CARBON-13 NMr

school of pharmaceutical science (sops, utd-rgpv, Bhopal (m.p.)

Prepared By-Sana iram

M.PHARM (Q.A)1st sem.

INTRODUCTION:-

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NUCLEAR MAGNETIC RESONANCE (NMR Spectroscopy)

• A spectroscopic technique that gives us information about the number and types of atoms in a molecule.• Nuclear magnetic resonance spectroscopy is a powerful analytical technique used to characterize organic molecules by identifying carbon-hydrogen frameworks within molecules.

MagneticNuclear Resonance

Involves Magnets

In the Nucleus

In the Nucleus

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NMR is the most powerful tool available for organic structure determination. It is used to study a wide variety of nuclei: 1. 1H

2. 13C 3. 15N 4. 19F 5. 31P

It is concerned with the magnetic properties of

certain atomic nuclei.

Involves change in the spin state at the nuclear level.

SPINNIG NUCLEUS: proton acts as a tiny spinning bar magnet and possesses both electrical charge and mechanical spin.

INTRODUCTION TO 13C-NMR

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• Proton NMR used often for the complete elucidation of the unknown compound.

• Carbon NMR can used to determine the number of non-equivalent carbons and to identify the types of carbon atoms(methyl, methylene, aromatic,carbonyl….) which may present in compound.

.

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• 12C has no magnetic spin. 13C has a magnetic spin, but is only 1% of the carbon in a sample. •The gyromagnetic ratio of 13C is one-fourth of that of 1H.•Signals are weak, getting lost in noise. Hundreds of spectra are taken, averaged

INTRODUCTION ( CONT : )

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• CMR is a noninvasive and nondestrutive method,i.e,especially used in repetitive In-vivo analysis of the sample without harming the tissues .

• CMR of biological materials allows for the assessment of the metabolism of carbon, which is so elementary to life on earth.

• CMR, chemical shift range(0-240 ppm) is wider compared to H-NMR(0-14 ppm), which permits easy seperation and identification of chemically closely related metabolites.

• The low natural abundance of 13C nuclei (1.1%) can be made use of tagging a specific carbon position by selective C-13 enrichment, which the signal intensities and helps in tracing the cellular metabolism.

• Labelling is more convenient means of followimg the metabolism specific carbons throughout the metabolism.

• C13 nuclei are a stable isotope and hence it is not subjected to dangers related to radiotracers.

• Labelling of 13C nucleus at multiple carbon sites in the same molecule was possible, as result homonuclear 13C-13C coupling provides novel biochemical information.

IMPORTANCE OF 13C NMR

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CHARACTERISTIC FEATURES OF 13C NMR

• The chemical shift of the CMR is wider(δ is 0-240ppm relative to TMS) in comparison to PMR(δ is 0-14ppm relative to TMS).

• C13-C13 coupling is negligible because of low natural abundance of C13 in the compound. Thus in one type of CMR

• spectrum(proton de coupled) each magnetically non equivalent carbon gives a single sharp peak that does undergo further splitting.

• The area under the peak in CMR spectrum is not necessary to be proportional to the number of carbon responsible for the signal. Therefore not necessary to consider the area ratio.

• In proton-coupled spectra, the signal for each carbon or a groupof magnetically equivalent carbon is split by proton bonded directly to that carbon and the n+1 rule is followed.

Types of 13c spectra 1) proton coupled 13c spectra 2) proton decoupled 13c spectra

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1) PROTON COUPLED 13c SPECTRAa) Homoannular coupling the probablity of finding 13c adjacent carbon is very less therefore homonuclear [carbon- carbon ]splitting is rearaly seen b) Hetronuclear coupling. it involving two different atoms [carbon- hydrogen]

Here splitting arises due proton attached directly to 13c carbon

The effect of attached protons on 13C resonances

n+1 = 4n+1 = 3

n+1 = 2 n+1 = 1

C13

3 protons 2 protons 1 proton 0 protons

H

H

H

C13

H

H

C13

H C13

Methylcarbon

Methylenecarbon

Methinecarbon

Quaternarycarbon

( n+1 rule applies )

COUPLING TO ATTACHED PROTONS

(J’s are large ~ 100 - 200 Hz)

2) proton decoupled 13c spectra

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Here the decouplig technique obliterates all the interaction between proton and c13nuclei thus singlet are observed in proton decoupled c-13 spectra

ETHYL PHENYLACETATE

13C coupledto the hydrogens

13C decoupledfrom the hydrogens

in some casesthe peaks of the multiplets willoverlap

this is aneasier spectrumto interpret

Problems of 13C-NMR

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13C-NMR signal is 6000 times weaker than 1H-NMR why???

NATURAL ABUNDANCE

GYRO MAGNETIC RATIO

COUPLING PHENOMENON

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NATURAL ABUNDANCE:13C natural abundance is very low (1.08%).

GYRO MAGNETIC RATIO:13C nucleus gyro magnetic ratio is much lesser than proton nucleus. C-1.404; H-5.585.This shows that CMR is more sensitive than PMR which is overcome by using FT-NMR technique

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Coupling phenomenon: 13C & 1H have I=1/2 so that coupling between them probability occur.

Extremely complex spectra and overlap of multiplets difficult to interprete.

13C-13C coupling no! Not probable

13C-1H coupling YES! Very common

Problems of 13C-NMR can overcome by

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FOURIER TRANSFORM TECHNIQUE.

DECOUPLING

TECHNIQUE.

NUCLEAR OVERHAUSER

PHENOMENON.

FTnmr• These involves irradiation of sample with all

the frequency simultaneously ,by supplying a powerful pulse of rf current for few milliseconds.

• The proton in each environment absorb there appropriate frequency from pulse and these frequency couple to give beats.

• At the end of excitation pulse the nuclei undergoes relaxation process and reemit the absorbed and coupled energies

• To give interferogrm in the time domin • The Fourier transform converts these same

into frequency domin as spectrum.

CH2 C

O

CH3

FREE INDUCTION DECAY( relaxation )

n1

n2

n3

n1, n2, n3 have different half lifes

COMPOSITE FID

“time domain“ spectrum

n1 + n2 + n3 + ......

time

FOURIER TRANSFORMA mathematical technique that resolves a complexFID signal into the individual frequencies that add together to make it.

COMPLEXSIGNAL n1 + n2 + n3 + ......

computer

FourierTransform

FT-NMR

individualfrequencies

TIME DOMAIN FREQUENCY DOMAIN

a mixture of frequenciesdecaying (with time)

converted to

converted to a spectrum

( Details not given here. )

FID NMR SPECTRUM

DOMAINS ARE MATHEMATICALTERMS

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FT-NMR

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FID

• ADVANTAGES OF FT-NMR TECHNIQUE

• The scanning takes place rapidly compared to continuous wave NMR.

• The sensitivity problems are eliminated in NMR, therefore which helps in

a) analysis the sample at very low concentration. b) NMR studies on nuclei with low natural abundance(c13) c) NMR studies on nuclei with low natural abundance and low magnetic moment (C13,N16).

Nuclear overhausner enhancement

13C-NMR and theNuclear Overhauser Effect NOE is an interaction between a targetnucleus and its radiatively-saturatedneighbors. Spin is transferred from the target nucleusto its saturated neighbors, increasing thepopulation of low-spin target nuclei. Thisincreases the signal of the target nucleus. As a result, during a standard H-decoupledexperiment, 13C atoms attached tohydrogens show enhanced signalscompared to quaternary 13C atoms.

DECOUPLING

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PROTON OR NOISE DECOUPLING

COHERENT OR BROAD BAND DECOUPLING

OFF-RESONANCE DECOUPLING

DECOUPLED SPECTRA

DECOUPLING THE PROTON SPINSPROTON-DECOUPLED SPECTRA

A common method used in determining a carbon-13NMR spectrum is to irradiate all of the hydrogen nuclei in the molecule at the same time the carbonresonances are being measured.

This requires a second radiofrequency (RF) source (the decoupler) tuned to the frequency of the hydrogen nuclei, while the primary RF source is tuned to the 13C frequency.

1H-13CRF source 2 RF source 1

continuouslysaturateshydrogens

pulse tuned tocarbon-13

13C signal (FID) measured

“the decoupler”

ETHYL PHENYLACETATE

13C coupledto the hydrogens

13C decoupledfrom the hydrogens

in some casesthe peaks of the multiplets willoverlap

this is aneasier spectrumto interpret

q

tt

s s

d d

d

SOME INSTRUMENTS SHOW THE MULTIPLICITIES OF THE PEAKS ON THE DECOUPLED SPECTRA

s = singlet t = tripletd = doublet q = quartetCODE :

This method gives the best of both worlds.

Broadband decouplingAlso called Noise decoupling or 1H

decouplingDouble irradiation at the resonance

frequency is carried outHetronuclear decoupling in which spin-spin

splitting of 13C lines by 1H nuclei is avoidedBroadband Radiofrequency signal

encompassing entire proton spectral region is irradiated

Off- resonnance decouplingDecoupling frequency is set at 1000-2000

Hz above the proton spectral regionPartially decoupled spectrum are obtainedPrimary carbon nuclei- quartetSeconadry carbon nuclei- tripletTertiary carbon nuclei- doubletQurternary carbon nuclei- single line

“Chemical shift is the difference between the absorption position of the sample proton and the absorption position of reference standard”

Variations of the positions of NMR absorptions due to the electronic shielding and deshielding.

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CHEMICAL SHIFT

Chemical Shifts….• Measured in parts per million (ppm).

• It is the ratio of shift downfield from TMS (Hz) to total spectrometer frequency (MHz).

• The chemical shift is independent of the operating frequency of the spectrometer.

• Same value for 60, 100, or 300 MHz machine.

• Common scale used is the delta (δ) scale.

April 11, 2023M.M.C.P.34

chemical shift is measured in frequency unit ‘Hertz’. Most of routine instruments operate at 60, 90, 100 MHz. More sophisticated instruments operate as high as 600MHz. The chemical shift recorded in Hz may vary with the spectrometer. To avoid this complication the chemical shift values are expressed in terms of delta or tau scale. Which are independent of field strength. Chemical shift in delta scale are expressed in parts per million (ppm).

Measurement of Chemical shift.

13C NMRcc cchekjjjhhffdmical shift ranges in chemical shifts

Interpreting 13C NMR

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• The number of different signals indicates the number of different kinds of carbon.

• The location (chemical shift) indicates the type of functional group.

• The peak area indicates the numbers of carbons (if integrated).

• The splitting pattern of off-resonance decoupled spectrum indicates the number of protons attached to the carbon.

Proton-decoupled 13C spectrum of 1-propanol (22.5 MHz)

200 150 100 50 0

1-PROPANOL

PROTONDECOUPLED

HO-CH2-CH2-CH3

c b a

Applications of c-13 nmrCmr is noninvasive and nondestructive method.i.e ,

especiallly used in repetitive in vivo analysis of the sample without harming the tissues.

Cmr of biological materials allows for the assesssment of the metabolism of carbon, which is so elementary to life on earth.

The low natural abundance of 13C nuclei (1.1%) can be made use of tagging a specific carbon position by selective C-13 enrichment, which the signal intensities and helps in tracing the cellular metabolism

Labelling is more conveinent means of following the metabolism specific carbons throughout the metabolism

Labelling of C-13 nucleus at multiple carbon sites in the same molecule was possible, as result homonuclear 13C-13C coupling provides novel biochemical information

REFERENCES

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William Kemp: Organic spectroscopy, 3rd edition.Robert M. Silverstein: Spectroscopic

identification of Organic compounds, 6th edition. Pavia: Introduction to spectroscopy, 3rd edition.Y.R. Sharma: Elementary organic spectroscopy,

principle and chemical applications. Instrumental Methods of Chemical Analysis , Gurdeep

R,Chatwal , Sham K. Anand

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