Application Note 21

Structural Elucidation by Benchtop
NMR Spectroscopy: IbuprofenIntroduction
One of the major uses of nuclear magnetic resonance(NMR) spectroscopy is for the structural elucidationof unknown chemical compounds, and the structuralconfirmation of known compounds.
In this application note, a series of one- and twodimensional NMR spectra obtained on theOxford Instruments X-Pulse Broadband Benchtop
NMR Spectrometer are analysed, showing how
the information obtained by NMR spectroscopy
corresponds to structural features in a known
molecule. In this case the chosen molecule isIbuprofen, and the spectra were obtained of a 1 mol/ℓ
solution in CDCl3.

Ibuprofen
Ibuprofen, 2-(4-(2-methylpropyl)phenyl)propanoicacid, is an anti-inflammatory drug that is commonlyused to treat pain, fever, and inflammation. Ibuprofen
was originally discovered in 1961 by Dr Stewart
Adams and Dr John Nicholson, working at Boots UKLtd., in Nottingham, U.K. It is on the World HealthOrganisation’s List of Essential Medicines, and isavailable without prescription worldwide. It waschosen as an example here due to it’s similarity inmolecular weight and structural complexity to a wide
range of common production pharmaceuticals thatare readily analysable by benchtop NMR.
The molecular structure of Ibuprofen is shown infigure 1, where the eight unique proton chemical
environments (1, 3, 4, 6, 7, 9 - 11), and the ten uniquecarbon environments (2 - 11) can be clearly identified.

Figure 1 Molecular Structure of Ibuprofen.

Structural Elucidation
One-dimensional NMR Spectra
Simple one-dimensional NMR spectroscopy entails theexcitation of nuclei of a single isotope to get information
about those atoms only. This produces spectra withsignal intensity against frequency. The chemical shift (δδ)
is plotted on the horizontal axis, in ppm (parts per million).
The plotting of chemical shift in ppm rather than Hertz (Hz)ensures comparability between data measured by different
spectrometers.

1	The chemical shift, δδH, of the signal in the spectrum, whichcorresponds to the local chemical environment of thenuclei giving rise to the signal.
2	The (integrated) area of each signal, which corresponds
to the number of nuclei associated with each signal. The
integral value shown in green below each signal in figure 2.
3	The multiplicity of the signal, which comprises thenumber of peaks in the signal, their relative intensities,
and the separation between the peaks. It also provides
information on other nearby NMR-active nuclei.

Proton (Hydrogen-1) NMR
The first NMR spectrum obtained of most known or
unknown samples, is usually a simple proton (hydrogen-1)
NMR spectrum, which for most samples can be obtainedin under 5 minutes. The 1H NMR spectrum of Ibuprofen isshown in figure 2.

By initially considering only the chemical shifts andintegrated areas of the signals, an initial assignment can bemade as follows:Signals in an NMR spectrum comprise one or moreindividual peaks, for example while the signal at δδH 11.9
ppm is a single peak, the signal centred at δδH 3.7 ppm
comprises four peaks (and commonly would be described
as a quartet). There are three major features that should beconsidered for each signal in a 1H NMR spectrum:• five signals, arising from thirteen aliphatic protons.

• one carboxylic acid proton.
• four aromatic protons.

Since there is only a single carboxylic acid proton inIbuprofen, 1, that can be assigned to the signal at δδH 11.9
ppm. The signal for four aromatic protons centred around δδH
7.2 ppm, can be assigned to protons 6 & 7.1
To further assign the five alkyl signals, their multiplicity
should be considered alongside their integrations andchemical shifts.

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Figure 2 1H NMR spectrum of Ibuprofen in CDCl3
1The multiplicity of this signal is consistent with a para substituted aromatic ring, however an explanation of this is beyond the scope of

this Application Note.

In simple cases, the number of peaks that make up an
1
H NMR signal correlate to the number of protons boundto adjacent carbon atoms, with one more peak that thenumber of carbon atoms. It is therefore possible to identify
the following facts for the remaining unassigned signals.
The signal at δδH 3.7 ppm is made up of four peaks, andintegrates as a single proton, implying a CH group adjacent
to a CH3 group, the only proton in Ibuprofen consistent withthis is 3.
There are three signals at δδH 2.4, 1.5 & 0.9 ppm, each madeup of two peaks (a doublet) implying they’re immediatelyadjacent to a single CH group. The signal at δδH 0.9 ppm
integrates as six protons, suggesting two CH3 groups, withthe two methyl groups 11, as the only possible protons inIbuprofen it could be. The signal at δδH 2.4 ppm integrates
as two protons, thereby corresponding to a CH2 bound toa CH, and consistent with 9 in Ibuprofen. The remaining
doublet at δδH 1.5 ppm overlaps with the multiplet (meaninga signal with a complex multiplicity), at δδH 1.9 ppm,
however close inspection of the integrations, identifies thedoublet as corresponding to three protons, hence as a CH3
bound to CH, and 3 in Ibuprofen.
By a process of elimination, the multiplet at δδH 1.9 ppm, canbe assigned to the CH group 10, which is bound to two CH3groups and one CH2 explaining the more complex coupling
pattern.
While unnecessary to assign the proton NMR spectrum
in this case, there’s one more piece of information which
may be obtained from a 1H NMR spectrum to aid in theassignment of signals.

When couplings occur between nuclei, resulting in observationof multiplets in the NMR spectrum, the magnitude of that
coupling can be measured (in Hz) from the separation of thepeaks in the multiplet. Since the coupling is a constant, thesame value will be measured whichever signal from a pairis chosen. This can be applied to pair up signals in the NMR
spectrum that are directly coupled. For example, the quartetat δδH 3.7 ppm, which has been assigned as 3, has a coupling
constant, 3JHH of 7.1 Hz, while the doublets at δδH 2.4, 1.5 &0.9 ppm, have coupling constants of 3JHH 6.7, 7.2, & 6.3 Hz
respectively, confirming that the signal at δδH 3.7 ppm, is coupledwith that at δδH 1.5 ppm (which has already been assigned as4). 2 This potentially time-consuming process of measuring andpairing couplings to aid assignment can be avoided with theuse of two-dimensional spectra, as shown later for the 1H-1H
COSY spectrum.
Carbon-13 NMRWhile proton is a high gyromagnetic ratio nucleus with near
100% natural abundance, making it the ‘best’ nuclei for NMR
spectroscopy, the situation for carbon is more complex. The
most abundant isotope of carbon, carbon-12, is NMR inactive.
Instead carbon NMR spectra are measured using carbon-13,
this has only 1.1% natural abundance along with a lower
gyromagnetic ratio than proton. Therefore, carbon-13 is1/5870th the receptivity of the proton; hence requiring higher
sample concentrations and longer measurement times.
The carbon-13 NMR spectrum is shown in figure 3, thespectrum appears simpler than the proton spectrum with all
signals appearing as a single sharp peak. This is because all
carbon-proton couplings have been removed by applying
proton-decoupling.

Figure 3 13C{1H} NMR spectrum of Ibuprofen in CDCl3.
2The magnitude of the J-coupling can also provide information about the relative positions of the nuclei; however there are nosuitable examples in Ibuprofen, putting this beyond the scope of this Application Note.

This spectrum is referred to as a proton-decoupledcarbon-13 NMR spectrum, simplified to 13C{1H}
NMR spectrum.3
However, as previously noted there are ten chemicallyunique carbon environments in Ibuprofen, yet there are
only nine signals observed in the NMR spectrum due to theIbuprofen, suggesting that two signal overlap appearing asa single peak.
Were this a proton NMR spectrum, it would be easy
to identify any overlapping signals, by considering theintegrated areas of the peaks. In general a protondecoupled carbon-13 NMR spectrum is an exception to therule that integration of peaks/signals directly corresponds
to the number of nuclei giving rise to that signal.4 There issome information which can be inferred from the intensityof signals in a 13C{1H} NMR spectrum. In general signals ofcarbon atoms not directly bound to any protons (quaternarycarbons), are considerably less intense than if the carbon
atom is directly bound to one or more protons.

3This can be observed for the signals at δδC 181.4, 140.9 &
137.2 ppm, along with the signal (a 1:1:1 triplet) arising forthe deuterated chloroform solvent.
By considering the chemical shift of these carbon signals,
it’s possible to assign the signal at δC 181.4 ppm as acarbonyl group, and hence to the carboxylic acid carbon 2.
While the signals at δC 140.9 & 137.2 ppm are consistent
with aromatic carbons, and therefore the quaternarycarbons 5 & 8.
The two considerably more intense signals in the aromaticregion, at δδC 129.5 & 127.4 ppm, can therefore be assigned
as the aromatic CH carbons 6 & 7.
The remaining four signals in the carbon-13 NMR
spectrum, are all consistent with alkyl, CHx carbons, withthose at a lower chemical shift likely to correspond withthe terminal methyl, CH3 groups. A complete assignment ofthese signals requires additional information which cannot
be obtained from a simple one-dimensional 13C{1H} NMRspectrum.

Carbon-carbon couplings are generally not observed, due to the low natural abundance of carbon-13, and hence the weak signalsthat would result.
4It is possible to obtain carbon-13 spectra that can be integrated, however that requires non-standard sequences, and even longer

experiment durations.

Figure 4 1H-1H gradient-selective Correlation Spectroscopy NMR spectrum of Ibuprofen in CDCl3.
Two-dimensional NMR Spectra
So far, only simple one-dimensional NMR spectra havebeen considered. However, one of the great strengths on
NMR spectroscopy, is the wide range of different pulse
sequences available, and the different spectra which may
be obtained. One major group of sequences are those thatgive two-dimensional correlation spectra. In these cases,
cross-peaks are observed corresponding to interactions(such as through J-couplings) between signals observed inthe corresponding one-dimensional spectra. The following
examples demonstrate how the introduction of a second
dimension can resolve complications due to overlap,
significantly simplify the assignment process, and result in
rapid identification of complex molecules.
H-1H Correlation Spectroscopy
The first two-dimensional spectrum to be considered in theproton-proton Correlation Spectroscopy (COSY) spectrum,
which is shown in figure 4 for Ibuprofen.
1Note that on the x and y axis, the simple proton spectrum
discussed previously is displayed. The actual twodimensional spectrum consists of cross-peaks shown in red
in figure 4.
As a homonuclear correlation spectrum, a COSY issymmetric along the diagonal, with off-diagonal crosspeaks present when signals directly couple. In general,cross-peaks will be observed in the COSY, for signal pairs
where J-coupling is observed in the one-dimensional
spectrum.
In the case of Ibuprofen, cross-peaks are observed in
the COSY spectrum for 4-CH3 & 3-CH; additionally crosspeaks are observed between the three signals 9-CH2,
10-CH & 11-CH3. This is also an example of where COSY
cross peaks can be observed when the J-coupling is notobserved in the one-dimensional spectrum, as in the casefor the four-bond interaction between 9-CH2 & 11-CH3.

Figure 5 1H-13C gradient-selective Heteronuclear Single-Quantum Correlation with multiplicity editing NMR spectrumof Ibuprofen in CDCl3.
H-13C Correlation Spectra
In addition to proton-proton correlation experiments
like the COSY, pulse sequences which allow for theobservation of proton-carbon correlations also aid theassignment process. Two of the most commonly used
are the Heteronuclear Single Quantum Coherence with
Multiplicity Editing (HSQC-ME) sequence which allow for
the observation of single bond proton-carbon correlations,and the Heteronuclear Multiple Bond Correlation (HMBC)
sequence which allows for the observation of multiple
bond proton-carbon correlations.

1The 1H-13C HSQC-ME and 1H-13C HMBC spectra (figures5 & 6) both show the proton spectrum in the horizontal
dimension, while the carbon spectrum is displayed in
the vertical dimension. The cross peaks in figure 5 for theHSQC-ME spectrum of Ibuprofen correspond to the single
bond proton-carbon correlations.

This spectrum was also obtained multiplicity edited,which provides information on the multiplicity of the CHxenvironments. With CH and CH3 giving cross-peaks withpositive phase (red in figure 5), and CH2 giving cross-peaks
with negative phase (blue in figure 5).
The first thing that should be noted from the HSQC-ME
spectrum is it explains the nine signals that are observedin the one-dimensional 13C{1H} spectrum when ten signals
were expected. The signal at δδC 45.2 ppm, correlates withtwo proton signals: 3-CH (δδH 3.69 ppm) & 9-CH2 (δδδH 2.44ppm), confirming that the two carbon atoms directly boundto the aromatic ring have indistinguishable 13C chemicalshifts.

Figure 6 1H-13C gradient-selective Heteronuclear Multiple-Bond Correlation NMR spectrum of Ibuprofen in CDCl3.

The 1H-13C HMBC spectrum of Ibuprofen is shown infigure 6, with the observed cross-peaks corresponding
to two or three bond proton-carbon correlations. TheHMBC spectrum allows for the full structural assignment /elucidation for Ibuprofen to be completed.
For example, the signal at δδH 3.69 ppm for 3-CH, correlateswith an aromatic CH at δδC 127.4 ppm and a quaternaryaromatic carbon at δδC 137.2 ppm, identifying the sourceof those signals as 6 and 5 respectively. By systematically
applying this for all the signals, the carbon-connectivity canbe deduced, and the full spectrum assigned / structure
elucidated.

elucidation of chemical compounds. The Total Correlation
Spectroscopy (TOCSY) sequence allows for the observationof not only the direct proton-proton couplings observed in
the COSY, but also indirect proton-proton couplings throughthe spin system; and is discussed in our Application Note
“Spin Locking: Total Correlation Spectroscopy (TOCSY)”.
While the Nuclear Overhauser Enhancement Spectroscopy(NOESY) shows cross-peaks arising from a through-spaceinteraction,5 and allows for the three-dimensional structureof molecules to be determined.

Additional Two-dimensional Correlation Spectra
The COSY, HSQC and HMBC are not the only twodimensional pulse sequences available on the X-Pulse
which are useful for the assignment of NMR spectra, and5

Rather than the through-bond interactions observed by COSY, TOCSY, HSQC & HMBC.

Ibuprofen Spectral Assignments
By analysing all these spectra, it’s possible to fully assign
the signals in the one-dimensional spectra. The complete
assignment of the proton and carbon-13 NMR spectra ofIbuprofen are detailed as follows.
δδ H (60 MHz, 40°C, CDCl3): 11.94 (1H, s, 1-OH), 7.46 – 6.83
(4H, m, 6,7-C6H4), 3.69 (1H, q, 3JHH 7.1, 3-CH), 2.44 (2H, d,3
JHH 6.7, 9-CH2), 1.89 (1H, qt, 3JHH 6.7, 3JHH 6.3, 10-CH), 1.48
(3H, d, 3JHH 7.2, 4-CH3), 0.88 (6H, d, 3JHH 6.3, 11-CH3).
δδC (15 MHz, 40°C, CDCl3): 181.36 (1C, s, 2-C=O), 140.93(1C, s, 8-CH), 137.19 (1C, s, 5-CH), 129.51 (2C, s, 7-CH),
127.43 (2C, s, 6-CH), 45.21 (2C, s, 3-CH & 9-CH2), 30.25(1C, s, 10-CH), 22.51 (2C, s, 11-CH3), 18.21 (1C, s, 4-CH3).

Summary
Using a range of one- and two-dimensional proton andcarbon-13 NMR pulse sequences, the Oxford Instruments
X-Pulse Broadband Benchtop NMR Spectrometer,
complete structural assignment / elucidation can be
performed on a wide range of small molecules. Acquisitionof the experiments demonstrated here can be automatedfor up to 25 samples at once when X-Pulse is used inconjunction with the X-Auto autosampler.

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