EXPERIMENT 4: NMR Analysis of a Constitutional Isomer

4.1 Abstract

4.2 Introduction

4.3 Objectives

4.4 Theory

4.5 Apparatus and materials

4.6 Procedure

4.7 Result

4.8 Discussion

16

4.9 Conclusion

17

4.10 References

17

4.11 Appendices

17

4.1

Abstract

The objective of this experiment is to identify compounds of constitutional isomer using

NMR analysis. There are 9 compounds (A-I) needed to be identified. The compounds are
constitutional isomers. The data analysis from 13C NMR and 1H NMR are given for each
compound. The constitutional isomers are made up from three different species element (C,
O, H). It is given the compounds having the following elemental composition (C: 64.3%, O:
28.6%, H: 7.2%) and a relative mass of 112.1 g/mol. From the percentage, the total molecular
mass and the relative atomic mass of C,O and H we get that the unknown compounds are
made up of C6H8O2. From the NMR data analysis of 1H NMR and 13C , it can be concluded
that Compound A was buta-1, 2-dien-1-yl acetate, Compound B was 2-methylcyclopentane-1,
3-dione, Compound C was Cyclohexane-1,4-dione, Compound D was ethenyl (2E)-but-2enoate, Compound E was methyl 2-methylidenebut-3-enoate, Compound F was
cyclobutylideneacetic acid, Compound G was (3E)-5-hydroxyhexa-3,5-dien-2-one,
Compound H was 6-methyl-2,3-dihydro-4H-pyran-4-one, and lastly Compound I was hex-2ynoic acid.

4.2

Introduction

In 1983, Isidor Rabi who is American physicist is the first person who described nuclear
magnetic resonance and measured it in molecular beams. On 1944, he was awarded the Nobel
Prize for his work. On 1946, Felix Blonch and Edward Mills Purcell are expanded the
techniques for use on liquids and solid and they are both shared the Nobel Prize in Physics in
1952. They found that 1H and 31P can absorb radio frequency energy such as radio wave when
it placed in a magnetic field at a specific strength (Wikipedia, 2011).
When the nuclei is absorb the radio frequency energy, the nucleus is being an resonance.
Different nuclei will resonate at different radio frequencies but in the same strength of
magnetic field. Nuclear Magnetic Resonance (NMR) is the effect where magnetic nuclei in a
magnetic field absorb energy from the electromagnetic and re-emit that energy. Energy that is
emitted is at a specific frequency which is depend on the strength of the magnetic field itself
and also depends on the other factors. If a strong magnetic field is applied, the energy
difference between two spin is larger and higher energy will required for a spin-flip but if a
weaker magnetic field is applied, the energy difference between two spin is smaller and less
energy is required McMurry (2006).
Atom consists of neutron, proton and electron. In the nucleus, it consists of subatomic
particles called neutron which is neutral and also proton which are positively charged. In this
case of NMR, all stables nuclides that contain an odd number of proton or neutron have an
intrinsic magnetic moment and also angular momentum. Or in the other words, nuclides that
contain odd number of proton or neutron have nonzero spin while nuclides that contain an
even number of proton or neutron have zero spin. This is because nuclides that contains even
number of proton and neutron will produce the two spins in an orbital which are opposite
to each other that produce zero spin. So, if this nuclides have zero spin, so there is no
magnetic moment, so it cannot react to the external electromagnetic field.
1

The most commonly nuclei that used in this NMR study are hydrogen , 1H and also carbon,
13
C. In the absence of magnetic field, the spin of nuclei are oriented randomly but when these
nuclei is placed in the magnetic field, the spin of nuclei will perform specific oriented.

magnet

gate
frequency
source

sample
and RF coil
amp

mixer
pre-amp
Figure 4.1: A simple figure of the NMR
Frequency source is used to generate a wave at frequency close to the resonance frequency.
The frequency is gated, amplified and applied to the probe to generate an excitation field.
Following the excitation and sample consists of a rotating bar magnet that couples into the
coil to produce an oscillating voltage across the coil which is detected and amplified. The
mixer is used to remove the excitation frequency so that only the offset frequency is recorded.
Nowadays, NMR spectroscopy is being using in many aspects of disciplines which are in
medicine, industries and also in scientific research. This is because NMR spectroscopy
becomes a powerful analytical technology in order to determine the physical and chemical
properties of atom or molecule. Table below shown some of the applications of NMR
spectroscopy in medicine:

Applications
Solution structure

How NMR spectroscopy works

This method is very useful in order to
determine biomacromolecules in aqueous
solutions.
This is the most powerful technique that can be
used to quantify motional properties of
biomacromolecules.
This technique is used to determine the residual
structure of unfolded protein and the structure
of folding intermediates.
This toll is used to detect the interactions of
interior water with biomacromolecules.
This technique is important because it can
identify drug leads besides it can determine the
conformations of the compound that bound to
enzymes, and other proteins.

Molecular dynamics

Protien folding

Protien hydration
Drug screening and design

Table 4.2 Application of NMR spectroscopy in medicine by Michigan State University

(www.chemistry.msu)

4.3

Objective

The main objective of conducting this experiment is to identify the structure of various
unknown compounds

4.4

Theory

NMR( Nuclear Magnetic Resonance) is based on the fact that some atomic nuclei have a
nuclear spin (I) and the presence of this spin enables them to act like a bar magnet to
determined by number of proton and neutron if both are even then no spin. In the presence of
an applied magnetic field the nuclear magnetic can oriented themselves in 2I+I ways. The
overall spin, I, is important. The presence of this spin enable them to act like a bar magnets.
The rules for determining the net spin of a nucleus are as follows;
1. If the number of neutrons and the number of protons are both even, then the nucleus
has NO spin.
2. If the number of neutrons plus the number of protons is odd, then the nucleus has a
half-integer spin (i.e. 1/2, 3/2, 5/2)
3. If the number of neutrons and the number of protons are both odd, then the nucleus
has an integer spin (i.e. 1, 2, 3)
Quantum mechanics tells us that a nucleus of spin I will have 2I + 1 possible orientations in
the presence of an applied magnetic field. For example, a nucleus with spin 1/2 will have 2
possible orientations. Since a nucleus is a charged particle in motion, it will develop a
3

magnetic field. In the absence of a magnetic field, these are randomly oriented but when a
field is applied they line up parallel to the applied field, either spin aligned or spin opposed.
Two schematic representations of these arrangements are shown below:

Figure 4.3
In NMR, electromagnetic radiation is used to "flip" the alignment of nuclear spins from the
low energy spin aligned state to the higher energy spin opposed state. Electromagnetic
radiation is used to flip the alignment of nuclear spins from low to high energy known as
resonance. The flip can be detected and used to produce spectrum, which relate directly to the
energy required to flip and the environment in which the nuclei are formed.

Figure 4.4
As this diagram shows, the energy required for the spin-flip depends on the magnetic field
strength at the nucleus. With no applied field, there is no energy difference between the spin
states, but as the field increases so does the separation of energies of the spin states and
therefore so does the frequency required to cause the spin-flip, referred to as resonance.

Figure 4.5
The difference in energy between two different spin states is:
E=h B0 /2
h = plancks constant
B = magnetic field strength
= magnetogyric ratio (strength at the nuclear magnet, unique)
The sample is positioned in the magnetic field. The sample get excites via pulsations in the
radio frequency input circuit. The pulse generates an oscillating magnetic field along the xaxis at the right angle to the applied magnetic field (B)-z-axis. The realigned magnetic fields
induce a radio signal in the output circuit which is used to generate the output signal. Fourier
analysis of the complex output produces the actual spectrum. The pulse is repeated as many
times as necessary to allow the signals to be identified from the background noise.
The Boltzmann distribution plays a very important role in the NMR. MaxwellBoltzmann
distribution describes the speed of particles where the particles do not constantly interact with
each other but move freely between short collisions. The difference between two energy level
states can be determined by Boltzmann distribution as followed:
E
N
=e kT
N

k = Boltzmann constant
T = absolute temperature
N , Higher energy
N , Lower energy
When the ratio frequency is applied to the system the distribution is changed; if the ratio
frequency matches the frequency at which the nuclear magnets naturally precess (glyrate) in
N
N
the magnetic field than more
will be promoted to
.
Chemical Shift
The magnetic field at the nucleus is not equal to the applied magnetic field; electrons around
the nucleus shield it from the applied field. The difference between the applied magnetic field
and the field at the nucleus is termed the nuclear shielding. The chemical shift is reported as a
relative measure from some reference resonance frequency.

An NMR spectrum is a plot of the radio frequency applied against absorption.

A signal in the spectrum is referred to as a resonance.

The frequency of a signal is known as its chemical shift.

The position at this resonance is referred to as chemical shift. The reference point is TMS
(tetramethylsilane). TMS has 0Hz frequency. TMS is an inert volatile nontoxic and cheap.
J-coupling
J-coupling also called indirect dipole dipole coupling is the coupling between two
nuclear spins due to the influence of bonding electrons on the magnetic field running between
the two nuclei. Nuclei which are close to one another could cause an influence on each other's
effective magnetic field. If the distance between non-equivalent nuclei is less than or equal to
three bond lengths, this effect is observable. This is called spin-spin coupling or J coupling.
Coupling combined with the chemical shift (and the integration for protons) tells us not only
about the chemical environment of the nuclei, but also the number of neighbouring NMR
active nuclei within the molecule. In more complex spectra with multiple peaks at similar
chemical shifts or in spectra of nuclei other than hydrogen, coupling is often the only way to
distinguish different nuclei.
Multiplicity
Singlet (s)
Doublet (d)
Triplet (t)
Quartet (q)
Quintet
Sextet
Septet

Intensity Ratio
1
1:01
1:02:01
1:3:3:1
1:4:6:4:1
1:5:10:10:5:1
1:6:15:20:15:6:1
Table 4.6

4.6

Procedures
1. The sample was prepared and placed in a container to be used for the experiment.
2. Sign in into the workstation. Then the TopSpin software was selected for the
experiment.
3. The sample that was prepared earlier was inserted into the NMR tube.
4. The NMR tube containing the sample was connected with the spinner, which was
later inserted to the sample depth gauge. The central line on the sample depth gauge
should be in the middle of the samples depth.
6

5. Then, the black cap on top of the magnetic bore was removed.
6. The LIFT button was pressed. At the same time, the sample depth gauge was
removed.
7. To lock on a specific substance, select the lock display window and select the
substance to lock from the solvent table window.
8. On instruments without the automatic tuning and matching, you have to manually
tune and match the sample by adjusting the screws.
9. Go through the NMR data processing set (figure 4.7) on the computer.

Figure 4.7 NMR data processing guide (www.mrl.ucsb.edu)

4.7

Results

Compounds
Compound A
Compound B
Compound C
Compound D
Compound E
Compound F
Compound G
Compound H
Compound I

NMR
13
C NMR
1
H NMR
13
C NMR
1
H NMR
13
C NMR
1
H NMR
13
C NMR
1
H NMR
13
C NMR
1
H NMR
13
C NMR
1
H NMR
13
C NMR
1
H NMR
13
C NMR
1
H NMR
13
C NMR
1
H NMR
Table 4.8

Analysis
205.6, 189.8, 103.5, 82.8, 16.9 and 16.3
5.4 (1,s), 4.5 (1, qrt), 2.2 (3,s) and 1.4 (3,d)
194.0, 111.6, 30.1 and 5.7
2.4 (4,t), 2.2 (1, qrt) and 1.5 (3,d)
208.3 and 37.6
2.7 (8,t)
163.2, 147.0, 141.3, 121.7, 97.8 and 18.2
7.4 (1,t), 7.1 (1, qnt), 5.9 (1,d), 4.7 (2,d) and 1.9 (3,d)
164.3, 141.5, 135.5, 127.2, 97.8 and 18.2
7.3 (1, t), 6.0 (2,s), 4.7 (2,d) and 2.0 (3,s)
193.0, 104.3, 70.0, 32.3 and 21.1
11.3 (1,s), 5.5 (1,s), 2.5 (2,t), 2.4 (2,t) and 2.0 (2,qnt)
152.6, 152.1, 108.6, 106.3, 57.1 and 13.5
6.1 (1,d), 5.9 (1,d), 4.5 (2,s), 2.3 (1,s) and 2.3 (3,s)
203.8, 149.5, 145.9, 32.1, 27.3, and 14.4
6.7 (1,s), 2.4 (2,t), 2.3 (2,t) and 2.0 (3,s)
158.5, 92.6, 72.9, 21.0, 20.7 and 13.4
9.7 (1,s), 2.3 (2,t), 1.6 (2, sxt) and 1.0 (3,t)

13

C and 1H NMR analysis of compounds (A-I)

4.8

Discussion

The objective of this experiment is to identify compounds of constitutional isomer using

NMR analysis. There are 9 compounds (A-I) needed to be identified. The compounds are
constitutional isomers. The data analysis from 13C NMR and 1H NMR are given for each
compound. The constitutional isomers are made up from three different species element (C,
O, H). It is given the compounds having the following elemental composition
C 64.3%
O 28.6%
H 7.2 %
and a relative mass of 112.1 g/mol.

112.1 g/mol X %E = MWE

; let E to be the subject element

MWC = 112.1 g/mol X 64.3% = 72.08 g/mol

MWO = 112.1 g/mol X 28.6% = 32.06 g/mol
MWH = 112.1 g/mol X 7.2% = 8.07 g/mol
From the percentage and the total molecular mass we know that the compounds are made up
of 72.08 g/mol of C, 32.06 g/mol of O and 8.07 g/mol of H. It is known that the relative
atomic mass for; C is 12.011 g/mol, 15.999 g/mol and 1.008 g/mol.
NC =

72.08 g /mol
12.011 g /mol

= 6C

NO =

32.06/mol
15.999 g/mol

= 2O

NH =

8.07 g /mol
1.0888 g/mo l

= 8H

From the two sets of data, we can get the ratio of the compounds to compose of C 6H8O2. In
identifying the structure of the compounds, graph of 1H NMR chemical shifts and 13C NMR
chemical shifts are used to determine and justified the structure constructed. The results are:

Compound A:

Buta-1,2-dien-1-yl acetate
13

C NMR

205.6
189.8
103.5
82.8
16.9
16.3

H NMR

Description

Description

ketone,
double bond,
double bond,
double bond,

5.4 (1,s)
4.5 (1,qrt)
2.2 (3,s)
1.4 (3,d)
sat. alkane,
sat. alkane

ester
double bond
sat. alkane
sat. alkane

13

C:

H:

Compound B:
2-methylcyclopentane-1,3-dione
13

C NMR

194.0
111.6
30.1
5.7

H NMR

Description

Description

ketone,
sat. alkane
sat. alkane

2.4 (4,t)
2.2 (1,qrt)
1.5 (3,d)
sat. alkane

sat. alkane
sat. alkane
sat. alkane

10

13

C:

H:

Compound C:
Cyclohexane-1,4-dione
13

C NMR

Description

208.3
37.6

ketone,

H NMR

2.7 (8,t)
sat. alkane

Description
sat. alkane

13

C:

11

H:

Compound D:
Ethenyl (2E)-but-2-enoate
13

C NMR

163.2
147.0
141.3
121.7
97.4
18.2

Description
ester
double bond
double bond
double bond
double bond
sat alkane.

H NMR

7.4 (1,t)
7.1 (1,qnt)
5.9 (1,d)
4.7 (2,d)
1.9 (3,d)

Description
double bond,
double bond,
double bond,
double bond,
sat alkane.

13

C:

12

Compound E:
Methyl-2-methylidenebut-3-enoate
13

C NMR

Description

164.3
141.5
135.5
127.2
97.8
18.2

ester
double bond
double bond
double bond
double bond,
Sat. alkane

H NMR

Description

7.3 (1,t)
6.0 (2,s)
4.7 (2,d)
2.0 (3,s)

double bond
double bond
double bond
sat. alkane

13

C:

H:

Compound F:

Cyclobutylideneacetic acid
13

C NMR

H NMR

Description

Description

193.0
104.3
32.3
21.1
97.8
18.2

carboxylic acid
double bond
sat. alkane
sat. alkane
double bond

11.3 (1,s)
5.5 (1,s)
2.5 (2,t)
2.4 (2,t)
2.0 (2,qnt)
sat. alkane

carboxylic acid
double bond
sat. alkane
sat. alkane
sat. alkane

13

13

C:

H:

Compound G:

(3E)-5-hydroxyhexa-3,5-dien-2-one
13

C NMR

H NMR

Description

Description

152.6
152.1
108.6
106.3
57.1
13.5

ketone
double bond
double bond
double bond
C-OH

6.1 (1,d)
5.9 (1,d)
4.5 (2,s)
2.3 (1,s)
2.3 (3,s)
sat. alkane

double bond
double bond
double bond
ROH
double bond

13

C:

14

H:

Compound H:

6-methyl-2,3-dihydro-4H-pyran-4-one
13

C NMR

203.8
149.5
145.9
32.1
27.3
14.4

H NMR

Description

Description

ketone,
double bond
double bond
sat. alkane

6.7 (1,s)
2.4 (2,t)
2.3 (2,t)
2.0(3,s)
sat. alkane
sat. alkane

double bond
sat. alkane
sat. alkane
sat. alkane

13

C:

H:

15

Compound I:

Hex-2-ynoic acid
13

C NMR

158.5
92.6
72.9
21.0
20.7
13.4

H NMR

Description

Description

carboxylic acid
triple bond
triple bond
sat. alkane

9.7 (1,s)
2.3 (2,t)
1.6 (2,sxt)
1.0 (3,t)
sat. alkane
sat. alkane

carboxylic acid
sat. alkane
sat. alkane
sat. alkane

13

C:

H:

4.9 Conclusion
From the percentage, the total molecular mass and the relative atomic mass of C,O and H we
get that the unknown compounds are made up of C6H8O2. From the NMR data analysis of 1H
NMR and 13C , it can be concluded that Compound A was buta-1, 2-dien-1-yl acetate,
Compound B was 2-methylcyclopentane-1, 3-dione, Compound C was Cyclohexane-1,4dione, Compound D was ethenyl (2E)-but-2-enoate, Compound E was methyl 2methylidenebut-3-enoate, Compound F was cyclobutylideneacetic acid, Compound G was
(3E)-5-hydroxyhexa-3,5-dien-2-one, Compound H was 6-methyl-2,3-dihydro-4H-pyran-4one, and lastly Compound I was hex-2-ynoic acid.

16

4.10 References

Application of NMR Spectroscopy in Medicine, 2010. Michigan State University.

[online]
Available
at:
<http://www2.chemistry.msu.edu/facilities/nmr/900Mhz/MCSB_NMR_applications.h
tml> [Accessed 3 April 2011]
Maxwel
Boltzman,
2009.
Chemistry
101.
[online]
Available
at:
<http://www.webchem.net/notes/how_far/kinetics/maxwell_boltzmann.htm>
[Accessed 6 April 2011]
McMurry, John, 2006. Organic Chemistry, 7th ed. Oxford: Oxford University Press.
NMR Data Processing Guide, 2010. UCSB. [online] Available at:
<http://www.mrl.ucsb.edu/mrl/centralfacilities/spectroscopy/1D_Solid_NMR_Proc.pd
f> [Accessed 6 April 2011]
Nuclear Magnetic Resonance, 2012. Wikipedia [online] Available at: <
http://en.wikipedia.org/wiki/Nuclear_magnetic_resonance> [Accessed 16 march
2011]

4.11 Appendices

17