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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. 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 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. 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 . 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. 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''. 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. CORRELATION SPECTROSCOPY 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. 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. DEPT SPECTRA ]] DEPT stands for Distortionless '''E'''nhancement by '''P'''olarization '''T'''ransfer. It is a very useful method 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 45o, 90o or 135o. The value chosen dictates the result (as said before, quaternary and deprotonated carbons are always suppressed!):
NMR SPECTROSCOPY APPLIED TO PROTEINS ''Main article: Protein NMR '' 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 Experiements 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. EXTERNAL LINKS
By Dr.Joseph Hornak, Professor of Chemistry at RIT ftp://ftp.chem.pku.edu.cn/sslin/NMR/Softwares/Mestrec/ REFERENCES |
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