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
April 11, 2023 M.M.C.P. 21
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.
April 11, 2023M.M.C.P.33
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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THANKYOU
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