In the Laboratory
NMR Analysis of Unknowns:
An Introduction to 2D NMR Spectroscopy
David E. Alonso* and Steven E. Warren
Department of Chemistry and Biochemistry, Andrews University, Berrien Springs, MI 49104-0430; *alonso@andrews.edu
The use of modern FT-NMR instruments in under-
graduate laboratories has increased significantly in recent years
(1–4). NMR is a ubiquitous and very powerful tool for struc-
tural characterization. Unfortunately, 1H NMR spectra can
be fairly complicated even for relatively simple molecules.
Nonequivalent proton groups can have nearly the same
chemical shift or complex splitting patterns that can com-
plicate spectral interpretation and lead to erroneous assign-
ments. This significantly limits the number of molecules
studied at the undergraduate level, particularly at the first-
and second-year levels. Access to modern NMR instruments
allows students to use a combination of one-dimensional (1D)
and more powerful two-dimensional (2D) NMR techniques
to solve sophisticated structural problems.
Unfortunately, some individuals in the educational com-
munity have concluded that 2D NMR experiments are non-
essential for introductory courses (3). This is most likely due
to the logistics of running 2D experiments in a large class or
a hesitancy to introduce the technique to students without
the proper background to fully understand the theory be-
hind experiments. This prevents the introduction of the tech-
nique to a large number of students that would greatly benefit
from the exposure. These include biology, biochemistry, and
other students interested in the study of larger molecules for
undergraduate research.
At Andrews University we found that early exposure to
various NMR techniques has facilitated research at our pre-
dominantly undergraduate institution. Furthermore, one can
argue that in many cases a 2D NMR spectrum can be more
valuable for deciphering molecular structural information as
compared to more traditional 1D techniques (e.g., analysis
of coupling constants, homonuclear decoupling, etc.). It is
not necessary to expose introductory students to the theory
behind the experiment at this stage in their development.
Instead, experiments should focus on the simplicity of 2D
NMR data for solving complex structural problems. These
techniques could be used for early research experiences for
students attending predominantly undergraduate institutions.
The objectives of this experiment are to combine 1D
NMR spectroscopy with 2D NMR spectroscopy to solve
structural organic problems. Three structural isomers were
chosen as unknowns for the experiment: 2-, 3-, and 4-
heptanone. These compounds were specifically chosen to
demonstrate the need for a combination of 1D and 2D NMR
techniques as structural complexity increases from 4- to 2-
heptanone. The experiment is designed for a second-year or-
ganic chemistry course and is presented as an alternative to
traditional qualitative techniques such as derivatization of un-
knowns for melting point determination and comparison
with literature values (5). These techniques often can be cum-
bersome owing to time consuming derivative formation and
purification. Furthermore, the derivatives do not possess ad-
equate purity for accurate melting point determinations. This
www.JCE.DivCHED.org • Vol. 82 No. 9 September 2005
W
experiment should follow typical 1D NMR lectures and one
or two 1D NMR laboratory experiments.
Experiment
Prelab Lecture
During the prelab lecture, students review 1D proton
and carbon-13 NMR spectroscopy. 1H and 13C NMR prob-
lems are presented during the lecture and students use their
laboratory handouts, laboratory manual, and textbook to
solve NMR problems.
Proton decoupled 13C NMR data are used during the
prelab lecture, experiment, and postlab lecture. Following the
review, students are given a short tutorial on 2D data interpre-
tation. For example, the 1H–13C correlation data for ethyl ben-
zene is interpreted simply as an xy plot. Including mass spectral
and infrared data for the unknowns rather than providing stu-
dents with the molecular formula enhances prelab lecture.
Organization
Organic chemistry students acquire and interpret the 1H,
13
C, and 2D NMR spectra of three unknowns with the for-
mula C7H14O. They work in groups of 3 or 4 students. Labo-
ratory time is divided into three parts: (i) sample preparation,
(ii) 1H and 13C NMR data acquisition, and (iii) NMR data
interpretation. 2-Heptanone, 3-heptanone, and 4-heptanone
are the unknowns for the lab. Students prepare solutions of
the three unknowns (5% solution of the unknown in CDCl3,
0.3% TMS). They load the samples and acquire the NMR
spectra for each unknown. After they acquire the 1D NMR
data, they hand their tubes to a teaching assistant for the over-
night acquisition of 2D NMR data. Alternatively, copies of
standard 2D NMR spectra can be provided to students at
the end of the laboratory session.
Student groups spend the rest of the laboratory period
interpreting 1D NMR data and proposing structures for the
three unknowns. They obtain their correlation spectroscopy
(COSY) and heteronuclear correlation (HETCOR) data the
following lecture period. During this time, the class goes over
the 1D and 2D NMR data and corrects their assignments.
Reagents and Instrumentation
Chloroform-d, 2-heptanone, 3-heptanone, and 4-hepta-
none were purchased from Aldrich Chemical Company and
used without further purification. NMR data were acquired
using a 400 MHz JEOL FT-NMR spectrometer, equipped
with an autosampler, auto-tune probe, and gradient shim unit.
An autosampler, auto-tune probe, and gradient shim unit are
essential for running a large number of samples in short peri-
ods of time. In addition, JEOL’s Delta NMR software is rela-
tively easy to use and has been installed on several department
computers for data processing. The software is free and is avail-
able for both Windows XP and Linux operating systems (6).
• Journal of Chemical Education 1385
 In the Laboratory

Hazards
O
H2 H2
Chloroform-d is a carcinogen (LD50 0.908 g兾kg). It C C C
H3C C C CH 3
should be collected and properly disposed of after the experi- H2 H2
ment. 2-, 3-, and 4-Heptanone are flammable. NMR samples
should be prepared in a fume hood. protons: a b c c b a
carbons: 1 2 3 4 3 2 1
Results
Figure 1. Carbon and proton assignments in 4-heptanone.
The NMR data for symmetrical 4-heptanone (Figure 1)
are relatively simple. 4-Heptanone’s 1H NMR spectrum con-
sists of three signals for protons a, b, and c. Students com- O
pletely assign all signals in the proton, carbon and 2D spectra. H2
However, the spectral data for 3-heptanone (Figure 2) is more H3C C C CH3
C C C
complex. Its 1H NMR spectrum consists of six signals a–f H2 H2 H2
and to complicate matters signals for protons b and c over-
lap making them difficult to assign. There a total of seven protons: a b c d e f
signals in the 13C NMR spectrum. Students incorrectly as- carbons: 1 2 3 4 5 6 7
signed three out of the seven carbon signals (carbons 1, 6,
and 7). However, students are not aware of this problem un- Figure 2. Carbon and proton assignments in 3-heptanone.
til they obtain copies of the 2D NMR spectra. They are typi-
cally shocked at the unexpected reversal in carbon chemical
shifts. The 13C signals do not appear in order of distance from O
the electronegative oxygen atom. An explanation of the con- H2 H2
tributions to deshielding (diamagnetic and paramagnetic C C C
H3C C C CH3
shielding) in 13C NMR spectroscopy is given during the post- H2 H2
lab lecture period.
The 1H NMR spectrum of 2-heptanone (Figure 3) con- protons: a b c d e f
sists of six signals a–f. Proton signal assignments for 2- carbons: 1 2 3 4 5 6 7
heptanone are challenging owing to the overlap of signals for
d and e. Integral values, chemical shifts, and splitting pat- Figure 3. Carbon and proton assignments in 2-heptanone.
terns of the other signals provided sufficient information to
characterize 2-heptanone, although the relative chemical shifts
for protons are unclear. Fortunately, the COSY NMR spec-
trum helps students correctly assign chemical shifts for pro- Acknowledgment
tons d and e.
As with 3-heptanone, students make 2-heptanone 13C This work was supported in part by the National Sci-
signal assignments based on the relative distance of the car- ence Foundation (DUE-9750876).
bons from the electron withdrawing carbonyl group. Unfor-
tunately, their signal assignments are not entirely correct. 2D WSupplemental Material
NMR data helps students unequivocally assign all proton and
carbon-13 signals. NMR data for 2-, 3-, and 4-heptanone, Instructions for the students, including prelab and post-
as well as, a complete interpretation of the data can be found lab question, notes for the instructor, and NMR data for 2-,
in the Supplemental Material.W 3-, and 4-heptanone are available in this issue of JCE Online.

Conclusion Literature Cited

1
H NMR and 13C NMR signal assignments for 4-
heptanone are straightforward because of the molecule’s sym-
metry. However, signal assignments for 2- and 3-heptanone
are more challenging owing to the overlap of signals and un-
expected 13C NMR chemical shifts. Students are provided
with an explanation for the unexpected 13C NMR chemical
shifts. Correct chemical shift assignments can be made by
using the 2D COSY and HETCOR NMR spectra. The com-
bination 1D and 2D NMR greatly improves the ability of
students to elucidate the structure of unknowns. Computa-
tional methods for shift predictions are helpful in confirm-
ing signal assignments.
1. Davis, D. S.; Moore, D. E. J. Chem. Educ. 1999, 76, 1617–
1618. Gaede, H. C.; Stark, R. E. J. Chem. Educ. 2001, 78,
1248–1250.
2. Vaughn, J. B., Jr. J. Chem. Educ. 2002, 79, 306–307.
3. Alexander, C. W.; Asleson, G. L.; Doig, M. T.; Heldrich, F. J.
J. Chem. Educ. 1999, 76, 1294–1296.
4. Baer, C.; Cornely, K. J. Chem. Educ. 1999, 76, 89–90.
5. Lehman, J. W. Operational Organic Chemistry, 3rd ed.; Prentice
Hall: Upper Saddle River, NJ, 1999; pp 88–94.
6. JEOL Web site. http://www.jeol.com/nmr/nmrprods/delta.html
(accessed Jun 2005). Visit http://www.redhat.com for the Linux
operating system (accessed Jun 2005).

1386 Journal of Chemical Education • Vol. 82 No. 9 September 2005 • www.JCE.DivCHED.org