RShowcasing benchtop NMR of small organic molecules
Example: Ibuprofen

Ibuprofen

H3C

H3C
Example of benchtop
NMR on small organic molecules
CH3 Ibuprofen (C13H18O2) is a non-steroidal anti-

Showcasing benchtop NMR of small organic molecules

inflammatory
drugand(NSAID)and is usedcommonlyIbuprofen (C13H18O2) is a non-steroidal antiinflammatory
drug (NSAID)
is commonly
for pain
Example:Ibuprofen
used
for
pain
relief,fever
reductionandagainstrelief, fever reduction and against inflammation.
inflammation.
H3C

O
H3C

OH

CH3

O

Ibuprofen (C13H18O2) is a non-steroidal antiinflammatory drug (NSAID) and is commonlyused for pain relief, fever reduction and against
inflammation.

1H
OHNMR spectrum
1H NMRThe 1H NMR spectrum of 200 mM ibuprofen in CDCl3 is shownin Figure1. The spectrum was recorded
spectrum1H NMR spectrum of 200 mM ibuprofen in CDCl
The
is
shown
in Figure1. The andspectrum1
1
in a single scan, taking 7 seconds to acquire. All peaks and H- H3couplings arewell resolved,
can
1
1
1
Thetaking
H NMR7spectrumof 200ibuprofenAll
in CDCl
1.couplings
The spectrum
H- H
was recorded
in a single
scan,
seconds
tomM
acquire.
peaks
andin Figure
3 is shown
be assigned
to the molecular
structure.
1
1
was recorded in a single scan, taking 7 seconds to acquire. All peaks and H- H couplings

are well resolved, and can beareassigned
toand
thecanmolecularwell resolved,
be assigned tostructure.
the molecular structure.

Figure 1: Proton NMR spectrum of 200 mM ibuprofen in CDCl3.

Figure 1: Proton NMR spectrum of 200 mM ibuprofen in CDCl3.

Figure 1: Proton NMR spectrum of 200 mM ibuprofen in CDCl3.

1H NMR RELAXATION

1H NMR relaxation

1H NMR
The relaxation
time measurements are shown in Figures 2 - 4. Note the long T and short T
relaxation
1

2

value of the exchanging hydroxyl proton. The amplitude of the first data point scales with
the number of protons for the corresponding peak.

The relaxation time measurements are shown in Figures 2 - 4. Note the long T1 and short T2

0.9 s The amplitude of the first data point
0.9 scaless
The relaxation timevaluemeasurementsof the exchangingarehydroxyl proton.
with
0.9 s
0.9 s
1
the
numberofprotonsforthecorrespondingpeak.
H NMR relaxation 1.3 s
shown in Figures 2 - 4. Note the long
1.3 s
2.5 s
2.3 s
1.4 s
1.3 s 2.1 s
1.3 s
T1 and short T2 value
1.3 s
0.9 s of the exchanging
0.9 s 1.3 s
The relaxation
timeofmeasurements
are shown
T and short T2
3.5 sin Figures 2 - 4. Note the longhydroxyl proton. The
amplitude
the0.9 s
0.9 1s 0.13 svalue of the exchanging hydroxyl proton. The amplitude of the first data point scales with
first data point scales
withofthe
number
1.3 s peak. 1.4 s
1.3 s
2.5
s for the corresponding
2.3 s
the2.1
numberprotonss
Figure2: Proton
T1 (left)and T
T2
relaxationtimeeach proton
positionof the molecule
ProtonT1 (left)and(right)relaxationfor each
proton2 (right)
of protons for the corresponding peak.1.3 s1.3
1.3forstime
s
1.3 s
1.3 s

0.9 s

3.5 s

0.9 s

position of the0.9molecules 0.13 s

0.9 s

1.3 s
1.3 s
2.5 s
2.3 s
2.1 s
1.4 s
relaxation time for each proton position1.3of theFigure 2: Proton T1 (left) and T1.3ss molecule
2 (right)
3.5 s

0.13 sFigure 2: Proton T1 (left) and T2 (right) relaxation time for each proton position of the molecule

Figure 3: Proton T1 relaxation time measurement of 200 mM ibuprofen in CDCl3.

Figure 3: Proton T1 relaxation time measurement of 200 mM ibuprofen in CDCl3.

Figure 2: Proton T1 relaxation time measurement of 200 mM ibuprofen in CDCl3.
Figure 3: Proton T1 relaxation time measurement of 200 mM ibuprofen in CDCl3.
Figure 4: Proton T2 relaxation time measurement of 200 mM ibuprofen in CDCl3.

Figure 4: Proton T2 relaxation time measurement of 200 mM ibuprofen in CDCl3.

Figure 4: Proton T2 relaxation time measurement of 200 mM ibuprofen in CDCl3.

Figure 3: Proton T2 relaxation time measurement of 200 mM ibuprofen in CDCl3.

1.3 s

2D COSY

2D COSY
The 2D COSY spectrum is shown in Figure 4. It clearly shows two spin systems (2,13) and(9,10,11,12). For example, the methyl group at position 13 only couples to the CH group at
position 2, whilst the methyl groups at positions 11 and 12 couple to CH and CH2 groups at
positions 10 and 9. There is no coupling of positions (9,10,11,12) to either 2 or 13.

2D COSY

The 2D COSY
spectrumis shown
The 2D COSY spectrum
is shown
in Figure4.in Figure 4. It clearly shows two spin systems (2,13) and(9,10,11,12).
For
example,themethylgroup at position 13 only couples to the CH group at
It clearly shows two spin systems (2,13) andposition 2, whilst the methyl groups at positions 11 and 12 couple to CH and CH2 groups at(9,10,11,12). For example, the methyl group at
positions 10 and 9. There is no coupling of positions (9,10,11,12) to either 2 or 13.
position 13 only couples to the CH group at
position 2, whilst the methyl groups at positions
11 and 12 couple to CH and CH2 groups at
positions 10 and 9. There is no coupling of
positions (9,10,11,12) to either 2 or 13.

Figure 4: COSY spectrum of 200 mM ibuprofen in CDCl3. The cross-peaks andcorresponding exchanging protons are marked by colour-coded ellipses and arrows.

Figure 4: COSY spectrum of 200 mM ibuprofen in CDCl3. The cross-peaks andcorresponding exchanging protons are marked by colour-coded ellipses and arrows.
Figure 4: COSY spectrum of 200 mM ibuprofen in CDCl3. The cross-peaks and corresponding
exchanging protons are marked by colour-coded ellipses and arrows.

2D HOMONUCLEAR J-RESOLVED SPECTROSCOPY
homonuclear
spectroscopy
spectrum.
Vertical traces through the peaks in the
In the 2D homonuclear2D
j-resolved
spectrum the j-resolved
2D spectrum yield the peak multiplicities, as shown
chemical shift is along the direct (f2) direction
In the 2D homonuclear j-resolved spectrum the chemical shift is along the direct (f2)by the green lines in Figure 5, and enables
and the effects of proton-proton coupling along
direction and the effects of proton-proton coupling along the indirect (f1) dimension. This
the measurement of proton-proton coupling
the indirect (f1) dimension. This allows the fullallows the full assignment of chemical shifts of overlapping multiplets, and can allow
frequencies. By comparing the coupling
assignment of chemical shifts of overlapping
otherwise unresolved couplings to be measured. The projection along the f1 dimension
frequencies between different peaks, it is possible
multiplets, and can allow otherwise unresolved
yields a “decoupled” 1D proton spectrum. Figure 5 shows the 2D homonuclear j-resolved
to extract information about which peaks arecouplings to be measured. The projection along
spectrum of ibuprofen, along with the 1D proton spectrum as blue line. The vertical
coupled to each other. For example, both thethe f1 dimension
yieldsa“decoupled”
1D
protonprojection shows how the multiplets collapse into a single peak, which greatly simplifies
multiplet at 1.90 ppm and the doublet at 3.78 ppm
spectrum.theFigure5 shows the 2D homonuclear
1D spectrum.
haveyield
a splitting
ofmultiplicities,
6.5 Hz, suggestingthat thesej-resolvedVertical
spectrumofibuprofen,
along
with
the
traces through the peaks in the 2D spectrum
the peakas showngroups are ofcoupledto eachcouplingother. These couplings
1D protonby
spectrumblue
Thethe greenas
lines
in line.
Figure5, verticaland enables the measurement
proton-proton
confirmthefindings
of
the
COSY
experiment inprojectionfrequencies.
shows howBy
thecomparing
multipletsthe
collapse
coupling frequencies between different peaks, it is possible
Figureinto a single
peak, which
greatlysimplifies
the 1Dare coupled
to extract
information
about
which peaks
to 4.
each other. For example, both the

6.5 Hz6.5 Hzmultiplet at 1.90 ppm and the doublet at 3.78 ppm have a splitting of 6.5 Hz, suggestingthat these groups are coupled to each other. These couplings confirm the findings of the
COSY experiment in Figure 4.

Figure 5: Homonuclear j-resolved spectrum of 200 mM ibuprofen in CDCl3. The multiplet
splittingfrequencies fordifferentspectrum
couplingsofare
asininCDCl
Figure4. multiplet splitting
Figure5: Homonuclear
j-resolved
200colour-codedmM ibuprofen
3. Thefrequencies for different couplings are colour-coded as in Figure 4.

2D homonuclear j-resolved spectroscopy

2D homonuclear
j-resolved spectroscopy
One unusual and often neglectedfeature of this experiment is that second order coupling
2D HOMONUCLEAR
J-RESOLVED
SPECTROSCOPY
effects show up in the indirect (f1) direction as extra peaks equidistant from the couplin

partners
wellexperimentremoved fromthe zerofrequency
the indirect dimension. These peaks a
One unusual and often neglected featureof thisis thatsecondorder incoupling
often neglected as artefacts, but provide direct evidence of second order coupling partne
effects show up in the indirect (f1) direction as extra peaks equidistant from the coupling
These extra peaks and coupling partners are marked by colour-coded ellipses and arrow
One unusual partners
and often
neglected
featurethis
thisdimension.
spectrumTheseis based
the same datawell
removed fromthe ofzero
frequency Notein the thatindirectpeaksonare
Figure 6. Note that this spectrum is based on the same data as Figure 5, only the scaling
as artefacts,
but provide
directevidenceofsecondorder
coupling
partners.
experiment isoftenthat neglected
second ordercoupling
effectsas
Figure5,
onlythescalinghaschanged.
changed.
These
extra(f1)peaks
and coupling
partners are marked by colour-coded ellipses and arrows inshow up in theindirectdirection
as extraFigurefrom
6. Notethis spectrum
is basedpeaks equidistantthethatcoupling
partners
well on the same data as Figure 5, only the scaling haschanged.
removed from the zero frequency in the indirect

dimension. These peaks are often neglected as
artefacts, but provide direct evidence of second
order coupling partners. These extra peaks andcoupling partners are marked by colour-coded
ellipses and arrows in Figure 6.

Figure 6: Homonuclear j-resolved spectrum of 200 mM ibuprofen in CDCl3 showing theextra peaks due to strong couplings.

Figure 6: Homonuclear j-resolved spectrum of 200 mM ibuprofen in CDCl3 showing theextra peaks due to strong couplings.

Figure 6: Homonuclear j-resolved spectrum of 200 mM ibuprofen in CDCl3 showing theextra peaks due to strong couplings.

1D 13C spectra

spectra1D 13C SPECTRA 1D 13CTheC NMR spectra of 2 M ibuprofen in CDCl are shown in Figure 7. The
133experiment is sensitive to all 13C nuclei in the sample. It clearly resolves 9 res
small triplet between 70 and 80 ppm is from the solvent.
The 13C NMR spectra of 2 M ibuprofen The
in CDClin Figure7. The 1DCarbon
13C DEPT
3 are shown
experimentuses polarisationtransfer between proton and carb
13
13
but
the
CH
groupsappearasnegativein to protons areexperimentsensitive
to all Cinnuclei
insample.
It
clearly
resolves
9
resonances;the peaks
The C NMR
spectrais of
2 M ibuprofen
CDClcan2
3 the
be used for spectral editing. Only carbons directly
attached
the
DEPT-135.
Throughlinearcombinationofthetriplet7.between
and 80experimentppm is fromthesolvent.
are shownsmallin FigureThe 1D 70
Carbonthese experiments. Since the peaks at 181, 140 and 137 ppm do not show in t
The
DEPT
experimentuses polarisationtransfer
betweenprotonandnucleiThe
and
three
DEPT
spectraonecarbon
cancarbons.
producesubspectra
is sensitive
to 13
allC13
C nucleiin the sample.
It clearly
spectrathey
mustbelongto quaternary
DEPT-90 experiment giv
be used forthe
spectralOnly carbons
directlyattached
toprotonsarevisibleinof thewhilst
CH3, theCHDEPT-45
andCH
groupsalone,
asshownof CH, CH2 anof CH groups,
and
DEPT-135
give
signals
resolves 9canresonances;smallediting.
triplet between
213
but140the CH
groupsappearas show
negative
peaks
inevident
the DEPT-135.
Since theThe
peaksCatDEPT
181,
and
137
ppm
do notin featurethe
DEPT
in 2Figure
7.
Aninterestingin theseThrough linea
70 and 80these
ppmexperiments.
is from the solvent.
of the three
spectraonethecanpeakproducesubspectraof the CH3, CH2 and C
spectramust belong
to quaternary
TheDEPT
DEPT-90gives
only
signalsubspectra
is experiment
that
at 45
ppm
is madeexperimentuses theypolarisationtransfer
between carbons.
alone, asup
shown
FigureAn
featureevident
in these subspectra
ofcarbonCH groups,
whilstgive
signalsof7.
groups,of ainCH
andaCH,
CHinteresting
resonance
identical
proton andnucleiand thecanDEPT-45
be used andfor DEPT-135
2 and CH3with
2CH
peak
at
45
ppm
is
made
up
of
a
CH
and
a
CH
resonancewithidentical chembut the Only
CH2 groups
appearas negative
DEPT-135.
Through linear combination2chemical
shifts.
spectral editing.
carbons
directlyattachedpeaks
to in theof visible
the three
one canSince
produce
protons are
in DEPT
these spectra
experiments.
the subspectra of the CH3, CH2 and CH groups
alone,140
as shown
in ppmFiguredo7.not
An show
interestingpeaks at 181,
and 137in feature evident in these subspectra is that the
peak
at
45
ppm
is
made
up
of
a
CH
and
a CH2 resonance with identical chemical shifts.
the DEPT spectra they must belong to quaternarycarbons. The DEPT-90 experiment gives only signalof CH groups, whilst the DEPT-45 and DEPT-135
give signals of CH, CH2 and CH3 groups,

Figure 7: Carbon spectra of 2 M ibuprofen in CDCl3.

Figure 7: Carbon spectra of 2 M ibuprofen in CDCl3.
Figure 7: Carbon spectra of 2 M ibuprofen in CDCl3.

HETCORHETCORsignal along the direct dimension and the proton
Similar to the 2D COSY experiment, which detects
signalproton-proton
along the indirect
dimension.
proton-proton
coupling
partners,aseriesof
Similar to the 2D COSY experiment, which detects
coupling
partners,Thea HETCOR
2 M ibuprofen
in CDCl3 is shown inheteronuclear
2DofNMR
experiments
seriesheteronuclear
2D have
NMRbeenexperiments spectrum
have been ofdevisedto detect coupling
Figure
8,
with
the
1D
protonand carbon
spectradevised topartners
detect coupling
partnersofdifferentof different nuclei. The Heteronuclear Correlation (HETCOR) experimentis usedfrom Figures
and7 asverticalTheand horizontal
nuclei. ThetoHeteronuclear
Correlation
correlate proton
resonances(HETCOR)
to the carbons directly
bonded1to
those
protons.
traces.
The
peaks
in
the
2D
spectrumshow which
experimentHETCOR
is used tocorrelate
protonresonances
experiment detects the carbon signal along the direct dimension and the proton
proton is bonded to which carbon.
to the carbons
bondedto dimension.
those protons.
signaldirectly
along theindirect
The HETCOR
experimentdetectsthecarbonThe HETCOR spectrum of 2 M ibuprofen in CDCl3 is shown in Figure 8, with the 1D

proton and carbon spectra from Figures 1 and 7 as vertical and horizontal traces. The peaksin the 2D spectrum show which proton is bonded to which carbon.

Figure 8: HETCOR spectrum of 2 M ibuprofen in CDCl3.
Figure 8: HETCOR spectrum of 2 M ibuprofen in CDCl3.

HMQC

HMQC

The HMQC spectrum of 2 M ibuprofen in CDCl3 is
Another heteronuclear 2D correlation experiment
Another heteronuclear 2D correlation experimentis the Heteronuclear Multiple Quantum
shown in Figure 9, with the 1D proton and carbon
is the Heteronuclear Multiple Quantum Coherence
Coherence (HMQC) experiment. Similar to HETCOR,
is usedto correlate
spectra it
from
Figures
1 and 7proton
as horizontal and(HMQC) experiment. Similar to HETCOR, it is used
resonances to the carbons directly bonded to those
protons.
However,in theverticaltraces.
The peaks
inHMQCthe 2D spectrum show
to correlate proton resonances to the carbons
experiment the carbon signal appears along thewhichindirectdimension,and theprotoncarbon.
signal A similar
protonis bonded
to whichdirectly bonded to those protons. However, inalong the direct dimension.
analysis as with the HETCOR spectrum can be
the HMQC experiment the carbon signal appears
in Figure9, withtheassignment.
1D proton
The
HMQC
spectrumof
2
M
ibuprofen
inCDCl
3 is shownfor
performed
conclusive
peak
along the indirect dimension, and the proton signal
carbonspectra from Figures 1 and 7 as horizontal and vertical traces. The peaks in thealong the anddirectdimension.

2D spectrum show which proton is bonded to which carbon. A similar analysis as with theHETCOR spectrum can be performed for conclusive peak assignment.

Figure 9: HMQC spectrum of 2 M ibuprofen in CDCl3.

Figure 9: HMQC spectrum of 2 M ibuprofen in CDCl3.

HMBC

The HMQC experiment shown on the previous page was designed to correlate pro
carbons which are connected through a one bond coupling. To obtain long-range pcarbon correlations through two or three bond couplings, the Heteronuclear Multip
Correlation (HMBC) experiment can be used. Like in the HMQC experiment the csignal appears along the indirect dimension, and the proton signal along the direct
dimension.
HMBC
The HMBC spectrum of 1 M ibuprofen in CDCl3 is shown in Figure 10, with the 1
HMQC
additionallycouplings
to
The HMQC experiment shown on the andprevious spectrathe
Figures
1 andto7correlateasshows
horizontal
The HMQC experiment showncarbon
on the previous frompage wasdesigned
protonsand
andvertical traces. The pea
quaternarycarbons,
which arenotvisiblein thepage was designed
to whichcorrelate
protons
2Dandspectrumshow
which
protons
are connected
toprotonwhichcarbons
via a long-range
carbons
are connected
througha one
bond
coupling.
To obtain
long-range
COSY
or
HMQC.
For
example,
there
are
clear
carbons which areconnected
throughatwo
oneorbondcarboncorrelations
throughthreecouplings,
the Heteronuclear
Multiple Bond
coupling.
Thebondcouplings
betweenmolecular positions
look similar to the ones founcouplings
from theprotons
at positions
coupling. To obtain
long-range
protoncarbon
Correlation(HMBC)
experimentcan beused.multibond
Like
the HMQC
HMQC
experimenttheshows
carboncouplings
the COSY
spectrum,butintheadditionallyto quaternary ca
2
and
13
to
the
carbonat
position1,
ascorrelations throughor threecouplings,
signal two
appears
along bondthe indirect
dimension,and theprotonsignaloralong
the directwhich
are not visible
in theCOSY
HMQC.
For example, marked
there areinclear multibo
dimension.
10.atItpositions
is also interesting
notethat atthere
is 1, as ma
the Heteronuclear
Multiple Bond Correlation
couplings from theFigure
protons
2 and 13 totothecarbonposition
is
shown
in
Figure
10,
with
the
1D
protonThe
HMBC
spectrumof
1M
ibuprofeninCDClacorrelation
betweencarbons(11,12)
toprotons(HMBC) experiment can be used. LikeFigurein the 10.
3and carbon
spectrasignal
from Figures
1 and
7 as horizontal
andverticalThe peaks inthe(11,12).
This
isthereduetraces.
couplingfrom 11(11,12) to p
HMQC experiment
the carbon
appearsalong
Itprotons
is alsoareinteresting
to
note
that
istoaathree-bondcorrelation between
carbons2D
spectrumshow
whichconnectedtowhichcarbonsvia
long-range
to
12
and
vice
versa
(lightgreen).
the indirect dimension, and the proton(11,12).
signal along
This is dueto three-bondcoupling
fromfound
11 tofrom
12 and vice versa (light gree
coupling. The couplings between molecular
positionslook similar
to the ones

HMBC

the direct dimension. The HMBC spectrum of 1 M
the COSY spectrum, but the HMQC additionally shows couplings to quaternary carbons,ibuprofen in CDClshown
in Figurewithorthe
3 is arewhich
not visible
in the10,
COSY
HMQC. For example, there are clear multibond
1D proton and carbon
spectrafrom
Figures
1 and2 and 13 to the carbon at position 1, as marked incouplings from the protons at positions
7 as horizontal andverticaltraces. The peaks in the
Figure10.
2D spectrum showwhich
protonstoare
It is alsointeresting
noteconnectedthat there is a correlation between carbons (11,12) to protons(11,12).
This
is
due
to
three-bondcoupling from 11 to 12 and vice versa (light green).
to which carbons via a long-range coupling. The
couplings between molecular positions look similar
to the ones found from the COSY spectrum, but

Figure 10: HMBC spectrum of 2 M ibuprofen in CDCl3, with some of the long-ran
couplings marked.
Figure 10: HMBC spectrum of 2 M ibuprofen in CDCl3, with some of the long-range

Figure10: HMBC
spectrum of 2M ibuprofen in CDCl3.
couplings
marked.

R

Specifications• Frequency: 42.5 MHz Proton, 10.8 MHz Carbon• Resolution: 50% linewidth < 0.7 Hz (16 ppb)
• Lineshape: 0.55% linewidth < 20 Hz• Dimensions: 58 x 43 x 40 cm• Weight: 55 kg• Magnet: Permanent and cryogen free
• Stray field: < 2 G all around system

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