2DNMRSpectroscopy Advancedspectralanalysis

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2D NMR SPECTROSCOPY_1-D and 2-D NMR, NOESY and COSY, HETCOR,

INADEQUATE techniques
Presentation · August 2021

DOI: 10.13140/RG.2.2.33970.94407
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ADVANCED SPECTRAL ANALYSIS (MPC201T) UNIT-I_NMR SPECTROSCOPY
TABLE OF CONTENT
• 1-D
• 2-D NMR,
• COSY,
• NOESY
• INADEQUATE techniques
• HETCOR,
Prepared by-Subham Kumar Vishwakarma, Guided by- Dr. S Raja_Gitam University 1 |P a g e

Email: - Shubhamkumarvishwakarma7@gmail.com
NMR SPECTROSCOPY
The basis of NMR: -
The principle behind NMR is that many nuclei have spin and all nuclei are electrically charged. If an
external magnetic field is applied, an energy transfer is possible between the base energy to a higher
energy level (generally a single energy gap). The energy transfer takes place at a wavelength that
corresponds to radio frequencies and when the spin returns to its base level, energy is emitted at the
same frequency. The signal that matches this transfer is measured in many ways and processed in
order to yield an NMR spectrum for the nucleus concerned.
above, relates to spin-½ nuclei that include the most commonly used NMR nucleus, proton (H or
hydrogen-1) as well as many other nuclei such as C, N and P. Many nuclei such as deuterium (H or
hydrogen-2) have a higher spin and are therefore quadrupolar and although they yield NMR spectra,
their energy diagram and some of their properties are different.
Chemical shift: -
The precise resonant frequency of the energy transition is dependent on the effective magnetic field
at the nucleus. This field is affected by electron shielding which is in turn dependent on the chemical
environment. As a result, information about the nucleus chemical environment can be derived from
its resonant frequency. In general, the more electronegative the nucleus is, the higher the resonant
frequency. Other factors such as ring currents (anisotropy) and bond strain affect the frequency shift.
It is customary to adopt tetra methyl silane (TMS) as the proton reference frequency. This is because
the precise resonant frequency shift of each nucleus depends on the magnetic field used. The
frequency is not easy to remember (for example, the frequency of benzene might be 400.132869
MHz) so it was decided to define chemical shift as follows to yield a more convenient number such
as 7.17 ppm.
(𝜈 − 𝜈˳)
𝛿=
𝜈˳
The chemical shift, using this equation, is not dependent on the magnetic field and it is convenient to
express it in ppm where (for proton) TMS is set to ν thereby giving it a chemical shift of zero.
For other nuclei, ν is defined as Ξ ν where Ξ (Greek letter Xsi) is the frequency ratio of the nucleus
(e. g., 25.145020% for C). In the case of the H NMR spectrum of ethyl benzene, the methyl (CH)
group is the most electron withdrawing (electronegative) and therefore resonates at the lowest
chemical shift. The aromatic phenyl group is the most electron donating (electropositive) so has the
highest chemical shift. The methylene (CH) falls somewhere in the middle. However, if the chemical
shift of the aromatics were due to electro positivity alone, then they would resonate between four and
five ppm. The increased chemical shift is due to the delocalized ring current of the phenyl group.
para
meta ortho

Spin-spin coupling: -
The effective magnetic field is also affected by the orientation of neighboring nuclei. This effect is
known as spin-spin coupling which can cause splitting of the signal for each type of nucleus into two
or more lines.
The size of the splitting (coupling constant or J) is independent of the magnetic field and is therefore
measured as an absolute frequency (usually Hertz). The number of splitting’s indicates the number of
chemically bonded nuclei in the vicinity of the observed nucleus.


Coupling constant (J): -

The distance between the peaks in a simple multiplet is called the coupling constant J. The coupling
constant is a measure of how strongly a nucleus is affected by the spin states of its neighbor. The
spacing between the multiplet peaks is measured on the same scale as the chemical shift, and the
coupling constant is always expressed in Hertz (Hz).
The coupling constant is a measure of how strongly a nucleus is affected by the spin states of its
neighbor. The spacing between the multiplet peaks is measured on the same scale as the chemical
shift, and the coupling constant is always expressed in Hertz (Hz). In ethyl iodide, for instance, the
coupling constant J is 7.5 Hz.
The spectrum in Figure 5.26 was determined at 60 MHz; thus, each ppm of chemical shift (d unit)
represents 60 Hz. Inasmuch as there are 12 grid lines per ppm, each grid line represents (60 Hz)/12 =
5 Hz. Notice the top of the spectrum. It is calibrated in cycles per second (cps), which are the same as
Hertz, and since there are 20 chart divisions per 100 cps, one division equals (100 cps)/20 = 5 cps =
5 Hz. Now examine the multiplets. The spacing between the component peaks is approximately 1.5
chart divisions, so
That is, the coupling constant between the methyl and methylene protons is 7.5 Hz. When the protons
interact, the magnitude (in ethyl iodide) is always of this same value, 7.5 Hz. The amount of coupling
is constant, and hence J can be called a coupling constant.

One-dimensional NMR
1D NMR defines the types of anomeric configuration (a, b) present, provides information about non-
carbohydrate substituents (e.g., acetyl or pyruvyl moieties) and quantify degree of substitution.
1D NMR provides information about the purity of the carbohydrate. For polysaccharides with a
repeating sequence, both 13C-NMR and 1HNMR give information regarding the number of
monosaccharide residues contained in the repeating glycosyl. 13C-NMR can provide some
information regarding glycosyl linkage and can detect the presence of unusual or rarely observed
sugars. We can also acquire 31P-NMR spectra. (In a one-dimensional experiment, the signal is
presented as a function of a single parameter, usually

the chemical shift.)

1H-NMR: In 1H-NMR spectroscopy, spin transitions of only hydrogen nuclei are noticed.
Interpretation of 1H-NMR spectra can be well understood from data presented in table 1 representing
different δ values, couplings, coupling constants and chemical shifts of 1H nuclei processing in
different chemical environments. Commonly, δ value scale of 1H-NMR ranges from 0-10 ppm with
respect to Tetra methyl Silane (TMS) as internal standard.
A two-dimensional variation of NMR was first proposed by Jean Jeener in 1971; since then, scientists
such as Richard Ernst have applied the concept to develop the many techniques of 2D NMR. Although
traditional, one-dimensional NMR is sufficient to observe distinct peaks for the various functional
groups of small molecules, for larger, more complex molecules, many overlapping resonances can
make interpretation of an NMR spectrum difficult. Two-dimensional NMR, however, allows one to
circumvent this challenge by adding additional experimental variables and thus introducing a second
dimension to the resulting spectrum, providing data that is easier to interpret and often more
informative.
Basics of 2D NMR: -
• The first two-dimensional experiment was proposed by Jean Jenner.
• 2D NMR is a set of nuclear magnetic resonance spectroscopy (NMR) methods which give data
plotted in a space defined by two frequency axes rather than one.
• 2D NMR spectra provide more information about a molecule than one-dimensional NMR
spectra.
• In two-dimensional experiments, both the x and the y axes have chemical shift scales, and the
two-dimensional spectra are plotted as a grid like a map. Information is obtained from the
spectra by looking at the peaks in the grid and matching them to the x and y axes.
OR
• In a two-dimensional experiment, there are two coordinate axes. Generally, these axes also
represent ranges of chemical shifts. The signal is presented as a function of each of these
chemical shift ranges. The data are plotted as a grid; one axis represents one chemical shift
range, the second axis represents the second chemical shift range, and the third dimension
constitutes the magnitude (intensity) of the observed signal. The result is a form of contour
plot in which contour lines correspond to signal intensity.
Experimental Set-up: -
In traditional 1D Fourier transform NMR, a sample under a magnetic field is hit with a series of RF
pulses, as seen in the pulse sequence below, and the Fourier transform of the outgoing signal results
in a 1D spectra as a function chemical shift.

A 2D NMR experiment, however, adds an additional dimension to the spectra by varying the length
of time (T) the system is allowed to evolve following the first pulse. The result is an outgoing signal
f (T, t2), which, when Fourier transformed, gives a 2D spectrum of F (ω1 ω2).
The use of two-dimensional NMR allows the researcher to better resolve signals which would
normally overlap in 1D NMR. Depending on the size of your molecule, different variations or
combinations of 2D and multidimensional NMR experiments are utilized.
The Spin Hamiltonian
The spin of a given nuclei during any NMR experiment is Governed by the spin Hamiltonian. If long-
range spin interactions are ignored, the spin Hamiltonian for a one-spin system is given the equation
Ĥ=Ĥ0 + ĤRF ……………………(1)
The magnetic field along the z-axis, shielding, and J-coupling with nearby nuclei are all constant and
are accounted for in H°. HRF is the induced magnetic field resulting from an RF pulse. For a system
where two spins are coupled, the H° is
……………………(2)
Where is the Larmor frequency, I is the net magnetization vector of the given nucleus or nuclei, and
J is the observed J coupling between nuclei. is directly related to the chemical shift (δ) by the
equation
……………………(3)
Where γ is the gyromagnetic ratio of the given isotope. If nuclei 1 and 2 are of the same element and
isotope, the system is referred to as homonuclear. If they are different, it is a heteronuclear spin
system.
Homonuclear through-bond correlation methods
• Correlation spectroscopy (COSY)
• Exclusive correlation spectroscopy (ECOSY)
• Total correlation spectroscopy (TOCSY)
• Incredible natural-abundance double-quantum transfer experiment
(INADEQUATE)
Heteronuclear through-bond correlation methods
• Heteronuclear single-quantum correlation spectroscopy (HSQC)
• Heteronuclear multiple-bond correlation spectroscopy (HMBC)
Through-space correlation methods
• Nuclear Overhauser effect spectroscopy (NOESY)
• Rotating-frame nuclear Overhauser effect spectroscopy (ROESY)
1. Correlation spectroscopy (COSY): -

COSY (Correlated Spectroscopy) is a useful method for determining which signals arise from
neighboring protons (usually up to four bonds). Correlations appear when there is spin-spin coupling
between protons, but where there is no coupling, no correlation is expected to appear.
The most basic form of 2D NMR is the 2D COSY (pulse sequence shown below) experiment, a
homonuclear experiment with a pulse sequence similar to the procedure discussed above. It consists

of a 90° RF pulse followed by an evolution time and an additional 90° pulse. The resulting oscillating
magnetization (symbolized by decaying the sinusoidal curve) is then acquired during t2.
COSY is a homonuclear 2D NMR correlation spectroscopy. COSY correlates chemical shift of two
hydrogen nuclei located on two different carbons that are separated by a single bond via J coupling.
Thereby detects the chemical shift for hydrogens on both F1 and F2 axis. COSY experiment is
categorized as follows:
• Simple COSY
• DQF COSY (Double Quantum Filtered Correlation Spectroscopy)
• TQF COSY (Triple Quantum Filtered Correlation Spectroscopy)
• MQF COSY (Multiple-Quantum Filtered Correlation Spectroscopy)
Simple COSY
Simple COSY technique involves simple pulse sequence in which firstly a (π/2) x pulse is
introduced in 1H channel to create an evolution phase. There after some time a second (π/2) y
pulse is introduced to create an acquisition phase. In 1H-1H COSY pulse sequence contains
variable relaxation delay time (t1) and acquisition time (t2). Experiment is repeated with
different values of t1 and t2. So that value of t1 is increased at regular intervals, to generate a
series of different FID data during t2. COSY offers three bond coupling (3JH-H). The COSY
interpretation can be best understood from COSY spectrum of Ethyl-2-butenoate given below.
The off-diagonal peaks at point 1, 2, and 3 represents coupling of protons of hydrogens on ‘a’
with ‘b’, ‘e’ with ‘f’, and ‘e’ with ‘d’ protons of molecule of Ethyl-2-butenoate. There is no cross
peak for c because it does not possess any hydrogens.

DQF COSY (Double Quantum Filtered COSY)

1H-1H DQF COSY is a modified technique of COSY that incorporates a typical pulse sequence,
in which firstly a (π/2)x pulse is introduced in 1H channel. Nextly, after first pulse a second
(π/2)y pulse with just immediate third (π/2)z pulse is introduced to eliminate singlet peaks. This
simplifies the complex COSY spectrum. The DQF COSY interpretation can be best understood
from Ethyl acetate (liquid) in CDCl3, simple COSY and DQF COSY spectra given in figure 15.
In simple COSY spectrum of ethyl acetate, the isolated ‘d’ hydrogen produces a singlet cross
peak at spot ‘1’. But in case of 1H-1H DQF COSY spectrum of ethyl acetate spot ‘1’ is removed
and the spectra is simplified.
We use the gradient enhanced DQF-COSY pulse sequence shown below
TQF COSY (Triple Quantum Filtered Correlation Spectroscopy)

1H-1H TQF COSY employs further typical pulse sequence, in which initially a (π/2)x pulse is
introduced in 1H channel. Which is followed by introduction of a second (π/2)y pulse along with
just immediate third (π/2)z and fourth (π/2)z1 pulse. This eliminates singlet and doublet peaks.
MQF COSY (Multiple-Quantum Filtered Correlation Spectroscopy)

1H-1H MQF COSY employs a pulse sequence, in which firstly a (π/2)x pulse is introduced in
1H channel. This initial pulse is followed by introduction of a second (π/2)y pulse along with a

just immediate multiple number of (π/2)zm pulse (as desired). This eliminates triplets or any
unwanted multiple peaks and simplifies complex COSY spectrum.
Other Example: -
The COSY spectrum as shown in fig.1 for ethylbenzene contains a diagonal and cross peak (signals
that are not on the diagonal and correspond to other signals on the same horizontal and vertical
projections). The cross peaks indicate couplings between two mutliplets up to three, or occasionally
four, bonds away. The diagonal consists of the 1D spectrum with single peaks suppressed. The most
apparent cross-peak in the spectrum is between H1' and H2' at 2.65 and 1.24 ppm. A much weaker
four-bond correlation (see the figure below) appears between H1' and H2 at 2.65 and 7.20 ppm. All
the desired signals are antiphase. Half the multiplet is positive and half negative. In addition, artifacts
(undesired signals) appear in the spectrum as vertical streaks (interference and f noise) and along the
inverted 'V' (fig.2) whose tip is on the top axis of the sepctrum. These artifacts are rarely in phase
with the desired signals and appear in specific locations.
Fig.1. 2D COSY spectrum of ethylbenzene

Fig. 2. Artifacts in the COSY spectrum of ethylbenzene

A more useful representation of 2D data is called a correlation map. The correlation map of the steroid
progesterone is shown.
In this representation, the x- and y-axes correspond

to the frequencies resulting from the Fourier
transforms, and the intensity of shade at each
frequency coordinate indicates the peak intensity.
Two types of peaks are observed in a homonuclear
correlation map—diagonal peaks and cross peaks.
Diagonal peaks are found along the diagonal of the
map where the x- and y-axes have equal frequency
values and simply correspond to the absorptions
from a one-dimensional NMR experiment. Because
heteronuclear NMR does not involve the same
isotope, diagonal peaks are not observed. Cross
peaks, on the other hand, give information on the
coupling of two nuclei and are seen in both homo-
and heteronuclear spectra. Fig. 2D COSY spectrum of
progesterone

Nuclear Overhauser Effect Spectroscopy (NOESY)

One of the major applications of 2D NMR spectroscopy is the NOESY method, used to determine
the spatial structure of biomolecules. For example, several NOESY spectra cross peaks, which are
the result of interactions between different nuclear spins, can indicate several close hydrogen atoms
as in globular proteins or double helical nucleic acids.
This information can be used to infer the presence of loops when paired with sequence-specific
resonance assignments. Statistical models can be employed to find arrangements of the sequence that
are in congruence with the distance constraints.
The nuclear Overhauser effect (NOE) is the transfer of nuclear spin polarization from one
population of spin-active nuclei (e.g. 1H, 13C, 15N etc.) to another via cross-relaxation. A
phenomenological definition of the NOE in nuclear magnetic resonance spectroscopy (NMR) is the
change in the integrated intensity (positive or negative) of one NMR resonance that occurs when
another is saturated by irradiation with an RF field. The change in resonance intensity of a nucleus is
a consequence of the nucleus being close in space to those directly affected by the RF perturbation.
The NOE is particularly important in the assignment of NMR resonances, and the elucidation and
confirmation of the structures or configurations of organic and biological molecules. The two-
dimensional NOE Spectroscopy (NOESY) experiment is an important tool to identify stereochemistry
of proteins and other biomolecules in solution, whereas in solid form crystal x-ray diffraction must
be used to identify the stereochemistry.
NOESY is a homonuclear correlation spectroscopy that correlates between the nuclei which are
physically close to each other regardless of whether there is a bond between them exists or not. The
interpretation can be understood from NOESY and COSY spectrum of a quinoline derivative given
in figure 21. The two experiments differentiate on the diastereotopic protons of the CH2 group.
NOESY spectrum, shows two NOE correlations at (4.29, 1.28) and (4.29, 3.13) ppm. There are no
NOE’s to the proton signal at 2.68 ppm. The DQF-COSY below shows 2J and 3J correlations at
(4.29,3.13), (4.29,1.28) and (3.13, 2.68) ppm. There are no four-bond correlations present as the 4J
coupling constants are close to zero.

We use regular or the gradient enhanced NOESY pulse sequence as they give very similar results.
Just leave out the gradients for the regular sequence. The mixing time (tm) should be between half T1
and T1 to achieve good sensitivity for spectral assignment by NOESY. However, for quantitative
NOESY, short mixing times and long relaxation delays are required. The strength of the NOE signal
is proportional to the inverse sixth power of the distance between the atoms, I μ 1/r6. Comparison of
the cross-peak integrals in a quantitative NOESY is used as a measure of the distance between the
protons. A quantitative EXSY is used to calculate the rate of exchange of two or more nuclei.
Sensitivity of NOESY is increased by increasing the longitudinal relaxation time either by choosing
a low-viscosity solvent such as acetone-d and/or removing dissolved oxygen from the sample.
Fig. Pulse sequence for gradient NOESY

The NOESY spectrum as shown in figure below for ethylbenzene (fig.) contains a diagonal and cross
peak. The diagonal consists of the 1D spectrum. The cross peaks signals arising from protons that are
close in space. All the desired signals are pure phase, either positive or negative. NOE signals are
positive for small molecules in low-viscosity solvents and negative for very large molecules or very
viscous solvents relative to a negative diagonal. By convention one should plot the diagonal as
negative but most people plot the diagonal positively so the cross-peaks appear in the opposite sign
to the convention: negative for small molecules and positive for large molecules. Here, we plot the
diagonal as positive.

Incredible natural-abundance double-quantum transfer experiment

(INADEQUATE)
• 2D C- C INADEQUATE (Incredible Natural Abundance Double Quantum Transfer
Experiment) is useful for determining which signals arise from neighboring carbons.
• it is very insensitive as 0.01% of the carbons are excited at natural abundance. Use this
experiment as a last resort when all else fails. If there are protons within six bonds of the
carbons of interest, consider using 2D-ADEQUATE instead.
In the 2D INADEQUATE pulse sequence (figure) DELTA should be set to 1/(4J) where J is the one-
bond CC coupling. The one-bond coupling constants can be used to estimate carbon-carbon bond
order. The constants are usually 35 to 45 for a single bond and about 65 Hz for a double bond and are
increased by electronegative substituents. If the only purpose is to measure coupling constants then
1D-INADEQUATE is a more suitable experiment.
Fig. Pulse sequence for 2D INADEQUATE
The spectrum is laid out differently than other homonuclear 2D spectra. The cross-peaks are
distributed symmetrically and horizontally either side of the diagonal (red dashed line in fig.). The
connection between each pair of carbons is shown as a solid red line (figure). Each cross-peak is a
doublet split by carbon-carbon coupling. In the 2D-INADEQUATE spectrum (fig. 2) of ethylbenzene,
the carbon connectivity can be followed through the molecule from 2' to 1' to 1 to 2 to 3 to 4.

Fig.2D INADEQUATE spectrum of ethylbenzene in C6D6
When two neighboring carbons have similar chemical shifts, the cross-peak is weaker and the doublets
form a second order AB coupling pattern as shown below for the correlation between C2 and 3 of
ethylbenzene (figure).

Fig. 2D INADEQUATE spectrum of ethylbenzene showing second order coupling

HETCOR TECHNIQUE
• Heteronuclear Correlation Spectroscopy
• proton nmr spectra on one axis and the 13C nmr spectra on the other.
• The HETCOR spectra matches the H to the appropriate C.

• The information on how the H are C are matched is obtained by looking at the peaks inside the
grid. Again, these peaks are usually shown in a contour type format, like height intervals on a
map.
• In order to see where this information comes from, let's consider an example shown below, the
HETCOR of ethyl 2-butenoate.
• First look at the peak marked A near the middle of the grid. This peak indicates that the H at
4.1 ppm is attached to the C at 60 ppm. This corresponds to the -OCH2- group.
• Similarly, the peak marked B towards the top right in the grid indicates that the H at 1.85 ppm
is attached to the C at17 ppm. Since the H is a singlet, we know that this corresponds to the
CH3- group attached to the carbonyl in the acid part of the ester and not the CH3- group
attached to the -CH2- in the alcohol part of the ester.
• Notice that the carbonyl group from the ester has no "match" since it has no H attached in this
example.

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2D NMR SPECTROSCOPY_1-D and 2-D NMR, NOESY and COSY, HETCOR,

Presentation · August 2021

Subham Kumar Vishwakarma

The user has requested enhancement of the downloaded file.

Prepared by-Subham Kumar Vishwakarma, Guided by- Dr. S Raja_Gitam University 1 |P a g e

Prepared by-Subham Kumar Vishwakarma, Guided by- Dr. S Raja_Gitam University 3 |P a g e

Prepared by-Subham Kumar Vishwakarma, Guided by- Dr. S Raja_Gitam University 4 |P a g e

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Coupling constant (J): -

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the chemical shift.)

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1. Correlation spectroscopy (COSY): -

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DQF COSY (Double Quantum Filtered COSY)

TQF COSY (Triple Quantum Filtered Correlation Spectroscopy)

MQF COSY (Multiple-Quantum Filtered Correlation Spectroscopy)

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Fig.1. 2D COSY spectrum of ethylbenzene

Prepared by-Subham Kumar Vishwakarma, Guided by- Dr. S Raja_Gitam University 12 |P a g e

Fig. 2. Artifacts in the COSY spectrum of ethylbenzene

In this representation, the x- and y-axes correspond

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Nuclear Overhauser Effect Spectroscopy (NOESY)

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Fig. Pulse sequence for gradient NOESY

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Incredible natural-abundance double-quantum transfer experiment

Fig. Pulse sequence for 2D INADEQUATE

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Fig.2D INADEQUATE spectrum of ethylbenzene in C6D6

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Fig. 2D INADEQUATE spectrum of ethylbenzene showing second order coupling

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