Basic Two Dimensional NMR

Spectroscopy

M. Manickam

M. Manickam@ bham. ac. uk

Semester- 1; Week-4

Second year: CHM2C3

 First and Second Semester
Basic of 2D NMR
1H-1H Correlation spectra

1H-13C Correlation spectra

13C DEPT Spectra

HMBC, HSQC and NOESY

Few examples
2h Workshop and Pro-Forma

NOTE
Assessment

Given the expected compound-interpret the spectra

Pro-forma- 1 week to hand in with usual penalties for late submission

Haworth Room No 214 (administration office, between 12 noon and 2 pm)
 Application of Organic Spectroscopy
Why is it needed? What is it used for?
Structure Determination

Chemists synthesises new and known materials and they need to know the structure
To characterise materials
Is the structure as expected? Or is it different? Some interesting new reactions and
materials have been discovered from unexpected results
To enable the physical and chemical properties to be related to the structure. This
facilitates the synthesis of better materials, for example, drugs, liquid crystals,
pesticides, polymers etc
Safety reasons dictate that the structure of a materials is known so that any hazards can
be related to structure and so that a material can be safely used and disposed of
The progress of reactions can be monitored by spectroscopy, usually NMR and this
technique allows the perfect timing of reactions to provide optimum results and can
prevent unwanted further reaction
Reaction can be carried out in the NMR instrument to enable instant analysis of
structure which allows the structure of intermediates to be determined and reaction
mechanisms to be established
The purity of materials can be determined by NMR, routine checking of structures
 Why NMR
A + B Product

To find out required and side products

To analysis and confirm the natural products structures.

We use a variety of spectroscopic techniques

Mass spectroscopic: Gives a compounds mass (little

information)

IR spectroscopic: Functional groups information

UV spectroscopic: Chromospheres and conjugated systems

NMR spectroscopic :Gives great detailed structural information

and the most powerful spectroscopic method used by organic
chemists
 NMR: Nuclear Magnetic
Resonance
Basic principle

1H NMR spectroscopy provides information about the

environments of the H atoms in a molecule

It is based on the same principles as in 13C NMR spectroscopy

The 1H nucleus has nuclear spin 1/2, so when placed in a strong

magnetic field, it can exist in higher or lower energy states.
NMR: Nuclear Magnetic Resonance

When the nucleus is irradiated, it absorbs radio frequency

radiation, and nuclei in the lower energy spin states are
promoted to higher energy spin states

There are important differences between 1H and 13C NMR

spectroscopy:

The 1H atom has 99.98% abundance in naturally occurring H, so

1H NMR spectra can usually be measured by a single scan, so

FT methods are only used in exceptional circumstances

As a result, the peaks are proportional to the number of H

atoms that the peak represents - this is very valuable when
analysing spectra.
 Carbon 13 ( 13C) NMR Spectrum

Basically the same in principle to proton NMR obviously,

precessional frequencies of carbons are different to those of
protons but this is no problem
low sensitivity
Major problem is that 13C is only 1.1% of carbon additionally the
magnetic moment of 13C is 4x weaker than for 1H
Overall 13C signals are 6000x weaker than 1H signals
However, using pulse FT-NMR 30,000 pulses can be made
reasonably quickly to give an excellent spectrum
high resolution
Useful advantage is that typically 13C signals are spread over 200
units and so there is less chance of coincidence-hence-
13CNMR is 20x more resolved than 1H NMR

Chemical shift ( ) values are determined in the same way as for

proton signals-shielding and deshielding.
 NMR Spectrum

From each signal you should be able to obtain three pieces information:

From the Chemical shift, the environment of the proton-

containing group;

From the integration, the relative number of protons in the

proton- containing group;

From the splitting, the number of protons on an adjacent

carbon atom.
 1 Dimensional NMR
These are the most essential NMR spectra

Spectra have one frequency axis and one intensity axis (see spectra)

1H and 13C NMR spectra must contain all the required resonances for
the expected compound.

1 H NMR: - Integration-all protons must be accounted for

- Chemical shifts must be correct.

- Protons on adjacent carbon atoms will couple to

produce multiplets.

13C NMR: number of peaks shows number of carbon atoms

(accounting for overlap and equivalency)

Oxygen atoms, nitrogen atoms do not appear in NMR spectra but

their presence is implied in the chemical shift
General regions of Chemical
Shifts
13
C Chemical Shifts
 1
H- 1H COSY (Correlation
Spectroscopy)
2-D NMR spectra have two frequency axes and one
intensity axis. The most common 2-D spectra
involve 1H-1H shift correlation; they identify protons
that are coupled (i.e., that split each others signal).
This is called 1H-1H shift- correlated spectroscopy,
which is known by the acronym COSY.

1H-1H correlation spectra

2-D dimensional plot with 1H spectrum along each axis and
on the diagonal
Protons coupling to one another produce off diagonal correlations
This allows assignment of proton groups that are connected
in the molecule
Shows connectivity in the compound
COSY spectrum of Ethyl Vinyl
Ether
Fig:1 Stack plot Fig: 2 Contour plot

x
 1
H- 1H COSY Spectrum of Ethyl Vinyl
Ether
Fig:1. It looks like a mountain range viewed from the air because intensity
is the third axis.

These mountain-like spectra (known as stack plots) are not the spectra
actually used to identify a compound.

Instead, the compound is identified using a contour plot Fig:2, where

each mountain in Fig:1 is represented by a large dot (as if its top had
been cut off). The two mountains shown in Fig:1 correspond to the
dots labelled B and C in Fig: 2

Fig:2, the usual one-dimensional 1H NMR spectrum is plotted on both the

x- and y- axes.

To analyze the spectrum, a diagonal line is drawn through the dots that
bisect the spectrum.
 1
H- 1H COSY Spectrum of Ethyl
Vinyl Ether

The dots that are not on the diagonal (A, B, C) are called cross
peaks. Cross peaks indicate pairs of protons that are coupled.

For example, if we start at the cross peak labeled A and draw a

straight line parallel to the y-axis back to the diagonal, we hit
the dot on the diagonal at ~ 1.1 ppm produced by the Ha
protons

If we next go back to A and draw a straight line parallel to the x-

axis back to the diagonal, we hit the dot on the diagonal at ~
3.8 ppm produced by the Hb protons. This means that the Ha
and Hb protons are coupled.
 1
H- 1H COSY Spectrum of Ethyl
Vinyl Ether
If we then go to the cross peak labelled B and draw two
perpendicular line back to the diagonal, we see that the H c and
He protons are coupled; the cross peak labelled C shows that
the Hd and He protons are coupled.

Notice that we used only cross peaks below the diagonal; the
cross peaks above the diagonal give the same information.

Notice also that there is no cross peak due to the coupling of H c

and Hd, consistent with the absence of coupling for two
protons bonded to an sp2 carbon.
 HETCOR Spectrum or ( 1H- 13
C COSY)
2-D NMR spectra that show 13C-1H shift correlation are called HETCOR
from heteronuclear correlation) spectra. HETCOR spectra indicate
coupling between protons and the carbon to which they are
attached.

Example: 2-methyl-3-pentanone
The 13C NMR spectrum is shown on the x-axis and the 1H NMR
spectrum is shown on the y-axis. The cross peaks in a HETCOR
spectrum identify which hydrogens are attached to which carbons.

For example, cross peak A indicates that the hydrogens that shows a
signal at ~ 0.9 ppm in the 1H NMR are bonded to the carbon that
shows a signal at ~ 6 ppm in the 13CNMR spectrum.
Cross peak C shows that the hydrogens that show a signal at ~ 2.5
ppm are bonded to the carbon that shows a signal at ~ 34pp
HETCOR spectrum of 2-methyl-3-
pentanone
CH3
CH CH2 CH3
x

y
 DEPT 13
C NMR SPECTRA

13 C DEPT spectra enable different carbon

(CH3, CH2, CH, and quaternary)

Types to be identified

DEPT 90: only CH peaks visible?

DEPT 135: -CH2 peaks negative

-CH and CH3 peaks positive

PENDANT: -CH2 and quaternary peaks negative

-CH3 and CH peaks positive
 DEPT 13
C NMR SPECTRA
DEPT: stands for distortionless enhancement by polarization
transfer.

This technique to distinguish among CH3, CH2, and CH group

It is now much more widely used than proton coupling to

determine the number of hydrogens attached to a carbon.

DEPT 13C spectrum does not show a signal for a carbon that is
not attached to a hydrogen.

For example: 13C NMR spectrum of 2-butanone shows 4 signals

because it has 4 nonequivalent carbons, whereas the DEPT 13C
NMR of 2-butanone shows only three signals because the
carbonyl carbon is not bonded to a hydrogen, so it will not
produce a signal.
4 3 2 1 Normal 13C NMR gives 4 signals
CH3-CH2-CO-CH3 DEPT 13C NMR gives 3 signals
DEPT 13
C NMR Spectra of Ipsenol
In CDCl3 at 75.6 MHz:
6
Subspeectrum A, CH up. 5 7

Subspectrum B, CH3 and CH up, CH2 down. 4

8
The conventional 13C NMR spectrum is at HO 3
2
the bottom. 1

3X CH-

3X CH, 2X CH3

4X CH2
 Other Types Of 1H- 13C COSY

1. HMBC: 1H-13C several bond correlation

2. HSQC: 1H-13C carbon and protons direct correlation

3. HMQC: correlation between protons and other nuclei

such as 13C or 15N
HMBC SPECTRUM (Hetronuclear Multiple-
Bond CH Correlation)
This is a 2D experiment used to correlate, or connect, 1H and 13C
peaks for atoms separated by multiple bonds (usually 2 or 3).
The coordinates of each peak seen in the contour plot are the 1H
and 13C chemical shifts. This is extremely useful for making
assignments and mapping out covalent structure.

Points
Heteronuclear Multiple Bond Correlation
13C-1H Correlations over Several Bonds
Typically over 2 or 3 bonds can be seen.
Possible because of sensitivity of the powerful magnets of today's
NMR spectrometers.
Can be used to establish connectivity across barriers such as O
atoms or quaternary carbon atoms

1
H-C-13C (Two-bond) H-C-C-13C (Three- bond)
1
 HMBC of Codeine
H-8
H-9

C-2

C-3

C-4

C-6

C-1

H-8 to aromatic carbons C-1 H-9 to aromatic carbons C-1,

and C-6 ( both are three bond C-3 and C-4 ( both are three
coupling bond coupling
 HSQC (Heteronuclear Single-
Quantum Coherence)

13
C-1H correlation spectra

2 Dimensional plot -1H spectrum on one axis, 13C on the other

Shows Correlations between carbons and protons

directly attached to one another

allows further connectivity within the molecule to be established

 Nuclear Overhauser (NOESY) Spectrometry
Proximity Through Space

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

the NOE whether or not it is coupled to the irradiated proton; if it
is coupled, it remains at least partially coupled because the
irradiation is week in comparison with that used for a decoupling
experiment.

NOESY for very large molecules, ROESY for mid-size molecules

These spectra are used to locate protons that are close together in space

Can be a 1D or 2D NOESY technique

nOe is a through space effect

It has nothing to do with connectivity in the molecule

 Nuclear Overhauser
Enhancement
Applications

Elucidation of molecular constitution and conformation

Is used to solve geometric problems within a molecule

Relative stereochemistry can be seen
Regiochemistry can be seen
Powerful technique for 3D study of proteins and other
macromolecules

Aiding assignments

Investigating molecular motions

 Nuclear Overhauser
Enhancement
Which methyl signal belongs
+45%
to which group?
+18%
Ha OCOCH3 +17%
Cl Hb
Cl H3C H
N C
Cl H3C H3C H
O C C
Cl H3C COOH
Cl - 2%
Cl - 4%
semiclathrate Dimethylformamide 3-methylcrotonic
two Me groups are non- acid
equivalent owing to hindered
rotation about the C-N bond.
Both Me signals are therefore found
At 2.79 and 2.94, together with a singlet
at 8.0 for the formyl proton. If one now saturates
the Me signal at 2.94, the intensity of the formyl
proton signal increases by 18%. When instead the
other methyl signal is saturated, a decrease of 2%
is observed.
 Nuclear Overhauser Effect Difference
(NOESY) Spectrometry, 1H 1H Proximity
Through Space
O O O
2 readily
H 3C R H 3C 3
O O O1 available
6
H H H H H4 5 H
R CH3 CH3

natural product 2 3
1

NOE difference spectrometry determined the substitution pattern of a

natural product, whose structure was either 1 or 2.

NOE difference spectrometry of compound 3 will help us to

settle the final structure
 NOE difference spectrometry for
Compound 3
O
2
H3 C 3
O1
6
H4 5 H
CH3
3

Irradiation of the 5-Me group resulted in enhancement of both H-4 and H-6,
whereas irradiation of the 3-Me group enhanced only H-4; the assignments of
these entities to the absorption peaks is now clear.
2-D 13
C- 13C Correlations: INADEQUATE
Spectra

(Incredible Natural Abundance DoublEQUAntum Transfer Experiment)

2-D INADEQUATE provides direct carbon connectivities

enabling us to sketch the carbon sheleton unambiguously

2-D INADEQUATE has very limited applicability because of

its extremely low sensitivity
 Further Reading

1. Spectroscopic Methods in Organic Chemistry (fifth

edition): Dudley H. Williams and Ian Fleming
2. Spectrometric Identification of Organic
compounds (six edition): Robert M. Silverstein and
Francis X. Webster
3. Basic One- and Two- Dimensional NMR
Spectroscopy : Horst Friebolin