1. CARBON-13 NMr
school of pharmaceutical science
(sops, utd-rgpv, Bhopal (m.p.)
Prepared BySana iram
M.PHARM (Q.A)
1st sem.
2. INTRODUCTION: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.
Nuclear
In the Nucleus
Magnetic
Resonance
Involves
Magnets
In the
Nucleus
2
3. 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.
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
3
5. 31P
4. INTRODUCTION TO 13C-NMR
• 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.
4
5. INTRODUCTION ( CONT : )
• 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
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6. IMPORTANCE OF 13C NMR
• 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 HNMR(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.
6
7. 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.
7
8. Types of 13c spectra
1) proton coupled 13c spectra
2) proton decoupled 13c spectra
1) PROTON COUPLED 13c SPECTRA
a) 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
8
9. COUPLING TO ATTACHED PROTONS
3 protons
2 protons
H
13
0 protons
H
C
H
13
H
n+1 = 4
Methyl
carbon
1 proton
C
H
n+1 = 3
Methylene
carbon
13
C
H
n+1 = 2
Methine
carbon
The effect of attached protons on 13C resonances
( n+1 rule applies )
(J‟s are large ~ 100 - 200 Hz)
13
C
n+1 = 1
Quaternary
carbon
10. 2) proton decoupled 13c spectra
Here the decouplig technique obliterates all the
interaction between proton and c13nuclei thus
singlet are observed in proton decoupled c-13
spectra
10
11. ETHYL PHENYLACETATE
in some cases
the peaks of the
multiplets will
overlap
13C
coupled
to the hydrogens
13C
decoupled
from the hydrogens
this is an
easier spectrum
to interpret
12. Problems of 13C-NMR
13
C-NMR signal is 6000 times weaker than
NMR why???
NATURAL ABUNDANCE
GYRO MAGNETIC RATIO
COUPLING PHENOMENON
12
1
H-
13. NATURAL ABUNDANCE:
13
C natural abundance is very low (1.08%).
GYRO MAGNETIC RATIO:
13
C 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
13
14. Coupling phenomenon:
13
C & 1H have I=1/2 so that coupling between them
probability occur.
13
C-13C coupling no!
13
1
C- H coupling
YES!
Not probable
Very commo
Extremely complex spectra and overlap of
multiplets difficult to interprete.
14
15. Problems of 13C-NMR can overcome by
FOURIER
TRANSFORM
TECHNIQUE.
DECOUPLING
TECHNIQUE.
NUCLEAR
OVERHAUSER
PHENOMENON.
15
16. 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.
20. FOURIER TRANSFORM
A mathematical technique that resolves a complex
FID signal into the individual frequencies that add
( Details not given here. )
together to make it.
TIME DOMAIN
converted to
FID
FREQUENCY DOMAIN
NMR SPECTRUM
DOMAINS ARE
MATHEMATICAL
TERMS
FT-NMR
computer
COMPLEX
SIGNAL
Fourier
Transform
a mixture of frequencies
decaying (with time)
1
+
2
+
3
+ ......
individual
frequencies
converted to a spectrum
23. •
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).
25. 13C-NMR and the
Nuclear Overhauser Effect
NOE is an interaction between a target
nucleus and its radiatively-saturated
neighbors.
Spin is transferred from the target
nucleus
to its saturated neighbors, increasing the
population of low-spin target nuclei. This
increases the signal of the target
nucleus.
As a result, during a standard Hdecoupled
experiment, 13C atoms attached to
28. DECOUPLING THE PROTON SPINS
PROTON-DECOUPLED SPECTRA
A common method used in determining a carbon-13
NMR spectrum is to irradiate all of the hydrogen
nuclei in the molecule at the same time the carbon
resonances 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.
RF source 2
“the decoupler”
1H-13C
continuously
saturates
hydrogens
13C
RF source 1
pulse tuned to
carbon-13
signal (FID) measured
29. ETHYL PHENYLACETATE
in some cases
the peaks of the
multiplets will
overlap
13C
coupled
to the hydrogens
13C
decoupled
from the hydrogens
this is an
easier spectrum
to interpret
30. SOME INSTRUMENTS SHOW THE MULTIPLICITIES
OF THE PEAKS ON THE DECOUPLED SPECTRA
s = singlet
d = doublet
CODE :
d
t = triplet
q = quartet
d
q
s
s
t
t
d
This method gives the best of both worlds.
31. Broadband decoupling
Also called Noise decoupling or 1H decoupling
Double irradiation at the resonance frequency is
carried out
Hetronuclear decoupling in which spin-spin
splitting of 13C lines by 1H nuclei is avoided
Broadband Radiofrequency signal encompassing
entire proton spectral region is irradiated
32. Off- resonnance decoupling
Decoupling frequency is set at 1000-2000 Hz
above the proton spectral region
Partially decoupled spectrum are obtained
Primary carbon nuclei- quartet
Seconadry carbon nuclei- triplet
Tertiary carbon nuclei- doublet
Qurternary carbon nuclei- single line
33. CHEMICAL SHIFT
“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.
33
M.M.C.P.
20 December 2013
34. 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.
34
M.M.C.P.
20 December 2013
35. Measurement of Chemical shift.
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).
39. Interpreting 13C NMR
• 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.
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42. Applications of c-13 nmr
Cmr 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
43. REFERENCES
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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