Espectroscopia NMR: diferenças entre revisões

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Revisão das 19h32min de 23 de setembro de 2011

Espectroscopia de ressonância magnética nuclear, mais conhecida como espectroscopia NMR, é uma técnica de pesquisa que explora as propriedades magnéticas de certos núcleos atômicos para determinar propriedades físicas ou químicas de átomos o the molecules in which they are ucontained. 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.

A 900MHz NMR instrument with a 21.2 T magnet at HWB-NMR, Birmingham, UK

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 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.

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 spectroscopy 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^[^{carece de fontes?]}. This experiment was later implemented by Walter P. Aue, Enrico Bartholdi and Richard R. Ernst, who published their work in 1976.^[1]

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 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.

Biomolecular NMR

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 35 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. It is now a common tool for the determination of Conformation Activity Relationships 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.

Nucleic acids

Nucleic acid NMR is the use of NMR spectroscopy to obtain information about the structure and dynamics of nucleic acid molecules, such as DNA or RNA. Desde 2003^[update], nearly half of all known RNA structures had been determined by NMR spectroscopy.^[2]

Nucleic acid NMR uses similar techniques as protein NMR, but has several differences. Nucleic acids have a smaller percentage of hydrogen atoms, which are the atoms usually observed in NMR, and because nucleic acid double helices are stiff and roughly linear, they do not fod back on themselves to give "long-range" correlations.^[3] 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.^[4]

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 angles (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-loops and pseudoknots. 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.^[4]

Carbohydrates

Carbohydrate NMR is the application of NMR to structural and conformational analysis of carbohydrate molecules. The study of carbohydrate chemistry today relies heavily on NMR spectroscopy. It is a tool that allows the carbohydrate chemist to determine the structure of monosaccharides and oligosaccharides from synthetic and natural sources. It is also a useful tool for determining sugar conformations.

Modern high field strength NMR instruments used for carbohydrate samples, typically 500 MHz or greater, are able to run a suite of 1D and 2D experiments to determine primary structure and conformation of carbohydrate compounds.

References

↑ Martin, G.E; Zekter, A.S., Two-Dimensional NMR Methods for Establishing Molecular Connectivity; VCH Publishers, Inc: New York, 1988 (p.59)
↑ PMID 14523911. doi:10.1002/cbic.200300700 Em falta ou vazio |título= (ajuda)
↑ Addess, Kenneth J.; Feigon, Juli (1996). «Introduction to ¹H NMR Spectroscopy of DNA». In: Hecht, Sidney M. Bioorganic Chemistry: Nucleic Acids. New York: Oxford University Press. ISBN 0195084675
↑ ^a ^b Wemmer, David (2000). «Chapter 5: Structure and Dynamics by NMR». In: Bloomfield, Victor A., Crothers, Donald M., and Tinoco, Ignacio. Nucleic acids: Structures, Properties, and Functions. Sausalito, California: University Science Books. ISBN 0935702490

External links

James Keeler. «Understanding NMR Spectroscopy» (reprinted at University of Cambridge). University of California, Irvine. Consultado em 11 de maio de 2007
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.

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

[furtig-2] PMID 14523911. doi:10.1002/cbic.200300700 Em falta ou vazio |título= (ajuda)

[addess-3] Addess, Kenneth J.; Feigon, Juli (1996). «Introduction to ¹H NMR Spectroscopy of DNA». In: Hecht, Sidney M. Bioorganic Chemistry: Nucleic Acids. New York: Oxford University Press. ISBN 0195084675

[wemmer-4] Wemmer, David (2000). «Chapter 5: Structure and Dynamics by NMR». In: Bloomfield, Victor A., Crothers, Donald M., and Tinoco, Ignacio. Nucleic acids: Structures, Properties, and Functions. Sausalito, California: University Science Books. ISBN 0935702490

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