Two-Dimensional NMR Spectroscopy

The two-dimensional nuclear magnetic resonance

spectroscopy (2D NMR) is a set of nuclear magnetic resonance
spectroscopy (NMR) methods which give data plotted in a space
defined by the two frequency axes rather than one.

The types of 2D-NMR spectroscopy include correlation

spectroscopy (COSY), J-resolved spectroscopy, exchange
spectroscopy (EXSY) and nuclear overhauser effect spectroscopy
(NOESY).

Two-dimensional NMR spectra provide more information about a

molecule than one-dimensional NMR spectra and are especially
useful in determining the structure of a molecule, particularly for
molecules that are too complicated to work with using one-
dimensional NMR spectroscopy.
The first two-dimensional experiment, COSY, was proposed
by Jean Jeener, a Professor at the Université Libre de
Bruxelles of Belgium in 1971.

This experiment was implemented by the Swiss Scientists,

Walter P. Aue, Enrico Bartholdi and Richard R. Ernst who
published their work in 1976.

Later, Atta-ur-Rahman , James Keeler, James Nowick, Garey

E. Martin and J. Schram and others authored a number of
books in the field of 2D-NMR Spectroscopy.
Fundamental Concepts

Each of the 2D-NMR experiments consists of

a sequence of radio frequency (RF) pulses with
delay periods between them.

The timing, frequencies, and intensities of

these pulses distinguish different NMR
experiments from one another.
Almost all two-dimensional experiments have four stages:

1. The preparation period, where a magnetization coherence is

created through a set of RF pulses;

2. The evolution period, a determined length of time during which

no pulses are delivered and the nuclear spins are allowed to freely
precess (rotate);

3. The mixing period, where the coherence is manipulated by

another series of pulses into a state which will give an observable
signal; and

4. The detection period, in which the free induction decay signal

from the sample is observed as a function of time, in a manner
identical to one-dimensional FT-NMR.
The two dimensions of a two-dimensional NMR experiment
are two frequency axes representing a chemical shift. Each
frequency axis is associated with one of the two time variables,
which are the length of the evolution period (the evolution
time) and the time elapsed during the detection period
(the detection time).

They are each converted from a time series to a frequency

series through a two-dimensional Fourier Transform.

A single two-dimensional experiment is generated as a series

of one-dimensional experiments, with a different specific
evolution time in successive experiments, with the entire
duration of the detection period recorded in each experiment.
The end result is a plot showing an intensity value for each
pair of the frequency variables.

The intensities of the peaks in the spectrum can be represented

using a third dimension.

More commonly, intensity is indicated using contour lines or

different colors.
Chemical Shift (ppm)
Radio Frequency Pulses: The atomic nuclei exhibit ‘nuclear
spin’ which absorbs specific wavelengths of microwave
radiations to resulting the nuclear spin axis ‘flip’ to the
opposite direction.

The RF pulses are used in the spin preparation phase of a pulse

sequence to prepare the spin system for the ensuing
measurement.

The magnetization vector is moved by an angle proportional to

the length and intensity of the pulse.

Many preparation pulses are required to get desired signals

spectrum and to also suppress signals of unwanted spins.
Free Induction Decay (FID): The FID refers to a short lived
sinusoidal electromagnetic signal which appears immediately
following the 90o pulse. It is induced in the receiver coil by the
rotating component of the magnetization vector in the x-y plane
which crosses the coil loops perpendicularly.

Fourier Transformation (FT): The is a mathematical technique

that transforms a function of time, x(t) to a function of frequency,
x(ω). It is an important image processing tool which is used to
decompose an image into its sine and cosine components.

The out come of the transformation represents the image in the

Fourier or frequency domain.
 1 Excitation 2 Evolution FID

RF Pulse Frequency 1

Sample Nucleus 1
As both nuclei Transfer of
are coupled Magnetization

Signals

2D FT FID 4 Acquisition
Signals Frequency

Frequency 2 Nucleus 2
Frequency

2D NMR Spectrum

The 2D-NMR Process

Homonuclear Through-Bond Correlation Methods

In these methods, magnetization transfer

occurs between nuclei of the same type,
through J-coupling of nuclei connected by up
to a few bonds.
Correlation Spectroscopy (COSY):

The first and most popular two-dimension nuclear magnetic

resonance (2D-NMR) experiment is the homo-nuclear
correlation spectroscopy (COSY) sequence, which is used to
identify spins which are coupled to each other.

It consists of a single RF pulse (p1) followed by the specific

evolution time (t1) followed by a second pulse (p2) followed by
a measurement period (t2).
In standard COSY, the preparation (p1) and mixing (p2) periods each consists
of a single 90° pulse separated by the evolution time t1 and the resonance
signal from sample is read during the detection period over a range of times t2.
The two-dimensional spectrum that results from the COSY
experiment shows the frequencies for a single isotope, most
commonly hydrogen (1H) along both axes. Techniques have
also been devised for generating hetero-nuclear correlation
spectra in which the two axes correspond to different isotopes,
such as 13C and 1H.

Diagonal peaks correspond to the peaks in a 1D-NMR

experiment, while the cross peaks indicate couplings between
pairs of the nuclei (much as multiplet splitting indicates
couplings in one dimensional nuclear magnetic resonance (1D-
NMR) spectroscopy.
The cross peaks result from a phenomenon
called magnetization transfer and their presence indicates that
two nuclei are coupled which have the two different chemical
shifts that make up the cross peak's coordinates.

Each coupling gives two symmetrical cross peaks above and

below the diagonal. That is, a cross-peak occurs when there is
a correlation between the signals of the spectrum along each of
the two axes at these values.

An easy visual way to determine which couplings a cross peak

represents is to find the diagonal peak which is directly above
or below the cross peak, and the other diagonal peak which is
directly to the left or right of the cross peak. The nuclei
represented by those two diagonal peaks are coupled.
1H-COSY spectrum of progesterone. The spectrum that appears along both the horizontal and
vertical axes is a regular one dimensional 1H-NMR spectrum. The bulk of the peaks appear
along the diagonal, while cross-peaks appear symmetrically above and below the diagonal.
The COSY-90 is the most common COSY experiment. In
COSY-90, the p1 pulse tilts the nuclear spin by 90°. Another
member of the COSY family is COSY-45. In COSY-45 a 45°
pulse is used instead of a 90° pulse for the second pulse, p2.

The advantage of a COSY-45 is that the diagonal-peaks are

less pronounced, making it simpler to match cross-peaks near
the diagonal in a large molecule.

Additionally, the relative signs of the coupling constants (J-

coupling/ magnitude of J-coupling) can be elucidated from a
COSY-45 spectrum. This is not possible using COSY-
90. Overall, the COSY-45 offers a cleaner spectrum while the
COSY-90 is more sensitive.
Two of the most common experiments for routine work are the
gradient magnitude COSY-90 and COSY-45 experiments. The
only difference between the two methods is the flip angle of
the second pulse (90 degrees for the COSY-90 and 45 degrees
for the COSY-45). For a concentrated sample, these
experiments can be acquired in a matter of minutes.

Although the signal to noise ratio is higher for a COSY-90, the

COSY-45 is usually the preferred experiment because the
diagonal signals are smaller and less intense allowing
correlations between close resonances to be resolved more
easily. The figure below shows magnitude gradient COSY-90
and COSY-45 spectra for 3-heptanone. Note the smaller
diagonal responses in the COSY- 45.
Another related COSY technique is double quantum filtered
(DQF) COSY. DQF COSY uses a coherence selection method
such as phase cycling or pulsed field gradients, which cause
only signals from double-quantum coherences to give an
observable signal.

This has the effect of decreasing the intensity of the diagonal

peaks and changing their line shape from a broad "dispersion"
line shape to a sharper "absorption" line shape. It also
eliminates diagonal peaks from uncoupled nuclei.

These all have the advantage that they give a cleaner spectrum
in which the diagonal peaks are prevented from obscuring the
cross peaks, which are weaker in a regular COSY spectrum.
Exclusive Correlation Spectroscopy (ECOSY)

Total Correlation Spectroscopy (TOCSY): The TOCSY

experiment is similar to the COSY experiment, in that cross peaks of
coupled protons are observed. However, cross peaks are observed
not only for nuclei which are directly coupled, but also between
nuclei which are connected by a chain of couplings.

This makes it useful for identifying the larger interconnected

networks of spin couplings.

This ability is achieved by inserting a repetitive series of pulses

which cause isotropic mixing during the mixing period. Longer
isotropic mixing times cause the polarization to spread out through
an increasing number of bonds
 Typical TOCSY Values for Amino Acids.

Typical TOCSY values for amino acids

In the case of oligosaccharides, each sugar residue is an
isolated spin system, so it is possible to differentiate all the
protons of a specific sugar residue.

A 1D version of TOCSY is also available, and by irradiating a

single proton the rest of the spin system can be revealed.

Recent advances in this technique include the 1D-CSSF

(chemical shift selective filter) TOCSY experiment, which
produces higher quality spectra and allows coupling constants
to be reliably extracted and used to help determine
stereochemistry.

The TOCSY is sometimes called homo-nuclear Hartmann-

Hahn (HOHAHA) spectroscopy.
Incredible Natural-Abundance Double-Quantum Transfer
Experiment (INADEQUATE):

The INADEQUATE is a method often used to find 13C couplings

between adjacent carbon atoms. Because the natural
abundance of 13C is only about 1%, only about 0.01% of molecules
being studied will have the two nearby 13C atoms needed for a signal
in this experiment.

However, correlation selection methods are used (similarly to DQF

COSY) to prevent signals from single 13C atoms, so that the
double 13C signals can be easily resolved.

Each coupled pair of nuclei gives a pair of peaks on the

INADEQUATE spectrum which both have the same vertical
coordinate, which is the sum of the chemical shifts of the nuclei; the
horizontal coordinate of each peak is the chemical shift for each of
the nuclei separately.
Heteronuclear Through-Bond Correlation Methods

The hetero-nuclear correlation spectroscopy gives signal based

upon coupling between nuclei of two different types. Often the
two nuclei are protons and another nucleus (called a ‘hetero-
nucleus’).

For historical reasons, experiments which record the proton

rather than the hetero-nucleus spectrum during the detection
period are called ‘inverse’ experiments.

This is because the low natural abundance of most hetero-

nuclei would result in the proton spectrum being overwhelmed
with signals from molecules with no active hetero-nuclei,
making it useless for observing the desired, coupled signals.
With the advent of techniques for suppressing these undesired
signals, inverse correlation experiments such as Hetero-nuclear
Single-Quantum Correlation (HSQC), Hetero-nuclear multiple-
quantum correlation (HMQC) and Hetero-nuclear Multiple-
Bond Correlation (HMBC) are actually much more common
today.

The normal hetero-nuclear correlation spectroscopy in which the

hetero-nucleus spectrum is recorded is known as the HETCOR.
In this experiment, two different nuclei (usually C and H) are
correlated through single bond spin-spin coupling, revealing
which proton and carbon group are bonded to each other.

This experiment is similar to the HSQC experiment; however,

HETCOR is a less sensitive experiment since it a carbon
detected experiment unlike HSQC which is a proton detected
experiment.
Hetero-Nuclear Single-Quantum Correlation
Spectroscopy (HSQC):

The HSQC detects correlations between nuclei of two different

types which are separated by one bond.

This method gives one peak per pair of coupled nuclei, whose
two coordinates are the chemical shifts of the two coupled
atoms
The HSQC used frequently in NMR spectroscopy of organic
molecules and is of particular significance in the field of
protein NMR. It correlates the nitrogen atom of an NHX group
with the directly attached protons. Each signal in the HSQC
spectrum represents a proton that is bound to nitrogen atom. It
detects correlations between nuclei of two different types
which are separated by one bond.

This method gives one peak per pair of coupled nuclei, whose
two coordinates are the chemical shifts of the two coupled
atoms. The edited HSQC provides the same information as the
DEPT 135 experiment but the HSQC is comparatively much
more sensitive. The HMBC gives correlation between carbon
and proton that are separated by two, three, some time in
conjugated system, four bonds.
1H–15N HSQC spectrum of a fragment of the protein NleG3-2. Each peak in the spectrum represents
a bonded N–H pair, with its two coordinates corresponding to the chemical shifts of each of the H and
N atoms. Some of the peaks are labeled with the amino acid residue that gives that signal.
The HSQC works by transferring magnetization from
the nucleus (usually the proton) to the S nucleus (usually the
heteroatom) using the INEPT pulse sequence; this first step is
done because the proton has a greater equilibrium
magnetization and thus this step creates a stronger signal.

The magnetization then evolves and then is transferred back to

the nucleus for observation.

An extra spin echo step can then optionally be used to

decouple the signal, simplifying the spectrum by collapsing
multiplets to a single peak.
The undesired uncoupled signals are removed by running the
experiment twice with the phase of one specific pulse
reversed; this reverses the signs of the desired but not the
undesired peaks, so subtracting the two spectra will give only
the desired peaks .

The Hetero-nuclear multiple-quantum correlation spectroscopy

(HMQC) gives an identical spectrum as HSQC, but using a
different method.

The two methods give similar quality results for small to

medium-sized molecules, but HSQC is considered to be
superior for larger molecules.
The hetero-nuclear single quantum coherence (HSQC) and
hetero-nuclear multiple quantum coherence (HMQC) yields
essentially the same information- one bond correlations between
protons and carbons. Yet they differ in the way the coherence
transfers from the 1H into 13C and back to 1H accomplished.

The HSQC in spite of the numerous pulses and delays is

conceptually simpler experiment of the two. It consists of a non-
refocused insensitive nuclei enhanced by polarization transfer
(INEPT) transfer from proton to carbon, followed by the 13C
evolution period (t1) with proton decoupling by the 180o 1H
pulse in its center, and ends with a reverse INEPT transfer back
to proton before signal acquisition under 13C coupling.
The HMQC detects the carbon-proton single bond
connectivity and helps build the structure of the
molecule on the basis of such connection.

It also helps to confirm the carbon and proton

chemical shift assignments in the molecule.
Heteronuclear Multiple-Bond Correlation Spectroscopy
(HMBC):

The HMBC detects hetero-nuclear correlations over longer

ranges of about 2-4 bonds. The difficulty of detecting multiple-
bond correlations is that the HSQC and HMQC sequences
contain a specific delay time between pulses which allows
detection only of a range around a specific coupling constant.

This is not a problem for the single-bond methods since the

coupling constants tend to lie in a narrow range, but multiple-
bond coupling constants cover a much wider range and cannot
all be captured in a single HSQC or HMQC experiment.
The 1H-13C hetero-nuclear multiple bond correlation (HMBC) shows the correlations between
carbons that are separated by multiple bonds and are incredibly useful for assigning carbons
that have no protons attached.
In HMBC, this difficulty is overcome by omitting one of these
delays from an HMQC sequence.

This increases the range of coupling constants that can be

detected, and also reduces signal loss from relaxation.

The cost is that this eliminates the possibility of decoupling the

spectrum, and introduces phase distortions into the signal.

There is a modification of the HMBC method which suppresses

one-bond signals, leaving only the multiple-bond signals
Through-Space Correlation Methods

These methods establish correlations between nuclei which are

physically close to each other regardless of whether there is a
bond between them.

They use the nuclear overhauser effect (NOE) by which

nearby atoms (within about 5 Å) undergo cross relaxation by a
mechanism related to spin-lattice relaxation.
Nuclear Overhauser Effect Spectroscopy (NOESY):

In NOESY, the nuclear overhauser cross relaxation between

nuclear spins during the mixing period is used to establish the
correlations.

The spectrum obtained is similar to COSY, with diagonal

peaks and cross peaks, however the cross peaks connect
resonances from nuclei that are spatially close rather than
those that are through-bond coupled to each other.
The NOESY spectra also contain extra axial peaks which do
not provide extra information and can be eliminated through a
different experiment by reversing the phase of the first pulse.

One application of NOESY is in the study of large

biomolecules, such as in protein NMR, in which relationships
can often be assigned using sequential walking. The NOESY
experiment can also be performed in a one-dimensional
fashion by pre-selecting individual resonances.

The spectra are read with the pre-selected nuclei giving a

large, negative signal while neighboring nuclei are identified
by weaker, positive signals.
This only reveals which peaks have measurable NOEs to the
resonance of interest but takes much less time than the full 2D
experiment. In addition, if a pre-selected nucleus changes
environment within the time scale of the experiment, multiple
negative signals may be observed.

This offers exchange information similar to the EXSY (exchange

spectroscopy) NMR method.

The NOESY experiments are important tool to identify

stereochemistry of a molecule in solvent whereas single crystal
XRD used to identify stereochemistry of a molecule in solid form.
Rotating-Frame Nuclear Overhauser Effect Spectroscopy
(ROESY):

The ROESY is similar to the NOESY, except that the initial

state is different. Instead of observing cross relaxation from an
initial state of z-magnetization, the equilibrium magnetization
is rotated onto the x axis and then spin-locked by an external
magnetic field so that it cannot precess.

This method is useful for certain molecules whose rotational

correlation time falls in a range where the nuclear overhauser
effect is too weak to be detectable, usually molecules with
a molecular weight around 1000 Daltons, because ROESY has
a different dependence between the correlation time and the
cross-relaxation rate constant.
Rotating-Frame Nuclear Overhauser Effect Spectroscopy
(ROESY): The 2D-ROESY (Rotating frame Overhauser
Spectroscopy experiment also called CAMELSPIN (Cross-
relaxation Appropriate for Mini-molecule Emulated by Locked
Spin experiments) offers a simple way to obtain NOE
information in a molecule by single experiment and without
prior knowledge of the spectral assignment or molecular
structure.

In ROESY experiment cross-relaxations are carried out in the

rotatory-frame with spin-locked magnetization and this means
that NOE in the transverse plane (ROE) is always positive (no
nulling condition as in the NOESY type experiments) and, in
addition, chemical exchange can always be distinguish.
The ROESY is similar to the NOESY, except that the
initial state is different. Instead of observing cross
relaxation from an initial state of z-magnetization, the
equilibrium magnetization is rotated onto the x axis and
then spin-locked by an external magnetic field so that it
cannot precess.

This method is useful for certain molecules

whose rotational correlation time falls in a range where
the nuclear overhauser effect is too weak to be
detectable, usually molecules with a molecular
weight around 1000 Daltons, because ROESY has a
different dependence between the correlation time and
the cross-relaxation rate constant.
In NOESY the cross-relaxation rate constant goes from
positive to negative as the correlation time increases, giving a
range where it is near zero, whereas in ROESY the cross-
relaxation rate constant is always positive.

The ROESY is sometimes called ‘cross relaxation appropriate

for mini-molecules emulated by locked spins’, the
CAMELSPIN.
Resolved-Spectrum Methods

Unlike correlated spectra, resolved spectra spread the peaks in a 1D-

NMR experiment into two dimensions without adding any extra
peaks. These methods are usually called J-resolved spectroscopy, but
are sometimes also known as chemical shift resolved spectroscopy
or δ-resolved spectroscopy.

They are useful for analyzing molecules for which the 1D-NMR
spectra contain overlapping multiplets as the J-resolved spectrum
vertically displaces the multiplet from each nucleus by a different
amount.

Each peak in the 2D spectrum will have the same horizontal

coordinate that it has in a non-decoupled 1D spectrum, but its
vertical coordinate will be the chemical shift of the single peak that
the nucleus has in a decoupled 1D spectrum.
Each peak in the 2D spectrum will have the same horizontal
coordinate that it has in a non-decoupled 1D spectrum, but its
vertical coordinate will be the chemical shift of the single peak that
the nucleus has in a decoupled 1D spectrum.

For the hetero-nuclear version, the simplest pulse sequence used is

called a Müller-Kumar-Ernst (MKE) experiment which has a single
90° pulse for the hetero-nucleus for the preparation period, no
mixing period and applies a decoupling signal to the proton during
the detection period.

There are several variants on this pulse sequence which are more
sensitive and more accurate, which fall under the categories of gated
de-coupler methods and spin-flip methods. Homo-nuclear J-resolved
spectroscopy uses the spin echo pulse sequence.
Significance of J-Resolved:

The J-coupling provides three parameters: the multiplicity of

signals (the number of lines), the magnitude of the coupling
(strong, medium, weak), and the sign of the coupling.
Multiplicity of Signals: The multiplicity provides information on the
number of centers coupled to the signal of interest, and their nuclear spin.
For simple systems, as in 1H-1H coupling in NMR spectroscopy, the
multiplicity reflects the number of adjacent, magnetically nonequivalent
protons. Nuclei with spins greater than 1/2, which are called quadrupolar,
can give rise to greater splitting, although in many cases coupling to
quadrupolar nuclei is not observed. Many elements consist of nuclei with
nuclear spin and without.

In these cases the observed spectrum is the sum of spectra for

each isotopomer. One of the great conveniences of NMR spectroscopy
for organic molecules is that several important lighter spin 1/2 nuclei are
either monoisotopic, e.g. 31P and 19F, or have very high natural
abundance, e.g. 1H. An additional convenience is that 12C and 16O have
no nuclear spin so these nuclei, which are common in organic molecules,
do not cause splitting patterns in NMR.
Magnitude of J-coupling: For 1H–1H coupling, the magnitude
of ’J’ provides information on the proximity of the coupling partners.
Generally speaking 2-bond coupling (i.e. 1H–C–1H) is stronger than three-
bond coupling (1H–C–C–1H). The magnitude of the coupling also provides
information on the dihedral angles relating the coupling
partners, as described by the Karplus relationship.

For heteronuclear coupling, the magnitude of J is related to the nuclear

magnetic moments of the coupling partners. 19F, with a high nuclear
magnetic moment, gives rise to large coupling to protons. 103Rh, with a very
small nuclear magnetic moment, gives only small couplings to 1H. To
correct for the effect of the nuclear magnetic moment (or equivalently the
gyromagnetic ratio γ), the "reduced coupling constant" K is often discussed,
where

K = 4π2J/ h γx γy
Sign of Coupling: The value of J also has a sign, and
couplings constants of comparable magnitude often have
opposite signs. A double resonance experiments in which a
weak perturbing radiofrequency field is applied on spectral
lines may be used to determine the sign of the coupling
constants.

It is found that the J=1.37 Hz geminal proton-proton coupling

in styrene sulfide is of different sign (presumably negative)
then the two vicinal couplings J= 5.55 Hz and J= 6.60 Hz
between protons in the thiirane ring.

Similar experiments on styrene imine showed that the J= 0.87

Hz geminal coupling is the same sign as two vicinal coupling
J= 3.29 Hz and J= 6.12 Hz in the aziridine ring.
 Styrene

Styrene imine Styrene sulfide

Coupling Constants (J)
Measure the Relative Sign of Coupling Constants
• The cross-peak patterns identifies the coupling constant sign and magnitude

Based on the slopes of the diagonal line

drawn through coupling pattern

3J and 3JBX have the same sign

AX
3J opposite sign of 3JAX and 3JBX
AB
Yellow-highlighted regions
are expanded
Higher-Dimensional Methods

The 3D and 4D experiments can also be done, sometimes by

running the pulse sequences from two or three 2D experiments
in series.

Many of the commonly used 3D experiments, however,

are triple resonance experiments; examples include
the HNCA and HNCOCA experiments, which are often used
in protein NMR.
References:
01. W. P. Aue, E. Bartholdi, R. R. Ernst, “Two-Dimensional Spectroscopy- Application to Nuclear Magnetic
Resonance", Journal of Chemical Physics, 64, 2229-46 (1976).

02. J. Schram, J. M. Bellama, “Two-Dimensional NMR Spectroscopy”, John Wiley , New York (1988).

03. G. E. Martin, A. S. Zekter, “Two-Dimensional NMR Methods for Establishing Molecular Connectivity”, John
Wiley, New York (1988).

04. K. Nakanishi, Ed., “One-Dimensional and Two-Dimensional NMR Spectra by Modern Pulse Techniques”,
University Science Books, California (1990).

05. G. D. Mateescu, A. Valeriu, “2D NMR Density Matrix and Product Operator Treatment”, Englewood Cliffs, New
Jersey, Prentice Hall (1993).

06. J. W. Akitt, B. E. Mann, “NMR and Chemistry”, Cheltenham, UK (2000).

07. Atta-ur-Rahman, “Nuclear Magnetic Resonance”, Springer (1986); “One and Two Dimensional NMR
Spectroscopy”, Elsevier (1989); “Solving Problems with NMR Spectroscopy”, Elsevier (2015) and “Applications
of NMR Spectroscopy”, Bentham (2015).

08. J. Keeler, Ed.,“Understanding NMR Spectroscopy (2nd Ed.)”, Wiley, USA (2010).

09. J. Keeler, Ed., “2D Homonuclear Correlation: TOCSY", Queen's University, Belfast (2011).

10. M. Clore and J. Potts, Eds., Recent Developments in Bimolecular NMR, RSC Publishers, UK (2012).