Application Note 1

Application of Nuclear Magnetic
Resonance (NMR) Spectroscopy
for the Characterisation of
Small Molecules
Background
There are a variety of spectroscopic techniques that will give information about the structure of a molecule.
Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful technique for providing information about
functional groups, molecular backbone and the chemical environment of the nuclei in the molecule. Whileother techniques such as infra-red and Raman spectroscopy can give information about the functionalgroups and molecular backbone respectively; they cannot give all of the information about the moleculeand the environment of the nuclei.
The principle of NMR is that the resonance frequency of a nucleus is determined by its gyromagnetic ratio
and the strength of the static magnetic field. If this was the only factor determining resonance then nucleiof the same type would have identical frequencies. However, the resonance frequency of a nucleus also
depends subtly on its location within a molecule. More precisely it depends on the electron distribution ina molecule and the shielding effect of the surrounding electrons. The shielding is the result of the static
magnetic field inducing electron orbital motion. This motion generates a small magnetic field in the opposite
direction to the main field. Thus each nucleus experiences a slightly different magnetic field dependingon their location in a molecule. This effect is referred to as chemical shift and is the basis for the chemical
specificity that is one of the great strengths of NMR spectroscopy.
Chemical shift is not the only information containedin a NMR spectrum. The magnetic interaction
between neighbouring nuclei mediated throughthe bonding network is referred to as J-coupling or
scalar coupling. This coupling between nuclei resultsin multiplets in the NMR spectrum. The numberof spectral lines and spacing between them in a
multiplet provides additional information about
the structure of a molecule.
In addition, NMR has the advantage that theamplitude of the NMR signal is directly proportional
to the concentrations of the contributing nuclei.
Therefore, the ratio of the area under the different
peaks corresponds to the number of nuclei permolecule contributing to a resonance. The spectral
peak integrals are useful additional information thathelps confirm spectral assignments.

Analysis
To demonstrate the quality ofspectra that can be obtained
at 1.4 T, corresponding to a 1H
resonance frequency of 60 MHz,the 1H spectrum from 5 small
molecules are shown in figure 1.
The molecules are isomers, allhaving the same chemical formulaC6H10O2; and contain a doublebond {−C=C−} and a carboxyl group{−C(=O)O−} in the form of an ester{R'−C(=O)O−R''} or a carboxylic acid{R−C(=O)OH}.

Figure 1: Spectra of 5small molecules with thechemical formula C6H10O2;(a) vinyl butyrate, (b) ethyl
methacrylate, (c) ethyl
crotonate, (d) trans-3hexenoic acid, (e) trans-2hexenoic acid.

a)b)c)d)e)

121110987651
δ H / ppm

43210

Detailed Interpretation of the Ethyl
Crotonate spectrum
The 1H spectrum of ethyl crotonate (figure 2)
acquired at 60 MHz is shown in figure 3. There are
five resonances, labelled 1 to 5, with a range of
coupling patterns which can be used for spectral
assignment. Resonance 1 centred at 1.18 ppm is a
triplet with a splitting of 7.1 Hz. Resonance 2 centredon 1.78 ppm is a doublet of doublets with splittings
6.8 and 1.6 Hz. Resonance 3 centred at 4.09 ppm isa quartet with splitting 7.1 Hz. Resonance 4 centred
5.73 ppm is a doublet of quartets splitting 15.5 Hz
and 1.6 Hz. Resonance 5 is a doublet of quartets
centred at 6.89 ppm with splittings 15.5 Hz and 6.8
Hz. The spectral information is summarised intable 1.

OO
Figure 2: Chemical structure ofethyl crotonate

Considering the chemical shifts only and comparing
them to typical values for 1H nuclei, resonances 1and 2 are likely to originate from the two methylgroups (−CH3), with resonance 3 originating fromthe methylene group (−CH2−) and the source ofresonances 4 and 5 are the two alkene 1H nuclei.
The splitting pattern of resonances 1 and 2 canbe used to assign the appropriate methyl groups.
The triplet pattern of resonance 1 and the single
splitting imply that the nuclei assigned to resonance
1 should have two identical neighbouring 1H nuclei,
while the doublet of doublets structure in resonance
2 implies two non-identical neighbouring 1H nucleiwith two different splittings. It is now possible toassign resonance 1 to the methyl 1H nuclei of theethyl group (CH3−CH2−). Further evidence for thisassignment is resonance 3 which has been assigned
to the methylene hydrogens of the ethyl group. The
quartet structure implies three identical neighbouring
1H nuclei with the same splitting as resonance 1. In
fact the triplet-quartet pair of resonances with anapproximately 7 Hz coupling, is typical of an ethylgroup. Resonance 2 can be assigned to the second
methyl group that is adjacent to the double bond,where the two alkene 1H nuclei are the source of thetwo different splittings.

Figure 3: 1H spectrum of ethyl crotonate in CDCl3 acquired at 60 MHz

Splittings across a double bond are typically larger
than those across a single bond and the mutual
coupling between the two alkene 1H nuclei accounts
for the 15.5 Hz splitting. The coupling between two1
H nuclei becomes weaker the greater the number ofbonds between them. Resonance 5 can be assigned
to the alkene 1H nuclei closest to the methyl group,
accounting for the three-bond 6.8 Hz splitting.
Resonance 4, therefore, can be assigned to thealkene hydrogen nuclei closest to the carboxylgroup with the weaker four-bond coupling to themethyl group.

It is notable in figure 3 that the multiplet patternsof the resonances are not symmetrical and in thecase of the ethyl groups (−CH2−CH3) do not conformto the binomial pattern, 1:3:3:1 and 1:2:1, of peak
amplitudes. The asymmetry is particularly obvious inresonances 4 and 5, although it is still noticeable in
the other resonances. The source of the asymmetryis strong coupling; at 60 MHz the differences inchemical shift between two neighbouring nuclei is
not necessarily much larger than the scalar coupling
between them. Under these conditions the weakcoupling assumption is no longer valid and coupling
patterns associated with weak coupling should not
be expected.

Further evidence for the assignments can beobtained by integrating the area under each of
the resonances. Normalising the sum of all fiveintegrals to a value of 10, it can be shown that eachresonance corresponds to the correct numberof nuclei.

𝞭H / ppm

Multiplicity

JHH / Hz

IntegralAssignment

1.18

triplet7.1

3.04 (3)ethyl, –CH3

2

1.78

doublet of doublets

6.8, 1.6

2.99 (3)crotonyl, –CH3

3

4.09

quartet7.1

2.00 (2)ethyl, –CH2–4

5.73

doublet of quartets

15.5, 1.6

0.99 (1)crotonyl, =CH–C(=O)–

5

6.89

doublet of quartets

15.5, 6.8

0.98 (1)crotonyl, –CH=

1

Table 1: Summary of the spectral information and peak assignments for ethyl crotonate
As with ethyl crotonate, by considering the chemicalshift, splitting patterns due to scalar coupling andpeak integrals, the resonances seen in the spectrumof the two carboxylic acids can be assigned to thedifferent 1H nuclei.

Comparison of trans-2-hexenoic andtrans-3-hexenoic acid spectra
The 1H spectra of trans-2-hexenoic acid (figure4, left) and trans-3-hexenoic acid (figure 4, right)
acquired at 60 MHz are shown in figure 5, with thespectral information summarised in table 2.

O
65

4

365
2

14

O
3
OH

2

6
15

OH

4

3
65
2

O
4
13

OH

O
2

1

Figure 4: Chemical structures of trans-2-hexenoic acid (left) and trans-3-hexenoicacid (right), showing molecular numbering schemeOH6a10a

There are a number of notable features in the twospectra. Shifting the double bond by one carbon
has quite a dramatic effect on the 1H spectrum. The
broad peak that is characteristic of a carboxylic acidgroup, shows a shift of around 2.5 ppm when thecarboxylic acid group is one carbon further awayfrom the double bond (10a & 10b). In addition thedouble bond in the trans-3-hexenoic acid is flanked
on either side by a methylene group, the result isthat the two alkene hydrogens, –CH= experience
very similar chemical environments and as aconsequence have very similar chemical shifts; inthe spectrum of trans-3-hexenoic acid they are
virtually superimposed (9a). In contrast the twoalkene hydrogen nuclei in trans-2-hexenoic acidhave markedly different chemical shifts and show
very different splitting patterns due to their different
neighbouring groups (9b & 9c).

8a9a7a10b6b9c

121110989b8b765
δ 1H / ppm

437b2

10

Figure 5: 1H spectrum of trans-2-hexenoic
acid (bottom) and trans-3-hexenoic acid(top) in CDCl3 acquired at 60 MHz

𝞭H (ppm)trans-2-𝞭δH (ppm)trans-3-Assignments

6a & 6b0.93 (3H)

0.98 (3H)

6-CH3

7a & 7b1.49 (2H)

2.05 (2H)

5-CH28a-3.06 (2H)

2-CH28b2.17 (2H)-

4-CH29a-5.57 (2H)

4-CH & 3-CH

9b

5.82 (1H)

-2-CH

9c

7.10 (1H)-

3-CH

10a & 10b12.21 (1H)

9.63 (1H)

1-COOH

Table 2: Comparison of Chemical Shifts for trans-2- and trans-3-hexenoic acid

Summary
NMR has been shown to be an extremely useful
analytical technique for the characterisationof these small molecules. It has been possible
to assign the peaks to particular nuclei inthe molecule and observe the effect thatthe environment of the nuclei can have on
the chemical shift of the peaks in the NMR
spectrum. This is particularly useful in thisexample where the molecules have thesame molecular formula. The differences in
the NMR spectra that would be observedwith totally different chemicals with different
chain lengths, functional groups and chemical
environments would be even greater,
although the same principles would apply:a) The chemical environment of the nucleus
will influence the chemical shift of the peaksin the spectrum;b) The peak integrals from the spectrum will indicate
the number of nuclei giving rise to that specificpeak or peak multiplet.

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