NMR SPECTROSCOPY.pptx

Submitted by,
Manimegalai.G
M.Pharm - I year
5/17/2023
Department of Pharmaceutical Chemistry
1

 Introduction
 Principle
 Quantum numbers
 Chemical shift
 Spin-spin coupling
 Coupling constant
 Instrumentation
 FT-NMR
 13C-NMR
 Applications
 Interpretation
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Department of Pharmaceutical Chemistry 2

 Nuclear magnetic resonance (NMR) spectroscopy is
the study of molecules by recording the interaction
of radiofrequency (Rf) electromagnetic radiations
with the nuclei of molecules placed in a strong
magnetic field.
 Technique that detects the energy absorbed by
changes in the nuclear spin state.
 It provides information, at the atomic level, on the
dynamics of proteins and nucleic acids.
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 Radio waves are regarded as the lowest energy form
of electromagnetic radiation that find valid
applications in analytical chemistry.
 The frequency of radio waves lies between 107 & 108
Hz.
 It involves transition of a nucleus from one spin state
to another with the resultant absorption of
electromagnetic radiation by spin active nuclei
(having spin quantum number > 0) under the
influence of magnetic field.
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 The spectrum drawn between peak intensities vs.
frequency of absorption (expressed as 𝛿) is called as
NMR spectrum and the methodology is called as
NMR spectroscopy.
 NMR is non-destructive technique.
 Finest technique for determining the structure of
organic compounds.
 Larger amounts of sample are needed for NMR
than mass spectroscopy.
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 Many nuclei have spin, and all nuclei are
electrically charged, according to the NMR
principle. An energy transfer from the base energy
to a higher energy level is achievable when an
external magnetic field is supplied.
 All nuclei are electrically charged and many have
spin.
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 Transfer of energy is possible from base energy to
higher energy levels when an external magnetic field
is applied.
 The transfer of energy occurs at a wavelength that
coincides with the radio frequency.
 Also, energy is emitted at the same frequency when
the spin comes back to its base level.
 Therefore, by measuring the signal which matches
this transfer the processing of the NMR spectrum
for the concerned nucleus is yield.
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 The spinning nucleus
 The effect of an external magnetic field
 Precessional Motion
 Precessional frequency
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Spin up Spin down
Each Spinning Proton behaves like a “Mini-
Magnet”

Proton (the nucleus of hydrogen atom)
behaves as tiny bar magnet why ?
 Electric charge
 Mechanical spin
 The spinning charged nucleus generates a magnetic
field, hence proton also generate magnetic field
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 Nuclear spin has two states (+1/2 α state) & (-1/2 β
state). In the absence of External magnetic filed it
can exist either in α state or in β state.
 Under the influence of external magnetic field, the
proton will tend to adopt two orientations
i. aligned (+1/2 α state) with the field (the lower
energy state) or parallel
ii. opposed (-1/2 β state) to the field (the higher
energy state) or antiparallel
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14
In presence of a magnetic field
Magnetic moments precess and
Orient with or against the field

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Department of Pharmaceutical
Chemistry 15

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Chemistry 16

 The value of spin quantum number I 0
 The magnetic moment should be large
 The natural abundance (1H,19F,31P is 100%) of
the given isotope should be high
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17


Mass
number
Atomic
number
I Nuclei
Odd Odd ½ 1H,19F,31P
Odd Odd 3/2 11B, 35Cl, 79Br, 81Br
Odd Odd 5/2 17O
Odd Even ½ 13C, 29Si
Even Even 0 12C, 16O, 18O, 32S
Even odd 1 2H, 14N
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 The set of numbers used to describe the position and
energy of the electron in an atom are called
quantum numbers. There are four quantum
numbers,
i. Principal quantum number, denoted by n.
ii. Orbital angular momentum quantum number (or
azimuthal quantum number), denoted by l.
iii. Magnetic quantum number, denoted by ml.
iv. The electron spin quantum number, denoted by
ms .
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 They designate the principal electron shell of the
atom. Since the most probable distance between the
nucleus and the electrons is described by it, a larger
value of the principal quantum number implies a
greater distance between the electron and the
nucleus.
 The value of the principal quantum number can be
any integer with a positive value that is equal to or
greater than one. The value n=1 denotes the
innermost electron shell of an atom, which
corresponds to the lowest energy state (or the
ground state) of an electron.
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20

 When a given electron is infused with energy (excited
state), it can be observed that the electron jumps
from one principle shell to a higher shell, causing an
increase in the value of n. Similarly, when electrons
lose energy, they jump back into lower shells and the
value of n also decreases.
 The increase in the value of n for an electron is called
absorption, emphasizing the photons or energy being
absorbed by the electron. Similarly, the decrease in
the value of n for an electron is called emission,
where the electrons emit their energy.
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 It describes the shape of a given orbital. It is
denoted by the symbol ‘l’ and its value is equal to
the total number of angular nodes in the orbital.
 A value of the azimuthal quantum number can
indicate either an s, p, d, or f subshell which vary in
shape. This value depends on the value of the
principal quantum number, i.e. the value of the
azimuthal quantum number ranges between 0 and
(n-1).
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 The magnetic quantum number distinguishes
the orbitals available within a subshell, and is used
to calculate the azimuthal component of the
orientation of orbital in space.
 Electrons in a particular subshell (such as s, p, d, or
f) are defined by values of ℓ (0, 1, 2, or 3). The
magnetic quantum number takes integer values in
the range from −ℓ to +ℓ, including zero.
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23

 External magnetic field induces magnetic field where
the induced magnetic field opposes applied magnetic
field. It is the ratio of the change of field necessary to
achieve resonance to the field strength that resonates
with a standard (or)
 It is the difference between the resonance frequencies of
a given nucleus and a standard reference nucleus
 It arises due to interactions of electrons with the applied
magnetic field that generate local magnetic fields
around atomic nuclei
 Measured in parts per million. Scale (δ, ppm, τ = 10 - δ)
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 =
 sample(Hz) –  Reference (Hz)
--------------------------------
 Reference (Hz)
X 106 ppm

 Electronegativity (inductive effect/field effect)-
deshielding
 Hybridization – deshielding sp2
 Magnetic Anisotropic effects- deshielding (Except
acetylenic protons)
 Aromatic Ring current - deshielding
 Mesomeric (resonance effect)- EDG shielding
 EWG- deshielding
 Steric effect or Vander Waals - deshielding
 Hydrogen bonding- deshielding
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Compound CH3X CH3 H CH3 I CH3 Br CH3 Cl CH3 F
Element X H I Br Cl F
Electronegativity of X 2.1 2.5 2.8 3.1 4.0
Chemical Shift δ 0.23 2.16 2.68 3.05 4.26
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 sp3 Hydrogens (S character approx is 25%) (less
electronegative due to less “S “character) sp3
hydrogens are shielded since they are less
electronegative hydrogens attached primary carbon
resonate between 0 - 1 ppm, sec 1-2 and tert 3 to 4
ppm.
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 sp2 Hydrogens (S character approx is 33%) (more
electronegative than sp3) Hydrogens attached to
sp2 hybridized carbon atoms resonate farther
downfield than for normal aliphatic protons.
 The sp2 hybridized carbon atom of the double
bond has increased s-character, and is therefore
more electronegative (bonding electrons are more
closer to carbon and away from protons, which
become deshielded) than an sp3 hybridized carbon
atom.
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 sp Hydrogens (S character approx is 50%) (More
electronegative than sp2 & sp3, but appears at
upfield)
 Acetylenic hydrogens resonate between 2 - 3 ppm
due to the anisotropy of the carbon-carbon triple
bond
 Aromatic protons – deshielded 7 to 8.5 due to sp2
hybrid & due to ring current
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31
Anisotropy refers to the property of the
molecule where a part of the molecule
opposes the applied field and the other
part reinforces the applied field
Alkene Anisotropy Increases Chemical Shifts

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 In a magnetic field, the six p electrons in benzene
circulate around the ring creating a ring current.
 The magnetic field induced by these moving
electrons reinforces the applied magnetic field in
the vicinity of the protons.
 The protons thus feel a stronger magnetic field and
hence they are deshielded and absorb downfield.
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34

 +M effect (Positive mesomeric effect)
When the electrons or the p electrons are transferred
from a particular group towards a conjugate system,
thus increasing the electron density of the conjugated
system then such a phenomenon is known as (+M)
effect or positive mesomeric effect.
Group showing +M effect (ERG/EDG)
–NH, –NH2,–NHR, –NR2, – O, – OH, –OR, – F, – Cl, –
O–COR, – NHCOR, –SH, – SR
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 M Effect (Negative mesomeric effect)
When the p -bond electrons are transferred from the
conjugate system to a particular group thus the
electron density of the conjugate system is decreased,
then this phenomenon is known as negative mesomeric
(–M) effect.
The group which shows –M effect include
–COOR ,- COOH, -COR, –SO3H, – CHO, –CONH2
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 Bulky group C(CH3)3 present adjacent to proton
repel the electron around the proton thereby
causing deshielding effect
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 Causes downfield shift, which depends on strength of
hydrogen bonding due to the electronegative atom to
which the proton is hydrogen bonded, electron cloud
around the proton is decreased and hence deshielded.
 greater the degree of hydrogen bonding greater
downfield shift
 Intramolecular hydrogen bonding does not
show any shift in absorption due to change in
concentration
 Intermolecular hydrogen bonding causes upfield shift
upon decrease in concentration
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=
>

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40

 Non equivalent protons on adjacent carbons have
magnetic fields that may align with or oppose the
external field.
 This magnetic coupling causes the proton to absorb
slightly downfield when the external field is
reinforced and slightly upfield when the external
field is opposed.
 All possibilities exist, so signal is split
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 First point, signal splitting only occurs between non-
equivalent hydrogens – in other words, Ha1 in 1,1,2-
trichloroethane is not split by Ha2, and vice-versa.
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 Second, splitting occurs primarily between hydrogens
that are separated by three bonds. This is why the Ha
hydrogens in ethyl acetate form a singlet– the nearest
hydrogen neighbors are five bonds away, too far for
coupling to occur.
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44
 Third point is splitting is most noticeable with
hydrogens bonded to carbon. Hydrogens that are
bonded to heteroatoms (alcohol or amino hydrogens)
are coupled weakly - or not at all - to their neighbors.
This has to do with the fact that these protons
exchange rapidly with solvent or other sample
molecules.

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March 28, 2023 76
H H H H
| | | |
C - Y C - CH C - CH2 C - CH3
SINGLET DOUBLET TRIPLET QUARTERT

 Distance between the peaks of multiplet or the
distance between the split peaks
 Denoted by “J”
 Measured in Hz or cps
 J value is usually in the range of 0-20 Hz
 Not dependent on strength of the external field
 Not dependent on the frequency of EMR caused by
magnetic field of another nuclei provides the
magnitude of splitting (Hz)
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Both these molecules show two signals in NMR?
Ha & Hb protons in cis and trans gives double doublet signal?
Then how to confirm the structure?
It can be confirmed by J value
J value of trans structure is 15 Hz
Cis isomer give double doublet J value of cis structure is 10 Hz

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Chemistry 48

 Sample holder
 Permanent magnet
 Magnetic coil
 Radiofrequency generator
 Radiofrequency receiver
 Readout system
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49

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 It is normally tube shaped and is therefore called
the sample tube.
 It must be transparent to RF radiation, durable and
chemically inert
 Glass or Pyrex tubes are commonly used.
 These are sturdy, practical and cheap.
 Usually about 6-8cm long, 0.3-0.5cm in diameter,
with a plastic cap to contain the sample.
 This type of tubes is used for obtaining spectra of
bulk samples and solutions.
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 Carbon tetrachloride CCl4
 Carbon disulphide CS2
 Deuterated chloroform CDCl3
 Deuterated benzene C6D6
 Deuterated acetone CD3COCD3
 Deuterium oxide D2O
 DMSO-d6 CD3SOCD3
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Nuclei Internal std
1H TMS
2H CDCl3
11B H3BO3,BF3,(CH3CH2)2O
13C TMS
14N & 15N NH4NO3,NH3,CH3NO2
19F CFCl3, CF2Cl, CFCl2
31P (CH3O)3P, H3PO4
35Cl NaCl
17O Water
29Si TMS
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 It is chemically inert
 It is nontoxic – safe to use
 It is soluble in most organic solvents
 It has low boiling point and hence can be removed
easily to get back the sample
 It gives a single sharp intense peak from twelve
magnetically equivalent protons (no need to use
much)

 The protons in TMS are more strongly shielded
than the protons in pure organic compounds
 It is magnetically isotropic
 It does not make any intermolecular association
with the sample.
 The low electro negativity of silicon and four
electron releasing CH3 groups produce maximum
electron density around all equivalent protons of
TMS
 It gives reference signal at δ=0
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 Permanent magnet or electromagnet magnet is used
in NMR instrument.
 It should give stable and homogenous magnetic field
i.e. the strength and direction of magnetic field
should not change point to point.
 Strength field should very high 20,000 Gauss.
Because the chemical shifts are proportional to the
field strength.
 The magnet size is 15inches in diameter.
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 There is a relationship between the resonance
frequency of nucleus and the strength of the
magnetic field in which the sample is placed.
 For the nucleus is resonate , the precessional
frequency is equal to the applied RF radiation.
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 In order to generate radio frequency radiation,
radio frequency oscillator is used.
 To achieve the maximum interaction of the RF
radiation with the sample, the coil of oscillator
would be around the sample container.
 The oscillator irradiates the sample with a RF
radiation.
 The oscillator coil is perpendicular to the applied
magnetic field.
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59

 It is perpendicular to both magnetic field and the
oscillator coil.
 It is tuned to the same frequency as transmitter.
 When precession frequency is match with RF
radiation the nucleus induces in detector coil and
this signal is amplified and sent to read out system.
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 It gives a spectrum as a plot of strength resonance
signal on Y axis and strength of magnetic field on X
axis.
 The strength of resonance signal is directly
proportional to number of nuclei resonating at that
particular field strength.
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 It is the mathematical operation in which the
complex waveform can be broken-down into simple
mathematical operations.
 It is the mathematical operation required to convert
a time domain spectrum to frequency domain
spectrum
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 FTNMR or pulse NMR, the sample is irradiated
periodically with brief, highly intense pulses of
radiofrequency radiation, following which the free
induction decay signal - a characteristic radio-
frequency emission signal stimulated by the
irradiation – is recorded as a function of time.
 The frequency- domain spectrum can be obtained
by a Fourier transform employing a digital
computer
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 The central component of the instrument is a highly
stable magnet in which the sample is placed.
 The sample is surrounded by the transmitter/receiver
coil.
 A crystal controlled frequency synthesizer having an
output frequency of Vc - produces radio-frequency
radiation.
 This signal passes into a pulse switch and power
amplifier, which creates an - intense and reproducible
pulse of RF current in the transmitter coil.
 Resulting signal is picked up by the same coil which now
serves a as - receiver.
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 The signal is then amplified and transmitted to a
phase sensitive detector.
 The detector circuitry produced the difference
between the nuclear signals Vn and the crystal
oscillator output Vc which leads to the low frequency
time-domain signal.
 This signal is digitalized and collected in the memory
of the computer for analysis by a Fourier transform
program and other data analysis software.
 The output from this program is plotted giving a
frequency domain spectrum.
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 FT-NMR is more sensitive and can measure weaker
signals.
 The pulsed FT-NMR is much faster (seconds instead of
min) as compared to continuous wave NMR.
 FT-NMR can be obtained with less than 0.5 mg of
compound. This is important in the biological chemistry,
where only μg quantities of the material may be
available.
 The FT method also gives improved spectra for
sparingly soluble compounds.
 Pulsed FT-NMR is therefore especially suitable for the
examination of nuclei that are magnetic or very dilute
samples.
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67

 Proton NMR used for study of number of
nonequivalent proton present in unknown
compound.
 Carbon NMR can used to determine the number of
nonequivalent carbons and to identify the types of
carbon atoms(methyl, methylene, aromatic,
carbonyl….) which may present in compound.
 13 C signals are spread over a much wider range
than 1 H signals making it easier to identify & count
individual nuclei.
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 The chemical shift of the CMR is wider(δ is 0-
220ppm relative to TMS) in comparison to PMR(δ
is 0-12ppm relative to TMS).
 13 C-13 C coupling is negligible because of low
natural abundance of 13 C 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.
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 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 under ratio.
 Proton coupled spectra the signal for each carbon or
a group of magnetically equivalent carbon is split by
proton bonded directly to that carbon & the n+1
rule is followed.
 13 C nucleus is about one-fourth the frequency
required to observe proton resonance.
 The chemical shift is greater for 13 C atom than for
proton due to direct attachment of the
electronegative atom to 13 C
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 Chemical Shifts are measured in ppm (are
measured in ppm (δ) from the carbons of TMS)
from the carbons of TMS
 The correlation chart is here divided into sections
1. the saturated carbon atom which appear at
Upfield nearest to TMS(8-60ppm)
2. effect of electronegative atom(40-80ppm)
3. Alkenes and aromatic carbon atom(100-170) 4) It
contain carbonyl carbon bond. which appear at
Downfield value
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 Natural abundance- 13 C natural abundance is very
low (1.1%).
 Gyro magnetic ratio- 13 C nucleus gyro magnetic
ratio is much lesser than proton nucleus. 13 C-
1.404; 1 H-5.585.
 Coupling phenomenon- 13 C & 1 H have I=1/2 so
that coupling between them probably occur.
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72

 Fourier Transform Technique
 Decoupling Technique-
1. Broad Band Decoupling
2. Off Resonance Decoupling
3. DEPT (Pulse) Decoupling
 Nuclear Overhauser Phenomena
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 Chemistry laboratories
Chemists rely on NMR Spectroscopy as a tool to chart
the complex molecular structures of matter.
 Food quality control and research
widely used across the industry to map protein
structures, profile amino acids, identify carotenoids
and quantify metabolites.
 MRI scans
Most people are familiar with Magnetic Resonance
Imaging (MRI) scans which use powerful magnetic
fields and radio waves to reveal detailed images of the
internal organs
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 Cancer diagnosis
The ability to analyse abnormal behaviour in the
cellular metabolism allows scientists to detect the
metabolite-based biomarkers associated with cancers.
 Drug discovery and development
From trialing new cancer therapies to perfecting
nutritional supplements, NMR Spectroscopy is a
mainstay in the drug discovery and development area.
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 Assign the of number of signals
Different kinds of protons present in a molecule can be
predicted
 Assign the position of signals
It gives us the magnetic (electronic) environment of each
kind of proton
 Measure and record the intensity of signals (or) area under
the peak
Intensity of the peak is proportional to the number of
protons of each kind
 Record the multiplicity (or) splitting of signal
It gives us about the environment of a nuclei (proton) with
respect to each other or nearby nuclei/proton
 Identify Exchangeable protons if any
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 Step 1: Draw the molecular structure
 Step 2: Count number of different protons
Three 1. CH3
2.CH2
3.OH
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77

 Protons found in different functional
groups or different structures will have different
chemical shifts
 Upfield chemical shift (closer to TMS signal) (lower
delta )
 shielding
 Downfield chemical shift (further away to the left
from TMS signal) (higher delta )
 "deshielding"
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79

 Relative area under the signals identifies the
number of protons having that same
electronic environment (or)
 The area under each peak is in direct
proportion to the number of protons
responsible for the absorption
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72
30
43
Integration 5:2:3
C8H10O2
Benzyl Acetate

 The three peaks of benzyl acetate can be assigned as
follows
 Total grids=72 +30+43=145
 From molecular formula total number of
protons=10
 Area that corresponds to each proton= 145/10 = 14.5
 Number of proton at δ 7.3= 72/14.5=5
 Number of proton at δ 5.1=30/14.5=2
 Number of proton at δ 2.3=43/14.5=3
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 Due to "neighboring protons" (protons bonded to carbons
adjacent to the carbon whose proton signal is being observed)
the magnetic field generation of these neighboring protons
interact with the proton signal which splits it
Kinds of splitting patterns
 singlet (one peak)= zero neighboring protons
 doublet (two peaks) = 1 neighboring proton
 triplet (three peaks) = 2 neighboring protons
 quartet (four peaks) = 3 neighboring protons
 quintet (five peaks) = 4 neighboring protons
 sestet (six peaks) = 5 neighboring protons
 Number of splits = n + 1 where n = total of neighboring
protons
on both sides of the carbon having the protons being observed
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 Chemical shift will depend on concentration and
solvent.
 The signals are often broad and usually show no
splitting pattern (undergo rapid exchange of protons
through hydrogen bonding and so no coupling with
adjacent protons).
 To verify that a particular peak is due to O-H or N-H,
first take spectrum without D2O
 Then take another spectrum after mixing the sample
with D2O. Deuterium will exchange with the O-H or N-
H protons.
 On a second NMR spectrum the peak(app at δ 4.5) will
be absent, or much less intense
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85

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