dynamic nuclear polarisation. ... In general, a molecule is the smallest particle of a pure chemical substance that still retains its composition and chemical properties. ... Richard Robert Ernst (born August 14, 1933) is a Swiss chemist and Nobel Laureate. ...

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. Solid-state NMR (SSNMR) spectroscopy is a kind of nuclear magnetic resonance (NMR) spectroscopy, characterized by the presence of anisotropic (directionally dependent) interactions. ...

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. The magic angle is an angle of about 54. ...

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.

DEPT spectra

DEPT spectra of propyl benzoate

DEPT stands for Distortionless Enhancement by Polarization Transfer. It is a very useful methods for determining the presence of primary, secondary and tertiary carbon atoms. The DEPT experiment basically differentiates between CH, CH₂ and CH₃ groups by variation of the selection angle parameter - that is the tip angle of the final ¹H pulse. The technique suppresses all quaternary carbons and carbons with no attached protons (as in deuterated solvents). The DEPT experiment basically uses polarization transfer from ¹H to ¹³C, in order to increase the sensitivity over the normal NOE (Nuclear Overhauser Effect) enhancement. The selection angle varies: it can be 45^o, 90^o or 135^o. The value chosen dictates the result (as said before, quaternary and deprotonated carbons are always suppressed!): Image File history File links DEPT_spectra. ... Image File history File links DEPT_spectra. ... General Name, Symbol, Number carbon, C, 6 Chemical series nonmetals Group, Period, Block 14, 2, p Appearance black (graphite) colorless (diamond) Atomic mass 12. ... Properties In physics, the proton (Greek proton = first) is a subatomic particle with an electric charge of one positive fundamental unit (1. ... Deuterium, also called heavy hydrogen, is a stable isotope of hydrogen with a natural abundance of one atom in 6500 of hydrogen. ... It has been suggested that this article or section be merged with Dynamic nuclear polarisation. ...

• 45^o angle gives all carbons with attached protons (regardless of number of the latter) in phase
• 90^o angle gives only CH groups, the others being suppressed
• 135^o angle gives all CH and CH₃ in a phase opposite to CH₂

NMR spectroscopy applied to proteins

Main article: Protein NMR Pacific Northwest National Laboratorys high magnetic field (800 MHz) NMR spectrometer being loaded with a sample. ...

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. The aim of these investigations are often to obtain a high resolution 3 dimensional structure of the protein, similar to what can be achieved by X-ray crystallography. In contrast to X-ray crystallography, NMR is primarily limited to relative small proteins usually smaller than 25 kDa, even though technical advances continually moves this boundary. NMR spectroscopy is often the only way to obtain high resolution information on partially or wholly intrinsically unstructured proteins. A representation of the 3D structure of myoglobin, showing coloured alpha helices. ... Pacific Northwest National Laboratorys high magnetic field (800 MHz) NMR spectrometer being loaded with a sample. ... Structural biology is a branch of molecular biology concerned with the study of the architecture and shape of biological macromolecules--proteins and nucleic acids in particular—and what causes them to have the structures they have. ... X-ray crystallography is a technique in crystallography in which the pattern produced by the diffraction of X-rays through the closely spaced lattice of atoms in a crystal is recorded and then analyzed to reveal the nature of that lattice. ... The Kampfgruppen der Arbeiterklasse (German: Combat Groups of the Working Class) was a paramilitary organisation in East Germany, founded in 1953 and abolished in 1990. ... Description Intrinsically unstructured proteins, often referred to as naturally unfolded proteins, are characterized by their lack of tertiary structure. ...

Proteins are orders of magnitude larger than the compounds described previously, but the same NMR theory applies. Because of the increased amount of nuclear spins, that basic 1D experiments becomes crowded to an extend 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 ¹²C and ¹⁴N isotopes are not NMR-active. The structure determination works by generating a set minimum distances between different atoms in the molecule. If sufficient distance restraints are obtained, a specially designed computer programs can calculate a high resolution 3 dimensional structure for the protein. An order of magnitude is the class of scale or magnitude of any amount, where each class contains values of a fixed ratio to the class preceding it. ... Isotopes are forms of an element whose nuclei have the same atomic number - the number of protons in the nucleus - but different mass numbers because they contain different numbers of neutrons. ...

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 ftp://ftp.chem.pku.edu.cn/sslin/NMR/Softwares/Mestrec/

References

1. ^  Martin, G.E; Zekter, A.S., ‘’Two-Dimensional NMR Methods for Establishing Molecular Connectivity’’; VCH Publishers, Inc: New York, 1988 (p.59)