NMR spectroscopy
Encyclopedia
Nuclear magnetic resonance spectroscopy, most commonly known as NMR spectroscopy, is a research technique that exploits the magnetic
properties of certain atomic nuclei
to determine physical and chemical properties of atom
s or the molecule
s in which they are contained. It relies on the phenomenon of nuclear magnetic resonance
and can provide detailed information about the structure, dynamics, reaction state, and chemical environment of molecules.
Most frequently, NMR spectroscopy is used by chemists and biochemists to investigate the properties of organic molecules, though it is applicable to any kind of sample that contains nuclei possessing spin
. Suitable samples range from small compounds analyzed with 1-dimensional proton
or carbon-13 NMR
spectroscopy to large protein
s or nucleic acid
s using 3 or 4-dimensional techniques. The impact of NMR spectroscopy on the sciences has been substantial because of the range of information and the diversity of samples, including solution
s and solid
s.
When placed in a magnetic field, NMR active nuclei (such as 1H or 13C) absorb electromagnetic radiation
at a frequency characteristic of the isotope
. The resonant frequency, energy of the absorption and the intensity of the signal are proportional to the strength of the magnetic field. For example, in a 21 tesla
magnetic field, proton
s resonate at 900 MHz. It is common to refer to a 21 T magnet as a 900 MHz magnet, although different nuclei resonate at a different frequency at this field strength in proportion to their nuclear magnetic moment
s.
. The chemical shift is reported as a relative measure from some reference resonance frequency. (For the nuclei 1H, 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 reference signal to give the chemical shift. The frequency shifts are extremely small in comparison to the fundamental NMR frequency. A typical frequency shift might be 100 Hz, compared to a fundamental NMR frequency of 100 MHz, so the chemical shift is generally expressed in parts per million (ppm). To detect such small frequency differences the applied magnetic field must be constant throughout the sample volume. High resolution NMR spectrometers use shims
to adjust the homogeneity of the magnetic field to parts per billion (ppb) in a volume of a few cubic centimeters. In general, chemical shifts for protons are highly predictable since the shifts are primarily determined by simpler shielding effects (electron density), but the chemical shifts for many heavier nuclei are more strongly influenced by other factors including excited states ("paramagnetic" contribution to shielding tensor).
The chemical shift provides information about the structure of the molecule. The conversion of the raw data to this information is called assigning the spectrum. For example, for the 1H-NMR spectrum for ethanol (CH3CH2OH), one would expect signals at each of three specific chemical shifts: one for the CH3 group, one for the CH2 group and one for the OH group. A typical CH3 group has a shift around 1 ppm, a CH2 attached to an OH has a shift of around 4 ppm and an OH has a shift around 2–3 ppm depending on the solvent used.
Because of molecular motion at room temperature, the three methyl protons average out during the course of the NMR experiment (which typically requires a few ms
). These protons become degenerate
and form a peak at the same chemical shift.
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.
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 curve. 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 experiments, 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 known - therefore, the integral of the NMR signal is very difficult to interpret in more complicated NMR experiments.
Some of the most useful information for structure determination in a one-dimensional NMR spectrum comes from J-coupling or scalar coupling (a special case of spin-spin coupling) between NMR active nuclei. This coupling arises from the interaction of different spin states through the chemical bonds of a molecule and results in the splitting of NMR signals. These splitting patterns can be complex or simple and, likewise, can be straightforwardly interpretable or deceptive. This coupling provides detailed insight into the connectivity of atoms in a molecule.
Coupling to n equivalent (spin ½) nuclei splits the signal into a n+1 multiplet with intensity ratios following Pascal's triangle
as described on the right. Coupling to additional spins will lead to further splittings of each component of the multiplet e.g. coupling to two different spin ½ nuclei with significantly different coupling constants will lead to a doublet of doublets (abbreviation: dd). Note that coupling between nuclei that are chemically equivalent (that is, have the same chemical shift) has no effect of the NMR spectra and couplings between nuclei that are distant (usually more than 3 bonds apart for protons in flexible molecules) are usually too small to cause observable splittings. Long-range couplings over more than three bonds can often be observed in cyclic
and aromatic compounds, leading to more complex splitting patterns.
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. In principle, 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.
Coupling to any spin ½ nuclei such as phosphorus-31 or fluorine-19 works in this fashion (although the magnitudes of the coupling constants may be very different). 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. For instance, coupling to deuterium (a spin 1 nucleus) splits the signal into a 1:1:1 triplet because the spin 1 has three spin states. Similarly, a spin 3/2 nucleus 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, coupling is often the only way to distinguish different nuclei.
although the integrated area under the peaks remains constant.
In most high-field NMR, however, the distortions are usually modest and the characteristic distortions (roofing) can in fact help to identify related peaks.
Second-order effects decrease as the frequency difference between multiplets increases, so that high-field (i.e. high-frequency) NMR spectra display less distortion than lower frequency spectra. Early spectra at 60 MHz were more prone to distortion than spectra from later machines typically operating at frequencies at 200 MHz or above.
For example, the 4 H sites of 1,2-dichlorobenzene divide into two chemically equivalent pairs by symmetry, but an individual member of one of the pairs has different couplings to the spins making up the other pair.
Magnetic inequivalence can lead to highly complex spectra which can only be analyzed by computational modeling. Such effects are more common in NMR spectra of aromatic and other non-flexible systems, while conformational averaging about C-C bonds in flexible molecules tends to equalize the couplings between protons on adjacent carbons, reducing problems with magnetic inequivalence.
is one of several types of two-dimensional nuclear magnetic resonance (NMR) spectroscopy or 2D-NMR. 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.
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.
NMR spectroscopy, an important technique in structural biology
. A 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 spectroscopy is usually limited to proteins smaller than 35 kDa
, although larger structures have been solved. NMR spectroscopy is often the only way to obtain high resolution information on partially or wholly intrinsically unstructured proteins
. It is now a common tool for the determination of Conformation Activity Relationship
s where the structure before and after interaction with, for example, a drug candidate is compared to its known biochemical activity. Proteins are orders of magnitude
larger than the small organic molecules discussed earlier in this article, but the basic NMR techniques and some of the NMR theory also applies. Because of the much higher number of atoms present in a protein molecule in comparison with a small organic compound, the basic 1D spectra become crowded with overlapping signals to an extent where direct spectra analysis becomes untenable. 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 by solving a distance geometry
problem.
s, such as DNA
or RNA
. , nearly half of all known RNA structures had been determined by NMR spectroscopy.
Nucleic acid and protein NMR spectroscopy are similar but differences exist. Nucleic acids have a smaller percentage of hydrogen atoms, which are the atoms usually observed in NMR spectroscopy, and because nucleic acid double helices
are stiff and roughly linear, they do not fold back on themselves to give "long-range" correlations. The types of NMR usually done with nucleic acids are 1H or proton NMR
, 13C NMR
, 15N NMR, and 31P NMR
. Two-dimensional NMR methods are almost always used, such as correlation spectroscopy (COSY) and total coherence transfer spectroscopy (TOCSY) to detect through-bond nuclear couplings, and nuclear Overhauser effect
spectroscopy (NOESY) to detect couplings between nuclei that are close to each other in space.
Parameters taken from the spectrum, mainly NOESY cross-peaks and coupling constants
, can be used to determine local structural features such as glycosidic bond
angles, dihedral angle
s (using the Karplus equation
), and sugar pucker conformations. For large-scale structure, these local parameters must be supplemented with other structural assumptions or models, because errors add up as the double helix is traversed, and unlike with proteins, the double helix does not have a compact interior and does not fold back upon itself. NMR is also useful for investigating nonstandard geometries such as bent helices, non-Watson–Crick basepairing, and coaxial stacking. It has been especially useful in probing the structure of natural RNA oligonucleotides, which tend to adopt complex conformations such as stem-loop
s and pseudoknot
s. NMR is also useful for probing the binding of nucleic acid molecules to other molecules, such as proteins or drugs, by seeing which resonances are shifted upon binding of the other molecule.
s.
Magnetism
Magnetism is a property of materials that respond at an atomic or subatomic level to an applied magnetic field. Ferromagnetism is the strongest and most familiar type of magnetism. It is responsible for the behavior of permanent magnets, which produce their own persistent magnetic fields, as well...
properties of certain atomic nuclei
Atomic nucleus
The nucleus is the very dense region consisting of protons and neutrons at the center of an atom. It was discovered in 1911, as a result of Ernest Rutherford's interpretation of the famous 1909 Rutherford experiment performed by Hans Geiger and Ernest Marsden, under the direction of Rutherford. The...
to determine physical and chemical properties of atom
Atom
The atom is a basic unit of matter that consists of a dense central nucleus surrounded by a cloud of negatively charged electrons. The atomic nucleus contains a mix of positively charged protons and electrically neutral neutrons...
s or the molecule
Molecule
A molecule is an electrically neutral group of at least two atoms held together by covalent chemical bonds. Molecules are distinguished from ions by their electrical charge...
s in which they are contained. It relies on the phenomenon of nuclear magnetic resonance
Nuclear magnetic resonance
Nuclear magnetic resonance is a physical phenomenon in which magnetic nuclei in a magnetic field absorb and re-emit electromagnetic radiation...
and can provide detailed information about the structure, dynamics, reaction state, and chemical environment of molecules.
Most frequently, NMR spectroscopy is used by chemists and biochemists to investigate the properties of organic molecules, though it is applicable to any kind of sample that contains nuclei possessing spin
Spin (physics)
In quantum mechanics and particle physics, spin is a fundamental characteristic property of elementary particles, composite particles , and atomic nuclei.It is worth noting that the intrinsic property of subatomic particles called spin and discussed in this article, is related in some small ways,...
. Suitable samples range from small compounds analyzed with 1-dimensional proton
Proton NMR
Proton NMR is the application of nuclear magnetic resonance in NMR spectroscopy with respect to hydrogen-1 nuclei within the molecules of a substance, in order to determine the structure of its molecules. In samples where natural hydrogen is used, practically all of the hydrogen consists of the...
or carbon-13 NMR
Carbon-13 NMR
Carbon-13 NMR is the application of nuclear magnetic resonance spectroscopy to carbon. It is analogous to proton NMR and allows the identification of carbon atoms in an organic molecule just as proton NMR identifies hydrogen atoms...
spectroscopy to large protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s or nucleic acid
Nucleic acid
Nucleic acids are biological molecules essential for life, and include DNA and RNA . Together with proteins, nucleic acids make up the most important macromolecules; each is found in abundance in all living things, where they function in encoding, transmitting and expressing genetic information...
s using 3 or 4-dimensional techniques. The impact of NMR spectroscopy on the sciences has been substantial because of the range of information and the diversity of samples, including solution
Solution
In chemistry, a solution is a homogeneous mixture composed of only one phase. In such a mixture, a solute is dissolved in another substance, known as a solvent. The solvent does the dissolving.- Types of solutions :...
s and solid
Solid-state chemistry
Solid-state chemistry, also sometimes referred to as materials chemistry, is the study of the synthesis, structure, and properties of solid phase materials, particularly, but not necessarily exclusively of, non-molecular solids...
s.
Basic NMR techniques
Electromagnetic radiation
Electromagnetic radiation is a form of energy that exhibits wave-like behavior as it travels through space...
at a frequency characteristic of the isotope
Isotope
Isotopes are variants of atoms of a particular chemical element, which have differing numbers of neutrons. Atoms of a particular element by definition must contain the same number of protons but may have a distinct number of neutrons which differs from atom to atom, without changing the designation...
. The resonant frequency, energy of the absorption and the intensity of the signal are proportional to the strength of the magnetic field. For example, in a 21 tesla
Tesla (unit)
The tesla is the SI derived unit of magnetic field B . One tesla is equal to one weber per square meter, and it was defined in 1960 in honour of the inventor, physicist, and electrical engineer Nikola Tesla...
magnetic field, proton
Proton
The proton is a subatomic particle with the symbol or and a positive electric charge of 1 elementary charge. One or more protons are present in the nucleus of each atom, along with neutrons. The number of protons in each atom is its atomic number....
s resonate at 900 MHz. It is common to refer to a 21 T magnet as a 900 MHz magnet, although different nuclei resonate at a different frequency at this field strength in proportion to their nuclear magnetic moment
Nuclear magnetic moment
The nuclear magnetic moment is the magnetic moment of an atomic nucleus and arises from the spin of the protons and neutrons. It is mainly a magnetic dipole moment; the quadrupole moment does cause some small shifts in the hyperfine structure as well....
s.
Chemical shift
Depending on their local chemical environment, different nuclei in a molecule absorb at slightly different frequencies. Since this resonant frequency is directly proportional to the strength of the magnetic field, the shift is converted into a field-independent dimensionless value known as the chemical shiftChemical shift
In nuclear magnetic resonance spectroscopy, the chemical shift is the resonant frequency of a nucleus relative to a standard. Often the position and number of chemical shifts are diagnostic of the structure of a molecule...
. The chemical shift is reported as a relative measure from some reference resonance frequency. (For the nuclei 1H, 13C, and 29Si, TMS (tetramethylsilane
Tetramethylsilane
Tetramethylsilane is the chemical compound with the formula Si4. It is the simplest tetraorganosilane. Like all silanes, the TMS framework is tetrahedral...
) 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 reference signal to give the chemical shift. The frequency shifts are extremely small in comparison to the fundamental NMR frequency. A typical frequency shift might be 100 Hz, compared to a fundamental NMR frequency of 100 MHz, so the chemical shift is generally expressed in parts per million (ppm). To detect such small frequency differences the applied magnetic field must be constant throughout the sample volume. High resolution NMR spectrometers use shims
Shim (magnetism)
A shim is a device used to adjust the homogeneity of a magnetic field. Originally, shims have their name from the purely mechanical shims that are used to adjust position and parallelity of the pole faces of an electromagnet...
to adjust the homogeneity of the magnetic field to parts per billion (ppb) in a volume of a few cubic centimeters. In general, chemical shifts for protons are highly predictable since the shifts are primarily determined by simpler shielding effects (electron density), but the chemical shifts for many heavier nuclei are more strongly influenced by other factors including excited states ("paramagnetic" contribution to shielding tensor).
The chemical shift provides information about the structure of the molecule. The conversion of the raw data to this information is called assigning the spectrum. For example, for the 1H-NMR spectrum for ethanol (CH3CH2OH), one would expect signals at each of three specific chemical shifts: one for the CH3 group, one for the CH2 group and one for the OH group. A typical CH3 group has a shift around 1 ppm, a CH2 attached to an OH has a shift of around 4 ppm and an OH has a shift around 2–3 ppm depending on the solvent used.
Because of molecular motion at room temperature, the three methyl protons average out during the course of the NMR experiment (which typically requires a few ms
Millisecond
A millisecond is a thousandth of a second.10 milliseconds are called a centisecond....
). These protons become degenerate
Degenerate energy level
In physics, two or more different quantum states are said to be degenerate if they are all at the same energy level. Statistically this means that they are all equally probable of being filled, and in Quantum Mechanics it is represented mathematically by the Hamiltonian for the system having more...
and form a peak at the same chemical shift.
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.
Software allows analysis of the size of peaks to understand how many protons give rise to the peak. This is known as integration
Integral
Integration is an important concept in mathematics and, together with its inverse, differentiation, is one of the two main operations in calculus...
—a mathematical process which calculates the area under a curve. 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 experiments, for instance, experiments typically used to obtain carbon-13
Carbon-13
Carbon-13 is a natural, stable isotope of carbon and one of the environmental isotopes. It makes up about 1.1% of all natural carbon on Earth.- Detection by mass spectrometry :...
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 known - therefore, the integral of the NMR signal is very difficult to interpret in more complicated NMR experiments.
J-coupling
| Multiplicity | Intensity Ratio |
|---|---|
| Singlet (s) | 1 |
| Doublet (d) | 1:1 |
| Triplet (t) | 1:2:1 |
| Quartet (q) | 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 |
Some of the most useful information for structure determination in a one-dimensional NMR spectrum comes from J-coupling or scalar coupling (a special case of spin-spin coupling) between NMR active nuclei. This coupling arises from the interaction of different spin states through the chemical bonds of a molecule and results in the splitting of NMR signals. These splitting patterns can be complex or simple and, likewise, can be straightforwardly interpretable or deceptive. This coupling provides detailed insight into the connectivity of atoms in a molecule.
Coupling to n equivalent (spin ½) nuclei splits the signal into a n+1 multiplet with intensity ratios following Pascal's triangle
Pascal's triangle
In mathematics, Pascal's triangle is a triangular array of the binomial coefficients in a triangle. It is named after the French mathematician, Blaise Pascal...
as described on the right. Coupling to additional spins will lead to further splittings of each component of the multiplet e.g. coupling to two different spin ½ nuclei with significantly different coupling constants will lead to a doublet of doublets (abbreviation: dd). Note that coupling between nuclei that are chemically equivalent (that is, have the same chemical shift) has no effect of the NMR spectra and couplings between nuclei that are distant (usually more than 3 bonds apart for protons in flexible molecules) are usually too small to cause observable splittings. Long-range couplings over more than three bonds can often be observed in cyclic
Cyclic compound
In chemistry, a cyclic compound is a compound in which a series of atoms is connected to form a loop or ring.While the vast majority of cyclic compounds are organic, a few inorganic substances form cyclic compounds as well, including sulfur, silanes, phosphanes, phosphoric acid, and triboric acid. ...
and aromatic compounds, leading to more complex splitting patterns.
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. In principle, 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.
Coupling to any spin ½ nuclei such as phosphorus-31 or fluorine-19 works in this fashion (although the magnitudes of the coupling constants may be very different). But the splitting patterns differ from those described above for nuclei with spin greater than ½ because the spin quantum number
Spin quantum number
In atomic physics, the spin quantum number is a quantum number that parameterizes the intrinsic angular momentum of a given particle...
has more than two possible values. For instance, coupling to deuterium (a spin 1 nucleus) splits the signal into a 1:1:1 triplet because the spin 1 has three spin states. Similarly, a spin 3/2 nucleus 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, coupling is often the only way to distinguish different nuclei.
Second-order (or strong) coupling
The above description assumes that the coupling constant is small in comparison with the difference in NMR frequencies between the inequivalent spins. If the shift separation decreases (or the coupling strength increases), the multiplet intensity patterns are first distorted, and then become more complex and less easily analyzed (especially if more than two spins are involved). Intensification of some peaks in a multiplet is achieved at the expense of the remainder, which sometimes almost disappear in the background noise,although the integrated area under the peaks remains constant.
In most high-field NMR, however, the distortions are usually modest and the characteristic distortions (roofing) can in fact help to identify related peaks.
Second-order effects decrease as the frequency difference between multiplets increases, so that high-field (i.e. high-frequency) NMR spectra display less distortion than lower frequency spectra. Early spectra at 60 MHz were more prone to distortion than spectra from later machines typically operating at frequencies at 200 MHz or above.
Magnetic inequivalence
More subtle effects can occur if chemically equivalent spins (i.e., nuclei related by symmetry and so having the same NMR frequency) have different coupling relationships to external spins. Spins that are chemically equivalent but are not indistinguishable (based on their coupling relationships) are termed magnetically inequivalent.For example, the 4 H sites of 1,2-dichlorobenzene divide into two chemically equivalent pairs by symmetry, but an individual member of one of the pairs has different couplings to the spins making up the other pair.
Magnetic inequivalence can lead to highly complex spectra which can only be analyzed by computational modeling. Such effects are more common in NMR spectra of aromatic and other non-flexible systems, while conformational averaging about C-C bonds in flexible molecules tends to equalize the couplings between protons on adjacent carbons, reducing problems with magnetic inequivalence.
Correlation spectroscopy
Correlation spectroscopyCorrelation spectroscopy
Two-dimensional nuclear magnetic resonance spectroscopy is a set of nuclear magnetic resonance spectroscopy methods which give data plotted in a space defined by two frequency axes rather than one. Types of 2D NMR include correlation spectroscopy , J-spectroscopy, exchange spectroscopy , and...
is one of several types of two-dimensional nuclear magnetic resonance (NMR) spectroscopy or 2D-NMR. This type of NMR experiment is best known by its acronym, COSY
Correlation spectroscopy
Two-dimensional nuclear magnetic resonance spectroscopy is a set of nuclear magnetic resonance spectroscopy methods which give data plotted in a space defined by two frequency axes rather than one. Types of 2D NMR include correlation spectroscopy , J-spectroscopy, exchange spectroscopy , and...
. Other types of two-dimensional NMR include J-spectroscopy, exchange spectroscopy (EXSY), Nuclear Overhauser effect
Nuclear Overhauser effect
The Nuclear Overhauser Effect is the transfer of nuclear spin polarization from one nuclear spin population to another via cross-relaxation. It is a common phenomenon observed by nuclear magnetic resonance spectroscopy. The theoretical basis for the NOE was described and experimentally verified...
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
Molecule
A molecule is an electrically neutral group of at least two atoms held together by covalent chemical bonds. Molecules are distinguished from ions by their electrical charge...
, 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
Richard R. Ernst
Richard Robert Ernst is a Swiss physical chemist and Nobel Laureate.Born in Winterthur, Switzerland, Ernst was awarded the Nobel Prize in Chemistry in 1991 for his contributions towards the development of Fourier Transform nuclear magnetic resonance spectroscopy while at Varian Associates, Palo...
, who published their work in 1976.
Solid-state nuclear magnetic resonance
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
Magic angle
The magic angle is a precisely defined angle, the value of which is approximately 54.7356°. The magic angle is a root of a second-order Legendre polynomial, P_2=0 \,, and so any interaction which depends on this second-order Legendre polynomial vanishes at 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.
Proteins
Much of the innovation within NMR spectroscopy has been within the field of proteinProtein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
NMR spectroscopy, an important technique in structural biology
Structural biology
Structural biology is a branch of molecular biology, biochemistry, and biophysics concerned with the molecular structure of biological macromolecules, especially proteins and nucleic acids, how they acquire the structures they have, and how alterations in their structures affect their function...
. A 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
X-ray crystallography
X-ray crystallography is a method of determining the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and causes the beam of light to spread into many specific directions. From the angles and intensities of these diffracted beams, a crystallographer can produce a...
. In contrast to X-ray crystallography, NMR spectroscopy is usually limited to proteins smaller than 35 kDa
KDA
KDA may refer to:* Karachi Development Authority* Kongsberg Defence & Aerospace* Kotelawala Defence Academy* Kramer Design Associates* Lithium diisopropylamide, KDA is the potassium analogue of lithium diisopropylamideOr kDa may refer to:...
, although larger structures have been solved. NMR spectroscopy is often the only way to obtain high resolution information on partially or wholly intrinsically unstructured proteins
Intrinsically unstructured proteins
Intrinsically unstructured proteins, often referred to as naturally unfolded proteins or disordered proteins, are proteins characterized by lack of stable tertiary structure when the protein exists as an isolated polypeptide chain under physiological conditions in vitro...
. It is now a common tool for the determination of Conformation Activity Relationship
Conformation activity relationship
Conformation-activity relationship is the relationship between the biological activity and the conformation or conformational changes of a biomolecule...
s where the structure before and after interaction with, for example, a drug candidate is compared to its known biochemical activity. Proteins are orders of magnitude
Order of magnitude
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. In its most common usage, the amount being scaled is 10 and the scale is the exponent being applied to this amount...
larger than the small organic molecules discussed earlier in this article, but the basic NMR techniques and some of the NMR theory also applies. Because of the much higher number of atoms present in a protein molecule in comparison with a small organic compound, the basic 1D spectra become crowded with overlapping signals to an extent where direct spectra analysis becomes untenable. Therefore, multidimensional (2, 3 or 4D) experiments have been devised to deal with this problem. To facilitate these experiments, it is desirable to isotopically
Isotope
Isotopes are variants of atoms of a particular chemical element, which have differing numbers of neutrons. Atoms of a particular element by definition must contain the same number of protons but may have a distinct number of neutrons which differs from atom to atom, without changing the designation...
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
Nuclear Overhauser effect
The Nuclear Overhauser Effect is the transfer of nuclear spin polarization from one nuclear spin population to another via cross-relaxation. It is a common phenomenon observed by nuclear magnetic resonance spectroscopy. The theoretical basis for the NOE was described and experimentally verified...
to measure distances between pairs of atoms within the molecule. Subsequently, the obtained distances are used to generate a 3D structure of the molecule by solving a distance geometry
Distance geometry
Distance geometry is the characterization and study of sets of points based only on given values of the distances between member pairs. Therefore distance geometry has immediate relevance where distance values are determined or considered, such as in surveying, cartography and...
problem.
Nucleic acids
"Nucleic acid NMR" is the use of NMR spectroscopy to obtain information about the structure and dynamics of polynucleic acidNucleic acid
Nucleic acids are biological molecules essential for life, and include DNA and RNA . Together with proteins, nucleic acids make up the most important macromolecules; each is found in abundance in all living things, where they function in encoding, transmitting and expressing genetic information...
s, such as DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
or RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
. , nearly half of all known RNA structures had been determined by NMR spectroscopy.
Nucleic acid and protein NMR spectroscopy are similar but differences exist. Nucleic acids have a smaller percentage of hydrogen atoms, which are the atoms usually observed in NMR spectroscopy, and because nucleic acid double helices
Nucleic acid double helix
In molecular biology, the term double helix refers to the structure formed by double-stranded molecules of nucleic acids such as DNA and RNA. The double helical structure of a nucleic acid complex arises as a consequence of its secondary structure, and is a fundamental component in determining its...
are stiff and roughly linear, they do not fold back on themselves to give "long-range" correlations. The types of NMR usually done with nucleic acids are 1H or proton NMR
Proton NMR
Proton NMR is the application of nuclear magnetic resonance in NMR spectroscopy with respect to hydrogen-1 nuclei within the molecules of a substance, in order to determine the structure of its molecules. In samples where natural hydrogen is used, practically all of the hydrogen consists of the...
, 13C NMR
Carbon-13 NMR
Carbon-13 NMR is the application of nuclear magnetic resonance spectroscopy to carbon. It is analogous to proton NMR and allows the identification of carbon atoms in an organic molecule just as proton NMR identifies hydrogen atoms...
, 15N NMR, and 31P NMR
Phosphorus-31 NMR
Phosphorus-31 NMR spectroscopy is an analytical technique. Solution 31P-NMR is one of the more routine NMR techniques because 31P has an isotopic abundance of 100% and a relatively high magnetogyric ratio. The 31P nucleus also has a spin of ½, making spectra relatively easy to interpret...
. Two-dimensional NMR methods are almost always used, such as correlation spectroscopy (COSY) and total coherence transfer spectroscopy (TOCSY) to detect through-bond nuclear couplings, and nuclear Overhauser effect
Nuclear Overhauser effect
The Nuclear Overhauser Effect is the transfer of nuclear spin polarization from one nuclear spin population to another via cross-relaxation. It is a common phenomenon observed by nuclear magnetic resonance spectroscopy. The theoretical basis for the NOE was described and experimentally verified...
spectroscopy (NOESY) to detect couplings between nuclei that are close to each other in space.
Parameters taken from the spectrum, mainly NOESY cross-peaks and coupling constants
J-coupling
J-coupling is the coupling between two nuclear spins due to the influence of bonding electrons on the magnetic field running between the two nuclei. J-coupling contains information about dihedral angles, which can be estimated using the Karplus equation...
, can be used to determine local structural features such as glycosidic bond
Glycosidic bond
In chemistry, a glycosidic bond is a type of covalent bond that joins a carbohydrate molecule to another group, which may or may not be another carbohydrate....
angles, dihedral angle
Dihedral angle
In geometry, a dihedral or torsion angle is the angle between two planes.The dihedral angle of two planes can be seen by looking at the planes "edge on", i.e., along their line of intersection...
s (using the Karplus equation
Karplus equation
The Karplus equation, named after Martin Karplus, describes the correlation between 3J-coupling constants and dihedral torsion angles in nuclear magnetic resonance spectroscopy:J = A \cos^2 \phi + B \cos\,\phi + C...
), and sugar pucker conformations. For large-scale structure, these local parameters must be supplemented with other structural assumptions or models, because errors add up as the double helix is traversed, and unlike with proteins, the double helix does not have a compact interior and does not fold back upon itself. NMR is also useful for investigating nonstandard geometries such as bent helices, non-Watson–Crick basepairing, and coaxial stacking. It has been especially useful in probing the structure of natural RNA oligonucleotides, which tend to adopt complex conformations such as stem-loop
Stem-loop
Stem-loop intramolecular base pairing is a pattern that can occur in single-stranded DNA or, more commonly, in RNA. The structure is also known as a hairpin or hairpin loop. It occurs when two regions of the same strand, usually complementary in nucleotide sequence when read in opposite directions,...
s and pseudoknot
Pseudoknot
A pseudoknot is a nucleic acid secondary structure containing at least two stem-loop structures in which half of one stem is intercalated between the two halves of another stem. The pseudoknot was first recognized in the turnip yellow mosaic virus in 1982...
s. NMR is also useful for probing the binding of nucleic acid molecules to other molecules, such as proteins or drugs, by seeing which resonances are shifted upon binding of the other molecule.
Carbohydrates
Carbohydrate NMR spectroscopy addresses questions on the structure and conformation of carbohydrateCarbohydrate
A carbohydrate is an organic compound with the empirical formula ; that is, consists only of carbon, hydrogen, and oxygen, with a hydrogen:oxygen atom ratio of 2:1 . However, there are exceptions to this. One common example would be deoxyribose, a component of DNA, which has the empirical...
s.
See also
- Distance geometryDistance geometryDistance geometry is the characterization and study of sets of points based only on given values of the distances between member pairs. Therefore distance geometry has immediate relevance where distance values are determined or considered, such as in surveying, cartography and...
- In vivo magnetic resonance spectroscopyIn vivo magnetic resonance spectroscopyIn vivo magnetic resonance spectroscopy is a specialised technique associated to magnetic resonance imaging ....
- Low field NMRLow field NMRLow field NMR is a branch of nuclear magnetic resonance that is either related to Earth's field NMR, or to NMR at a man-made very low magnetic field and shielding from the Earth's magnetic field. With magnetic fields on the order of μT or nT, SQUIDs are typically used as...
- Magnetic Resonance ImagingMagnetic resonance imagingMagnetic resonance imaging , nuclear magnetic resonance imaging , or magnetic resonance tomography is a medical imaging technique used in radiology to visualize detailed internal structures...
- NMR crystallography
- Nuclear Magnetic ResonanceNuclear magnetic resonanceNuclear magnetic resonance is a physical phenomenon in which magnetic nuclei in a magnetic field absorb and re-emit electromagnetic radiation...
- NMR spectra databaseNMR spectra databaseNuclear Magnetic Resonance spectra database is an electronic repository of information concerning NMR spectra. The repository can be stored as a complete self contained data set or as an online repository that can be accessed and searched remotely. The form in which the data is stored ranges...
- NMR tubeNMR tubeAn NMR tube is a thin glass walled tube used to contain samples in nuclear magnetic resonance spectroscopy. Typically NMR tubes come in 5 mm diameters but 10 mm and 3 mm samples are known. It is important that the tubes are uniformly thick and well-balanced to ensure that NMR tube...
- includes a section on sample preparation - Protein nuclear magnetic resonance spectroscopyProtein nuclear magnetic resonance spectroscopyNuclear magnetic resonance spectroscopy of proteins is a field of structural biology in which NMR spectroscopy is used to obtain information about the structure and dynamics of proteins. The field was pioneered by Richard R. Ernst and Kurt Wüthrich, among others...
- NMR spectroscopy of stereoisomersNMR spectroscopy of stereoisomersNMR spectroscopy techniques can determine the absolute configuration of stereoisomers such as cis or trans alkenes, R or S enantiomers, and R,R or R,S diastereomers....
- Earth's field NMREarth's field NMRNuclear magnetic resonance in the geomagnetic field is conventionally referred to as Earth's field NMR . EFNMR is a special case of low field NMR....
External links
- The Basics of NMR - A non-technical overview of NMR theory, equipment, and techniques by Dr. Joseph Hornak, Professor of Chemistry at RIT
- GAMMA and PyGAMMA Libraries - GAMMA is an open source C++ library written for the simulation of Nuclear Magnetic Resonance Specroscopy experiments. PyGAMMA is a Python wrapper around GAMMA.
- Vespa - VeSPA (Versatile Simulation, Pulses and Analysis) is a free software suite composed of three Python applications. These GUI based tools are for magnetic resonance (MR) spectral simulation, RF pulse design, and spectral processing and analysis of MR data.