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NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY
Among the various available non-destructive structure elucidation techniques, NMR is one of the most powerful and useful one. It is mainly an organic chemist's handy tool, though, it has an equal potential of users in the other branches of science and technology when it comes to testing non-paramagnetic samples.
Simply put, in a lay-man's language, the principle of conventional NMR technique can be explained as follows: The sample to be tested, taken in a special glass tube of diameter of 5 or 10 mm, generally dissolved in certain solvents, is kept in a high uniform magnetic field. Then, from a variable radio frequency (RF) source, RF of appropriate power is sent through shielded cable to a coil specially wound around the test sample. The frequency is varied in small steps of increments (which is generally known as scanning, in spectroscopic language) through a predetermined frequency range. Using appropriate electronic circuitry, the absorption of the RF is monitored and measured.
These two information, viz., frequency of the RF and the absorption are plotted as x- and y- axes. This graph, called the NMR spectrum, is characteristic of the test compound and some times can be considered as its "finger print". Thus the spectrum can be analyzed, typically, by comparison to get an idea of the structure of the compound. Or, the spectrum can be systematically studied and one can arrive at, in many cases, the structure of the compound." The above explanation, though sounds too simple, the actual mechanism of NMR phenomenon is too complicated to be explained in this write-up. Interested readers are advised to refer to some of the standard references and textbooks listed at the end of this.
Basically, the absorption of the RF energy occurs because of the presence of some nuclei like, 1H, 13C, 31P, 19F etc. all of which have got non-zero nuclear spin. Such nuclei are called magnetic nuclei. The NMR spectrum arising due to the presence of 1H is generally termed as proton NMR or PMR and that arising due to 13C NMR and incidentally the most useful tool for the organic chemists. When compound containing such atoms are kept in the magnetic field they "behave" in two or more different ways and are said to be different energy levels.
Protons and carbon nuclei split into 2 different energy levels each. The energy gap between these levels depend upon the many factors. The main factor is the magnetic field itself. Higher the magnetic field, higher the energy gap and hence higher RF frequency will be absorbed. However, during NMR measurements the field is kept constant. Even in a constant magnetic field, the energy level gap for the same nuclei within a molecule can be different due to the difference in shielding of the nuclei by the electrons surrounding the nuclei. In other words, since electrons around the nuclei of interest are involved in bond formation etc., they shield / deshield the nuclei to different extents resulting in different energy gaps and in turn different RF absorptions. These shifts in the absorption lines are called Chemical Shifts and are one of the most fundamental parameters of the NMR. Generally, the NMR signals are reported in "chemical shifts", which are the ratio of the frequency difference between a reference standard and the test signal to the frequency of the spectrometer. This is a dimensionless quantity and is generally multiplied by a factor of 106, and expressed in ppm to make the ratio a convenient number. These dimensionless chemical-shift quantity is generally termed as 'd'. To the solvent itself some times, the chemical shift reference standard is added so that one can directly measure/calculate the chemical shift. Thus, Tetramethyl silane (TMS), which is an inert, volatile compound is used as ppm marker for 1H, 13C and 29Si NMR measurements. The NMR signal arising out of the TMS is set to zero ppm in each case. Similarly for other nuclei such convenient standards are chosen. It is some times not possible to have these primary ppm markers or reference in the solvents. In such cases, secondary standards can be used, for which the ppm value is known, can be used. When one is not too much concerned about the exact ppm value, one can use external referencing. In this case, the spectrum of a known compound of known ppm value is measured first and the spectral chart is calibrated. Then without changing any instrumental experimental parameters, the NMR spectrum of the test compound is measured. By comparing the 'external' standard spectrum and the sample spectrum, one can get a reasonably accurate ppm value of the signals. Another important phenomenon in NMR is the interaction between neighboring magnetic nuclei. This is called spin-spin coupling. This causes otherwise single resonance line of each chemically shifted nucleus to be split into multiple lines in an ordered fashion. Though this can increase the complexity of the spectrum, one can, if correctly assigned, get a wealth of information of the group of atoms present in the immediate vicinity and their through-bond connectivities.
Instrumentation :
An NMR instrument consists of a Magnet (permanent magnet/elctromagnet or iron magnet/superconducting magnet). The sample is inserted into the centre of the magnetic field. The sample and the sample is surrounded by RF coils as mentioned earlier. Signals are detected by either the same coil or another coil depending upon the manufacturer's design. The main spectrometer consists of the RF generator, magnetic field monitor, RF signal receiving and detecting system recording device, necessary power supplies etc. Optionally there can be computer connected (interfaced) to the spectrometer to collect (acquire) the spectral data and store/process etc.
There are two classes of NMR spectrometers, viz., CW (continuous wave) and pulsed Fourier Transform (FT) spectrometers. The CW technique is old and is almost obsolete whereas almost all present manufacturers produce only FT NMR spectrometers, which are much more versatile. Though both type of spectrometers contain the magnet and the sample probe, the design of the RF transmitter and the detection circuitry are different. In FT NMR the sample in a resonant coil is subjected to an intense and short RF pulse. Since the short pulse contains a broad band of frequencies (Fourier components of the RF pulse) this system is subjected to a broad band excitation, and irrespective of the differences in the chemical shift spread ALL nuclei of given kind are simultaneously excited and gives a time-response which is acquired by a fast digital computer to give the so-called Free Induction Decay (FID).
The FID upon a mathematical Fourier transform produce a frequency spectrum which will be identical to the spectrum obtained by the conventional CW sweep of the frequency. The advantage is that several thousand responses from a sample can be coherently added increasing the signal to noise ratio enormously. This makes it possible to address by MR any Magnetic nucleus in the Periodic Table irrespective of the magnetic moment and natural abundance. Further pulsed FT techniques van be used to measure dynamic process in the system such as relaxation times and also leads to a newer class of multidimensional correlation spectroscopy That has led to important advances in the application of NMR to the elucidation of biomolecular structure. The details of such techniques are beyond the scope of this article, and the reader may refer to books cited at the end of this write-up. Advantages, applications and
limitations of NMR technique:
The proton and 13C
NMR spectral data are quite useful and well adapted for the structural
elucidation of most of the organic compounds. As mentioned earlier, a fairly
good idea of the structure of the compound can be arrived at using the NMR
spectrum. This is quite useful when identification of a new compound in
R&D, natural products separated from plants etc., and in Quality Control in
Industries where purity of a product is to be monitored.
However this is not an ultimate technique. One should be aware of the
limitations before resorting to NMR:
1. First, it should be noted than inherently NMR is a very insensitive
technique. This problem is still more acute when one wishes to do 13C
or 15N NMR of ordinary organic compounds.
2. Another point the user must keep in mind is that the test sample should be
pure, non-paramagnetic and should not be a mixture of many compounds. If the
sample is impure or a mixture, understandably, the spectrum also will show
signals from all the individual components present, adding more confusion and
problems in analyzing the results. Except in cases where the test sample
contains known impurities or known unwanted compounds for which the spectral
signals are known, the assignment becomes formidable. The presence of
paramagnetic impurities causes spectral pattern to become broad, feature-less
and uninformative.
3. Most of the routine NMR spectral measurements are done in dissolved/liquid
state. So it becomes mandatory to make sample in solution form. The solvent
itself has to be free from the nuclei one is interested in the sample. For
example, for taking proton NMR spectrum of a compound, the sample should be
freely soluble in Carbon tetrachloride (CCl4) or Carbon disulfide
(CS2), which do not contain any 'H' (proton). In case the sample is
soluble only in say, Chloroform(CHCl3) or any other solvent which
contains protons, then one has to use these solvents in the per-deuterated
form, i.e., all the 'H' atoms are replaced by deuterium in the solvent, so that
the solvent itself will not give any signal and mask the sample signal.
It is worth mentioning that solvent concentration generally will be much more than 99.9% in the solution. Needless to say that these deuterated solvents are very expensive and one can not afford to stock all these solvents in bulk quantities. For 13C measurements this problem will not be serious, though, there also it is advisable to use deuterated solvents. For other nuclei like 15N, 31P, 29Si etc., one can afford to use ordinary solvents, in as much as they do not contain the nuclei of measurement. In the recent years, techniques
to do NMR measurements of samples in the solid state have been developed.
However, this does not have such wide adaptability as a routine analytical
tool, since, the resolution of the spectra are inherently very poor thus
putting constraints in the information availability. These include multiple
pulse line narrowing sequences and cross polarization with 'magic angle'
spinning techniques. Please see appropriate references cited at the end.
Selected Bibliography:
1.A. Abragam, Principles of Nuclear
Magnetism, Clarendon Press, Oxford, 1961.
2.C.P. Slichter, Principles of Nuclear Magnetic Resonance,3rd edn.,Springer,New York,1990. 3.J.A.Pople,W.G.Schneider, and H.J.Bernstein,High-Resolution Nuclear Magnetic Resonance, Mcgraw-Hill,New York,1959 4.J.W.Emsely, J. Feeney,and L.H.Sutcliffe, High Resolution Nuclear Magnetic Resonance Spectroscopy ,2 vols.,Pergamon Press, Oxford,1968. 5.R..K. Harris, Nuclear Magnectic Resonance spectroscopy, Longman,Harlow,UK,1986 6.N.Chandrakumar,S.Subramanian, Modern Techniques in High Resolution FT NMR , Springerverlag,Newyork,1987 7.R.R.Ernst,G.Bodenhausen and A.Wokhaun , Principles of Nuclear Magnetic Resonance in One and Two Dimensions,Clarendon Press,Oxford,1987 8.Jermy K.M.Sanders and Brian K. Hunter, Modern NMR Spectroscopy, Oxford University Press,New York,1988 9.Eiichi Fuckushima, Stephen B.W. Roeder Addison, Experimental Pulse NMR, A Nuts and Bolts Approach, Wesley puublishing company, 1981 10.D.M.Grasnt & R.K.Harris (Ed) ,John Wiley & Sons,Chichester,UK,1996. |
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