STUDY ON NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY (HETCOR - SPECTROSCOPY)

 

 

STUDY ON NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY (HETCOR - SPECTROSCOPY)

O. Al – Ameen, R. Raju

National College of Pharmacy, Manassery, Calicut

Cite this: O. Al–Ameen, R. Raju "Study on nuclear magnetic resonance spectroscopy (Hetcor - Spectroscopy)", B. Pharm Projects and Review Articles, Vol. 1, pp. 121-168, 2006. (http://farmacists.blogspot.in/)

 

 

INTRODUCTION

NMR

 

    Nuclear magnetic resonance, or NMR as it is abbreviated by scientists, is a phenomenon which occurs when the nuclei of certain atoms are immersed in a static magnetic field and exposed to a second oscillating magnetic field.¹¹

 

SPECTROSCOPY

 

    Spectroscopy is the study of the interaction of electromagnetic radiation with matter. Nuclear magnetic resonance spectroscopy is the use of the NMR phenomenon to study physical, chemical and biological properties of matter. As a consequence, NMR spectroscopy finds applications in several areas of science. NMR spectroscopy is routinely used by chemists to study chemical structure using simple one-dimensional techniques. Two-dimensional techniques are replacing x-ray crystallography for the determination of protein structure.¹³ Time domain NMR spectroscopic techniques are used to probe molecular dynamics in solutions. Solid state NMR spectroscopy is used to determine the molecular structure of solids.

 

 

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 number of the nuclei as follows:

 

Mass number	Atomic number	Spin quantum number
odd	odd or even	, , , ...
even	even	0
even	odd	1,2,3

Table 1 : the spin quantum number 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.¹⁵

 

    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 B_o = 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 B_o, for common magnetic nuclei.

 

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

 

Table: Precessional frequencies (in MHz) as a function of increasing field strength

    

    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).¹²

 

What happens when protons absorb 60 MHz radiofrequency energy?

 

    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.¹⁸

 

How can the nuclei lose energy and undergo transitions from the high-to the low energy state?

 

    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

 

DETECTION OF RESONANCE

    

    A radio frequency source feeds energy at 60 MHz into a coil wound around the sample tube, the radio frequency detector tuned to 60 MHz, if nuclei of sample does not resonance with 60 MHz source, the detector will only record a weak signal coming directly from the source coil to the detector.¹⁶ An increased signal will be detected if nuclei in the sample resonate with the source, since energy will transferred from the source via sample nuclei to the detector coil.

HARDWARE

 

 

Figure 1: 600 MHz instrument

 

 

Figure 2: NMR spectrometer,schematic

 

    The graphics window displays a schematic representation of the major systems of a nuclear magnetic resonance spectrometer and a few of the major interconnections. This overview briefly states the function of each component.

 

MAGNET

    

    The NMR magnet is one of the most expensive components of the nuclear magnetic resonance spectrometer system. Most magnets are of the superconducting type. A superconducting magnet has an electromagnet made of superconducting wire. Superconducting wire has a resistance approximately equal to zero when it is cooled to a temperature close to absolute zero (- 273.15^o C or 0 K) by emersing it in liquid helium. Once current is caused to flow in the coil it will continue to flow for as long as the coil is kept at liquid helium temperatures. (Some losses do occur over time due to the infinitesimally small resistance of the coil. These losses are in theorder of a ppm of the main magnetic field per year).¹⁴

 

FIELD LOCK

    

    In order to produce a high resolution NMR spectrum of a sample, especially one which requires signal averaging or phase cycling, you need to have a temporally constant and spatially homogeneous magnetic field. Consistency of the B_o field over time will be discussed here; homogeneity will be discussed in the next section.¹¹ The field strength might vary over time due to aging of the magnet, movement of metal objects near the magnet, and temperature fluctuations.

 

SHIM COILS

    

    The purpose of shim coils on a spectrometer is to correct minor spatial inhomogeneitises in the B_o magnetic field. These inhomogeneities could be caused by the magnet design, materials in the probe, variations in the thickness of the sample tube, sample permeability, and ferromagnetic materials around the magnet. A shim coil is designed to create a small magnetic field which will oppose and cancel out an inhomogeneity in the B_o magnetic field. Because these variations may exist in a variety of functional forms (linear, parabolic, etc.), shim coils are needed which can create a variety of opposing fields.¹⁷

 

SAMPLE PROBE

    

    The sample probe is the name given to that part of the spectrometer which accepts the sample, sends RF energy into the sample, and detects the signal emanating from the sample. It contains the RF coil, sample spinner, temperature controlling circuitry, and gradient coils.

 

RF COILS

    

    RF coils create the B₁ field which rotates the net magnetization in a pulse sequence. They also detect the transverse magnetization as it processes in the XY plane. Most RF coils on NMR spectrometers are of the saddle coil design and act as the transmitter of the B₁ field and receiver of RF energy from the sample. You may find one or more RF coils in a probe.¹⁰

 

GRADIENT COILS

    

    The gradient coils produce the gradients in the B_o magnetic field needed for performing gradient enhanced spectroscopy, diffusion measurements, and NMR microscopy. The gradient coils are located inside the RF probe. Not all probes have gradient coils, and not all NMR spectrometers have the hardware necessary to drive these coils.

 

QUADRATURE DETECTOR

 

    The quadrature detector is a device which separates out the and signals from the signal from the RF coil. For this reason it can be thought of as a laboratory to rotating frame of reference converter. The heart of a qudrature detector is a device called a doubly balanced mixer. The doubly balanced mixer has two inputs and one output. If the input signals are Cos(A) and Cos(B), the output will be 1/2 Cos(A + B) and 1/2 Cs(A - B). for this reason the device is often called a product detector since the product of Cos(A) and Cos(B) is the output.

 

DIGITAL FILTERING

    

    Many newer spectrometers employ a combination of oversampling, digital filtering, and decimation to eliminate the wrap around artifact. Oversampling creates a larger spectral or sweep width, but generates too much data to be conveniently stored. Digital filtering eliminates the high frequency components from the data, and decimation reduces the size of the data set.

PRACTICAL CONSIDERATIONS

 

SAMPLE PREPARATION

 

1. Sample should be run as solutions in one of the deuterated solvents found in the cabinet in Room . In preparing the samples, it is best to use one of the high quality 8" NMR tubes, alghouth the 7" tubes can provide adequate spectra for routine samples.
2. For liquid samples, use a clean disposable pipette to add carefully one or two drops of sample to a clean, dry NMR tube. Then use the sample gauge to add deuterated solvent to a height of 4 cm from the bottom of the tube. (The mark on the short side of the gauge) Cap and mix the sample well.
3. For solid samples, use a microspatula to add carefully one or two spatula tips of sample to a clean dry NMR tube. Again, fill with solvent to 4 cm, cap and mix well.
4. Of course, problems such as air instability, water instability, or low solubility will require different approaches for preparing good samples.

1. Always wipe the outside of your tube with a clean Kimwipe before placing it into the spectrometer. This step will keep the spectrometer probe clean and free from contaminants and skin oils.

 

MAGNETIC MATERIALS AND SAFETY CONSIDERATIONS

 

1. If you have a pacemaker or other metallic implant, do not enter Room . The large magnetic field associated with a 300 MHz superconducting magnet can interfere with the operation of a pacemaker, or could possibly result in tissue tears at the site of a metallic (magnetic) implant.
2. Do not put any magnetic (mainly iron) items near the magnet. The magnet will strongly attract such items. For example, a moderately heavy item like a wrench or even a screwdriver could puncture the stainless steel vacuum dewar if it were dropped near the magnet.
3. A massive magnetic item like a gas cylinder could move the superconducting coil, even if the cylinder did not come in contact with the dewar.
4. And even small items like paper clips or staples can eventually find their way up into the magnet and thus interfere with the collection of spectra. So try not to drop such items in the room, and certainly do not take them past the yellow warning rope.
5. The magnetic field is strong enough to erase information from magnetic storage media. Such media (found on credit cards, ATM cards, computer diskettes, and audio tapes) should be removed from your person before you cross the yellow warning rope. It would be wise to remove your wallet and analog watch before crossing the yellow warning rope.
6. Do not work unsupervised.
7. Follow directions. The NMR is an expensive piece of equipment and can e damaged if used improperly.

 

 

 

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.⁷

 

    The relation between the number of signals or peaks in the spectrum and the number of different kinds of hydrogen atoms in the molecule. It is, therefore, possible to known different kinds of environment of the hydrogen atoms in the molecule.

 

    The number of signals or peaks signifies how many different kinds of protons are present in the molecule. The positions of the signals tell us about the electronic environment of the different types of protons present. For example, aliphatic or aromatic, primary, secondary or tertiary adjacent to oxygen or halogen etc. the shift in position is known as chemical shift. The intensities of signals tell us about the different kinds of protons present.

 

    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.

 

    Most protons in organic compounds absorb to the left of, or downfield from TMS in the NMR spectrum. The distance in d values from TMS to each signal (absorption or peak) in the NMR spectrum is called the chemical shift for the proton or protons giving that signal.

    Signals on the left of the spectrum are said to occur downfield and those on the right are said to occur upfield. The area under the signal for the methyl groups of p-xylene (6H) is larger than for the hydrogen atoms (4H) of benzene ring.

 

    The chemical shift or position of the line in NMR spectrum gives information on molecular environment of the nuclei from which it arises. The chemical shifts of nuclei in different molecules are similar if the molecular magnetic environments are similar. The intensity of the lines gives directly the relative number of magnetically active nuclei undergoing the different chemical shifts.

 

    The chemical shift is used for the identification of functional groups and as an aid in determining structural arrangements of groups. The proton is shielded when the induced field opposes the applied field. The proton is deshielded, when the induced field reinforces the applied field and the field experienced by the proton is increased.

 

    A shielded proton requires a stronger magnetic field and a deshielded proton requires a weaker field to give the effective strength at which absorption occurs. A deshielded proton would give the resonance signal upfield and a shielded proton would absorb downfield. These shifts in the position of NMR signals which arise from the shielding or deshielding of electrons are generally called chemical shifts.⁶

 

    Elelctron withdrawing substituents, e.g, halogens, on the carbon bearing the proton deshield the proton, while electron releasing substituents, e.g alkyl group shield the proton. Attachment of electronegative atom or group to the carbon bearing the proton causes a downfield shift (higher value of d) due to deshielding, because electronegative group or atom attracts electrons towards itself and hence electron density around the nearby proton decreases causing deshielding. Because of deshielding smaller values of applied field will be required to bring the proton to resonance. 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).

 

    Chemical shifts are usually expressed in cycles per second relative to TMs. There is yet another, and a more popular way of describing chemical shifts. Most NMR spectrometers operate with rf oscillators set at 40, 60 or 100 megacycles per second. Chemical shifts are proportional to the spectrometer frequency so that signals 100 cycles per second apart at 60 megacycles per second would appear at 167 cps at 100 Meps. In order to facilitate comparison between chemical shifts measured at different oscillator frequencies, shifts in cps are often divided by the oscillator frequency and reported as ppm (parts per million).⁵ Thus if a signal comes 100 cps at 60 Mcps downfield (towards higher frequency) relative to TMS, it can be reported as ppm relative to 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.

 

    The chemical shift of a proton is determined by its electronic environment. Equivalent protons have protons with the same environment and so in a molecule they have the same chemical shift. Non-equivalent protons with different electronic environment will have different chemical shifts. A proton with a particular environment will have the same chemical shift, irrespective of the molecule in which it is present.

 

    Chemical shift (d, in ppm) is the position of an absorption peak relative to that of a reference substance such as TMS. Chemical shift largely depends on the presence (shielding) or absence (deshielding) of electron density. A proton is said to be deshielded, when bonded to a group which withdraws part of the shielding electron density from around the proton. The more shielded a proton is, the farther down field it gives the peak.

 

    A proton is shielded when it is surrounded by electrons whose induced magnetic field opposes the externally applied magnetic field and shields the proton from its influence. The effective magnetic field at the shielded nuclus is less than the applied magnetic field. For bringing the proton into resonance the applied field is increased to overcome the diamagnetic shielding effect.

 

    Multiple bonded systems give rise to induced magnetic fields. These induced field lines curve around and consequently divide the immediate environment into two regions. In one region the proton is shielded and in the other the proton is deshielded from the applied field. In other words, in one region the induced field acts to oppose the external field, while in other the induced field adds to the external field.

 

    The induced magnetic field of circulting aromatic electrons is larger than in the alkenes because of large effective ring of electrons, eg., in benzene. Thus benzenoid protons and aromatic protons in general, have greater d values (6-8.5 ppm) than alkene protons.

 

    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.⁴

 

    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.

 

    The protons may be chemically equivalent, but magnetically non equivalent. Magnetically equivalent protons have the same coupling constant to every other nucleus in the system. Most of the protons in an organic compound experience a rapidly changing magnetic environment around the same values. In otherwise equivalent environment, the more the number of hydrogen atoms on one carbon, the more the magnetic shielding. Attachment of chlorine directly to the carbon bearing the proton leads to downfield shift.¹ The attachment of increasing number of halogens to the same carbon atom leads to an increase in the chemical shift unsaturation has a deshielding effect on chemical shift.

 

    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 compoundsd.

 

    The chemical shifts of hydrogen bonded protons, ie, O – H protons in alcohols and N – H protons in amines, depend on concentration. In concentrated solutions these protons are deshelded by hydrogen bonding and absorb at lower field (d 4.5 for an alcohol O – H and d 3.5 for an amine N – H). When an alcohol or amine is diluted with a solvent having no hydrogen bonding, the hydrogen bonding becomes less important and consequently these resonate around d 2.0. Hydrogen bonding and the proton exchange that accompanies it leads to a broadening of the signal corresponding to the resonance of a O – H or N – H proton. Broadening of peak is due to the fact that protons are exchanging from one molecule to another during NMR resonance.

 

    Circulation of electrons, especially p electrons about nearby nuclei generates a field that can either oppose or reinforce the applied magnetic field at the proton, depending on the location of the proton or space occupied by the proton. This shielding occurs in alkynes and deshielding occurs in alkene, aldehyde and benzene. These shielding and deshielding effects depend on the orientation of the nuclear spin w.r.t the induced field, caused due to the circulation of p electrons and are known as space or anisotropic effects. Because anisotropic effect causes shielding and deshielding, these effects are also responsible for influencing the chemical shift values.³

 

    The chemical shift positions of the protons attached to C = C in alkenes, aldehydic protons and aromatic protons is higher than predicted alone by electronegativity effects. The alkynes protons appear relatively at low positions. These chemical shifts are explained by anisotropic effect. Ansitropic effects occur in addition to the ever present molecular fields induced by sigma bond electrons. The aldehyde protons is shifted downfield both by anisotropic effects and by electron withdrawal by the carbonyl oxygen. p electrons, like sigma electrons are induced to circulate in the presence of the applied field to generate a molecular field. Generally the strength of the magnetic field generated by circulating p electrons is stronger than that generated by sigma electrons. Thus the presence of p electrons can have a pronounced effect on chemical shifts of nearby protons.

 

    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.

 

    The alkyne protons appear at high field, because circulation of the electron around the triple bond occurs in such a manner that the proton of an alkyne experiences a diamagnetic shielding effect. The alkyne molecule is linear and the p electrons in the alkyne are more free to circulate around the symmetry axis of the triple bond. The electrons associated with several atoms such as halogen, bonds such as C = C and groups like C = O and C₆H₅, effect the chemical shift of neighbouring protons.

 

    The shielding and deshielding effects of benzenoid ring current are more powerful that the p electrons in C = C bonds of alkene. Thus protons attached to isolated double bonds are observed to resonate between d 4.6 and 6.4. Benzenoid protons usually resonate between d 6.6 and 8.5 ppm. In general, the space surrounding a double bond or a benzenoid ring can be divided into shielding and deshielding regions where protons will be observed at relatively high and low fields respectively. The anisotropic effects of the p electrons of a C – C bond are small compared to the circulating p electrons and the axis of C – C bond is the axis of the deshielding cone.

 

    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.⁸

 

    Generally in saturated systems there should not be more than three and in unsaturated systems not more than four interconnecting bonds for significant coupling to occur. The coupling constant is maximum for a dihedral angle of 180⁰, a lesser maximum when this angle is 0⁰, and is about 0 for dihedral angle of 90⁰. in several cases the magnetic difference in, for example, protons in an organic molecule is large enough to be observed by NMR spectroscopy. The methylene protons, eg., in a compound near a source of chirality have different chemical shifts, split each others signals and have different coupling constants associated with their spin-spin interactions with protons on adjacent carbon atoms.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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.²

 

    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)

 

    ¹²C nucleus is not magnetically active because its spin number I = 0. ¹³C, nucleus, like the ¹H nucleus, has a spin number I = 1/2, but ¹³C NMR or CMR spectra are difficult to record than ¹H spectra because of the following important reasons.

 

    (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.⁵

 

    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.

 

    While splitting is often useful in the study of simple molecules, in various cases, it is more difficult to interpret the spectrum. For this reason, and in order to increase the signal strength, CMR spectra are generally recorded under conditions in which the protons are decoupled from the carbons. In NRM, we observe the most abundant isotope, ¹H, but in CMR, the most abundant isotope ¹²C has an even - even nucleus and has not net nuclear spin or magnetic moment. Thus in CMR, we observe the isotope ¹³C, which has a natural abundance of only 1.1%.

 

    The proton decoupled CMR spectrum is recorded by irradiating the sample at two frequencies. The first radiofrequency signal is used to effect carbon magnetic resonance, while the simultaneous exposure to the second signal causes all the protons to be in resonance at the same time and they flip their a– or b– spins very fast. When a specific proton is irradiated at its exact frequency at a lower power level than is used for off resonance decoupling, the absorption of the directly bonded ¹³C becomes a singlet, while the other ¹³C absorptions show residual coupling. This is called selective proton decoupling.²

 

    For ¹³C, common range of energy absorption is wider (d 0 – 200 relative to TMS) in comparison to PMR (d 0 – 15 relative to TMS). ¹³C – ¹³C coupling is negligible because of only 1.1% ¹³C carbon in the compound. Thus, in one type of CMR spectrum (proton decoupled) each magnetically non-equivalent carbon gives a single sharp peak that does not undergo further splitting.

 

    The areas under the peaks in CMR spectra are not necessary to be proportional to the number of carbons responsible for the signals. It is therefore not necessary to consider area ratios. In proton coupled spectra, the signal for each carbon or a group of magnetically equivalent carbons is split by the protons bonded directly to that carbon and (n + 1) rule is still followed.

 

 

   
 

 

 

 

 

 

 

 

 

 

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

CORRELATION SPECTROSCOPY

 

    The ability to present computed data in 'three dimensions' rests very much with graphics software, permitting such stack-plots. Alternatively, the same information can be presented in cross-section, effectively a contour map of the peaks.

 

Figure. 5. Three Dimensional view of Correlation spectrum

 

    A conventional NMR spectrum is a plot of intensity against frequ3ency, but for coupling nuclei (H –H or C–H, etc.) their interactions are also time-variable, as discussed above, by sampling these interactions as a function of time it is possible to separate out the interactions among the carbons and hydrogens of organic functions in such a way as to establish which protons couple with which carbons, or else which protons couple with which other protons. Since two frequency axes are involved, the method is called two-dimensional NMR, but the information is plotted in pseudo-three-dimensional form, with intensity as the third dimension.⁴

 

    There have been very many variations published using 2-D NMR, and these are dealt with in the specialist texts listed in 'Further Reading', but two of the most important are examples of correlation spectroscopy, either homonuclear or heteronuclear.

 

HOMO NUCLEAR CORRELATION SPECTROSCOPY (¹H – ¹H COSY)

 

    It sets out the proton NMR spectrum of an organic molecule such as ipsenol along the x axis, and repeats it along the y axis, with the signals repeated yet again in the contours of the diagonal peaks. Wherever a proton couples with another proton, this is indicted by the contour of an off-diagonal cross-peak.

 

HETERONUCLEAR CORRELATION SPECTROSCOPY

    

    C–H shift correlation spectrum for menthol. In this with the proton spectrum on the y axis and the carbon-13 spectrum on the x axis, wherever correlation exists, cross-peak appear in the correlation spectrum.⁷

 

    There are only three cases possible for each carbon atom. If a line drawn down encounters no cross peaks, then the carbon has no attached hydrogens. If the drawn line encounters only one cross peak, then the carbon may have either 1,2 or 3 protons attached; if 2 protons are attached, then they are either chemical-shift equivalent or they fortuitously overlap. If the dropline encounters two cross peaks then we have the special case of diastereotopic protons attached to a methylene group.

 

 

 

 

 

 

 

 

 

INTERPRETATION

 

    We can begin with either a carbon or a proton resonance and obtain equivalent results. We will use the carbon axis as our starting point because we usually have less overlap there. For example , aline drawn parallal to the proton axis at about 68 ppm on the carbon axis (the carbinol carbon) intersects five crosspeaks; none of the five correlations to the attached proton (1JCH) at 3.8 ppm. Four of the crosspeaks corresponds to the two pairs of diastereotopic methylene groups (2.48,2.22, 1.45,and1.28 ppm) and these represents (2JCH) two-bond couplings. The fifth inter action (3JCH) correlates this carbon atom (68 ppm) to the isopropyl methane proton (1.82 ppm), which is bonded to a carbon atom in the b-position. The other carbon atom in a b-position has no attached protons so we do not have a correlation to it from the carbinol carbon atom. Thus, we have indirect carbon connectivities to two a carbons and to one of two b carbons.³

 

    Another useful example can be found by drawing a line from the carbon resonance at 41 ppm. This carbon is the C-5 methylene and we first note that correlations to the attached protons at 2.48 and 2.22 ppm are absent. There is only one a carbon that has one or more attached protons; its corresponding correlation is found to the C-4 carbinol methane proton at 3.83 ppm. There are three b crbons and they all have attached protons. The C-3 methylene carbon shows indirect coorelation through both of its diastereotopic protons at 1.45 and 1.28 ppm. The C-7 olefinic methane proton gives a cross peak at 6.39 ppm, as do the protons of the olefinic methylene group attached to C-6 at 5.16 and 5.09 ppm. Other assignments are left to the reader as an exercise.

 

   
 

 

 

 

ADVANTAGES

 

1. Less time consuming
2. only very small amount of sample is needed
3. Prediction of structure before elucidation is possible

APPLICATIONS

 

1. 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

1. Elucidation of dynamic properties like conformational isomerism
2. Determination of optical purity
3. Study of molecular interactions

CONCLUSION

 

    From the above studies it can be estimated HETCOR 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, HETCOR, 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 with in 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. Douglus. A. Skoog, E. James Holler, Timothy. A. Nieman; Principles of Instrumental Analysis - Fifth Edition, Page No: 445 - 486.
2. Robert thornton Morrison & Robert Neibon Boyd; Organic Chemisry - Sixth Edition, Page No: 600 - 642.
3. Robert M, Silverstein, Francis X Webster; Spectrometric Identification of Organic Compounds - Sixth Edition, Page No: 144 - 273).
4. William Kemp; Organic Spectroscopy - Third Edition, Page No: 101 - 226.
5. H. Kaur; Spectroscopy - First Edition, Page No: 233 - 299.
6. Williard, Merrit, Dean; Instrumental Method of Analysis - Seventh Edition, Page No: 422 - 454.
7. B.K. Sharma; Instrumental Methods of Chemical Analysis - 22^nd Edition, Page No: S510 - S659.
8. Indian Journal of Chemistry - Section A March 2006, Page No: 581 - 586.
9. www.nmr.chem.uu.nl/~klaartje/bookmarks.html-19k
10. www.pittstate.edu/chem/instrumentation/nmr2.html
11. www.wfu.edu/~ylwong/nmr/c13
12. http://www.modgraph.co.uk/product_nmr_c13.htm
13. www.blackwell-synergy.com/doi/pdf/10.1111/j.1432-1033.1984.tb08419.x
14. http://www.chem.uic.edu/nmr/downloads/mercury6.pdf
15. http://en.wikipedia.org/wiki/Research
16. www.iic_nmr.com
17. www.newera_spectro.com

www.http//www.process_nmr.com

Cite this: O. Al–Ameen, R. Raju "Study on nuclear magnetic resonance spectroscopy (Hetcor - Spectroscopy)", B. Pharm Projects and Review Articles, Vol. 1, pp. 121-168, 2006. (http://farmacists.blogspot.in/)