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NMR Spectroscopy

By: Pharma Tips | Views: 3837 | Date: 15-Jun-2010

Nuclear Magnetic Resonance is a type of spectroscopy used to help determine the structure of specific molecules.

NMR SPECTROSCOPY
Nuclear Magnetic Resonance is a type of spectroscopy used to help determine the structure of specific molecules.

1. Introduction
NMR spectroscopy is currently the techniques capable of determing the structures of biological macromolecules like proteins and nucleic acids at atomic resolution. In addition, it is possible to study time dependent phenomena with NMR, such as intramolecular dynamics in macromolecules, reaction kinetics, molecular recognition or protein folding.
The basic phenomenon of NMR was discovered in 1945: The energy levels of atomic nuclei are split up by a magnetic field. Transitions between these energy levels can be induced by exciting the sample with radiation whose frequency is equivalent to the energy difference between the two levels. Since 1960 the field of NMR has seen an explosive growth which started with the development of pulsed Fourier-transform NMR and multidimensional NMR spectroscopy and still continues today.
Radio frequency waves induce transition between magnetic energy levels of nuclei of a molecule. It often concerned with nuclei with I=1/2.          

EX. 15N, 13C and 2H .
Spectra can not be obtained from nuclei with I=o, it can be obtained from nuclei when I≥1.
Frequency of radio wave lies between 10⁷ and 10⁸ cps.
Energy of Radio frequency calculated by 
E =h v 
Where h is plank constant and v is frequency.

The limitations of NMR spectroscopy result from the low inherent sensitivity of the technique and from the high complexity and information content of NMR spectra. These problems are partially alleviated by new developments: The sensitivity and resolution of NMR are increased by progress in spectrometer technology. Progress in the theoretical and practical capabilities of NMR lead to a increasingly efficient utilization of the information content of NMR spectra. Parallel developments in the biochemical methods (recombinant protein expression) allow the simple and fast preparation of protein samples. Heteronuclei like 15N, 13C and 2H can be incorporated in proteins by uniformly or selective isotopic labelling. Spectra from these samples can be drastically simplified. Additionally, some new informations about structure and dynamics of macromolecules can determined with these methods. 
All these developments currently allow the structure determination of proteins with a mass of up to 30 kDa. Spectroscopists hope to extend this limit to even larger values (perhaps 40-50 kDa) by further improvements.Recently,a group reported the backbone assignment of a protein complex with a mass of 64 kDa.

2. Theory of NMR Spectroscopy
Nuclear Spin and Quantization of Energy:
The phenomenon of magnetic resonance results from the interaction of the magnetic moment of an atomic nucleus (µ) with an external magnetic field. The cause of this magnetic moment is the quantum mechanical angular momentum (spin angular momentum) of all nuclei that are no nuclei (even number of protons and neutrons). In order to understand this, imagine the nucleus as a small charged particle which is spinning around its own axis thus representing an electric current. Due to this current the atomic nucleus behaves as a small electromagnet.
Of course, this picture is a classical model whis has nothing to do with reality. The quantum mechanical property 'spin' does not mean that the nucleus is spinning around its own axis (if it did its radial speed would be greater than the speed of light). It is therefore an unfortunate choice of words if we denominate the spin as the angular momentum of a nucleus, because spin is a pure quantum mechanical property which could as easily be called 'happiness' or 'peppermint flavor'.
The spin is quantized according to
J = h/(2π) × I(I+1)) ½
With J being the spin angular momentum, I the spin quantum number (which can have values of I=0,1/2,1,3/2,...,6. By convention it is simply called 'spin'.) and h the plank’s constant. The angular momentum and the magnetic moment are directly proportional:
µ = γ× J = γ × h/(2 π) ×(I(I+1)) ½
The constant gamma is characteristic for each isotope and is called the gyromagnetic ratio. The sensitivity of a nucleus in NMR depends on gamma (high gamma, high sensitivity).
In an external magnetic field the magnetic moment orients according to:
Jz = - m × h/(2 π) 
=> µz = m × γ × h/(2 π)
The magnetic quantum number m can be an integer number between -I and +I. Thus, the external field leads to a splitting of the energy levels. For spin 1/2 nuclei (e.g. protons, see table) two energy levels exist according to a parallel or antiparallel orientation of the magnetic moment with respect to the magnetic field: 
The energy of these levels is given by the classical formula for a magnetic dipole in a homogenous magnetic field of the strength B0:
E = - µz ×B0 = - m × γ × h/(2 π) × B0
The magnetic moment of each nucleus precesses around B0. The frequency of this precession is the larmor frequency (w0) which is equivalent to the resonance frequency of the nucleus and the energy difference between the two levels.
γ× h/(2 π) × B0 =Δ
= h × nu 
= h/(2 π) × w0 
=> w0 = γ  × B0

The larmor frequency depends on the gyromagnetic ratio and the strength of the magnetic field (see picture), i.e. it is different for each isotope. At a magnetic field of 18.7 T the larmor frequency of protons is 800 MHz.

Continuous wave spectroscopy:
Transitions between different energy levels occur if the frequency of radiation is equivalent to the energy difference between the two levels: In the old days of NMR, experiments were carried out by varying the frequency of radiation at constant magnetic field ('frequency sweep') and measuring the absorption of radiation by the different nuclei. Equivalently, the magnetic field strength could be varied at constant radiation frequency ('field sweep'). Until the nineteen-seventies all NMR spectrometers worked with this continuous wave technique.
Pulsed Fourier Transform NMR Spectroscopy:
A far better resolution and sensitivity in NMR was achieved by the introduction of pulsed Fourier transform techniques (FT-NMR). In FT-NMR the resonances are not measured one after another but all nucleis are excited at the same time by a radio frequency pulse: Normally a radio emitter works at fixed frequency nu0. However, if the radiation is emitted as a very short pulse (some µs in NMR) the pulse frequency becomes 'uncertain'. A short radio frequency pulse contains many frequencies in a broad band around nu0 and thus excites the resonances of all spins in a sample at the same time.
The excited spins emit the absorbed radiation after the pulse. The emitted signal is a superposition of all excited frequencies. Its evolution in time is recorded. The intensities of the several frequencies, which give the observed signal in their superposition, are calculated by a mathematical operation, the Fourier transformation, which translates the time data into the frequency domain. The resulting NMR spectrum looks like an ordinary cw spectrum but its resolution is several orders of magnitudes better.
The FT method can be compared to the tuning of a bell. In principle, you could measure each of the tones which make up the sound of a bell in a 'cw experiment': Excite the bell with all frequencies from the deepest tones to the edge of ultrasound and measure the reaction of the bell with a microphone. But this method is extremely complicated and every bell founder knows a much faster way: Take a little hammer (or perhaps a bigger one) and - BOIIINGGGG..... 
The sound of the bell contains each tone at the same time an every person can analyze it directly with his or her ears (which are a cleverly 'constructed' instrument for FT). The advantages of this 'pulse FT method' over the 'cw method' are clearly obvious.
Populations and equilibrium magnetization:
A NMR sample contains many identical molecules (usually in a concentration range of mM for proteins). The spins of these molecules align indepently of each other parallel or antiparallel to the external field. The ratio of parallel spins to the antiparallel ones is given by the Boltzmann distribution:
Np / Nap = exp (Δ E/(kT)) = exp (γ ×h/(2 π) × B0 / (kT))

Both energy levels are nearly equally populated, because the energy difference is in the order of magnitude of thermic movements (kT). At T=300 K and a magnetic field of 18.7 T (800 MHz) the excess in the lower energy level is only 6.4 of 10000 particles for protons. This is the main reason for the inherently low sensitivity of NMR when compared to optical spectroscopic methods.
The magnetic moments of the individual spins sum up to a macroscopic magnetization M0 which can estimated according to Curie's law:
M0 = N × γ 2 × (h/(2 π))2 ×B0 ×I(I+1) / (3kT) 
=> M0 = N × γ 2 × (h/(2 π))2 ×B0 / (4kT), with I=1/2
It is the evoution of this macroscopic magnetization which is recorded in the spectrometer. The classical theory of NMR also deals with this quantity. In thermal equilibrium only magnetization along the axis of the magnetic field exists (by definition z), because the x and y components sum up to zero.

The Bloch equations:
For the mathematical description of NMR spectroscopy a rotating coordinate frame is used, the rotation frequency of which equals the larmor frequency of the nuclei. All nuclei rotating with the larmor frequency are fixed in this coordinate frame. 
This concept should be very familiar to us, because we all live in a rotating coordinate frame - the earth. To an observer in a spaceship a person 'standing' on the equator is moving at a speed of about 1700 km/h. A ball which is thrown 'vertically' up in the air comes down again in a straight vertical line. However, our observer in space sees this ball moving on a complex parabola.
Mathematically, the time dependency of the macroscopic magnetization M is described by the Bloch equation: 
dM/dt = γ × [M × Beff] 
Beff = (B0 + (w0/ γ)) + B1 = B1
--------------

=0
The time dependency of the magnetization vector M results from the interaction of the magnetization with the effective external magnetic field Beff. In the rotating frame the contribution of B0 to Beff is cancelled out (for nuclei with the larmor frequency w0), i. e. Beff equals zero as long as only the static external field B0 is applied.                                                        
Transversal magnetization  can now be created by applying an additional magnetic field B1  which is perpendicular to B0. This B1 field is the radiofrequency pulse mentioned above. If the radiation frequency is equal to the larmor frequency of the nuclei the field causes a rotation of the equilibrium magnetization Mz  around the x axis (cross product, Beff=B1). You can completely transform the z magnetization to y magnetization if the duration of the pulse is sufficiently long. In this case the pulse is called an 'excitation pulse' or '90° pulse' (obviously because the rotation angle of the magnetization is 90°). 

The state of x (or y) magnetization can be explained in the single spin model: The two energy levels explained above are equally populated (Mz = 0). Additionally, the magnetization dipoles of the spins are not statistically distributed around the z axis. A small part of them precesses 'focussedly' in phase around the z axis. They sum up to the macroscopic x magnetization. Therefore, states with transversal magnetization are also called 'phase coherence'.
Relaxation:
The Bloch equation is not complete because it predicts an infinite precession of transversal magnetization. In reality, transversal magnetization is a non equilibrium state and the system returns to thermal equilibrium within short time. Therefore, Bloch introduced two empirical relaxation times (T1, T2) in his equation. He assumed that these relaxation processes are of first order:
dMz/dt = γ × [M × Beff]z + ((M0 - Mz)/T1) 
dMx, y/dt = γ × [M × Beff]x, y - (Mx,y/T2) 
T1 and T2 are called the longitudinal and transversal relaxation times, respectively. The transversal components of magnetization (Mx, My) approach zero whereas the longitudinal component Mz approaches M0 with time. Relaxation is caused by several time dependent interactions between different spins (T2) and between spins and the surrounding lattice (T1). Therefore, T1 is also called spin-lattice relaxation time and T2 spin-spin relaxation time.
3. BASICS OF NMR
Nuclear magnetic resonance spectroscopy, commonly referred to as NMR, has become the preeminent technique for determining the structure of organic compounds. Of all the spectroscopic methods, it is the only one for which a complete analysis and interpretation of the entire spectrum is normally expected. Although larger amounts of sample are needed than for mass spectroscopy, NMR is non-destructive, and with modern instruments good data may be obtained from samples weighing less than a milligram. To be successful in using NMR as an analytical tool, it is necessary to understand the physical principles on which the methods are based.
The nuclei of many elemental isotopes have a characteristic spin (I). Some nuclei have integral spins (e.g. I = 1, 2, 3 ....), some have fractional spins (e.g. I = 1/2, 3/2, 5/2 ....), and a few have no spin, I = 0 (e.g. 12C, 16O, 32S, ....). Isotopes of particular interest and use to organic chemists are 1H, 13C, 19F and 31P, all of which have I = 1/2. Since the analysis of this spin state is fairly straightforward, our discussion of NMR will be limited to these and other I = 1/2 nuclei. 
Spin Properties of Nuclei
Nuclear spin may be related to the nucleon composition of a nucleus in the following manner:
1.  Odd mass nuclei (i.e. those having an odd number of nucleons) have fractional spins.                                   Examples are I = 1/2 ( 1H, 13C, 19F ), I = 3/2 ( 11B ) & I = 5/2 ( 17O ).
2. Even mass nuclei composed of odd numbers of protons and neutrons have integral spins.     Examples are I = 1 ( 2H, 14N ).
3. Even mass nuclei composed of even numbers of protons and neutrons have zero spin                        ( I = 0 ). Examples are 12C, and 16O.
Spin 1/2 nuclei have a spherical charge distribution, and their NMR behavior is the easiest to understand. Other spin nuclei have nonspherical charge distributions and may be analyzed as prolate or oblate spinning bodies. All nuclei with non-zero spins have magnetic moments (μ), but the nonspherical nuclei also have an electric quadruple moment. Some characteristic properties of selected nuclei are given in the following table.
Isotope Natural %
Abundance Spin (I) Magnetic
Moment (μ)* Magnetogyric
Ratio (γ)†
1H 99.9844 ½ 2.7927 26.753
2H 0.0156 1 0.8574 4,107
11B 81.17 3/2 2.6880 --
13C 1.108 ½ 0.7022 6,728
17O 0.037 5/2 -1.8930 -3,628
19F 100.0 ½ 2.6273 25,179
29Si 4.700 ½ -0.5555 -5,319
31P 100.0 ½ 1.1305 10,840

* μ in units of nuclear magnetons = 5.05078•10-27 JT-1 
† γ in units of 107rad T-1 sec-1

The following features lead to the NMR phenomenon: 
1. A spinning charge generates a magnetic field, as shown by the animation on the right.
The resulting spin-magnet has a magnetic moment (μ) proportional to the spin. 

2. In the presence of an external magnetic field (B0), two spin states exist, +1/2 and -1/2.
The magnetic moment of the lower energy +1/2 state is alligned with the external field, but that of the higher energy -1/2 spin state is opposed to the external field. Note that the arrow representing the external field points north. 

3. The difference in energy between the two spin states is dependent on the external magnetic field strength, and is always very small. The following diagram illustrates that the two spin states have the same energy when the external field is zero, but diverge as the field increases. At a field equal to Bx a formula for the energy difference is given (remember I = 1/2 and μ is the magnetic moment of the nucleus in the field).
Strong magnetic fields are necessary for NMR spectroscopy. The international unit for magnetic flux is the Tesla (T). The earth's magnetic field is not constant, but is approximately 10-4 T at ground level. Modern NMR spectrometers use powerful magnets having fields of 1 to 20 T. Even with these high fields, the energy difference between the two spin states is less than 0.1 cal/mole. To put this in perspective, recall that infrared transitions involve 1 to 10 kcal/mole and electronic transitions are nearly 100 times greater.
For NMR purposes, this small energy difference (ΔE) is usually given as a frequency in units of MHz (106 Hz), ranging from 20 to 900 Mz, depending on the magnetic field strength and the specific nucleus being studied. Irradiation of a sample with radio frequency (rf) energy corresponding exactly to the spin state separation of a specific set of nuclei will cause excitation of those nuclei in the +1/2 state to the higher -1/2 spin state. Note that this electromagnetic radiation falls in the radio and television broadcast spectrum. NMR spectroscopy is therefore the energetically mildest probe used to examine the structure of molecules .
The nucleus of a hydrogen atom (the proton) has a magnetic moment μ = 2.7927, and has been studied more than any other nucleus. The previous diagram may be changed to display energy differences for the proton spin states (as frequencies) by mouse clicking anywhere within it.

4.  For spin 1/2 nuclei the energy difference between the two spin states at a given magnetic field strength will be proportional to their magnetic moments. For the four common nuclei noted above, the magnetic moments are: 1H μ = 2.7927, 19F μ = 2.6273, 31P μ = 1.1305 & 13C μ = 0.7022. These moments are in nuclear magnetons, which are 5.05078•10-27 JT-1. The following diagram gives the approximate frequencies that correspond to the spin state energy separations for each of these nuclei in an external magnetic field of 2.35 T. The formula in the colored box shows the direct correlation of frequency (energy difference) with magnetic moment (h = Planck's constant = 6.626069•10-34 Js).
The 14 π-electron bridged annulene on the right is an aromatic (4n + 2) system, and has the same anisotropy as benzene. Nuclei located over the face of the ring are shielded, and those on the periphery are deshielded. The ring hydrogens give resonance signals in the range 8.0 to 8.7 δ, as expected from their deshielded location (note that there are three structurally different hydrogens on the ring). The two propyl groups are structurally equivalent (homotopic), and are free to rotate over the faces of the ring system. On average all the propyl hydrogens are shielded, with the innermost methylene being the most affected. The negative chemical shifts noted here indicate that the resonances occur at a higher field than the TMS reference signal. A remarkable characteristic of annulenes is that antiaromatic 4n π-electron systems are anisotropic in the opposite sense as their aromatic counterparts. A dramatic illustration of this fact is provided by the dianion derivative of the above bridged annulene. This dianion, formed by the addition of two electrons, is a 16 π-electron (4n) system. In the NMR spectrum of the dianion, the ring hydrogens resonate at high field (they are shielded), and the hydrogens of the propyl group are all shifted downfield (deshielded). The innermost methylene protons (magenta) give an NMR signal at +22.2 ppm, and the signals from the adjacent methylene and methyl hydrogens also have unexpectedly large chemical shifts. By clicking on the above structure the dianion data will appear.
Compounds in which two or more benzene rings are fused together were described in an earlier section.
Examples such as naphthalene, anthracene and phenanthrene, shown in the following diagram, present interesting insights into aromaticity and reactivity.
The resonance stabilization of these compounds, calculated from heats of hydrogenation or combustion, is given beneath each structure.

Unlike benzene, the structures of these compounds show measurable double bond localization, which is reflected in their increased reactivity both in substitution and addition reactions. However, the 1Hnmr spectra of these aromatic hydrocarbons do not provide much insight into the distribution of their pi-electrons. As expected, naphthalene displays two equally intense signals at δ 7.46 & 7.83 ppm. Likewise, anthracene shows three signals, two equal intensity multiplets at δ 7.44 & 7.98 ppm and a signal half as intense at δ 8.4 ppm. Thus, the influence of double bond localization or competition between benzene and higher annulene stabilization cannot be discerned.
The much larger C48H24 fused benzene ring cycle, named "kekulene" by Heinz Staab and sometimes called "superbenzene" by others, serves to probe the relative importance of benzenoid versus annulenoid aromaticity. A generic structure of this remarkable compound is drawn on the left below, together with two representative Kekule contributing structures on its right. There are some 200 Kekule structures that can be drawn for kekulene, but these two canonical forms represent extremes in aromaticity. The central formula has two [4n+2] annulenes, an inner annulene and an outer annulene (colored pink and blue respectively). The formula on the right has six benzene rings (colored green) joined in a ring by meta bonds, and held in a planar configuration by six cis-double bond bridges. 

The coupled annulene contributor in the center has an energetically equivalent canonical form in which the single and double bonds making up the annulenes are exchanged. If these contributors dominate the aromatic character of kekulene, the 6 inside hydrogens should be shielded by the ring currents, and the 18 hydrogens on the periphery should be deshielded. Furthermore, the C:C bonds composing each annulene ring should have roughly equal lengths. 
If the benzene contributor on the right (and its equivalent Kekule form) dominate the aromaticity of kekulene, all the benzene hydrogens will be deshielded, and the six double bond links on the periphery will have bond lengths characteristic of fixed single and double bonds
The extreme insolubility of kekulene made it difficult to grow suitable crystals for X-ray analysis or obtain solution NMR spectra. These problems were eventually solved by using high boiling solvents, the 1Hnmr spectrum being taken at 150 to 200° C in deuterated tetrachlorobenzene solution. The experimental evidence demonstrates clearly that the hexa-benzene ring structure on the right most accurately represents kekulene. This evidence will be shown above by clicking on the diagram. The extremely low field resonance of the inside hydrogens is assigned from similar downfield shifts in model compounds.
It is important to understand that the shielding and deshielding terms used through our discussion of relative chemical shifts are themselves relative. Indeed, compared to a hypothetical isolated proton, all the protons in a covalent compound are shielded by the electrons in nearby sigma and pi-bonds. Consequently, it would be more accurate to describe chemical shift differences in terms of the absolute shielding experienced by different groups of hydrogens. There is, in fact, good evidence that the anisotropy of neighboring C-H and C-C sigma bonds, together with that of the bond to the observed hydrogen, are the dominate shielding factors influencing chemical shifts. The anisotropy of pi-electron systems augments this sigma skeletal shielding.
Sigma bonding electrons also have a less pronounced, but observable, anisotropic influence on nearby nuclei. This is seen in the small deshielding shift that occurs in the series CH3–R, R–CH2–R, R3CH; as well as the deshielding of equatorial versus axial protons on a fixed cyclohexane ring.
Solvent Effects
Some Typical 1H Chemical Shifts (δ values) in Selected Solvents 
Solvent
Compound
CDCl3 C6D6 CD3COCD3 CD3SOCD3 CD3C≡N D2O
(CH3)3C–O–CH3
C–CH3
O–CH3 1.19 
3.22 1.07 
3.04 1.13 
3.13  1.11
3.03 1.14 
3.13 1.21 
3.22
(CH3)3C–O–H
C–CH3
O–H 1.26 
1.65 1.05 
1.55 1.18 
3.10 1.11 
4.19 1.16 
2.18 ---
---
C6H5CH3
CH3
C6H5 2.36 
7.15-7.20 2.11 
7.00-7.10 2.32 
7.10-7.20 2.30 
7.10-7.15 2.33 
7.15-7.30 ---
---
(CH3)2C=O  2.17 1.55 2.09 2.09 2.08 2.22
Chloroform-d (CDCl3) is the most common solvent for NMR measurements, thanks to its good solubilizing character and relative underactive nature ( except for 1º and 2º-amines). As noted earlier, other deuterium labeled compounds, such as deuterium oxide (D2O), benzene-d6 (C6D6), acetone-d6 (CD3COCD3) and DMSO-d6 (CD3SOCD3) are also available for use as NMR solvents. Because some of these solvents have π-electron functions and/or may serve as hydrogen bonding partners, the chemical shifts of different groups of protons may change depending on the solvent being used. The following table gives a few examples, obtained with dilute solutions at 300 MHz.
For most of the above resonance signals and solvents the changes are minor, being on the order of ±0.1 ppm. However, two cases result in more extreme changes and these have provided useful applications in structure determination. First, spectra taken in benzene-d6 generally show small up field shifts of most C–H signals, but in the case of acetone this shift is about five times larger than normal. Further study has shown that carbonyl groups form weak π–π collision complexes with benzene rings that persist long enough to exert a significant shielding influence on nearby                                                   
groups. In the case of substituted cyclohexanones, axial α-methyl groups are shifted up field by 0.2 to 0.3 ppm; whereas equatorial methyls are slightly deshielded (shift downfield by about 0.05 ppm). These changes are all relative to the corresponding chloroform spectra.
The second noteworthy change is seen in the spectrum of tert-butanol in DMSO, where the hydroxyl proton is shifted 2.5 ppm down-field from where it is found in dilute chloroform solution. This is due to strong hydrogen bonding of the alcohol O–H to the sulfoxide oxygen, which not only deshields the hydroxyl proton, but secures it from very rapid exchange reactions that prevent the display of spin-spin splitting. Similar but weaker hydrogen bonds are formed to the carbonyl oxygen of acetone and the nitrogen of acetonitrile. 

Chemical shift
The magnetic field at the nucleus is not equal to the applied magnetic field; electrons around the nucleus shield it from the applied field. The difference between the applied magnetic field and the field at the nucleus is termed the nuclear shielding. 
Consider the s-electrons in a molecule. They have spherical symmetry and circulate in the applied field, producing a magnetic field which opposes the applied field. This means that the applied field strength must be increased for the nucleus to absorb at its transition frequency. This upfield shift is also termed diamagnetic shift.

Electrons in p-orbitals have no spherical symmetry. They produce comparatively large magnetic fields at the nucleus, which give a low field shift. This "deshielding" is termed paramagnetic shift.
In proton (1H) NMR, p-orbitals play no part, which is why only a small range of chemical shift (10 ppm) is observed. We can easily see the effect of s-electrons on the chemical shift by looking at substituted methanes, CH3X. As X becomes increasingly electronegative, so the electron density around the protons decreases, and they resonate at lower field strengths.
Chemical shift is defined as nuclear shielding / applied magnetic field. Chemical shift is a function of the nucleus and its environment. It is measured relative to a reference compound. For 1H NMR, the reference is usually tetramethylsilane, Si (CH3)4
Unlike infrared and uv-visible spectroscopy, where absorption peaks are uniquely located by a                      
frequency or wavelength, the location of different NMR resonance signals is dependent on both    the external magnetic field strength and the rf frequency. Since no two magnets will have exactly the same field, resonance frequencies will vary accordingly and an alternative method for characterising and specifying the location of NMR signals is needed. This problem is illustrated by the eleven different compounds shown in the following diagram. Although the eleven resonance signals are distinct and well separated, an unambiguous numerical locator cannot be directly assigned to each.
Method of solving this problem is to report the location of an NMR signal in a spectrum relative to a reference signal from a standard compound added to the sample. Such a reference standard should be chemically unreactive, and easily removed from the sample after the measurement. Also, it should give a single sharp NMR signal that does not interfere with the resonances normally observed for organic compounds. Tetramethylsilane, (CH3)4Si, usually referred to as TMS, meets all these characteristics, and has become the reference compound of choice for proton and carbon NMR.
Since the separation (or dispersion) of NMR signals is magnetic field dependent, one additional step must be taken in order to provide an unambiguous location unit. This is illustrated for the acetone, methylene chloride and benzene signals by clicking on the previous diagram. To correct these frequency differences for their field dependence, we divide them by the spectrometer frequency (100 or 500 MHz in the example), as shown in a new display by again clicking on the diagram. The resulting number would be very small, since we are dividing Hz by MHz, so it is multiplied by a million, as shown by the formula in the blue shaded box. Note that νref is the resonant frequency of the reference signal and νsamp is the frequency of the sample signal. This operation gives a locator number called the Chemical Shift, having units of parts-per-million (ppm), and designated by the symbol δ .Chemical shifts for all the compounds in the original display will be presented by a third click on the diagram.
The compounds referred to above share two common characteristics:
• The hydrogen atoms in a given molecule are all structurally equivalent, averaged for fast conformational equilibria.
• The compounds are all liquids, save for neopentane which boils at 9 °C and is a liquid in an ice bath.
The first feature assures that each compound gives a single sharp resonance signal. The second allows the pure (neat) substance to be poured into a sample tube and examined in a NMR spectrometer. In order to take the NMR spectra of a solid, it is usually necessary to dissolve it in a suitable solvent. Early studies used carbon tetrachloride for this purpose, since it has no hydrogen that could introduce an interfering signal. Unfortunately, CCl4 is a poor solvent for many polar compounds and is also toxic. Deuterium labeled compounds, such as deuterium oxide (D2O), chloroform-d (DCCl3), benzene-d6 (C6D6), acetone-d6 (CD3COCD3) and DMSO-d6 (CD3SOCD3) are now widely used as NMR solvents. Since the deuterium isotope of hydrogen has a different magnetic moment and spin, it is invisible in a spectrometer tuned to proto
From the previous discussion and examples we may deduce that one factor contributing to chemical shift differences in proton resonance is the inductive effect. If the electron density about a proton nucleus is relatively high, the induced field due to electron motions will be stronger than if the electron density is relatively low. The shielding effect in such high electron density cases will therefore be larger, and a higher external field (Bo) will be needed for the rf energy to excite the nuclear spin. Since silicon is less electronegative than carbon, the electron density about the methyl hydrogens in tetramethylsilane is expected to be greater than the electron density about the methyl hydrogens in neopentane (2,2-dimethylpropane), and the characteristic resonance signal from the silane derivative does indeed lie at a higher magnetic field. Such nuclei are said to be shielded. Elements that are more electronegative than carbon should exert an opposite effect (reduce the electron density); and, as the data in the following tables show, methyl groups bonded to such elements display lower field signals (they are deshielded). The deshielding effect of electron withdrawing groups is roughly proportional to their electronegativity, as shown by the left table. Furthermore, if more than one such group is present, the deshielding is additive (table on the right), and proton resonance is shifted even further downfield. 
Proton Chemical Shifts of Methyl Derivatives
Compound (CH3)4C (CH3)3N (CH3)2O CH3F
Δ 0.9 2.1 3.2 4.1
Compound (CH3)4Si (CH3)3P (CH3)2S CH3Cl
Δ 0.0 0.9 2.1 3.0
Proton Chemical Shifts (ppm)
Cpd. / Sub. X=Cl X=Br X=I X=OR X=SR
CH3X 3.0 2.7 2.1 3.1 2.1
CH2X2 5.3 5.0 3.9 4.4 3.7
CHX3 7.3 6.8 4.9 5. 
The general distribution of proton chemical shifts associated with different functional groups is summarized in the following chart. Bear in mind that these ranges are approximate, and may not encompass all compounds of a given class. Note also that the ranges specified for OH and NH protons (colored orange) are wider than those for most CH protons. This is due to hydrogen bonding variations at different sample concentrations.
Hydroxyl Proton Exchange and the Influence of Hydrogen Bonding
The last two compounds in the lower row are alcohols. The OH proton signal is seen at 2.37 δ in 2-methyl-3-butyne-2-ol, and at 3.87 δ in 4-hydroxy-4-methyl-2-pentanone, illustrating the wide range over which this chemical shift may be found. A six-membered ring intramolecular hydrogen bond in the latter compound is in part responsible for its low field shift, and will be shown by clicking on the hydroxyl proton. We can take advantage of rapid OH exchange with the deuterium of heavy water to assign hydroxyl proton resonance signals . As shown in the following equation, this removes the hydroxyl proton from the sample and its resonance signal in the NMR spectrum disappears. Experimentally, one simply adds a drop of heavy water to a chloroform-d solution of the compound and runs the spectrum again. The result of this exchange is displayed below. 
R-O-H   +   D2O   →  R-O-D   +   D-O-H

Hydrogen bonding shifts the resonance signal of a proton to lower field ( higher frequency. Numerous experimental observations support this statement, and a few of these will be described here.
i)   The chemical shift of the hydroxyl hydrogen of an alcohol varies with concentration. Very dilute solutions of 2-methyl-2-propanol, (CH3)3COH, in carbon tetrachloride solution display a hydroxyl resonance signal having a relatively high-field chemical shift (< 1.0 δ ). In concentrated solution this signal shifts to a lower field, usually near 2.5 δ. 
ii)   The more acidic hydroxyl group of phenol generates a lower-field resonance signal, which shows a similar concentration dependence to that of alcohols. OH resonance signals for different               percent concentrations of phenol in chloroform-d are shown in the following diagram (C-H signals are not shown).
iii)   Because of their favored hydrogen-bonded dimeric association, the hydroxyl proton of carboxylic acids displays a resonance signal significantly down-field of other functions. For a typical acid it appears from 10.0 to 13.0 δ and is often broader than other signals. The spectra shown below for chloroacetic acid (left) and 3,5-dimethylbenzoic acid (right) are examples. 
iv)   Intramolecular hydrogen bonds, especially those defining a six-membered ring, generally display a very low-field proton resonance. The case of 4-hydroxypent-3-ene-2-one (the enol tautomer of 2,4-pentanedione) not only illustrates this characteristic, but also provides an instructive example of the sensitivity of the NMR experiment to dynamic change. In the NMR spectrum of the pure liquid, sharp signals from both the keto and enol tautomers are seen, their               
mole ratio being 4 : 21 (keto tautomer signals are colored purple). Chemical shift assignments for these signals are shown in the shaded box above the spectrum. The chemical shift of the hydrogen-bonded hydroxyl proton is δ 14.5, exceptionally downfield. We conclude, therefore, that the rate at which these tautomers interconvert is slow compared with the inherent time scale of NMR spectroscopy.

Two structurally equivalent structures may be drawn for the enol tautomer (in magenta brackets). If these enols were slow to interconvert, we would expect to see two methyl resonance signals associated with each, one from the allylic methyl and one from the methyl ketone. Since only one strong methyl signal is observed, we must conclude that the interconversion of the enols is very fast-so fast that the NMR experiment detects only a single time-averaged methyl group (50% α-keto and 50% allyl).
Although hydroxyl protons have been the focus of this discussion, it should be noted that corresponding N-H groups in amines and amides also exhibit hydrogen bonding NMR shifts, although to a lesser degree. Furthermore, OH and NH groups can undergo rapid proton exchange with each other; so if two or more such groups are present in a molecule, the NMR spectrum will show a single signal at an average chemical shift. For example, 2-hydroxy-2-methylpropanoic acid, (CH3)2C(OH)CO2H, displays a strong methyl signal at δ 1.5 and a 1/3 weaker and broader 

OH signal at δ 7.3 ppm. Note that the average of the expected carboxylic acid signal and the alcohol signal is 7. Rapid exchange of these hydrogens with heavy water, as noted above, would cause the low field signal to disappear.
Hydrogen Bonding Influences                                                                                      
Hydrogen bonding of hydroxyl and amino groups not only causes large variations in the chemical shift of the proton of the hydrogen bond, but also influences its coupling with adjacent C-H groups. As shown on the right, the 60 MHz proton NMR spectrum of pure (neat) methanol exhibits two signals, as expected. At 30° C these signals are sharp singlets located at δ 3.35 and 4.80 ppm, the higher-field methyl signal (magenta) being three times as strong as the OH signal (orange) at lower field. When cooled to -45 ° C, the larger higher-field signal changes to a doublet (J = 5.2 Hz) having the same chemical shift. The smaller signal moves downfield to δ 5.5 ppm and splits into a quartet (J = 5.2 Hz). The relative intensities of the two groups of signals remains unchanged. This interesting change in the NMR spectrum, which will be illustrated by clicking the "Cool the Sample" button, is due to increased stability of hydrogen bonded species at lower temperature. Since hydrogen bonding not only causes a resonance shift to lower field, but also decreases the rate of intermolecular proton exchange, the hydroxyl proton remains bonded to the alkoxy group for a sufficient time to exert its spin coupling influence.
Under routine conditions, rapid intermolecular exchange of the OH protons of alcohols often prevents their coupling with adjacent hydrogens from being observed. Intermediate rates of proton exchange lead to a broadening of the OH and coupled hydrogen signals, a characteristic that is useful in identifying these functions. Since traces of acid or base catalyze this hydrogen exchange, pure compounds and clean sample tubes must be used for experiments of the kind described here.
Another way of increasing the concentration of hydrogen bonded methanol species is to change the solvent from chloroform-d to a solvent that is a stronger hydrogen bond acceptor. Examples of such solvents are given in the following table. In contrast to the neat methanol experiment described above, very dilute solutions are used for this study. Since chloroform is a poor hydrogen bond acceptor and the dilute solution reduces the concentration of methanol clusters, the hydroxyl proton of methanol generates a resonance signal at a much higher field than that observed for the pure alcohol. Indeed, the OH resonance signal from simple alcohols in dilute chloroform solution is normally found near δ 1.0 ppm.
The exceptionally strong hydrogen bond acceptor quality of DMSO is demonstrated here by the large downfield shift of the methanol hydroxyl proton, compared with a slight upfield shift of the methyl signal. The expected spin coupling patterns shown above are also observed in this solvent. Although acetone and acetonitrile are better hydrogen-bond acceptors than chloroform, they are not as effective as DMSO.
1H Chemical Shifts of Methanol in Selected Solvents 
Solvent CDCl3 CD3COCD3 CD3SOCD3 CD3C≡N
CH3–O–H
CH3
O–H 3.40 
1.10 3.31 
3.12 3.16 
4.01 3.28 
2.16
The solvent effect shown above suggests a useful diagnostic procedure for characterizing the OH resonance signals from alcohol samples. For example, a solution of ethanol in chloroform-d displays the spectrum shown on the left below, especially if traces of HCl are present (otherwise broadening of the OH and CH2 signals occurs). Note that the chemical shift of the OH signal (red) is less than that of the methylene group (blue), and no coupling of the OH proton is                                 apparent. The vicinal coupling (J = 7 Hz) of the methyl and methylene hydrogens is typical of ethyl groups. In DMSO-d6 solution small changes of chemical shift are seen for the methyl and methylene group hydrogens, but a dramatic downfield shift of the hydroxyl signal takes place because of hydrogen bonding. 

Coupling of the OH proton to the adjacent methylene group is evident, and both the coupling constants can be measured. Because the coupling constants are different, the methylene signal pattern is an overlapping doublet of quartets (eight distinct lines) rather than a quintet. Note that residual hydrogens in the solvent give a small broad signal near δ 2.5 ppm.
For many alcohols in dilute chloroform-d solution, the hydroxyl resonance signal is often broad and obscured by other signals in the δ 1.5 to 3.0 region. The simple technique of using DMSO-d6 as a solvent, not only shifts this signal to a lower field, but permits 1°-, 2 °- & 3 °-alcohols to be distinguished. Thus, the hydroxyl proton of 2-propanol generates a doublet at δ 4.35 ppm, and the corresponding signal from 2-methyl-2-propanol is a singlet at δ 4.2 ppm. The more acidic OH protons of phenols are similarly shifted – from δ 4 to 7 in chloroform-d to δ 8.5 to 9.5 in DMSO-d6.
π-Electron Functions
An examination of the proton chemical shift chart (above) makes it clear that the inductive effect of substituents cannot account for all the differences in proton signals. In particular the low field resonance of hydrogens bonded to double bond or aromatic ring carbons is puzzling, as is the very low field signal from aldehyde hydrogens. The hydrogen atom of a terminal alkyne, in contrast, appears at a relatively higher field. All these anomalous cases seem to involve hydrogens bonded to pi-electron systems, and an explanation may be found in the way these pi-electrons interact with the applied magnetic field.Pi-electrons are more polarizable than are sigma-bond electrons, as addition reactions of electrophilic reagents to alkenes testify. Therefore, we should not be surprised to find that field induced pi-electron movement produces strong secondary fields that perturb nearby nuclei. The pi-electrons associated with a benzene ring provide a striking example of this phenomenon, as shown below. The electron cloud above and below the plane of the ring circulates in reaction to the external field so as to generate an opposing field at the center of the ring and a supporting field at the edge of the ring. This kind of spatial variation is called anisotropy, and it is common to nonspherical distributions of electrons, as are found in all the functions mentioned above. Regions in which the isupports or adds to the external field are said to be deshielded, because a slightly weaker                             
External field will bring about resonance for nuclei in such areas. 

However, regions in which the induced field opposes the external field are termed shielded because an increase in the applied field is needed for resonance. Shielded regions are designated by a plus sign, and deshielded                        regions by a negative sign.The anisotropy of some important unsaturated functions will be displayed by clicking on the benzene diagram below. Note that the anisotropy about the triple bond nicely accounts for the relatively high field chemical shift of ethynyl hydrogens. The shielding & deshielding regions about the carbonyl group have been described in two ways, which alternate in the display.

 Examples of Anisotropy Influences on Chemical Shift
The compound on the left has a chain of ten methylene groups linking para carbons of a benzene ring. Such bridged benzenes are called paracyclophanes. The meta analogs are also known. The structural constraints of the bridging chain require the middle two methylene groups to lie over the face of the benzene ring, which is a NMR shielding region. The four hydrogen atoms that are part of these groups display resonance signals that are more than two ppm higher field than the two methylene groups bonded to the edge of the ring (a deshielding region). 
The following general rules summarize important requirements and characteristics for spin 1/2 nuclei :
1)   Nuclei having the same chemical shift (called isochronous) do not exhibit spin-splitting. They may actually be spin-coupled, but the splitting cannot be observed directly.
                                                                                                                                                                       2)   Nuclei separated by three or fewer bonds will usually be spin-coupled and will show mutual spin-splitting of the resonance signals, provided they have different chemical shifts. Longer-range coupling may be observed in molecules having rigid configuration of atoms .
3)   The magnitude of the observed spin-splitting depends on many factors and is given by the coupling constant J (units of Hz). J is the same for both partners in a spin-splitting interaction and is independent of the external magnetic field strength 
4)   The splitting pattern of a given nucleus can be predicted by the n+1 rule, where n is the number of neighboring spin-coupled nuclei with the same (or very similar) Js. If there are 2 neighboring, spin-coupled, nuclei the observed signal is a triplet (2+1=3); if there are three spin-coupled neighbors the signal is a quartet (3+1=4). In all cases the central line(s) of the splitting pattern are stronger than those on the periphery. The intensity ratio of these lines is given by the numbers in Pascal's triangle. Thus a doublet has 1:1 or equal intensities, a triplet has an intensity ratio of 1:2:1, a quartet 1:3:3:1 etc.
4. Theoretical principles
Nuclear Magnetic Resonance spectroscopy is a powerful and theoretically complex analytical tool. On this page, we will cover the basic theory behind the technique. It is important to remember that, with NMR, we are performing experiments on the nuclei of atoms, not the electrons. The chemical environment of specific nuclei is deduced from information obtained about the nuclei.

Nuclear spin and the splitting of energy levels in a magnetic field
Subatomic particles (electrons, protons and neutrons) can be imagined as spinning on their axes. In many atoms (such as 12C) these spins are paired against each other, such that the nucleus of the atom has no overall spin. However, in some atoms (such as 1H and 13C) the nucleus does possess an overall spin. The rules for determining the net spin of a nucleus are as follows; 
• If the number of neutrons and the number of protons are both even, then the nucleus has NO spin. 
• If the number of neutrons plus the number of protons is odd, then the nucleus has a half-integer spin (i.e. 1/2, 3/2, 5/2) 
• If the number of neutrons and the number of protons are both odd, then the nucleus has an integer spin (i.e. 1, 2, 3) 

The overall spin, I, is important. Quantum mechanics tells us that a nucleus of spin I will have 2I + 1 possible orientations. A nucleus with spin 1/2 will have 2 possible orientations. In the                                       absence of an external magnetic field, these orientations are of equal energy.                                                 If a magnetic field is applied, then the energy levels split. Each level is given a magnetic quantum number, m.
                                                                                                                                                                         
When the nucleus is in a magnetic field, the initial populations of the energy levels are determined by thermodynamics, as described by the Boltzmann distribution. This is very important, and it means that the lower energy level will contain slightly more nuclei than the higher level. It is possible to excite these nuclei into the higher level with electromagnetic radiation. The frequency of radiation needed is determined by the difference in energy between the energy levels.
 Calculating transition energy
The nucleus has a positive charge and is spinning. This generates a small magnetic field. The nucleus therefore possesses a magnetic moment, μ, which is proportional to its spin,I.

The constant, γ, is called the magnetogyric ratio and is a fundamental nuclear constant which has a different value for every nucleus. h is Plancks constant .The energy of a particular energy level is given by; 

Where B is the strength of the magnetic field at the nucleus.
The difference in energy between levels (the transition energy) can be found from

This means that if the magnetic field, B, is increased, so is ΔE. It also means that if a nucleus has a relatively large magnetogyric ratio, then ΔE is correspondingly large.
If you had trouble understanding this section, try reading the next bit (The absorption of radiation by a nucleus in a magnetic field) and then come back. 

The absorption of radiation by a nucleus in a magnetic field
In this discussion, we will be taking a "classical" view of the behaviour of the nucleus - that is, the behavior of a charged particle in a magnetic field. Imagine a nucleus (of spin 1/2) in a magnetic field.                                                                                                                                   
                                                                                                                                                                 This nucleus is in the lower energy level (i.e. its magnetic moment does not oppose the applied field). The nucleus is spinning on its axis. In the presence of a magnetic field, this axis of rotation will precess around the magnetic field;
The frequency of precession is termed the Larmor frequency, which is identical to the transition frequency.
The potential energy of the precessing nucleus is given by;
E = - B cos 
Where  is the angle between the direction of the applied field and the axis of nuclear rotation.
If energy is absorbed by the nucleus, then the angle of precession, , will change. For a nucleus of spin 1/2, absorption of radiation "flips" the magnetic moment so that it opposes the applied field (the higher energy state).
It is important to realise that only a small proportion of "target" nuclei are in the lower energy state (and can absorb radiation). There is the possibility that by exciting these nuclei, the populations of the higher and lower energy levels will become equal. If this occurs, then there will be no further absorption of radiation. The spin system is saturated. The possibility of saturation means that we must be aware of the relaxation processes which return nuclei to the lower energy state.
Relaxation processes

How do nuclei in the higher energy state return to the lower state? Emission of radiation is insignificant because the probability of re-emission of photons varies with the cube of the frequency. At radio frequencies, re-emission is negligible. We must focus on non-radiative relaxation processes (thermodynamics!). 
Ideally, the NMR spectroscopist would like relaxation rates to be fast - but not too fast. If the relaxation rate is fast, then saturation is reduced. If the relaxation rate is too fast, line-broadening in the resultant NMR spectrum is observed.
There are two major relaxation processes; 
• Spin - lattice (longitudinal) relaxation 
• Spin - spin (transverse) relaxation 
Spin- lattice relaxation
Nuclei in an NMR experiment are in a sample. The sample in which the nuclei are held is called the lattice. Nuclei in the lattice are in vibration and rotational motion, which creates a complex magnetic field. The magnetic field caused by motion of nuclei within the lattice is called the lattice field. This lattice field has many components. Some of these components will be equal in frequency and phase to the Larmor frequency of the nuclei of interest. These components of the lattice field can interact with nuclei in the higher energy state, and cause them to lose energy (returning to the lower state). The energy that a nucleus loses increases the amount of vibration and rotation within the lattice (resulting in a tiny rise in the temperature of the sample).
The relaxation time, T1 (the average lifetime of nuclei in the higher energy state) is dependent on the magnetogyric ratio of the nucleus and the mobility of the lattice. As mobility increases, the vibration and rotational frequencies increase, making it more likely for a component of the lattice field to be able to interact with excited nuclei. However, at extremely high mobilities, the probability of a component of the lattice field being able to interact with excited nuclei decreases.
Splin-splin relaxation
Spin - spin relaxation describes the interaction between neighboring nuclei with identical precessional frequencies but differing magnetic quantum states. In this situation, the nuclei can exchange quantum states; a nucleus in the lower energy level will be excited, while the excited nucleus relaxes to the lower energy state. There is no net change in the populations of the energy states, but the average lifetime of a nucleus in the excited state will decrease. This can result in line-broadening. 

Spin - spin coupling
Consider the structure of ethanol;

The 1H NMR spectrum of ethanol (below) shows the methyl peak has been split into three peaks (a triplet) and the methylene peak has been split into four peaks (a quartet). This occurs because there is a small interaction (coupling) between the two groups of protons. The spacings between the peaks of the methyl triplet are equal to the spacings between the peaks of the methylene quartet. This spacing is measured in Hertz and is called the coupling constant, J.

To see why the methyl peak is split into a triplet, let's look at the methylene protons. There are two of them, and each can have one of two possible orientations (aligned with or opposed against 
the applied field).
 This gives a total of four possible states;
In the first possible combination, spins are paired and opposed to the field. This has the effect of reducing the field experienced by the methyl protons; therefore a slightly higher field is needed to bring them to resonance, resulting in an upfield shift. Neither combination of spins opposed to each other has an effect on the methyl peak. The spins paired in the direction of the field produce a downfield shift. Hence, the methyl peak is split into three, with the ratio of areas 1:2:1.
Similarly, the effect of the methyl protons on the methylene protons is such that there are eight possible spin combinations for the three methyl protons;

Out of these eight groups, there are two groups of three magnetically equivalent combinations. The methylene peak is split into a quartet. The areas of the peaks in the quartet have the ration 1:3:3:1.
In a first-order spectrum (where the chemical shift between interacting groups is much larger than their coupling constant), interpretation of splitting patterns is quite straightforward; 
• The multiplicity of a multiplet is given by the number of equivalent protons in neighboring atoms plus one, i.e. the n + 1 rule
• Equivalent nuclei do not interact with each other. The three methyl protons in ethanol cause splitting of the neighboring methylene protons; they do not cause splitting among themselves
• The coupling constant is not dependant on the applied field. Multiplets can be easily distinguished from closely spaced chemical shift peaks. 
5. Different spectroscopy
5.1 Carbon NMR Spectroscopy
The power and usefulness of 1H NMR spectroscopy as a tool for structural analysis should be evident from the past discussion. Unfortunately, when significant portions of a molecule lack C-H bonds, no information is forthcoming. Examples include polychlorinated compounds such as chlordane, polycarbonyl compounds such as croconic acid, and compounds incorporating triple bonds (structures below, orange colored carbons).

Even when numerous C-H groups are present, an unambiguous interpretation of a proton NMR spectrum may not be possible. The following diagram depicts three pairs of isomers (A & B) which display similar proton NMR spectra. Although a careful determination of chemical shifts should permit the first pair of compounds (blue box) to be distinguished, the second and third cases might be difficult to identify by proton NMR alone.

These difficulties would be largely resolved if the carbon atoms of a molecule could be probed by NMR in the same fashion as the hydrogen atoms. Since the major isotope of carbon (12C) has no spin, this option seems unrealistic. Fortunately, 1.1% of elemental carbon is the 13C isotope, which has a spin I = 1/2, so in principle it should be possible to conduct a carbon NMR experiment. It is worth noting here, that if much higher abundances of 13C were naturally present in all carbon compounds, proton NMR would become much more complicated due to large one-bond coupling of 13C and 1H.
Many obstacles needed to be overcome before carbon NMR emerged as a routine tool :
1)  As noted, the abundance of 13C in a sample is very low (1.1%), so higher sample                  concentrations are needed.
2)  The 13C nucleus is over fifty times less sensitive than a proton in the NMR experiment, adding to the previous difficulty.                                                                                         
3)  Hydrogen atoms bonded to a 13C atom split its NMR signal by 130 to 270 Hz, further complicating the NMR spectrum.

The most important operational technique that has led to successful and routine 13C NMR spectroscopy is the use of high-field pulse technology coupled with broad-band heteronuclear decoupling of all protons. The results of repeated pulse sequences are accumulated to provide improved signal strength. Also, for reasons that go beyond the present treatment, the decoupling irradiation enhances the sensitivity of carbon nuclei bonded to hydrogen. 
When acquired in this manner, the carbon NMR spectrum of a compound displays a single sharp signal for each structurally distinct carbon atom in a molecule (remember, the proton couplings have been removed). The spectrum of camphor, shown on the left below, is typical. Furthermore, a comparison with the 1H NMR spectrum on the right illustrates some of the advantageous characteristics of carbon NMR. The dispersion of 13C chemical shifts is nearly twenty times greater than that for protons, and this together with the lack of signal splitting makes it more likely that every structurally distinct carbon atom will produce a separate signal. The only clearly identifiable signals in the proton spectrum are those from the methyl groups. The remaining protons have resonance signals between 1.0 and 2.8 ppm from TMS, and they overlap badly thanks to spin-spin splitting.

5.2 Proton NMR Spectroscopy

This important and well-established application of nuclear magnetic resonance will serve to illustrate some of the novel aspects of this method. To begin with, the NMR spectrometer must be tuned to a specific nucleus, in this case the proton. The actual procedure for obtaining the spectrum varies, but the simplest is referred to as the continuous wave (CW) method. A typical CW-spectrometer is shown in the following diagram. A solution of the sample in a uniform 5 mm glass tube is oriented between the poles of a powerful magnet, and is spun to average any magnetic field variations, as well as tube imperfections. Radio frequency radiation of appropriate energy is broadcast into the sample from an antenna coil (colored red). A receiver coil surrounds the sample tube, and emission of absorbed rf energy is monitored by dedicated electronic devices and a computer. An NMR spectrum is acquired by varying or sweeping the magnetic field over a small range while observing the rf signal from the sample. An equally effective technique is to vary the frequency of the rf radiation while holding the external field constant.

As an example, consider a sample of water in a 2.3487 T external magnetic field, irradiated by 100 MHz radiation. If the magnetic field is smoothly increased to 2.3488 T, the hydrogen nuclei of the water molecules will at some point absorb rf energy and a resonance signal will appear. 

An animation showing this may be activated by clicking the Show Field Sweep button. The field sweep will be repeated three times, and the resulting resonance trace is colored red. For visibility, the water proton signal displayed in the animation is much broader than it would be in an actual experiment.

Since protons all have the same magnetic moment, we might expect all hydrogen atoms to give resonance signals at the same field / frequency values. Fortunately for chemistry applications, this is not true. By clicking the Show Different Protons button under the diagram, a number of representative proton signals will be displayed over the same magnetic field range. It is not possible, of course, to examine isolated protons in the spectrometer described above; but from independent measurement and calculation it has been determined that a naked proton would resonate at a lower field strength than the nuclei of covalently bonded hydrogens. With the exception of water, chloroform and sulfuric acid, which are examined as liquids, all the other compounds are measured as gases.
Why should the proton nuclei in different compounds behave differently in the NMR experiment ? 

The answer to this question lies with the electron(s) surrounding the proton in covalent compounds and ions. Since electrons are charged particles, they move in response to the external magnetic field (Bo) so as to generate a secondary field that opposes the much stronger applied field. This secondary field shields the nucleus from the applied field, so Bo must be increased in order to achieve resonance (absorption of rf energy). As illustrated in the drawing on the right, Bo must be increased to compensate for the induced shielding field. In the upper diagram, those compounds that give resonance signals at the higher field side of the diagram (CH4, HCl, HBr and HI) have proton nuclei that are more shielded than those on the lower field (left) side of the diagram. 
The magnetic field range displayed in the above diagram is very small compared with the actual field strength (only about 0.0042%). It is customary to refer to small increments such as this in units of parts per million (ppm). The difference between 2.3487 T and 2.3488 T is therefore about 42 ppm. Instead of designating a range of NMR signals in terms of magnetic field differences, it is more common to use a frequency scale, even though the spectrometer may operate by sweeping the magnetic field. Using this terminology, we would find that at 2.34 T the proton signals shown above extend over a 4,200 Hz range (for a 100 MHz rf frequency, 42 ppm is 4,200 Hz). Most organic compounds exhibit proton resonances that fall within a 12 ppm range (the shaded area), and it is therefore necessary to use very sensitive and precise spectrometers to resolve structurally distinct sets of hydrogen atoms within this narrow range. In this respect it might be noted that the detection of a part-per-million difference is equivalent to detecting a 1 millimeter difference in distances of 1 kilometer. 

6. APPLICATION OF NMR
Chemical Applications of NMR

1. Structure Diagnosis by NMR or Qualitative Analysis:
 The number of main NMR signals should be equal to the number of equivalent protons in the unknown compound.
 The chemical shift indicate that what type of hydrogen atoms are present e.g. methylene, methyl group, olefins, ethers etc.
 From the area of the peaks ; one can draw the conclusion about the number of hydrogen nuclei; present in each group .For Example the relative area of methyl (CH3) and methelene (CH2) peaks in CH3 –CH2-CH3 would be 6:2. In butane, it would be 6:4.
2. Qantitative Analysis:
 NMR spectroscopy has been used to determine the molar ratio of the component in a mixture.
3. Hydrogen Bonding:
 The NMR can be used to study the hydrogen bonding in metal chelates as well as in organic compounds. Proton signal is shifted towards low field in the case of hydrogen bonding.
4. Structure Determination.
 NMR spectroscopy is very useful in the study of polyethylene which has wide applications in industry.
 NMR spectroscopy has been used to study the ligand isomerisation and disproportionation of some unsymmectrically substituted metal acetylacetonate .
 Structure of SOF6, CIF3 ,HF¬2- can be determine¬.
5. Keto- enol Tautomerism.
 The NMR spectroscopy is very useful in studing Keto- enol tautomerism.
6. Elemental Analysis :
 NMR spectroscopy can be used for the determination of the total concentration of a given kind of magnetic nucleus in a sample.
Petroleum Exploration
Whole Crude Analysis ,Custody Transfer , Produced and Waste Water Analysis , Olefins Analysis.
Food Applications
Butter , Cream Cheese , Sour Cream, Milk, Cheeses, Yogurt ,  Beverages (Juices and Alcoholic)
Baby Food and Soups , Sodium Analysis.
 LNG and Power Industry
BTU Analysis and Limited Speciation of Gas Components.
 Protein Structure Determination
The emphasis has been on identification of the observed signals in the spectra and their correlation with the amino acid protons giving rise to the signals. 

7. LIMITATION OF NMR SPECTROSCOPY

1. One of the serious problems with NMR is its lack of sensitivity. The minimum sample size is about 0.1 ml having minimum concentration of about 1%.

2. In some compounds, two different types of hydrogen atoms resonate at similar resonance frequencies .This results in an overlap of spectra and make such spectra difficult to interpret.

3. While characterizing, the organic compounds, no information about molecular weight is given but the relative number of different protons present are only known.

4. In most of cases, only liquids can be studied by NMR spectroscopy, although polymers, when preheated with various solvents, frequently become fluids which can be treated as liquids.

REFERENCE
1.James Keeler. "Chapter 2: NMR and energy levels" (reprinted at University of Cambridge). Understanding NMR Spectroscopy. University of California, Irvine. http://www-keeler.ch.cam.ac.uk/lectures/Irvine/chapter2.pdf. Retrieved on 2007-05-11. 

2. Martin, G.E; Zekter, A.S., Two-Dimensional NMR Methods for Establishing Molecular Connectivity; VCH Publishers, Inc: New York,

3. "Protein NMR Spectroscopy", J. Cavanagh, W.J. Fairbrother, A.G. Palmer III, N.J. Skelton (Academic Press, 1996
4. Principles of Nuclear Magnetic Resonance in One and Two Dimensions", R.R. Ernst, G. Bodenhausen, A. Wokaun (Clarendon Press, 1987). 
5. "Principles of Magnetic Resonance", C.P. Slichter (Springer-Verlag, 1990). 
6. "High Resolution NMR: Theory and Chemical Applications", E.D. Becker (Academic Press, 1999). 
7. "Experimental Pulsed NMR: A Nuts and Bolts Approach", E. Fukushima, S.B.W. Roeder (Addison, 1981). 
8. "Why just NMR?, R.R. Ernst, Israel Journal of Chemistry 1992, 32, 135-136. 
9. "Solid state NMR: Some personal recollections", A. Pines, Israel Journal of Chemistry 1992, 137-144. 

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