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NMR-2
Type of
NMR Experiments

Type of NMR Experiments

1D Experiments Homonuclear

2D Experiments Heteronuclear

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One Dimensional NMR

Spectroscopy

Each 1D NMR experiment consists of two sections:

preparation and detection. During preparation the spinsystem
is set to a defined state. During detection the resulting signal
is recorded. In the simplest case the preparation is a 90o rf
pulse. After this pulse each spin precesses with its own
(Larmor) frequency and induces a signal in the receiver coil.

The signal decays due to

relaxation and is therefore
called free induction decay
(FID).

Usually, the experiment is
repeated several times and
the data are summed up to
increase the signal to noise
ratio.

After summation the data
are fourier transformed to
yield the final 1D spectrum.

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An FID signal containing four

different frequencies

Spectral Parameters:

Chemical shift

Integration

Scalar coupling ( multiplicity)

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1H NMR Experiment

This is the simplest, most
straight-forward
homonuclear NMR
experiment

The triplet at 1.25 ppm with integral of 3

corresponds to the protons Ha in the methyl group,
whereas the quadruplet at 2.65 ppm with integral of
2 corresponds to the methylene protons, Hb. The
multiplets at 7.25 ppm with integral of 5 correspond
to the aromatic protons Hc. The singlet at 0 ppm is
due to the internal standard, tetramethyl silane
(TMS).

Note that the chemical shifts, when expressed in

ppm of applied magnetic field, do not change with
field strength. The spin-spin coupling (J-constants)
are not field-dependent, either.

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13C Experiment without decoupling

Note that both 13C and 1H are spin 1/2 nuclei. We expect to see a
signal for each magnetically-unique carbon, split into n+1 peaks,
where n is the number of protons attached to the carbon.

Of course, the signal from 13C is much weaker than that of a

proton because only 1.1% of all carbons present in a molecule
are 13C--the balance are 12C, which does not give an NMR signal.
This also explains why we don't see spin-spin coupling from
adjacent carbons--the chance of two adjacent carbons both
being 13C are 0.012%! Also, the magnetogyric ratio for 13C is only
23% that of the proton. All things considered, this is a difficult and
time-consuming experiment to attempt, which explains why we
don't do it very often.

Note the triplet at 77ppm due to CDCl3 (solvent) in the spectrum

below. This signal is very strong because the sample is mostly
CDCl3. The carbon in CDCl3 is split into a triplet because the D
attached to it is a spin +1 nucleus: the D can be in any one of
three spin states (-1, 0 or +1). Thus, any given carbon in the
solvent "sees" one of three distinct environments, depending
upon the spin state of its attached D.

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13C Experiment with PGD

(heteronuclear experiment)

In PGD (Programmed Gated Decoupling)
technique, we decouple the protons from the
carbon by means of radio-frequency pulse
sequence directed at the protons.

This is the fastest 13C experiment to carry

out. As a result, it is the "standard" 13C
experiment on newer spectrometers.

In the spectrum, the peak at 15 ppm is the methyl carbon Ca

and the peak at 28 ppm is the methylene carbon Cb. The
quarternary carbon in the benzene ring Cc (the one without an
attached proton) is at 144 ppm. The remaining three peaks at
125, 127, and 128 ppm are the remaining aromatic carbons.
Note that even though there are five additional carbons in the
ring, there are only three magnetically unique signals.

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13C DEPT and APT

(heteronuclear)

This DEPT experiment (Distortionless
Enhancement by Polarization Transfer) is an
example of a carbon-editing pulse sequence.
Systematic changes in the internal delays in
the complex pulse program make different
carbons respond in different fashions, based
upon the number of protons attached.

Pulse program
for DEPT:

Signals observed:
amplitude

Pulse length

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13C-DEPT45

In this variant of the DEPT experiment, all CH, CH2, and CH3 are
visible (positive).

13C-DEPT90

In this variant of the DEPT experiment, only CH yields

peaks; CH0, CH2, and CH3 are invisible. In our example
we see only three lines due to Cd, Ce, and Cf in the
aromatic range from 126 to 129 ppm.

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13C-DEPT135

In this variant of the DEPT experiment, CH2 yields negative

peaks, whereas CH and CH3 are positive. Thus, we see Ca, Cd,
Ce, and Cf as positive peaks, while Cb is negative.

APT
Whereas the APT (Attached Proton Test) experiment is not as
sensitive as a DEPT experiment, the APT has the advantage of
seeing all carbons present: CH0 at 145 ppm and CH2 at 29 ppm
are negative, while CH3 at 16 ppm and CH in the range of 126-
129 ppm are positive. Thus Cc and Cb are negative, while Ca, Cd,
Ce, and Cf are positive.

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Two Dimensional NMR

Spectroscopy

Why 2D NMR ?

1D protein and other macromolecules spectra are far too complex

for interpretation as most of the signals overlap heavily. By the
introduction of additional spectral dimensions these spectra are
simplified and some extra information is obtained.

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Anatomy of a 2D experiment:

1D experiment

The construction of a 2D experiment is simple: In

addition to preparation and detection which are
already known from 1D experiments the 2D
experiment has an indirect evolution time t1 and a
mixing sequence. This scheme can be viewed as:

Do something with the nulcei (preparation),

let them precess freely (evolution),

do something else (mixing),

and detect the result (detection).

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After preparation the spins can precess freely for a

given time t1. During this time the magnetization is
labelled with the chemical shift of the first nucleus.
During the mixing time magnetization is then
transferred from the first nucleus to a second one.
Mixing sequences utilize two mechanisms for
magnetization transfer: scalar coupling or dipolar
interaction (NOE). Data are acquired at the end of
the experiment (detection, often called direct
evolution time); during this time the magnetization is
labelled with the chemical shift of the second
nucleus.

Cross-peak could comes from:

Connection through bond (scalar coupling)

Connection through space (dipolar
coupling/Nuclear Overhouse Effect)

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2D NMR Spectroscopy

• Homonuclear 2D Experiments
• Heteronuclear 2D Experiments

Homonuclear 2D Experiments

Two dimensional FT
yields the 2D
spectrum with two
frequency axes. If
the spectrum is
homonuclear (signals
of the same isotope
(usually 1H) are
detected during the
two evolution
periods) it has a
characteristic
topology:

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A diagonal of signals (A and B) divides the spectrum in two equal

halves. Symmetrical to this diagonal, there are more signals (X),
called cross signals. The diagonal results from contributions of
the magnetization that has not been changed by the mixing
sequence (equal frequency in both dimensions) i.e. from
contributions which remained on the same nucleus during both
evolution times.

The cross signals originate from nuclei that exchanged

magnetization during the mixing time (frequencies of the first and
second nucleus in each dimension, respectively). They indicate
an interaction of these two nuclei. Therefore, the cross signals
contain the really important information of 2D NMR spectra.

Homonuclear 2D Experiments

There are three 2D spectra which are widely

used for the structure determination of
proteins with a mass of up to 10 kD: 2D
COSY, 2D TOCSY and 2D NOESY.

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2D COSY:

In the COSY experiment, magnetization is transferred by scalar

coupling. Protons that are more than three chemical bonds apart give
no cross signal because the 4J coupling constants are close to 0.
Therefore, only signals of protons which are two or three bonds apart
are visible in a COSY spectrum (red signals).

2D TOCSY:
long range
cross peak

In the TOCSY experiment, magnetization is dispersed over a

complete spinsystem by successive scalar coupling (long range
coupling). The TOCSY experiment correlates all protons of a spin
system. Therefore, we can also observed additional signals which
originate from the interaction of all protons of a spin system that are
not directly connected via three chemical bonds.

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2D NOESY

The NOESY experiment is crucial for the determination of protein
structure. It uses the dipolar interaction of spins (the nuclear
Overhauser effect, NOE) for correlation of protons. The intensity
of the NOE is in first approximation propotional to 1/r6, with r
being the distance between the protons: The correlation between
two protons depends on the distance between them, but normally
a signal is only observed if their distance is smaller than 5 Å. The
NOESY experiment correlates all protons which are close
enough.

It also correlates protons which are distant in the amino acid
sequence but close in space due to tertiary structure. This is the
most important information for the determination of protein
structures.

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NOESY spectrum of Ethyl Benzene

Heteronuclear 2D Experiments:

Apart from protons a protein contains other magnetic active
nuclei. For NMR of proteins, 15N and 13C are of special
importance. The use of these hetero nuclei allows some new
features in NMR which facilitate the structure determination
especially of larger proteins (> 100 AA). The natural abundance
of 15N and 13C is very low and their gyromagnetic ratio is
markedly lower than that of protons. Therefore, two strategies
are used for increasing the low sensitivity of these nuclei:
Isotopic enrichment of these nuclei in proteins and enhancement
of the signal to noise ratio by the use of inverse NMR
experiments in which the magnetization is tranferred from
protons to the hetero nucleus.

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The HSQC Experiment:

The most important inverse NMR experiment
is the HSQC (heteronuclear single quantum
correlation). 13C-HSQC experiment correlates
the carbon atom of an CHx group with the
directly attached proton. Each signal in a
HSQC spectrum represents a proton that is
bound to a carbon atom.

An analogous experiment (15N-HSQC) can
be performed for 15N and 1H and is very
important as well.

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HSQC spectrum of Ethyl Benzene

Exercise
The sample is 18 mg of codeine in 0.65 ml CDCl3,
assign all the 13C signals !

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APT-Codein

Edited DEPT-Codein

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HMBC

Heternuclear correlation emphasizing
long range couplings

Not all the nuclei of the correlating nucleus
are necessarily directly connected to a
proton. Long-range heteronuclear correlation
can yield signals for these nuclei while
suppressing one-bond correlations.

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The most common application of this

technique is for 1H-13C. ∆1 is set to 1/(2Jone-
bond) and ∆2 to 1/(2Jlong-range). As the long
range couplings are not all the same and
although a value of 10 Hz is usually a good
compromise, it is best to run the experiment
for a few different values of ∆2.

HMBC spectrum of Ethyl Benzene

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