NMR Spectroscopy

Spin-spin coupling- mechanism and sign of J coupling- AX, AB, ABC, AMX, AABB, AA′BB′
systems - Karplus relationship- second order effects- chemical shift reagents- double
irradiation experiments- 13CNMR –chemical shifts and line intensities- Spin decoupling-
Nuclear Overhouser effect.
NMR spectrometer

Superconducting magnet

NMR console
Workstation Rf generation and
Spectrometer amplification
control

NMR probe
 NMR Spectroscopy General trend of chemical shift.

Alkene Alkyne
COOH CHO Aromatic -OCH3 -NCH3 -CH2CH3
 Spin-Spin Coupling

Two types of magnetic coupling interactions of nuclei with non-zero spin quantum
number:
(1) Direct coupling (dipole-dipole coupling)
(2) Indirect coupling (spin-spin splitting)

The direct interaction is about 1000 times as large as the scalar coupling. Example: at 2
Å distance H-H dipolar coupling is ca 30,000 Hz.

These direct couplings make the observation and interpretation of high-resolution

NMR spectra in solids and very viscous liquids difficult,

In isotropic liquids the random motion of molecules completely averages the dipolar
coupling, so no direct effects are seen.

There are however, indirect effects, such as the Nuclear Overhauser Effect (NOE)
which have important consequences for NMR spectroscopy
 Spin-Spin Coupling
The scalar or indirect coupling J is a through bond interaction. How?

The spin of one nucleus perturbs (polarizes) the spins of the intervening electrons, and
the energy levels of neighboring magnetic nuclei are therefore perturbed by the
polarized electrons. So, the principal mechanism for J-coupling is through bond
polarization.

Two neighboring nucleus influence the energy levels of each other. The J coupling
(always reported in Hz) is field-independent (i.e. J is constant at different external
magnetic field strength), and is mutual (i.e. JAX = JXA).

Because the effect is usually transmitted through the bonding electrons, the
magnitude of J falls off rapidly as the number of intervening bonds increases.

HA HX
Why the signal for proton A and X split into doublet?
C C
 Spin-Spin Coupling
Why the signal for proton A and X split into doublet? HA HX

C C
Simplest Explanation:

The resonance position of ‘A’ depends on its total magnetic environment which is
influenced by the two different orientations of the magnetic moment of nucleus ‘X’.
When the nuclear magnet of X is aligned with ‘A’ the NMR signal of HA is deshielded.
Similarly when nuclear magnet of X is opposed with ‘A’ the HA shielded. Hence a
doublet of HA is observed.

Similarly the influence of the orientations of the nuclear magnetic moment of HA on

HX causes a doublet of HX proton.

Remember that this mutual magnetic influence between protons A and X is not
transmitted through space, but through the polarization of the electron in the
intervening bonds.

The nuclear spin of A couples with the electron spin the C-HA bonding electrons; these
in turn couple with C-C bonding electrons and then with the C-HX bonding electrons.
 HA HX

Spin-Spin Coupling C C

Therefore, this spin-spin splitting interaction is more strong for coupling via one or two
bonds and less strong through three bonds and rather weak through four or more
bonds.

Because the effect is usually transmitted through the bonding electrons, the
magnitude of J falls off rapidly as the number of intervening bonds increases.

Coupling over one (1 J), two (2 J) and three (3 J) bonds usually dominates the fine
structure of NMR spectra, but coupling across four (4 J) and five (5 J) bonds is often
seen, especially through π bonds (double and triple bonds, aromatic carbons).
 HA HX
Sign of Spin-Spin Coupling C C

The coupling constant J is a measure of the interaction between neighboring nuclei.

Now, because of spin-spin coupling interactions, for each nuclei there are four energy
levels involved in NMR transitions.

The coupling constant, J can be either positive or negative depending on how the
energy levels of the nuclei is influenced by the nuclear spin orientation of the other
nuclei

J is positive if the energy of HA is lowered by the

interactions with the opposite spin of HX (i.e. lowering
of energy of HA by interaction between HA, ∝ and HX, 𝛽
or HA, 𝛽 and HX, ∝) [right side of the figure]

J is negative the energy is lowered by the interaction

between the same spins of HA and HX.
 HA HX
Sign of Spin-Spin Coupling C C

Coupling constants can be either positive or negative, defined as follows: coupling

constants are positive if the energy of A is lower when X has the opposite spin as A (αβ
or βα), and negative if the energy of A is lower when X has the same spin as A (αα or
ββ).

The signs of couplings shows some consistency.

• 1 JC-H and many other one-bond couplings are positive.

• 2JH-H in sp3 CH2 groups are negative.

• 3JH-H is always positive.

 HA HX
Sign of Spin-Spin Coupling C C

The sign of J is hardly ever of importance to organic chemical applications of NMR.

For first order patterns the signs of the couplings have no effect on the appearance of
the spectrum.

For second-order patterns (e.g. ABX or AA'BB'), the relative signs of coupling constants
often have dramatic effects on the appearance of the spectrum.
 HA HX
Sign of Spin-Spin Coupling C C

It is known from spectroscopy of the hydrogen radical (H. ) that the more stable
orientation is:
when the angular momentum vectors of the nucleus and the electron are antiparallel.

Since the gyromagnetic ratio of the nucleus is positive, and that of the electron is
negative, this means that the magnetic vectors are parallel.

For the mechanism of spin-spin coupling, the bonding electrons for a H-13C fragment
will become polarized as shown, so that the more stable orientation of the 13C-nucleus
will be down, when the proton is up. This corresponds to a positive one-bond C-H
coupling.
 Sign of Spin-Spin Coupling HA HX

C C

If we continue down the bond sequence, the polarization of the C-H electrons will
cause polarization of the C-C electron pair. Again, parallel spins are the more stable
orientation (triplets are more stable than singlets -- Hund's rule). Thus the two-bond
coupling (2 J) is predicted to be negative, and the three-bond coupling (3 J) positive.
Interpret the spectrum?
First Order Coupling Rules:

1. Nuclei must be chemical shift nonequivalent to show obvious coupling to each

other. Thus the protons of CH2Cl2 , Si(CH3 )4 , Cl-CH2 -CH2 -Cl, H2C=CH2 and
benzene are all singlets.

2. J coupling is mutual, i.e. JAX = JXA always. Thus there is never just one nucleus
which shows J splitting - there must be two, and they must have the same splitting
constant J.

3. Two closely spaced lines can be either chemically shifted or coupled. It is not
always possible to distinguish J from δ by the appearance of the spectrum
For a simple example see the spectrum of 3-acetoxy-2-butanone below. Here it is pretty easy
to identify one of the doublets as the 4-methyl group, the other "doublet" (with a
separation of 9 Hz, which could easily be a coupling) actually corresponds to the two
CH3C(=O) groups.

How to identify whether doublet or closed spaced lines?

For difficult cases, (e.g. two closely spaced singlets in the methyl region) there are
several possibilities:

Obtain it at a different field strength (measured in Hz, coupling constants are field
independent, chemical shifts are proportional to the magnetic field)

Measure the spectrum in a different solvent (chemical shifts are usually more solvent
dependent than coupling constants, benzene and chloroform are a good pair of
solvents).

For multiplets with more than two lines, areas, intensities, symmetry of the pattern and
spacing of the lines generally make it easy to distinguish chemical shift from coupling.
4. Chemical shifts are usually reported in δ (units: ppm) so that the numeric values will
not depend on the spectrometer frequency (field-independent units). Coupling
constants are always reported in Hz (cycles per second).

Chemical shifts are caused by the magnetic field. Couplings are field-independent, the
coupling is inherent in the magnetic properties of the molecule.
5. Protons two (2J, geminal) or three bonds (3J, vicinal) apart are usually coupled to each
other, more remote protons (4J, 5J) may be if geometry is right, or if π-systems (multiple
bonds) intervene. Long range couplings (4J or greater) are usually small, typically <0.5 Hz,
but up to 3 Hz in some cases where there are intervening π bonds.
Multiplicity for first order patterns:

If all couplings to a particular proton are the same there will be 2nI+1 lines, where I is the
spin and n is the number of neighboring nuclei (n + 1 for 1H I = 1/2). The intensities will
follow Pascal's triangle.
If all couplings are different:

Then the number of peaks is 2n for 1H, and the intensities are 1:1:1: . . ..

Thus a proton coupled to two others by different couplings gives a dd (doublet of

doublets, see Figure).

This pattern is never called a quartet. As the number of couplings gets larger, accidental
superpositions of lines will sometimes occur.

The intensities are also often distorted by leaning effects (see AB/AX patterns), as seen
in several examples below.
More typically, some of the couplings are the same, others different:

We get a variety of patterns. In favorable cases, these patterns can be analyzed and all
couplings extracted. The number and size of couplings (J-values) provide important
structural information.
Second Order Effects:

Protons or groups of protons form simple multiplets only if the chemical shift differences
between the protons (∆ν) are large compared to the coupling constants between them
(J).

If ∆ν /J (all in Hz) is <5 then second order effect appear which complicate the analysis.
Rule of First Order Multiplets:

First, the chemical shift of the observed proton must be far away from any of the
protons it is coupled to (far away means ∆ν >> J). In practice, multiplets can be
treated in a first order fashion if ∆ν > 3J

Structure of First Order Multiplets.

The fundamental rule governing multiplet intensities for spin 1/2 nuclei with all
couplings identical is Pascal's triangle (n = number of equivalent couplings).

A very characteristic and diagnostic intensity relationship is that between the first
and second lines - the intensity ratio is 1/n, where n is the number of equivalent
coupling partners.
A first order multiplet consists of the product (not the sum) of several such multiplets. In
other words, a single line will first be split into one of the symmetrical multiplets (1:1 d,
1:2:1 t, 1:3:3:1 q, etc), then each line of this multiplet will be again split into d, t, q, or
higher multiplet.
Recognizing a First Order Multiplet:

All truly first order multiplets are centrosymmetric - there is a mirror plane in the middle
(in real spectra, this is usually not strictly true because of leaning and other distortions)

If the small outermost peaks are assigned intensity 1, then all other peaks must be an
integral multiple intensity of this 1 (one) (1x, 2x, 3x, 4x in height).

The total intensity of all peaks must be a power of 2 (2, 4, 8, 16, 32, etc).

The intensity of each of the two outermost lines is 1/2n of the total multiplet intensity,
where n is the number of protons which are coupled with the proton signal being
analyzed. There can be no lines smaller than the outermost one.

Note, however, that if n is large, the outermost peaks may not be distinguishable from
noise. Intensity assignments and determination of n cannot be easily made for such
multiplets.
There is a strict regularity of spacing in a first order multiplet: if you have correctly
identified a coupling constant J, then every peak in the multiplet must have a partner J Hz
away to the left or to the right of it.

Most first order multiplets integrate to a single proton, a few may be 2 or 3 protons in
area. It is rare to have more than 3 protons, unless there is symmetry in the molecule (e.g.,
(CH3 )2CH- gives a 6-proton doublet for the methyl groups)
Analysis of First Order Multiplets:
First order multiplets are analyzed by constructing a reverse coupling tree, by "removing"
each of the couplings in turn, starting with the smallest.

1. “Take out" the smallest couplings first. The separation between the two lines at the edge
of the multiplet is the smallest coupling. Each time you remove a coupling you generate a
new, simpler multiplet, which can then be analyzed in turn. Remember that each line of the
multiplet participates in each coupling.

2. Watch line intensities (i.e., peak areas or peak heights) carefully--when you "take out" a
coupling, the intensities of the newly created lines should be appropriate (i.e., each time
you "take out" a coupling, also "take out" the proper intensity). When a coupling has been
taken out completely, all intensity should be accounted for. Keep track of your analysis by
using a "coupling tree".

3. The couplings may be removed one at a time as doublets, or as triplets, quartets and
higher multiplets. The intensity ratio of the first two lines signals the number of protons
involved in the coupling: 1:1 means there is only one proton, 1:2 means that there are 2
protons (triplet), etc. Be especially careful to keep track of intensities when you "take out"
triplets (1:2:1) or quartets (1:3:3:1). Each time you completely remove a coupling you
generate a new simpler multiplet which follows first order rules, and can be analyzed in
turn.
When you have finished your analysis, all peaks and all intensity in the multiplet must be
accounted for. You can check the analysis as follows: the separation of the two outermost
peaks of the multiplet is the sum of all the J's (i.e., for a dt, J = 8, 3 Hz the outermost lines
are separated by 8 + 3 + 3 = 14 Hz).

Reporting a First Order Multiplet.

Multiplets are reported starting with the largest coupling, and the symbols must be in
the order of the reported numbers: δ 2.10, 1H, qt, J = 10, 6 Hz means: a single proton q
of 10 Hz, t of 6 Hz with a chemical shifts of 2.10 ppm.
First Order Analysis:
Pople Nomenclature for Coupled Spin Systems
(AX, AB, ABC, AMX, AABB, AA′BB′ systems)

The analysis of complex NMR patterns is assisted by a general labelling method for
spin systems

Each set of chemically equivalent protons (or other nuclei) is designated by a letter of
the alphabet.

1. Nuclei are labeled AX or AMX if their chemical shift differences are large
compared to the coupling between them. (∆δ > 5J). Here, A, X, M, these are non-
equivalent with different chemical shifts

2. Nuclei are labeled with adjacent letters of the alphabet (AB, ABC, MN or XYZ) if
they are close in chemical shift compared to the coupling between them (i.e. if
the different nuclei are strongly coupled).
Pople Nomenclature for Coupled Spin Systems
(AX, AB, ABC, AMX, AABB, AA′BB′ systems)

1. If groups of nuclei are magnetically equivalent, they are labeled AnXn or AnBn

2. A group of magnetically equivalent nuclei must have identical chemical shifts,

and all members of the group must be coupled equally to nuclei outside the
group.

3. If nuclei are chemical shift equivalent but not magnetically equivalent, then they
are labeled AA', BB'B'' or XX'.
Chemical Shift Equivalent and Magnetically Equivalent:

If nuclei are interchangeable by a symmetry operation or a rapid

mechanism (that is, rapid on the NMR time scale), they are
chemical shift equivalent (isochronous); that is, they have the same
chemical shift under achiral or racemic conditions.

Symmetry Operations:
The symmetry operations are
(1) rotation about a symmetry axis (Cn),
(2) inversion at a center of symmetry (i),
(3) reflection at a plane of symmetry (σ), or
(4) higher orders of rotation about an axis followed by reflection in
a plane normal to this axis (S„).
The symmetry element (axis, center, or plane) must be a symmetry element for the
entire molecule.

Nuclei that are interchangeable through an axis of symmetry (Cn) are chemical shift
equivalent in any environment (solvent or reagent) whether achiral, racemic, or chiral
(Y—CH2—Y)

Nuclei that are interchangeable through any other symmetry operation (where there
is no Cn operation) are chemical shift equivalent in an achiral or racemic environment
but not in chiral environments (Y—CH2—Z).

Nuclei that are not interchangeable through a symmetry operation (nor through a
rapid mechanism) are not chemical shift equivalent in any environment.

Rapid mechanisms involve inter- or intramolecular interchange between functional

groups (for example, hydroxylic-carboxylic acid proton interchange), conformational
interchange (for example, axial-equatorial or rotational), or inversions (for example,
RNH2); these are obviously temperature-dependent phenomena and are usually rapid
on the NMR under ordinary conditions
Symmetry in NMR Spectra:

Protons and other nuclei in NMR spectra can be classified as heterotopic,

diastereotopic, enantiotopic and homotopic.

Heterotopic and diastereotopic protons will have different chemical shifts and
couplings to neighboring magnetic nuclei.

Enantiotopic and homotopic protons will have identical chemical shifts. They may or
may not have identical couplings to other nuclei. Distinction can be made by the
substitution test.
The Substitution Test for Equivalance of Protons:

For a pair of protons to be tested, first replace one and then the other with another group
(one not present in the molecule). Compare the two structures formed.

Homotopic Protons:

If they are identical, the protons are homotopic.

Enantiotopic Protons:
If they are enantiomers, the protons are enantiotopic.

Homotopic and enantiotopic protons have identical chemical shifts.

Diastereotopic Protons:

If they are diastereomers then the protons are diastereotopic.

A typical situation where diastereotopic protons are seen is a CH2 group in a chiral
molecule

In the following example, even though diethyl acetal has no asymmetric centers, the CH2
group is diastereotopic.

Thus the ethyl group forms an ABX3 pattern

Heterotopic Protons:

If they are structural isomers, the protons are constitutionally heterotopic.

The protons of the CH2Cl group are diastereotopic. However, the protons of the cyclopropane
CH2 group are not, since they are related by a plane of symmetry.

Not all CH2 groups in chiral molecules are diastereotopic - in the chiral molecules below the
CH2 is on a C2 axis of symmetry, and the protons are homotopic.
Magnetic equivalent nuclei:

If nuclei in the same set (i.e. chemical shift equivalent nuclei) couple equally to each
nucleus (probe nucleus) in every other set of the spin system, they are magnetic
equivalent, and the designations A2, X2, etc. apply.

However, if the nuclei are not magnetic equivalent, but they are chemical shift equivalent
then the designations AA', XX' are used.
Magnetic Equivalence:

Protons that are enantiotopic or homotopic will have the same chemical shift, but they
will not necessarily be magnetically equivalent.

For two protons to be magnetically equivalent they not only have to have the same
chemical shift, but they must also each have the same J coupling to other magnetic nuclei in
the molecule.

Example: The spectrum is much more complicated than two

triplets, and both sets of protons will be identical.

The two vinyl and two allylic protons in cyclopropene are each magnetically equivalent. On
the other hand, the two pairs of equivalent protons in trans-
bis(carbomethoxy)cyclopropane are NOT magnetically equivalent, because each of the A
protons is coupled differently to the two X protons (one is a trans coupling, the other a
cis).
In general any system which contains chemical shift equivalent but magnetically
inequivalent nuclei of the AA' type will not give first order splitting patterns.

However, sometimes the spectra may appear to be first order ("deceptively simple"
spectra).
Pople Nomenclature for Coupled Spin Systems
(AX, AB, ABC, AMX, AABB, AA′BB′ systems)

Thus in an A2X2 system the A nucleus must have identical couplings to the two X
nuclei.

In an AA'XX' system, on the other hand, JAX ≠ JAX’.

Pople Nomenclature for Coupled Spin Systems
(AX, AB, ABC, AMX, AABB, AA′BB′ systems)

There are usually profound differences in the appearance of A2X2 compared to AA'XX'
patterns.

Two-Spin Systems:

AX:

First order. Significant parameters: JAX. A and X are each doublets.

AB:
Intensities are distorted: the doublets are not 1:1; the inner lines are larger, the outer
lines smaller.
Pople Nomenclature for Coupled Spin Systems
(AX, AB, ABC, AMX, AABB, AA′BB′ systems)

Three-Spin Systems:

AX2:

First order. Significant parameters: JAX. First order. A is a triplet, X is a doublet

AB2:

Second order. Both JAB and νAB must be calculated - neither can be directly measured
from the spectrum. Significant parameters: JAB, νAB.
AMX:
First order. Significant parameters: JAM, JAX, JMX. A, M and X are each doublet of
doublets (assuming all three couplings are large enough to detect). Typical systems are
trisubstituted benzenes, vinyl groups, and monosubstituted furans and thiophenes.

ABX:
Second order. This is a very common pattern. JAB is directly measurable. The
parameters JAX, JBX, νA and νB can be calculated from the line positions of the spectrum
once it has been properly analyzed.

ABC:
Second order. This pattern can only be accurately solved using computer simulation
methods. Manual analysis as a distorted ABX or even AMX pattern will lead to
approximate values of coupling constants, which in severe cases can be drastically
wrong.
Pople Nomenclature for Coupled Spin Systems
(AX, AB, ABC, AMX, AABB, AA′BB′ systems)

Four-Spin Systems:

AX3: First order. Significant parameters: JAX. Commonly seen in CH3CHXY groups.

AA'XX’:
Second order. Common pattern. Can be solved by hand, but there are several
ambiguities. For example, one cannot distinguish JAA' from JXX’.

Significant parameters: JAA', JXX', JAX, JAX'. The AA' and XX' patterns are each
centrosymmetric, and they are identical to each other, hence the inability to
distinguish A from X parameters. A common type is X-CH2-CH2-Y, which is often just
two triplets. Also common are p-disubstituted benzenes: and dioxolanes:
Pople Nomenclature for Coupled Spin Systems
(AX, AB, ABC, AMX, AABB, AA′BB′ systems)

Four-Spin Systems:

AA'BB' : Second order. This is a common pattern. Requires computer simulation to

solve. Seen in X-CH2CH2-Y groups where X and Y have similar shift effects. The entire
multiplet is centrosymmetric (i.e., the AA' part is a mirror image of the BB' part).
Second Order Effects in Coupled Systems

For first order systems J and δ values are directly measurable from line positions. In these
cases the chemical shift differences between the protons (∆ν) are large compared to the
coupling constants

When ∆ν /J (all in Hz) is < 5 then second order effects appear. When ∆ν /J < 1 then
second order effects become very pronounced, often preventing detailed manual
interpretation of multiplets.

If the spectrum is measured at higher spectrometer frequency the chemical shifts (in Hz)
become larger, whereas the coupling constants stay the same, so the spectrum usually
gets simpler.
The aromatic signals go from nearly a singlet at 60 MHz to a reasonably resolved set of
peaks at 600 MHz (spectra courtesy of Kris Kolonko). This increase in information content
and greater ease of interpretation of NMR spectra at higher magentic field strength is the
main justification for the additional expense of more powerful magnets.
A nice example is provided by the compound below. For the BrCH2CH2O group the two
methylenes at δ 3.48 and δ 3.81 have a relatively large chemical shift separation, and they
form recognizable triplets, although with a little leaning. For the MeOCH2CH2O group the
chemical shift between the CH2 groups is small, and the signals are a complicated multiplet
with only a vague resemblance to a triplet.
Thus Me3Si-CH2-CH2-OH is not just two triplets, since the CH2CH2 is an AA'XX' system. These
patterns do not get simpler at higher field strengths.
AX and AB Spectra
More Than One Value of J:

Analysis of the splitting pattern to extract the information about the different J values
has been discussed.
1H and 13C NMR:

Both give us information about the number of chemically nonequivalent nuclei

(nonequivalent hydrogens or nonequivalent carbons)

Both give us information about the environment of the nuclei (hybridization state,
attached atoms, etc.)

The signal for the NMR of a 13C nucleus is 10-4 times weaker than the signal for a
hydrogen nucleus

A signal for a 13C nucleus is only about 1% as intense as that for 1H because of the
magnetic properties of the nuclei.

At the "natural abundance" level only 1.1% of all the C atoms in a sample are 13C (most
are 12C)
13Csignals are spread over a much wider range than 1H signals making it easier to identify
and count individual nuclei

It is much easier to identify the compound as 1-chloropentane by its 13C spectrum than by
its 1H spectrum.

ClCH2 CH3

ClCH2CH2CH2CH2CH3
13C chemical shifts are measured in ppm (δ) from the carbons of TMS

13C Chemical shifts are most affected by:

• Hybridization state of carbon

• Electronegativity of groups attached to carbon

sp3 hybridized carbon is more shielded than sp2

An electronegative atom deshields the carbon to which it is attached

 Alkyne

Alkene
CHO, ketone Aromatics
(sp2)
-Alkane

200 180 160 140 120 100 80 60 40 20

COOH
derivative C-N in nitrile C-X
C-O
C-N
The correlation chart is divided into four sections. Saturated carbon atoms appear at
highest field, nearest to TMS (8 to 60 ppm).

The next section of the correlation chart demonstrates the effect of electronegative
atoms (40 to 80 ppm).

The third section of the chart includes alkene and aromatic ring carbon atoms (100 to
175 ppm).

Finally, the fourth section of the chart contains carbonyl carbons, which appear at the
lowest field values (155 to 220 ppm).

Electronegativity, hybridization, and anisotropy all affect 13C chemical shifts in nearly
the same fashion as they affect 1H chemical shifts; however l3C chemical shifts are
about 20 times larger.

Electronegativity produces the same deshielding effect in carbon NMR as in proton

NMR-the electronegative element produces a large downfield shift.

The shift is greater for a 13C atom than for a proton since the electronegative atom is
directly attached to the 13C atom, and the effect occurs through only a single bond, C-
X.
Steps towards deducing the structure of an organic compound using 13C NMR:

1. Number of signal – this will give you the number of non-equivalent carbon atom (do
not count the signal for solvent)

2. Use the chart to assign the types of carbon based on the region (δ = 0 – 80, 80 – 150,
160 – 200(carbonyls)

3. Intensity of the signals: Non proton bearing carbons give lower intensity, higher the
number of equivalent carbons the higher is the intensity
13C Chemical Shifts:

It is possible to predict the chemical shift of almost any 13C atom using the table.

Starting with a base value for the molecular skeleton and then adding increments
that correct the value for each substituent.

Corrections for the substituents depend on both the type of substituent and its
position relative to the carbon atom being considered.

The ipso carbon is the one to which the substituent is directly attached. The
calculations for m-xylene start with the base value and add these increments as
follows:
Spin-Spin Splitting of 13C signal:

The spins of protons attached directly to 13C atoms do interact with the spin of carbon
and cause the carbon signal to be split ac-cording to the n + 1 Rule

Both 13C and 1H have I = ½, so that we should expect to see coupling in the spectrum
between (a) 13C – 13C and (b) 13C – 1H

The probability of two adjacent 13C atoms being together in the same molecule is so
low that this coupling is not observed

However, the coupling between 13C and 1H interactions is observed in 13C spectra.

These coupling makes the spectrum more complicated and therefore they have been
eliminated by decoupling.
Splitting Pattern of 13C signal:

This is heteronuclear (carbon-hydrogen) coupling involving two different types of

atoms.

Since the hydrogens are directly attached to the carbon-13 (one-bond couplings), the
coupling constants are quite large, with J values of about 100 to 250 Hz.

Compare the typical three-bond H-C-C-H couplings that are common in NMR spectra,
which have J values of about 1 to 20 Hz.
Proton Decoupling:

Only singlet peaks are observed in a decoupled l3C NMR spectrum. Although this
technique simplifies the spectrum and avoids overlapping multiplets, it has the disadvantage
that the information on attached hydrogens is lost.

Proton decoupling is accomplished in the process of determining a l3C NMR spectrum by

simultaneously irradiating all of the protons in the molecule with a broad spectrum of
frequencies in the proper range.

Most modern NMR spectrometers provide a second, tunable radiofrequency generator,

the decoupler, for this purpose.

Irradiation causes the protons to become saturated, and they undergo rapid upward and
downward transitions, among all their possible spin states.

These rapid transitions decouple any spin-spin interactions between the hydrogens and
the 13C nuclei being observed.

In effect, all spin interactions are averaged to zero by the rapid changes. The carbon nucleus
"senses" only one average spin state for the attached hydrogens rather than two or more
distinct spin states.
Decoupling and Nuclear Overhauser Enhancement (NOE):

When proton-decoupled 13C spectrum is recorded, the intensities of many of

the carbon resonances increase significantly

Carbon atoms with hydrogen atoms directly attached are enhanced the
most.

This effect is known as the nuclear Overhauser effect, and the degree of
increase in the signal is called the nuclear Overhauser enhancement (NOE).

The NOE effect is heteronuclear in this case, operating between two dissimilar
atoms (carbon and hydrogen).
Nuclear Overhauser Effect (NOE):

A change in the integrated NMR absorption intensity of a nuclear spin when

the NMR absorption of another spin is saturated.

The introduction of a second irradiation frequency, i.e. irradiation of a nucleus

other than the one being observed has two direct consequences: decoupling
and saturation.

Irradiation of a signal at the resonance frequency interferes with any coupling

of the nucleus to others in the molecule.

The effects of decoupling are almost instantaneous - once the decoupler is

turned on coupling disappears on the order of fractions of a millisecond, when
the decoupler is turned off, the coupling reappears on a similar time scale
If the frequency of a 1H is on resonance and the power is high enough, then
coupling of that nucleus can be completely suppressed.

At weaker powers, however, complicated effects arise.

The most common experiment of this type is homonuclear decoupling in

proton NMR spectra (HOMODEC), which is a simple and effective technique
for establishing coupling relationships among protons.

The experiment provides similar information to the 2D COSY experiment.

Similarly, heteronuclear decoupling provides information about the

correlation between, e.g. proton and carbon signals, much in the way that
CH-COSY experiments do
Saturation:

When a proton is irradiated transitions between α and β states are induced,

and the populations of the two states will tend to be equalized.

The rate at which this occurs is a function of the strength of the decoupling
field, but will in general be faster than T1 relaxation.

If the field is powerful enough (i.e., if the induced transitions greatly exceed the
rate of normal T1 relaxation), the populations of the α and β states will become
identical and the signal will disappear (become saturated).

If the decoupler is turned off, normal signal intensity will return as a function
of T1 (the coupling will return to normal on a much shorter time scale).
Nuclear Overhauser Effect (NOE): How does it happen?

The alteration of normal spin population of a nucleus X by irradiation will cause

the populations (and hence signal intensities) of other (non-irradiated) nuclei
(A) to change provided that X is causing T1 relaxation of A by the dipole-dipole
mechanism.

This is known as the Nuclear Overhauser Effect (NOE).

Distinction between Decoupling and the NOE experiment:

In a decoupling experiment (HOMODEC) the irradiation of a specific nuclei must

be on during acquisition of the FID.

In an NOE experiment the decoupler is on during a delay period, but may be

turned off during the acquisition of the FID.
Origin of the NOE Effect:

When a pair of protons are close in space, their magnetic dipoles interact
(Dipole-Dipole interaction, DD).

This interaction is distinct from J coupling, which is not a through space effect,
but is mediated by polarization of bonding electrons in the molecule.

The effects of DD interactions between molecules on the appearance of NMR

spectra is completely averaged by the normal tumbling of molecules in solution if
the medium is isotropic.

The DD interactions between protons do, however, dominate the 1H T1

relaxation processes in most molecules that contain more than one proton.
Decoupling and Nuclear Overhauser Enhancement (NOE)
Experiment:

Continuous irradiation
during spectrum
recording

Signal presaturation
before spectrum
recording
The NOE effect can be either positive or negative, depending on which atom
types are involved.

In the case of carbon-13 interacting with hydrogen-1, the effect is positive;

irradiating the hydrogens increases the intensities of the carbon signals. The
maximum enhancement that can be observed is given by the relationship

NOEmax is the enhancement of the signal, and it must be added to the

original signal strength:
This indicates that the 13C signals can be enhanced up to 200% by irradiation
of the hydrogens.

Because NOE increases the intensity of the carbon signals, it substantially

increases the sensitivity (signal-to-noise ratio) in the 13C spectrum.

Due to the interaction of spin dipoles, the spins of the carbon nuclei "sense"
the spin imbalance of the hydrogen nuclei and begin to adjust themselves to
a new equilibrium state.

This new equilibrium state has more spins in the lower state. This increase
of population in the lower spin state of carbon increases the intensity of the
NMR signal.
The interaction of the spin-spin dipoles operates through space, not through
bonds, and its magnitude decreases as a function of the inverse of r3, where
r is the radial distance from the hydrogen of origin.

Example:

Irradiation of the aldehyde hydrogen leads to a larger NOE for the carbon of
the syn methyl group (36.2 ppm) than for that of the anti methyl group (31.1
ppm), allowing the peaks to be assigned. The syn methyl group is closer to
the aldehyde hydrogen.
Nuclear Overhauser Enhancement (NOE):

The nuclear Overhauser effect also occurs in 1H – 1H spin decoupling, but the
effect is small

We can determine 1H-1H proximity within a molecule by means of the through

space NOE effect which can be brought about using a much weaker irradiation
by ν2 that used for decoupling.

A proton that is close in space to the irradiated proton is affected by NOE

whether or not it is coupled (J- coupling is not a requirement) to the irradiated
nuclei

In case a proton that is in close proximity as well as J-coupled to the irradiated

proton, the NOE experiment will cause partially decouple the proton
In very large molecules other factors intervene and these results may be a
decrease in signal intensity as NOE.

The through-space distances involved in 1H-1H proximity determination are

quite small and the effect decreases as the inverse of the sixth power of the
distance through space between the protons.

The usual observable enhancement is leass than 20%.

To increase the sensitivity, we use the NOE difference experiment, in which a

conventional 1H spectrum is subtracted from a specific proton – irradiated
spectrum.

This subtraction leaves only the enhanced absorptions.

Under these conditions a measurable effect can be expected between 1H
nuclei over a distance of up to about 4 A0

For example, the distance between 1,3-diaxial protons in the cyclohexane

chair form is 2.6 A0

NOE builds up slowly during irradiation and persists during acquisition while
decoupling effects are instantaneous.

Since, relaxation rates are very sensitive to the presence of paramagnetic

substances, dissolved oxygen should be removed while performing NOE
experiment
 After irradiation at
After irradiation at
3CH3 frequency 
Interpretation: H4 No change after this peak the No change after
this H4 intensity is
and CH3 proton irradiation – so enhanced  the intensity become irradiation – so
are in close zero intensity in subtracted zero  so same zero intensity in
proximity subtracted spectrum intensity intensity in the subtracted
undergoing NOE spectrum is the enhanced subtracted spectrum
amount spectrum

After irradiation at After irradiation at

After irradiation at
Interpretati 5CH3 frequency  No change after this peak (5CH3
5CH3 frequency 
on: H4 and this H4 intensity is irradiation – so nucleus) the
this H4 intensity is
H6 protons enhanced  the intensity become
enhanced  the zero intensity in
subtracted spectrum
both are in intensity is the
subtracted spectrum subtracted zero  so same
close intensity is the intensity in the
enhanced amount spectrum
enhanced amount
proximity subtracted spectrum
with 5CH3

undergoing
NOE
NMR Shift Reagents:

NMR “shift reagent” is a reagent which is added to a compound containing an

appropriate functional group to spread out the NMR absorption pattern of the
compound

Most of the commercially available shift reagents are paramagnetic chelates of

europium

The chelate forms a complex with the functional group of the substrates and shifts
the NMR peaks
Karplus Equations:
Diaxial protons have coupling constants around 10 – 13 Hz and this is due to 1800
orientation

Diequatorial protons or those with axial/equatorial relationship, have coupling

constants around 2 - 5 Hz corresponding to about 600 orientaiton
Nuclear magnetic resonance II: Polarization transfer schemes- APT/INEPT/DEPT-
dynamic processes by NMR- restricted rotation (DMF, DMA, biphenyls, annulenes),
cyclohexane ring inversion, degenerate rearrangements (bullvalene and related systems)
- Coalescence temperature- Multi nuclear NMR- 19F, 31P, and 11B spectra- 2-D methods
COSY-HETCOR - HSQC - HMQC – TOCSY – INADEQUATE - Interpretation of
spectra
Polarization Transfer Schemes: (APT/INEPT/DEPT)
Polarization Transfer:

Change of equilibrium spin population distribution. It will have big effect on

the NMR signal such as, intensity.

In decoupling experiment the irradiation of a nearby 1H-nuclei of 13C – 1H

affect the spin population distribution of 13C.

So, it causes polarization transfer from 1H to 13C nucleus and thereby results in
an increase of 13C signal intensity.

NOE experiment is based on the polarization Transfer. The total NOE

enhancement depends on the extent of polarization transfer i.e. the number
of H atoms attached to the 13C nuclei

NOE experiment is therefore only performed to increase the peak intensities

of less sensitive nuclei (e.g. 13C) by irradiating nearby sensitive nuclei (1H)
Attached Proton Tests (APT):

The disadvantage of obtaining fully decoupled 13C NMR spectra is that all
information about how many protons are coupled to each carbon is lost.

Information about the number and position of C, CH, CH2 and CH3 carbons
is valuable in making structure and spectral assignments.

Un-decoupled spectra are not usually used because of extensive signal

overlap and poor signal-to-noise.

More efficient methods are various APT tests, in particular the DEPT pulse
sequences

Such techniques depend on the size of the 1JCH

The key to this type of experiments (APT, DEPT, INEPT) are that the signal
of the nucleus that we observe in somehow modulated by the chemical
shift of one or more other nearby nuclei through the polarization transfer.
These spin-polarization scheme based experimental techniques
(APT/INEPT/DEPT) use complex multiple pulse sequences:

Experiments:

APT (Attached proton test) – It works by using the concept of J-

modulated Spin Echo

APT Distinguishes (quarternary C and CH2) from (CH and CH3)

INEPT:

DEPT (Distortionless enhancement by polarization transfer): It allows to

identify CH, CH2, and CH3
Different Pulses:

Pulses are generally described by this angle of rotation (also called flip angle)
of the magnetization vector.

The angle of rotation is dependent on the intensity of the pulse and the width
of the pulse .
Application of a short Rf pulse at the appropriate frequency will rotate the
bulk magnetization by a specific angle

A 90º pulse width is defined as the amount of time the pulse of Rf energy is
applied to the particular sample in order to flip the bulk magnetization from
the Z-axis precisely into the X-Y plane.

For example, if it takes 8 µs to rotate 90º, then using the same Rf intensity it
will take 4*8µs = 32 µs to rotate 360º.
Single Pulse Sequence Experiments: (Normal 1D NMR spectrum)

The single pulse experiment is the simplest one-dimensional NMR

experiment. It contains three parts:

pulse delay (pd in seconds), pulse width (pw in microseconds) and

acquisition time (in seconds).

The parameter “pulse delay” specifies the time that should be about 3T1 or
longer for a complete relaxation.
One pulse experiment without decoupling:
One pulse experiment with decoupling:
One pulse experiment with decoupling and NOE:
Complex Multi-Pulse Sequences:

All of the advanced NMR techniques and MRI technology have been
developed after the advent of higher field pulse-mode spectrometers and
with the realization of careful manipulation of the ν1 and ν2 pulse
sequences

After the first 90 degree pulse is applied along x axis at ν1, the Z
magnetization flips its orientation to the transverse direction

This magnetization initially generated along the Y-axis will start dephasing
through various relaxation mechanisms

With time the Y-magnetization will be decaying and Z-magnetization will

be regained and if the signal is detected along Y axis a FID curve will be
obtained

However, if a sequence of pulses is applied at different time intervals

before the timescale of decaying Y-magnetization, it is possible to redirect
(refocus) the magnetization vector M
This idea of multiple pulse modulated spin echo provides an almost infinite
variety of interesting spectroscopic effects

https://www.youtube.com/watch?v=gqgC2XD0Oe8

https://www.youtube.com/watch?v=EDyxBWXp6IU
Effect of pulses applied at different directions on the orientation
of net magnetization vector:
Effect of pulses on the direction of net magnetization vector:
Right hand thumb rule:

The direction in which the magnetization is bent by a particular pulse may be predicted by
a simple "right-hand thumb" rule: If the thumb of your right hand represents the direction
along which a pulse is applied, then the partly bent fingers of that hand will show you the
direction in which the magnetization will be bent. Let us consider three different situations
and see if we can predict the direction of bending of the magnetization vector in each
case.
The Y-magnetization can be restored or refocused by using multiple
pulse sequences

This restoring the Y-magnetization or the signal (by not letting it

decay) is called spin-echo
Suppose we give a 90 rf pulse to a set of identical uncoupled nuclei.
Magnetization is developed in the xy plane. After a period τ a 180 ° pulse is
given. An echo is observed at 2 τ
The J modulated spin echo and the APT experiment:

Spin echo technique is based on the multiple pulse sequences

The pulse sequences focus on the events that occurs before FID
degeneration/acquition

The interval between 𝜈1 pulse and the FID detection is divided into a
preparation time and the initiating pulse, an evolution time (tau), followed
by one or more additional 𝜈1 pulses and evolution times

During this entire period, pulses and evolution time, and the subsequent FID
collection, the 𝜈2 pulse (irradiating) channel can also be activated
What happens when we alter the normal 13C-1H experiment by using the J-
modulated spin echo pulse sequence
 Step 1: Usual 90 degree pulse of 𝜈1  rotates magnetization to +y’ axis.

 Next with the decoupling channel off, the two Magnetization component
components of the doublet begin to diverge (modulated by J)

 One moving clockwise with +J/2 frequency and other one is moving
anticlock wise with -J/2 frequency (in the rotating frame).

Freqency J/2 means, it requires 2/J seconds to complete one cycle. This
also corresponds that it requires 1/J seconds to complete half of a cycle

 After the desired evolution time (tau) has elapsed, a 180 degree pulse is
delivered, reflecting each component of M through y’z plane.

 Next is decoupler is on causing all the component to freeze in the rotating

frame as well as generating additional enhancement

This can be repeated at different time evolution (tau) values

 With the decoupler still on, we collect the FID and carry out the FT leading
to the frequency domain spectra of 13C signal

 Note that the sign and intensity of the 13C-H signal is a function of which
evolution time (tau) selected.

An evolution time (tau) = 0 leads to a normal positive signal for the CH

carbon, and at evolution time (tau) = 1/J gives perfectly inverted signal
Sign and intensity of the 13C-H signal is a function of which evolution time
(tau) selected.
The reason this pulse sequence is so useful is that 13C signals of different
multiplicities (e.g. singlets for quarternary carbons, doublets for CH carbons,
triplets fro CH2 carbons and quartets for CH3 carbons) have predictably
different responses to the evolution time as shown in the equations below:
When these equation are plotted graphically it shows that:

A quarternary carbon signal is always positive through the range Tau = 0 to

Tau = 1/J

CH signal varies from +1 to -1

CH2 signal varies from +1 to 0 and then +1

CH3 goes from +1 through an inflection at zero and then -1

So, the J-modulated spin echo spectrum with tau = 1/J will have all signals
from quarternary C and CH2 carbons positive, while all signals from CH and
CH3 are negative
This is known as APT

It determines the parity (odd/even) number of hydrogens attached to a

given carbon
Although the APT experiment readily differentiates CH and CH3 signals
from C and CH2 signals

But, it is difficult to differentiate CH signals from CH3 signals and

quarternary C signals from CH2 signals unless we run a number of
experiment with differing (tau) values.

This method also requires that all 1J CH values are approximately equal

There is a simpler way to distinguish all four classes of carbons using

DEPT analysis.
DEPT (Distortionless enhancement by population transfer):

Like an APT spectrum, a DEPT 13C spectrum is designed to display separate sub-
spectra fro CH, CH2, and CH3 carbon signals

The pulse sequences used for DEPT analysis is as follows:

2-Dimensional NMR Experiment:

COSY-HETCOR - HSQC - HMQC – TOCSY – INADEQUATE

2D NMR basically provides information about connectivity and proximity

2 Dimensional NMR Experiments:

In 2D-NMR technique multi-pulse sequences are applied

2D NMR spectroscopy provide correlations between proton (or other

NMR-active nuclei) signals based on some interaction between them,
most frequently J-coupling (H-H. H-C or even C-C)

It basically provides information about connectivity and proximity

Why 2D-NMR is needed?

Because (1) it is not possible to correlated between two chemical shifts

using 1D NMR and (2) it is difficult to analyze overlapping spectra of a
large molecule
The most common types of 2D experiments are listed below.

COSY: Homonuclear correlated spectroscopy. Correlation between protons that are coupled
to each other.

TOCSY: Total Correlation Spectroscopy. Uses a spin-lock for coherence transfer. During the
spin-lock all protons of a coupled system become "strongly coupled," leading to cross peaks
between all resonances of a coupled system. Has some advantages over COSY in that small
couplings can be detected

HETCOR: Heteronuclear correlation, usually between 1 H and 13C resonances mediated by JC-
1 JC-H or longer range couplings. It has poor
H. The experiment can be run using either
sensitivity because the observed nucleus is 13C, and has been largely replaced by the inverse
detection experiments HMBC and HMQC.
During the preparation period, the equilibrium state has been established
in the pulse delay period, apply a pulse to perturb the system to an initial
state.

In the evolution period, the system is allowed to evolve freely and it will be
carried over with some information to the next period. This period is
responsible for providing the information encoded into the second
frequency domain.

The mixing time is for observing coherence transfer, cross relaxation and
chemical exchange, not mandatory for all 2D experiments.

The detection period is free from any pulses of observed nucleus. However
a decoupling pulse of nucleus other than the observed nucleus may be
used.
2- Dimensional NMR: –based on multiple pulse sequences

 Difference between 1-D and 2-D NMR?

1D NMR:

First pulse (900) –– Acquisition of FID immediately after Pulse –– Then again
Pulse

The FID collection is repeated multiple times to get better resolution of the 1D NMR
spectrum
2- Dimensional NMR: –based on multiple pulse sequences

 Difference between 1-D and 2-D NMR?

2D NMR:
First pulse (900) –– Delay period (t1 varies) during spin evolution––Second
pulse (900) –– Acquisition of FID (t2)

The delay time is varied and in each delay time the FID is collected
Collection of a series of FID (over a relaxation time period of t2) at several different
time delays of t1

 t2 is the normal relaxation time period and the FID acquisition time

 t1 is the delay (gap) between the two 900 pulses. This time-delay t1 is varied to
obtain a series of FID

 During mixing the information from one part of spin system is transferred to the
other part of the spin through bond coupling (magnetization transfer)
  A 2-Dimensional array of FIDs is obtained as collected in two
time-domains;
 2-D array of FID in t2 timescale is obtained by collecting the
FIDs at varying delay time t1

FT can be performed on the vertical data sets. During t1 time delay (which is the
spin evolution time) individual spins (HA and HB) are evolved as per their chemical
shifts and couplings. And the signals can be labelled by the chemical shifts and
coupling.

However, after the second 90°pulse the component of the magnetization which
used to be in the y' direction is now in the z-direction, and becomes undetectable.

Only the component in the x' direction evolves and is detected during collection of
the FID.
What cannot be shown in vector diagrams is that during t2 period the
magnetization in the x-y plane contains components from both the nucleus
observed as well from the frequency of its coupling partner, mediated by
the coupling between them.

When the spectra are FT transformed in the t1 dimension, the normal

coherence is detected, such signals appear along the diagonal of the
resulting 2D spectrum.

In addition the coherence from the frequency of the coupled partner is

also detected, and results in cross peaks at that position.
The signals can be easily assigned, provide one can find a signal whose chemical
shift or coupling pattern makes it unique to allow a firm assignment. Suitable
signals are the SH proton (coupled only to one other) or the H5 proton (coupled to
three other protons. On the following page is the 1D spectrum of the same sugar.
The signals can mostly be assigned, but even a careful consideration of leaning
leaves some ambiguities in the assignment.
COSY spectrum
COSY: COrrelation SpectroscopY

It allows to correlate all coupled protons

 Along the diagonal of the resulting 2D spectrum, the peak positions represent the
normal coherence of the signals are detected

 The cross peak positions represent the coherence from the frequency of the coupled
partners.
HETCOR: HETeronuclear CORrelation spectroscopY

It allows to draw correlation between the protons and the attached carbon

 This is complimentary to DEPT

 Peak positions appear from direct correlation of 13C and attached 1H

 Particularly useful for identification of diastereotopic protons.

Structure elucidation by 2D-NMR

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CT 1_Key

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RURhNHBBTUpRX2hB