nmr pradip

13C NMR

Presented by Pradip Ghori

Maratha mandal`s college of pharmacy, Belgaum

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CONTENTS

INTRODUCTION IMPORTANCE DIFFICULTIES INVOLVED IN 13C-NMR FT-NMR INTERPRETETION NOE NMR PULSE SEQUENCE DECOPLING & RELAXATION PHENOMENON 2 D NMR APPLICATION REFERENCES

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INTRODUCTION:-

13C is a natural, stable isotope of carbon.

13C NMR is analogous to proton NMR.

NMR spectroscopy is based on the measurement of absorption of EMR in radiofrequency region of roughly 4 to 900 MHz with applied magnetic field.

Nuclei of atoms are involved.

NMR technique can be classified as

PMR:- 1H NMR

Isotopic NMR:- 12C NMR, 19F NMR, 31P NMR. 3

The 13C-NMR spectra are recorded by the pulsed –FT-NMR method, with the sensitivity enhanced by several spectra.

The spin quantum number of 12C =zero, therefore it is non-magnetic and hence does not give NMR signal.

Both 13C and 1H have spin quantum number i.e. I = ½, so we can expect to see coupling in the spectrum between:

a) 13C – 13C

b) 13C – 1H

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The probability of the two 13C atoms being together in the same molecule is so low that 13C – 13C coupling are not observed.

Only 1.1% of carbon atoms in 13C are magnetic and these nuclei split the protons in 13C-H groups into doublet and hence the 13C-H coupling is seen in the spectra.

However these couplings make the 13C spectra extremely complex and can be eliminated by decoupling.

The spectroscopy that is done using this nucleus 13C NMR gives the information about the carbon chains in the compound.

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This information is complimentary to that obtained from 1H NMR spectroscopy:- The number of signals tells us how many different carbons or different sets of equivalent carbons are present in a molecule.

The splitting of a signal tells us how much hydrogen is attached to each carbon.

The chemical shift tells us the hybridization (sp3,sp2, & sp) of each carbon.

The chemical shift tells about the electronic environment of each carbon with respect to other, nearby carbon or functional groups.

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Importance of 13C-NMR / why should 13C-NMR be recorded when PNMR is present:

13C NMR is a non-destructive and non-invasive method.

13C NMR can be used in biological systems and easy assessment of the metabolism of carbon and its pathway.

Chemical shift for 13C NMR ranges from (δ = 0-240) when compared to proton NMR (δ =0-14). Since chemical shift gives information regarding the physico-chemical environment of compound, i.e. when chemically closely related metabolites are under NMR scan, they are often well separated and resolved to obtain clearly identifiable spectra.

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As 13C nuclei have low abundance, thus tagging the specific carbon position by selective 13C enrichment, thus 13C labeling increases the signal intensities and often helps to trace the cellular metabolism

Labeling with 13C helps to know the fate of specific carbon throughout the metabolism with out need for tedious isolation and purification.

The danger involved in using radioactive isotopes in tracing is avoided as 13C nuclei are stable carbon isotope.

Labeling at multiple carbon sites in the same molecule and homonucleus 13C-13C spin coupling provides novel biochemical information.

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Difficulties of recording 13C spectra than 1H spectra are because of the following reasons:

1. Natural low abundance of 13C .

2. Magnetic movement and Gyro magnetic ratio.

3. Chemical shift.

4. Decoupling phenomenon pronounced C-13 and H-1

spin-spin interactions.

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1. Natural abundance:- The natural abundance of 13C is only 1.1% that of

12C, which is not detectable by NMR, that renders CMR less sensitive that PMR.

2. Magnetic movement and Gyro magnetic ratio: -

The 13C nucleus has only a weak magnetic movement and consequently a small gyromagnetic ratio.

Sensitivity of CMR is much reduced due to the presence of only 1.1% magnetic isotope (13C) in the sample.

13C sensitivity is 1/4th that of C(Overall sensitivity of 13C compared with 1H is about 1/5700).

The gyromagnetic ratio of 13C is 1.4043 as compared to 5.5854 of a proton 1H

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Carbon-13 NMR

These factors show that 13C CMR is much less sensitive than PMR.

The weak signals observed in 13C-CMR are therefore scanned i.e., recorded routinely by Pulse-irradiation, signal-summation and Fourier transforms.

The low sensitivity of CMR is overcome by the use of large samples, upon 2ml in 15mm tubes and by enhancement and decoupling techniques in conjunction with highly stable spectrometers operating at high fields

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The problems arises during recording CMR can be readily eliminated by adopting following methods:-

a) Fourier transform technique

b) NOE

C) DECOUPLING

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FT NMR to record the spectra:

It is a Fourier Transform NMR Spectroscopy.

TYPES OF FT NMR: Multi-Dimensional:

The use of pulses of different shapes, frequencies and durations in specifically designed patterns or pulse sequences allows the spectroscopist to extract different types of information about the molecule.

Multi-dimensional nuclear magnetic resonance spectroscopy is a kind of FT-NMR in which there are at least two pulses and, as the experiment is repeated, the pulse sequence is varied.

In multidimensional nuclear magnetic resonance there will be a sequence of pulses and, at least, one variable time period.

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In three dimensions, two time sequences will be varied. In four dimensions three will be varied.

2-dimensional and multidimensional FT-NMR into a powerful technique for studying biochemistry,in particular for the determination of the structure of biopolymers such as proteins or even small nucleic acids.

Pulsed radiofrequency-fourier transforms NMR spectroscopy:

The NMR spectrometer operates by exciting the nuclei of the isotope under observation only one type at a time.

In the case of 1H nuclei each distinct type of proton (phenyl, methyl, and vinyl) is excited individually and its resonance peak is observed and recorded independently of all the others. Scanning is done individually until all types have come into resonance. 15

FT NMR is an alternate approach to use powerful but short of energy called pulse that excites all of the magnetic nuclei in the molecule simultaneously for example, all of the 1H nuclei are induced undergo resonance at the same time.

An instrument with T magnetic fields uses a short burst of 90 MHz energy to accomplish this.

The source is turned on and off very quickly and generates a pulse.

Similarly FT NMR operates in case of carbon also. 12C nucleus is not magnetically active because spin number I=0 but the 13C nucleus like the 1H nucleus has a spin number of ½, however since the natural abundance of 13C is only about 1.1% that of 12C and its sensitivity is only about 1.6% that of 1H, the overall sensitivity of 13C compared with 1H is about 1/5700. Pulsed FT NMR permits simultaneously irradiation of all 13C nuclei and hence 13C spectra.

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A pulse is a powerful but short burst of energy. According to the variation of the Heisenberg Uncertainly principle, even though the frequency of the oscillator generating this pulse is set to 90 MHz, if the duration of the pulse is very short, the frequency content of the pulse is uncertain because the oscillator was on long enough to establish a solid fundamental frequency.

Therefore, the pulse actually contains a range of frequencies centered about the fundamental. This range of frequencies is great enough to excite all of the distinct types of the carbons in the molecule at once with this single burst of energy.

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When pulse is discontinued, the excited nuclei begin to lose their excitation energy and return to their original spin state or relax.As each excited nucleus relaxes, it emits the electromagnetic radiation. Since the molecule contains many different nuclei, many different frequencies of the electromagnetic radiation are emitted simultaneously, this emission is called “free induction decay” signal.

The intensity of FID decays with time as all of the nuclei eventually lose their excitation. This FID is complex and it is a superimposed combination of all the frequencies emitted. The individual frequencies due to different nuclei can be extracted by using a computer and by Fourier transform analysis. 18

An Advantages of Fourier Transforms: -

FT NMR spectroscopy is one of the principal techniques used to obtain physical, chemical, electronic and structural information about a molecule. It is the only technique that can provide detailed information on the exact three-dimensional structure of biological molecules in solution. Also, FT nuclear magnetic resonance is one of the techniques that have been used to build elementary quantum computers.

Fourier transform is more sensitive.

It takes few seconds to measure FID.

With computer and fast measurement, it is possible to repeat and average the measurement of the FID signal.

This is a real advantage when the sample is small in which case the FID is weak in intensity and has a great amount of noise associated with

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NOISE:-

Noise is random electronic signals that are usually visible as fluctuations of the baseline in the signal. Since noise is random; it normally cancels out of the spectrum after many interactions of the spectrum are added together. Using this procedure one can show that signal to noise ratio improves as a function of the square root of the number of the scans, n.

S/N = f√ n

Therefore pulsed FT-NMR is especially suitable for examination of the nuclei that are not very abundant in nature.

Nuclei that is not strongly magnetic.

Or very dilute sample 20

INTERPRETATION OF C-13 NMR SPECTRA: -

Chemical shifts in C-13 NMR Spectra: -

The range of shifts generally encountered in routine C-13 studies is about 240 ppm. Therefore C-13 chemical shifts represent the spread of chemical shifts of about 12 times that of the proton

The peak assignment or chemical shifts in CMR are made on the basis of reference compounds.

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S

N

N S

NH2

CH3

Molecular Formula: C11H11N3S2

CH3

13C NMR spectrum of 2-Amino-5-(4-methylphenyl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazole (1b)

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S

N

N

S

NH

CH3

O

NH2

CH3

3c

Molecular Formula: C14H16N4OS2

C=O

CH3

CH

13C NMR spectrum of 2-(Alanyl)-Amino-5-(4-methylphenyl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazole (3c)

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2a

N

S

N

NH

Cl

O

NH2

Molecular Formula: C11H11ClN4OS

CH3

CHC=O

13C NMR spectrum of 2-(Alanyl)-Amino-5-(4-chlorophenyl)-1,3,4-thiadiazole (2a)

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S

N

NS

N

H3C

3d

M olecular F orm ula = C 1 8H 1 5N 3S 2

CH3

CH

13C NMR spectrum of 5-(4-methylphenyl)-N-[(1E)-phenylmethylene][1,3]thiazolo[4,3-b][1,3,4]thiadiazol-2-amine (3d)

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S

N

N

S

N

O

ClH3C

4d

M olecular F orm ula = C 20H 16C lN 3O S 2

C=O

CH3

CH-Cl

CH

13C NMR spectrum of 3-chloro-1-[5-(4-methylphenyl)[1,3]thiazolo[4,3-b][1,3,4]thiadiazol-2-yl]-4-phenylazetidin-2-one (4d)

Factors Influencing Chemical Shifts :-

Shifts are mainly related to hybridization and substituent electronegativity. Solvent effects are also very important as in proton 1H spectra.

Chemical shifts for 13C are affected by substituents as far removed as the δ position. Pronounced shifts for 13C are caused by substituents at the ortho, Meta, and para positions in the benzene ring.

Steric compression causes 13C chemical shifts to move up field significantly.

Up field shifts my also occur on dilution.

Hydrogen bonding effects may cause downfield especially with polar solvents.

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TABLE 13C SHIFT PARAMETERS IN LINEAR & BRANCHED HYDROCARBONS

13 C Shift (ppm) A

α +9.1

β +9.4

γ -2.5

δ +0.3

ε +0.1

1º (3º) -1.1

1º (4º) -3.4

2º (3º) -2.5

2º (4º) -7.2

3º (2º) -3.7

3º (3º) -9.5

4º (1º) -1.5

4º (2º) -804

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Calculation of chemical shifts using the correlation data: -

ALKANES: -

e.g: shifts for c-atoms of 3-methyl pentane:

CH3

CH3 CH2 CH CH2 CH3

δ -calculations are made using the formula:

δ= -5.2+ ∑nA.

Where,

δ= predicted shift for a C atom.

A= additive shift parameter

n= number of C-atoms for each shift parameter.

-5.2= the shift of C-13 of methane.

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ALKENES: -

The alkenes Cs give signals in the range of δ 80-145. The base value for –CH2=CH2 is δ 123. in case of alkenes the influence of nearest substituent (α,β,γ) differ from the influence of the most distant substituent (α1,β1,γ1) as shown below.

Chemical shifts δ=123+Σ (increments for carbon atoms)

C – C – C – C =C – C – C - C

γ--β--α γ--β—γ

Increments -2 7 10 -8 -2 2

δ = 123

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E.g.: predict the 13C-chemical shift values for the alkenes in 2-pentene

CH2-CH=CH-CH2-CH3 base value : δ = 123

C2 1α,1α’,1β’ C3 1α,1β, 1α’

δ =123+10-8-2=123. δ 123+10+7-8=132.

ALKYNES: -

E.g.: - HC ≡ C - O - CH2 - CH3; H3C - C ≡ C - O - CH3

23.2 89.4 28.0 88.4

Base value to HC ≡ CH is δ =72.

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Influence of Functional Group substituents on Alkene & Aromatic Chemical Shifts:-

Alkene δ values will be affected by substitution at points further along the carbon chain (as in allyl alcohol CH2 = CH-CH2-OH). But, systematic correlations have not been compiled. However the major influence on the alkene Cs will be the direct substituent (in case of allyl alcohol, it is the –CH2 group). So we can say, if a substituent is identifiable as –CH2X, it should simply be treated as –CH3.

The same principles hold good in predicting the shifts in the aromatic δ values. The deviation from predicted values is often due to or associated with H- bonding and steric effects.

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E.g.: - compounds related to salicylic acid show such deviation because of the strong intramolecular H-bonding between the –OH group and the ortho-carbonyl group.

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Using the correlation data:

BASE ISOPROPYL(R) NITRO TOTAL

C1 128 +21 +1 150

C2 128 0 -5 123

C3 128 0 +20 148

C4 128 -2 -5 121

C5 128 0 +1 129

C6 128 0 +6 134

To predict the δ values of isopropyl group carbon atoms the benzene ring is considered to be the substituent isopropyl as alkane.

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CARBONYL GROUP CHEMICAL SHIFTS:

Major strength of C-13 NMR is the ability to observe the NMR characteristics of carbonyl C directly. The carbonyl resonance is at very high frequency and also, different classes appear within narrow ranges (advantage). So that quite fine distinctions can be made, in the knowledge that, the influences of unaccounted factors will be minimal.

Introduction of alkyl group on the Cs directly attached to- CO usually shifts the –CO signal by 2-3 ppm. conjugation with –CO group causes –CO resonance’s shift upfield (lower frequency). The anions of carboxylic acids are not much shifted in range from the free acids, inspite of the fact that the C-O bonding in carboxylate anions is weaker than the true C=O bond in acid. This theory fails to offer a convincing explanation.

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READING THE C-13 SPECTRUM:

The first steps in deducing the structure of an organic compound, using the C-13 NMR spectrum are; -

Count the number of signals in the spectrum; tis is the number of non-equivalent C environments in the molecule. (Identify and discount the signals from solvent).

Use figure (δ values table) to assign signals approximately the regions δ 0-80, δ 80-150 and δ 160-220(carbonyl carbons).

Note the intensities of the peaks: non-proton bearing Cs give lower intensity signals, and groups of two or more equivalent Cs give higher intensity signals.

Take account of any multiplicity into (q, t, d or s).

Use the correlation tables to predict the chemical shifts of all Cs inn each putative structure.

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Use of Correlation Tables: -

Two principle predictable influences that we can quantify in determining the chemical shift positions of a C atom:

1. The number of other carbon atoms attached to it (and whether these are CH3, CH2, CH, and C groups).

2. Natural of all other substituents attached (or nearby along a chain of other C atoms). But it is important to compute 1 before 2.

LIMITATIONS OF 13C NMR STUDIES: -

Sensitivity of C-NMR compared to PMR, chromatography, spectrophotometry, radiochemical studies, etc. is poor.

Limitation factors of C-NMR like intrinsically low sensitivity of magnetic resonance techniques, low gyromagnetic ratio of 13C and low natural abundance of 13C.

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NUCLEAR OVER HAUSER EFFECT (NOE):

In NMR spectroscopy, changes brought about in the energy populations of one nucleus by the decoupling of a neighboring nucleus are named the Nuclear Overhauser Effect of (NOE).

Two conditions that always apply to NOE are

It arises only during the double irradiation of one nucleus, and affects another nucleus which must be close but not necessarily coupled with the irradiated nucleus.

It is associated with dipolar relaxation mechanisms.Maximum NOE operates on CH3, CH2 and CH carbons, whereas no enhancement arises for 4 carbons (includes carbons on aromatic rings with substituent's attached.)

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THEORY OF NOE

Consider a hypothetical molecule in which 2 protons are in close proximity . In such compound, if we double irradiate Hb,then this proton gets stimulated and the stimulation is transferred through space to the relaxation mechanism of Ha.

C C

Ha Hb

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dell

Thus, due to increase in spin lattice relaxation of Ha, its signal will appear more intense by 15 to 50%. So, if the intensity of absorption of Ha signal is increased by double irradiating Hb, then protons Ha and Hb must be in close proximity in a molecule

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The carbon-13 spectrum from CH3I.

The NMR spectrum from the carbon-13 nucleus will yield one absorption peak in the spectrum.

If the hydrogen spin system is saturated, the four lines collapse into a single line having an intensity which is eight times greater than the outer peak in the 1:3:3:1 quartet since 1+3+3+1=8 .

Adding the nuclear spin from one hydrogen will split the carbon-13 peak into two peaks.

Adding one more hydrogen will split each of the two carbon-13 peaks into two, giving a1:2:1 ratio.

The final hydrogen will split each of the previous peaks, giving a 1:3:3:1 ratio.

In reality, we see a single line with a relative intensity of 24.

The Nuclear Overhauser Effect (NOE)

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If the hydrogen spin system is saturated, the four lines collapse into a single line having an intensity which is eight times greater than the outer peak in the 1:3:3:1 quartet since 1+3+3+1=8 .

In reality, we see a single line with a relative intensity of 24.


This is because of the Nuclear Overhauser Effect (NOE).

The NOE is one of the ways that spin system can release energy.

Magnetization transfer between spins is mediated by dipolar coupling.

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To describe the NOESY experiment, consider a pair of spin I and S, which are in close spatial proximity so as to have the dipolar interaction.

The first 900 pulse brings the magnetization of spin I down to the x-y plane.

After the evolving period t1, the second pulse flips the magnetization of I back to the z-axis.

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During the delay tM, cross relaxation between spin I and S occurs and some of the spin I magnetization is transferred to S.

In the detection period t2, magnetization of spin S is detected but the signal (at the frequency of spin S) is amplitude-modulated at the frequency of spin I.

The result is the cross peak in the NOESY spectrum. By adjusting the mixing time tM, the maximum distance between spins for which cross peaks will be seen can be adjusted.

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Another description of the NOE using energy level diagrams:

Here is a 2 spin system. In the diagram, Wrepresents the transition probability (therate at which certain transitions can occur).

At equilibrium, single quantum transitionsare allowed (i.e. W1I and W1S).

Double quantum transitions (W01S and W21S) are forbidden.The W1I and W1S transitions are related to spin-lattice relaxation.

Relaxation due to dipolar coupling takes place when the spins give offenergy close to the Larmor frequency.

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In pulse acquire experiment the x and y components of the free induction signalcould be computed by thinking about the evolution of the magnetizationduring the acquisition time. we assumed that the magnetizationstarted out along the −y axis as this is where it would be rotated to bya 90◦ pulse. For the purpose we are going to assume that themagnetization starts out along x; we will see later that this choice of startingposition is essentially arbitrary.

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From fig we can easily see that the x and ycomponents of the magnetization are:

.The signal that we detect is proportional to these magnetizations. The constantof proportion depends on all sorts of instrumental factors which need notconcern us here; we will simply write the detected x and y signals, Sx (t ) and

where S0 gives is the overall size of the signal and we have reminded ourselvesthat the signal is a function of time by writing it as Sx (t ) etc.It is convenient to think of this signal as arising from a vector of length S0rotating at frequency ; the x and y components of the vector give Sx and Sy,as is illustrated in Fig. 4.3.

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NMR Pulse Sequences

The 90o-FID Sequence

In the 90-FID pulse sequence, net magnetization is rotated down into the X'Y' plane with a 90o pulse.

The net magnetization vector begins to precess about the +Z axis.

The magnitude of the vector also decays with time.

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NMR Pulse SequencesThe 90o-FID Sequence

A timing diagram is a multiple axis plot of some aspect of a pulse sequence versus time. A timing diagram for a 90-FID pulse sequence has a plot of RF energy versus time and another for signal versus time. When this sequence is repeated, for

example when signal-to-noise improvement is needed, the amplitude of the signal (S) will depend on T1 and the time between repetitions, called the repetition time (TR), of the sequence.

In the signal equation below, k is a proportionality constant and is the density of spins in the sample.

S = k ( 1 - e-TR/T1 ) 52

NMR Pulse Sequences

The Spin-Echo Sequence

In the spin-echo pulse sequence, a 90o pulse is first applied to the spin system.

The 90o degree pulse rotates the magnetization down into the X'Y' plane. The transverse magnetization begins to dephase.

At some point in time after the 90o pulse, a 180o pulse is applied. This pulse rotates the magnetization by 180o about the X'axis.

The 180o pulse causes the magnetization to at least partially rephase and to produce a signal called an echo.

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NMR Pulse Sequences

The Spin-Echo Sequence

A timing diagram shows the relative positions of the two radio frequency pulses and the signal.

The signal equation for a repeated spin echo sequence as a function of the repetition time, TR, and the echo time (TE) defined as the time between the 90o pulse

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NMR Pulse Sequences

The Inversion Recovery Sequence

In this sequence, a 180o pulse is first applied. This rotates the net magnetization down to the -Z axis.

The magnetization undergoes spin-lattice relaxation and returns toward its equilibrium position along the +Z axis.

Before it reaches equilibrium, a 90o pulse is applied which rotates the longitudinal magnetization into the XY plane. In this example, the 90o pulse is applied shortly after the 180o pulse.

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NMR Pulse Sequences

The Inversion Recovery Sequence

Once magnetization is present in the XY plane it rotates about the Z axis and dephases giving a FID.

The timing diagram shows the relative positions of the two radio frequency pulses and the signal.

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Decoupling phenomenon/spin-decoupling methods:

Non decoupled (proton coupled) 13C spectra usually show complex overlapping multiplets that are very difficult to interpret, but some spectra are simple and can be interpreted easily.

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Various decoupling methods are as follows: -

a) Multiplicity & Proton (1H) Decoupling- Noise

Decoupling.

b) Coherent & Broadband Decoupling.

c) Off-Resonance Decoupling.

d) Selective Proton Decoupling.

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a) Multiplicity & Proton (1H) Decoupling- Noise Decoupling: -

Both 13C & 1H have I=1/2, so that we would expect to see coupling in the spectrum between

a) 13C-13C

b) 13C-1H

However the probability of 2 C13 atoms being together in the same molecule is so low that 13C-13C couplings are not usually observed.

The complicating effects of proton coupling in 13C spectra i.e., in 13C-1H coupling can be eliminated by decoupling the 1H nuclei by double irradiation at their resonant frequencies. this is an example of Heterounuclear De-coupling.

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Here specific protons are not decoupled but all protons are simultaneously decoupled by double irradiation while recording the 13 C spectrum. A decoupling signal is used that has all the 1H frequencies spread around 80-100 Hz & is therefore a form of radio frequency noise. Spectra derived thus are 1H decoupled or nose decoupled.

The proton-decoupled spectrum is recorded by irradiating the sample at 2 frequencies.

The First radio frequency signal is used to effect carbon magnetic resonance (CMR), while simultaneous exposure to the second signal causes all the protons in resonance at the same time and flip their α & β spins very fast. 61

In the noise decoupled spectrum of diethyl phthalate:-

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b) Coherent and Broadband Decoupling: -

The most widely used spin-decoupling technique involves simply broadband decoupling of all proton resonances to reduce the 13C spectrum (of most organic compounds) to a set of sharp peaks each directly reflecting a 13C chemical shift.

The requirements for broadband decoupling are: -

1. A Sufficiently strong decoupling field strength.

2. Method of modulation that will “spread” the decoupling field over the range of proton chemical shifts.

Satisfying the requirement of sufficiently strong decoupling method strength requires use of an radiofrequency power amplifier that is capable of supplying several watts of radiofrequency power of the decoupler coil in the probe. However the limitation here is the ability remove heat from the problem and the sample with a reasonable airflow.

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Here the decoupling frequency is phrase modulated with a 50% duty cycle, 100Hz square wave. Residual broadening of decoupled off-resonance 13C peaks is significantly reduced using this method in comparison to the former method. This method is now being widely used in broadband (1H) decoupling.

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C) Off Resonance Decoupling: -

The off-resonance coupling not only simplifies the spectrum but also retains the residual 13C-H coupling information.

This is a deliberately inefficient double irradiation of the proton frequencies.

The decoupler is offset by 1000-2000Hz upfield or about 2000-3000Hz downfield from the frequency of TMS without using the nose generator.

In off-resonance decoupling, while recording the CMR spectrum, the sample is irradiated at a frequency close (but not identical) to the resonance frequency of protons.

Consequently, the multiples become narrow and not removed altogether as in fully decoupled spectra i.e., the weak C-H coupling are decoupled and strong couplings remain though somewhat distorted.

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The residuals coupling constant Jr is < true J.

Jr =2J/γB2

= difference between decoupled frequencies and Resonance frequencies of 1H of interest.

J = true coupling constant.

B2= strength of rotating magnetic field generated by the decoupler frequencies.

= gyro-magnetic ratio.

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d) Selective Proton Decoupling: -

•When a specific proton is irradiated at its exact frequency at a lower power level than is used for off-resonance decoupling, the absorbance of the directly bonded 13C becomes a singlet, while the other 13C absorptions show residual coupling.

DEUTERIUM COUPLING: -

The number of orientations, which any magnetic nucleus can adopt in magnetic field, is (2I+1). I = spin quantum number.

Thus for 1H & 13C where, I = ½,2 orientations arise either +I or –I. But for deuterium whose I = 1, 3 orientations arise:

a) Aligned with the magnetic field most stable will augment Bo

b)Across the field on a plane

Deuterium nucleus is précessing on a plane cutting across Bo (magnetic field) & will not change field strength (1H frequencies unchanged). 69

c) Antiparallel/non-aligned with the Bo least stable will diminish Bo proton frequency reduced

(a) (b) (c)

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Protons coupled with one deuterium nucleus come to resonance at three different frequencies i.e., the 1H signal appears as a triplet; the line separation correspond to JH-D .

If a group of protons signal is coupled to more than one Deuterium then the Multiplicity of the proton signal is found from the general formula (2nI=1).

Thus two (equal) deuterium couplings give rise to quintets, & three deuterium gives septets & so on.

Deuteriated solvents (deuteriochloroform CDCL3, deuteriobenzene C6D6, deuterioacetone CD3COCD3 , or dexadeuteriodimethyl sulphoxide CD3SOCD3 ) give rise to 13C signals, which are split by coupling to deuterium.

Thus in molecules with one deuteron attached to each carbon (as in CDCL3 & C6D6) the C-13 signal form the solvent are a 1:1:1 triplet. For CD3 groups (CD3COCB, CD3SOCD3 ), the solvent gives rise to a septet with line intensities 1:3:6:7:6:3:1.

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Relaxation Phenomenon: -

What happens when protons absorb energy? Nuclei in the lower energy state undergo, transitions to the higher energy state; the populations of the tow states may approach equality, and if this arises, no further net absorption of energy can occur and the observed resonance signal will fade out saturation of the signal.

However, during a normal NMR run, the populations in the 2 spin states do not become equal, because higher E nuclei are constantly returning to the lower energy spin state

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E

Bo

Opposed

aligned

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How do the nuclear lose energy and undergo transition from the high to the low-energy state?

The energy difference E can be re-emitted as radio frequency E that is monitored by a radio frequency detector as evidence of resonance condition having been reached.

However 2 important radiation-less processed exist, which enable high-energy nuclei to lose energy.

Ø Spin-Lattice Relaxation

Ø Spin-Spin Relaxation 75

1) Spin-Lattice Relaxation

The high energy nuclear can undergo energy loss (or relaxation) by transferring E to some electromagnetic vector present in the surrounding environment e.g.: a nearby solvent molecule undergoing continuous vibration and rotational changes, will have associated electrical and magnetic changes, which might just be properly oriented and of the correct dimension to absorb E. since the nuclear may be surrounded by a whole array of neighboring atoms either in the same molecule or in solvent molecules, etc., this relaxation process is termed spin-lattice relaxation.

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2) Spin-Spin Relaxation: -

A 2nd relaxation process involves transferring E to c neighboring nucleus, provided that the particular value of E is common to both nuclei this mutual exchange of spin energy is termed spin-spin relaxation. While one nucleus loses energy, the other nucleus gains energy, so that no net change in the population of the 2 spin states is involved.

Relaxation phenomenon in terms of magnetization and vectors:-

Aligned with the field

One nucleus is an either

applied either field or

precesses

Opposed to the field

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When the system of nuclear spins relaxes, two different processes are identified:

(a) the reduced z-axis component eventually increases back to Mo

(b) the y-axis component reduces to zero.

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APPLICATIONS

13C-NMR is mainly used to study the metabolism in humans

1. Brain function.

2. Glucose metabolism and Glycogen quantification.

3. Glucose metabolism in the muscle.

4. Mechanism of hepatic glycogen repletion.

5. Disease status.

6. Characteristics of body fluids and isolated tissues.

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2D NMRAll 2D experiments are a simple series of 1D experiments collected with different timing.

2D NMR differ from the conventional NMR in that response intensity would be function of two frequency rather than a single frequency. 1D one time variable one intensity variable

2D two time variables two intensity variables 75

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1-D NMR - ONE OR TWO-DIMENSIONS?

1-D NMR COMPRISES TWO DIMENSIONS (ONE FREQUENCY

AND ONE INTENSITY AXES)

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2-D NMR• 2-D NMR CONSISTS OF TWO

FREQUENCIES AND ONE INTENSITY AXES - INTENSITY NOT COUNTED

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The two dimension of NMR based on dimension of time. One of the dimension is time domain with which we can collect the free induction decay (FID) output from the spectrophotometer and which contain frequency &intensity information .

The second dimension is refer to the time pass away / lapsing between application of some distribution to the system and the onset of collection of data in the first time domain.

The second time period is varied in regular way and series of FID response collected corresponding to each period chosen .

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Stack of several 1D spectra

Each 1D is different from the next by a Small Change in the evolution time t1

Parameters for each successive experiment in the series are constant except thephase of the pulses

FT of the two timedomains provides a mapof spin-spin correlations

WHAT…?

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WHY 2D-NMR…?

The various 2D-NMR techniques are useful when 1D-NMR is insufficient, as the signals start overlapping because of their resonant frequencies are very similar.

2D-NMR techniques can save time especially when interested in connectivity between different types of nuclei (e. g., proton and carbon).

This method is useful when the multiplets overlap or when extensive second order coupling complicates in the 1D spectrum.

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2D SPECTRUM-ACTUAL VIEW

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STACKED PLOT CONTOUR PLOT

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THEORYThe basic 2D NMR experiment consists of a pulse sequence that excites the nuclei with two pulses or groups of pulses.

The groups of pulses may be purely radiofrequency (rf) or include magnetic gradient pulses. The acquisition is carried out many times, incrementing the delay (evolution time - t1) between the two pulse groups.

The first aim of the system (pulse) will be the preparation of the spin system.

The variable Td is renamed as evolution time, T1.89

Secondly mixing event, in which information from one part of the spin system is relayed to other parts.

Finally, an acquisition period (T2) as with all 1D experiments.

Schematically, it is presented as following:

T1 is the variable delay time, and T2 is the normal acquisition time. This can be envisioned having f1 and f2, for both frequencies.

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BASIC SEQUENCES OF 2D-NMR

PREPARATION PERIOD:

During this period, magnetization is prepared by application of a pulse or a series of pulses (generally 900 pulse and 1800 refocusing pulse) to the spin system for evolution process.

The nuclei is allowed to relax to their equilibrium state.

For this reason, the actual time is usually set to about five times the average relaxation time of the nuclei(about 2 seconds)

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EVOLUTION PERIOD:

The preparation period is followed by evolution phase during which the spin system evolves, sometimes under the influence of chosen experimental conditions.

The evolution period is critical as its duration T1 will affect the FID acquired during the detection time T2.

The time interval serves as a variable whose value changes the phase and amplitude of the peaks.

The components of magnetization on the Y-axis depends on the length of time allowed for the evolution of magnetization before detection.

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MIXING:

Evolution phase is followed by mixing phase in which one or more radio frequency pulse are applied and to generate observable transverse magnetization.

The mixing period may be of zero or finite duration and during detection period it do not fixed the FID is acquired.

ACQUISITION TIME:

The essence of 2D experiments is that the time period T1 is used to modulate the FID. 93

Fourier transformation of the FID acquired during the fixed time T2 yields a series of spectra, each corresponding to a different value; a second transformation is then carried out over the period T1

which gives the two dimensional spectrum.

Finally there is a detection phase in which the correlated NMR signal is recorded.

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Classification of 2D NMR

2D NMR

J-resolved 2D NMR

COSY

Homonuclear Heteronucler Homonuclear Heteronucler

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J-RESOLVED SPECTROSCOPY (ROSY)In this technique, the scalar coupling are spread out along one axis of the plot whereas the other axis represents chemical shift.

This is thus, a useful method for separating crowded spectra with overlapping multiplets.

In spectra, the chemical shift on one axis is plotted against the multiplicity on the other axis but the graph obtained indicates that the mid points of the multiplets lie on the middle row of the stack plot.

It is represented using stacked plots which representing signal intensity perpendicular to plan of pages.

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ADVANTAGE:

J-Resolved 2D-NMR spectra allow identification of- 1.chemical shift position

2.Multiplicity3.coupling constant-J

DISADVANTAGE:

It do not necessarily establish proton coupled with proton or carbons

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J-RESOLVED SPECTRUM

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HOMONUCLEAR ROSY

The separate presentation of chemical shift and coupling information is the basic of homonuclear ROSY.

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E.g. Ethyl acetate

1. The normal ROSY spectra for ethyl acetate is at (a) and its simplicity does not require 2D treatment although it is a representative model.

2. At (b), the chemical shift is plotted at one axis and the multiplicity on other.

3. The additional information with its presentation reveals the projection spectrum at (c).

ADVANTAGES: It helps in separation of overlapping multiplets. The decoupled projection spectrum can be much

more facilitated by ROSY.

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HETERONUCLEAR ROSY

•In this spectrum, the multiplicity information for the carbon-proton coupling is plotted against the carbon is chemical shift.

•E.g. Decalin

•The projection spectrum in the case of trans Decalin would be the broad band C-H NMR spectrum which is in any event easily recorded by simpler means.

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CORRELATED 2D NMR (COSY)

•Here, correlation is plotted in second dimension with the classical chemical shift in the other dimension. It is represented by using counter plot which represents peak intensity

•COSY help to establish - proton couple with proton - proton couple with carbon

•While determine molecular structure from a high resolution NMR spectrum . It is important to establish signal which is comes from nuclei couple via the scalar interaction . 103

COSY

While determine molecular structure from a high resolution NMR spectrum . It is important to establish signal which is comes from nuclei couple via the scaler interaction . The scaler interaction allows to inter the location of nucei in molecule because the coupling constant j- depend on - the no of chemical bond are separating from those nuclei - whether the bonds are single or double - the angle they form with other bands

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APPLICATION OF NMR

QUANTITATIVE ANALYSISThe concentration of species can be determined directly by making use of signal area per proton and the area of that identifiable peak of one of the constituent for e.g. if the solvent present in known amount were benzene, cyclohexane or water, the area of single proton peak for these compound could be used in order to set the required information.

ANALYSIS OF MULTICOMPONENT MIXTUREHollis has described a method for the determination of aspirin, phenacetin and caffeine in commercial analgesic preparation.

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Chamber lain and kolthoff have described a method for the rapid analysis of benzene, ethylene glycol and water in mixture.

ELEMENTAL ANALYSIS

The total concentration of a given kind of magnetic nucleus in sample can also be determine by NMR for e.g. the integrated NMR intensities of Proton peak for a large no. of organic compound have successfully determined by Jungnikel and forbes.

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IDENTIFICATION OF COMPOUND

The structure of unknown compound from its NMR can be easily decided by certain principles, some of them are The no. of main NMR signal should be equal to the no of equivalent protons in interested compound.

The type of methylene hydrogen atom, methyl group hydrogen, ether hydrogen etc. is indicated by chemical shift.

The possible arrangement of group in the molecule is indicated by spin-spin splitting.

The area under NMR is directly proportional to the no. of nuclei present in each group.

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HYDROGEN BONDING

Hydrogen bonding causes a decreasing the electron shielding on the proton. Breaking of intermolecular hydrogen bond is indicated by an up field shift of the signal.

The downfield shift depends upon the strength of hydrogen bonding.

KETOENOL TAUTOMERISM

The keto-enol tautomerism has also been studied by NMR spectroscopy.

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STRUCTURAL DETERMINATION

NMR spectroscopy is very helpful in studying and establishing the structure of complexes, organic and inorganic compounds. For e.g.

A) structure of SOF4 - only one resolution field signal is obtained while 19F spectrum of SOF4 is recorded indicating that all the four fluorine in the molecule of SOF4 are equivalent.

B) Structure of HF2 if 19F magnetic resonance spectrum of HF2 is recorded, only one signal is recorded showing that HF2 has linear structure. 111

INTERMOLECULAR CONVERSION

EXCHANGE EFFECTS

The physical state of the sample and the type of nucleus are two important factors upon which the width of absorption band in NMR depends.

The width is small (2-3Hz) for most of the liquids: although broad bands have also been observed in the NMR spectra of liquids and this fact may be accounted for in terms of exchange effects.

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QUESTION20 MARK

1(A).Explain the techniques used for decoupling its interpretation between 13C NMR & 1H NMR interaction in carbon-13 NMR.

1.(B)Describe the concept of NMR Spectroscopy. What are the factor affecting Chemical Shifts. (April 2008, Sept 2007)

2.What are Decoupling methods? What is significance in 13C NMR Spectroscopy?

10 MARK 1.What is Decoupling? What is its significance in 13C NMR

Spectroscopy?(May 2010) 2.Discuss 13C NMR Spectroscopy & its application

(May 2012)

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5 MARK 1.Give Principles of 13C NMR Spectroscopy?(OCT

2010). 2.Explain Chemical Shifts in NMR.(2004) 3.Explain Brief account on 2-D NMR(May 2011) 4.Explain brief account on Nuclear overhouser

effect.(2006,2008,April 2009) Give detail on NMR pulse sequense.

(1996,2003,2006)

REFERENCES

1. James Keeper. In: Understanding of NMR spectroscopy; Wiley VCH, NY.2002

2. Joseph B. Lambert, Eugene P. Mazzola. In: NMR spectroscopy; Pearson Education Inc. NJ.

3. Jag Mohan. In: Organic spectroscopy; Narosa publication house.

4. Skoog, Holler, Nieman. In: Principles of instrumental analysis; Harcourt asia pte ltd.

5. G. Ganglitz, T. Vo-Dinh. In: Handbook of spectroscopy; Wiley VCH, NY.2003.

6. Sharma BK. Instrumental methods of chemical analysis; GOEL publishing House, Meerut

7. Some internet sources115

References : -

8.Organic spectroscopy by William Kemp.

9.Spectroscopy of organic compounds by P.S.Kalsi.

10.Spectrometer identification of organic compounds by Silverstein.

11.Elementary organic spectroscopy by Y.R.Sharma.

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