About Authors:
Reshma. K*, M.Muthukumaran*, B.krishnamoorthy, Amreen Nishat
Montessori Siva sivani Institute of Science&Technology- College Of
Pharmacy
Vijayawada, Andhrapradesh-521 230
reshmakaaja@gmail.com
Abstract
Nuclear magnetic resonance (NMR) has progressed rapidly over the last decade as a result of improved experimental technology and development of novel approaches. NMR spectroscopy has evolved into an important technique in support of structure-based drug design. It was most useful as a technique to provide structural information regarding protein drug targets and target–ligand interactions. More recently, it has been shown that NMR may be used as an alternative method for identification of small molecule ligands that bind to protein drug targets. High throughput implementation of these experiments to screen small molecule libraries may lead to identification of potent and novel lead compounds. NMR as a probe of microscopic dynamic behaviour through relaxation and direct diffusion measurements over a wide temperature range is examined.
REFERENCE ID: PHARMATUTOR-ART-2065
Introduction
Nuclear magnetic resonance spectroscopy, most commonly known as NMR spectroscopy, is a research technique that exploits the magnetic properties of certainatomic nuclei to determine physical and chemical properties of atoms or the molecules in which they are contained. It relies on the phenomenon of nuclear magnetic resonanceand can provide detailed information about the structure, dynamics, reaction state, and chemical environment of molecules. Most frequently, NMR spectroscopy is used by chemists and biochemists to investigate the properties of organic molecules, though it is applicable to any kind of sample that contains nuclei possessing spin. Suitable samples range from small compounds analyzed with 1-dimensional proton or carbon-13 NMR spectroscopy to large proteins ornucleic acids using 3 or 4-dimensional techniques. The impact of NMR spectroscopy on the sciences has been substantial because of the range of information and the diversity of samples, including solutions and solids.
Basic NMR Techniques
When placed in a magnetic field, NMR active nuclei (such as 1H or 13C) absorb electromagnetic radiation at a frequency characteristic of the isotope. The resonant frequency, energy of the absorption, and the intensity of the signal are proportional to the strength of the magnetic field. For example, in a 21 Tesla magnetic field, protons resonate at 900 MHz. It is common to refer to a 21 T magnet as a 900 MHz magnet, although different nuclei resonate at a different frequency at this field strength in proportion to their nuclear magnetic moments.
PRINCIPLE
The only nuclei that exhibit the NMR phenomenon are those for which the spin quantum number I is greater than 0: the spin quantum number I is associated with the mass number and atomic 1number of the nuclei as follows:
TABLE 1: SPIN QUANTUM NUMBER
|
Mass number |
Atomic number |
Spin quantum number |
|
Odd |
odd or even |
- |
|
Even |
Even |
0 |
|
even |
odd |
1,2,3 |
I is associated with the mass number and atomic number of the nuclei as follows:
The nucleus of 1H, the proton, has I = , whereas 12C and 16O have I = 0 and are therefore nonmagnetic. If 12C and 16O had been magnetic, the NMR spectra of organic molecules would have been much more complex.
Other important magnetic nuclei that have been studied extensively by NMR are 1B, 13C, 14N and 15N, 17O, 19F and 31P. Both deuterium (2H) and nitrogen - 14 have l = 1, and the consequences of this observation will become apparent later.
Under the influence of an external magnetic field, a magnetic nucleus can take up different orientatiuons with respect to that field; the number of possible orientations is given by (2l + 1), so that for nuclei with spin 1/2(1H, 13C, 19F, etc.) only two orientations are allowed. Deuterium and 14N have l = 1 and so can take up three orientations: these nuclei do not simply possessing electric quadrupoles can interact with both magnetic and electric field gradients, the relative importance of the two effects being related to their magnetic moments and electric quadrupole moments, respectively.[1]
In an applied magnetic field, magnetic nuclei like the proton precess at a frequency v, which is proportional to the strength of the applied field. The exact frequency is given by where Bo = strength of the applied external field experienced by the proton g = magnetogyric ratio, being the ratio between the nuclear magnetic moment, m, and the nuclear angular momentum, I: g is also called the gyromagnetic ratio.Typical approximate values for v are shown in Table for selected values of field strength Bo, for common magnetic nuclei.
Table 2: Precessional frequencies (in MHz)
|
Bo/tesla |
1.4 |
1.9 |
2.3 |
4.7 |
7.1 |
11.7 |
14.1 |
|
Nucleus |
|
|
|
|
|
|
|
|
1H |
60 |
80 |
100 |
200 |
300 |
500 |
600 |
|
2H |
9.2 |
12.3 |
15.3 |
30.6 |
46 |
76.8 |
92 |
|
11B |
19.2 |
25.6 |
32 |
64.2 |
96.9 |
159.8 |
192 |
|
13C |
15.1 |
20.1 |
25.1 |
50.3 |
75.5 |
125.7 |
151 |
|
15N |
6.1 |
8.1 |
10.1 |
20.3 |
30.4 |
50.7 |
61 |
|
17O |
8.1 |
10.8 |
13.6 |
27.1 |
40.7 |
67.8 |
81 |
|
Free electron |
3.9 ´ 104 |
|
|
|
|
|
|
The strength of the signal, and, hence, the sensitivity of the NMR experiment for a particular nucleus, are related to the magnitude of the magnetic moment, m. The magnetic moments of 1H and 19F are relatively large, and detection of NMR with these nuclei is fairly sensitive. The magnetic moment for 13C is about one-quarter that of 1H; these nuclei are less sensitively detected in NMR. (In contrast, the magnetic moment of the free electron is nearly 700 times that of 1H, and resonance phenomena for free radicals can be studied in extremely dilute solutions).[2]
Nuclei in the lower energy state undergo transitions to the higher energy state; the populations of the two states may approach equality, and if this arises, no further net absorption of energy can occur and the observed resonance signal will fade out. We describe this situation in practice as saturation of the signal. In the recording of a normal NMR spectrum, however, the populations in the two spin states do not become equal, because higher energy nuclei are constantly returning to the lower energy spin state.[3]
The nuclei loss energy - by two radiation less process:
a) spin lattice or longitudinal relaxation process - where the energy is lost by means of translational/vibrational/rotational energy.
b) spin or transverse relaxation process - where the energy is lost to the neighbouring nuclei
Spin lattice relaxation = T1
Spin-spin relaxation = T2
If T1 and T2 are small we will get broad peaks and if T1 and T2 are large (one second order) sharp peaks are obtained.
Sample Preparation
NMR samples are prepared by dissolving an analyte in a deuterium lock solvent. Several deuterium lock solvents are available. Some of these solvents will readily absorb moisture from the atmosphere and give water signal in your spectrum. It is therefore advisable to keep bottles of these solvents tightly capped when not in use.
Most routine high resolution NMR samples are prepared and run in 5 mm glass NMR tubes. Always fill your NMR tubes to the same height with lock solvent. This will minimize the amount of magnetic field shimming required. The animation window depicts a sample tube filled with solvent such that it fills the RF coil.
Sample Probe Tuning
Variations in the polarity and dielectric constant of the lock solvent will affect the probe tuning. For this reason the probe should be tuned whenever the lock solvent is changed. Tuning the probe entails adjusting two capacitors on the RF probe. One capacitor is called the matching capacitor and the other the tuning capacitor. The matching capacitor matches the impedance of the loaded probe to that of the 50 Ohm cable coming from the spectrometer. The tuning capacitor changes the resonance frequency of the RF coil.
Determinining a 90o Pulse
As pointed out in the previous section of this chapter, changes in the polarity and dielectric constant of the lock solvent affect the bandwidth of the RF probe which in turn affects the amount of RF power needed to produce a 900 rotation. Most NMR spectrometers will not allow you to change the RF power, but they will permit you to change the pulse length. Therefore, if the bandwidth of the RF probe increases, you will need to increase the RF pulse width to produce a 900 pulse
Field Shimming
The purpose of shimming a magnet is to make the magnetic field more homogeneous and to obtain better spectral resolution. Shimming can be performed manually or by computer control. It is not the intent of this section to teach you a step-by-step procedure for shimming, but to present you with the basic theory so that you can, with the aid of your NMR instruction manual, shim your magnet. The reader is encouraged to write down or save the current shim settings before making changes to any of the current shims coil settings.
Phase Cycling
There are a few artifacts of the detection circuitry which may appear in your spectrum if you record a single FID and Fourier transform it. Phase cycling is the technique used to eliminate these artifacts
TYPES OF NMR SPECTROSCOPY
Continuous wave NMR instruments
Continuous wave NMR spectrometers are similar in principle to optical spectrometers. The sample is held in a strong magnetic field, and the frequency of the source is slowly scanned (in some instruments, the source frequency is held constant, and the field is scanned).
Fourier transform NMR instruments
The magnitude of the energy changes involved in NMR spectroscopy are small. This means that sensitivity is a major limitation. One way to increase sensitivity would be to record many spectra, and then add them together; because noise is random, it adds as the square root of the number of spectra recorded. For example, if one hundred spectra of a compound were recorded and summed, then the noise would increase by a factor of ten, but the signal would increase in magnitude by a factor of one hundred - giving a large increase in sensitivity. However, if this is done using a continuous wave instrument, the time needed to collect the spectra is very large (one scan takes two to eight minutes).
In FT-NMR, all frequencies in a spectrum are irradiated simultaneously with a radio frequency pulse. Following the pulse, the nuclei return to thermal equilibrium. A timedomain emission signal is recorded by the instrument as the nuclei relax. A frequency domain spectrum is obtained by Fourier transformation.
Proton NMR Spectroscopy
All protons in an organic molecule, at a given radiofrequency, may give NMR signals at different applied field strengths. It is this applied field strength that is measured and against which the absorption is plotted. As a result, an NMR spectrum is obtained which shows many absorption signals or peaks, whose relative positions give vital information regarding the molecular structure. By studying a compound by proton NMR spectroscopy, there are various important types of information that can be obtained.[4]The splitting of a signal into several peaks or the hyperfine structure tell us the number of protons in the adjacent positions (ie., environment of a proton w.r.t other nearby protons). Usually the (n + 1) rule operates for proton-proton influence where the presence of n protons in the neighbouring position splits the main peak into (n + 1) components.
Greater is the electronegativity of the atom, greater is the deshielding caused to proton. Thus deshielding of proton attached to different halide groups decreases in the order HC–F> HC–Cl > HC – Br > HC – I, because electronegativity decreases in the order F > Cl > Br > I.
As the distance of the proton from electronegative atom increases, the deshielding effect due to it decreases. For example,
(1) CH3 - Cl (2) CH3 - C - Cl
d 3.0 d 1.5
In (2), the distance of electronegative atom increases and hence CH3 proton in (2) experiences less deshielding in comparison to CH3 proton in 1. This is also evident from their d values. Chemical shifts are measured with reference to a standard, tetramethyl silane (TMS).
The t (Tau scale) and the d (delta) scale are commonly used to describe chemical shifts in terms of ppm. On d-scale, the position of TMS signal is taken as 1.0 ppm, while on t scale TMS signal is taken to appear at 10.0 ppm. Most chemical shifts lie between 0 and 10 ppm and two scales are related as
t = 10 – d.
This forms the basis of the use of NMR spectroscopy in detecting aromaticity. The hydrogens which are in the same or identical environment have the same chemical shift. Thus, these absorb at the same frequency and called as chemically equivalent. Protons are equivalent if these are bonded to the same carbon which can freely rotate. For example, three protons of – CH3 group are equivalent. Similarly, the two protons of free rotating methylene group (-CH2 group) are also identical. Those protons on different carbon atoms are also chemically equivalent, if these are structurally not distinguishable. In p-xylene, eg., the six methyl hydrogens are equivalent. Similarly four aromatic hydrogens are also equivalent because of identical environment.[5]
The methylene protons of a freely rotating methylene group when adjacent to a chiral centre are not equivalent and such protons are called diastereotopic. The same is true for any pair of identical groups such as – C (CH3)2. The protons of methyle group in a cyclic system where rotation is restricted are not identical. Disasterotopic protons can be distinguished by NMR spectroscopy because these are not equivalent. Enantiotopic protons can not be distinguished by NMR spectroscopy, this being an achiral technique. The non equivalent protons are known as accidentially equivalent when these absorb at the same chemical shift. The protons with the same chemical shift do not split each other.
Terminal alkene hydrogens appear at higher fields (d = 4.6 – 5.0 ppm) than their internal counterparts (d = 5.2 – 5.9ppm). The electron density in the vicinity of alkenic protons is influenced by the electron withdrawing nature of carbonyl group, eg., in a, b-unsaturated carbonyl compounds.
The molecular field surrounded by an aldehyde proton is affected by the combined effects of an electronegative oxygen of carbonyl group and p electrons of carbonyl group. This proton is sufficiently shielded. The deshielding of external aromatic protons which occurs from the circulating p electrons (ring current) is most important evidence to show p electron delocalization in benzene rigns. Low field strength, proton absorption, is this used as an evidence for aromaticity in conjugated cyclic systems which obey Huckel's (4n + 2) p electron rule.
Spin spin splitting or magnetic coupling is the interaction of the magnetic fields of two or more nuclei, both through their connecting bonds are space. Spin spin splitting causes NMR signals to be split and to appear as two or more peaks, ie., as a multiplet. A signal that is being split by n equivalent protons appears as multiplet with n + 1 individual peaks, and is known as n + 1 rule. There is no spin spin splitting of chemical shift equivalent hydrogens. The magnitude of the coupling constant or magnitude of the separation of peaks depends on the link between the interacting protons.
Non equivalent protons mutually split each other. Thus the presence of one split absorption necessitates the presence of another split signal in the spectrum, provided the coupling constants for these patterns are same. Spin spin splitting is generally observed between hydrogens which are immediate neighbours, that is either bound to the same carbon (geminal coupling) or to two adjacent carbons (vicinal coupling). The magnitude of coupling constant J depends on the link between the interacting protons and decreases rapidly as the number of interconnecting bonds increases.[6]
INTERPRETATION
The protons of each of the two aliphatic CH2 are diastereotopic at about d 1.28, 1.42, 2.21, and 2.48 ppm. Most of the protons can be assigned on the basis of chemical shifts, integration ratios, and coupling patterns as follows.
The entry points for analysis of he spectrum are protons that have distinctive chemical shifts and/ or coupling such as the methyl groups just discussed. The proton multiplet at d 3.82 ppm must be the deshielded CHOH proton that is coupled to two sets of diasterotropic protons. If all couplings were equal, the multiplicity would be 5; obviously they are not equal.[7]
The conjugated olefinic protons are distinctively at the deshielded end of the spectrum. The isolated =CH2 group is at d 5.08 ppm. The CH=CH2 moiety accounts for the remainder of the patterns with the CH= protons centred at about d 6.40 and the =CH2 protons centred AMX system in which the AM coupling is barely detectable at the expansion shown.
At the shielded end of the spectrum, from right to left, we see the identified diastereotopic methyl groups, each of the H-3 diastereotopic protons, a mysterious two-proton multiplet at d 1.8, and the individual H-5 protons, each of which consists of the highly coupled H-2 protons superimposed on the broad OH absorption. The CH, CH2, and CH3 peaks thus identified are confirmed by the 13C/DEPT spectrum.
CARBON-13 NMR SPECTROSCOPY (CMR)
(a) The natural abundance of 13C is only 1.1% that of 12C, which is not detectable by NMR, since 12C is isotope has an even number of protons and even number of neutrons and hence no magnetic spin (I = 0). The less abundandant isotope 13C, has an odd number of neutrons and as a result it has a magnetic spin of 1/2, but its sensitivity is only about 1.6% that of 1H. The overall sensitivity of 13C compared with 1H is about 1/5700. This sensitivity is so low that unaided standard NMR spectrometers are not adequate for its study.[8]
13C NMR or CMR spectra are much more difficult to record than 1H spectra because, the most abundant isotope of carbon, 12C is not detectable by NMR, as this isotope has even number of protons and even number of neutrons (no magnetic spin). 13C is less abundant isotope (1.1%) and has an odd number of neutrons (magnetic spin I = 1/2). The magnetic resonance of 13C is much weaker, because of its 1.1% abundance (1.1%of carbon atoms in a sample are magnetically active). The gyromagnetic ratio of 13C is only about 25% that of a proton as the 13C resonance frequency is only one fourth of that for PMR at a given magnetic field. These reasons indicate that 13 CMR or CMR is less sensitive than PMR or NMR.
INTERPRETATIONS
Peak at d22,23 ppm are of 1st carbon & C of –CH3 on C2
Peak at d25 ppm is 2nd carbon
Peak at d40.5 ppm is 5th carbon
Peak at d47 ppm is 3rd carbon
Peak at d68 ppm is 4th carbon
Peak at d116 ppm is 8th carbon
Peak at d120 ppm is the isolated –CH2- carbon
Peak at d139 ppm is 7th carbon
Peak at d143 ppm is 6th carbon
APPLICATIONS
* Structure elucidation of organic compounds
a. Types of atoms (1H, 13C ...)
b. Environment of atoms
c. No. of atoms of each types
d. No. of adjescent atoms
* Elucidation of dynamic properties like conformational isomerism
* Determination of optical purity
* Study of molecular interactions
ADVANTAGES
* Less time consuming
* Only very small amount of sample is needed
*Prediction of structure before elucidation is possible
CONCLUSION
From the above studies it can be estimated NMR is having high importance in the prediction and elucidation of structure of organic compounds. In comparison to other spectroscopic methods like U.V, I.R and mass spectroscopy, NMR, not only gives the information about the presence of group and side chains but also their position in the compound.The traditional structure elucidation techniques like chemical reaction, U.V, I.R and mass spectroscopy will take months to years to predict the structure of compounds but NMR (HETCOR) predicts the structure of the compound within few hours. Thus it reduces the time taken for the identification and elucidation of the compound. The sample preparation is very easy, the method is reliable, convenient and easy to study.
REFERENCES
1. en.wikipedia.org/wiki/Research
2. modgraph.co.uk/product_nmr_c13.htm
3. process_nmr.com
4. B.K. Sharma; Instrumental Methods of Chemical Analysis - 22nd Edition, Page No: S510 - S659
5. Williard, Merrit, Dean; Instrumental Method of Analysis - Seventh Edition, Page No: 422 - 454.
6. H. Kaur; Spectroscopy - First Edition, Page No: 233 - 299.
7. William Kemp; Organic Spectroscopy - Third Edition, Page No: 101 - 226.
8. Robert thornton Morrison & Robert Neibon Boyd; Organic Chemisry - Sixth Edition, Page No: 600 - 42.
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