Understanding NMR spectroscopy

1. -TATHAGATA
CHAKRABORTY
B.Pharm , 4th Year , 1st
Semester

2. INTRODUCTION
• Nuclear magnetic resonance spectroscopy is a powerful
analytical technique used to characterize organic
molecules by identifying carbon-hydrogen frameworks
within molecules.
• Two common types of NMR spectroscopy are used to
characterize organic structure: 1H NMR is used to
determine the type and number of H atoms in a molecule;
13C NMR is used to determine the type of carbon atoms in
the molecule.
• The source of energy in NMR is radio waves which have
long wavelengths, and thus low energy and frequency.
• When low-energy radio waves interact with a molecule,
they can change the nuclear spins of some elements,
including 1H and 13C.

3. Mechanism of NMR
• When a charged particle such as a proton spins on its axis, it
creates a magnetic field. Thus, the nucleus can be
considered to be a tiny bar magnet.
• Normally, these tiny bar magnets are randomly oriented in
space. However, in the presence of a magnetic field B0, they
are oriented with or against this applied field. More nuclei
are oriented with the applied field because this
arrangement is lower in energy.

4. Mechanism of NMR (2)
• In a magnetic field, there are now two energy states for
a proton: a lower energy state with the nucleus aligned
in the same direction as B0, and a higher energy state in
which the nucleus aligned against B0.
• When an external energy source (h) that matches the
energy difference (E) between these two states is
applied, energy is absorbed, causing the nucleus to
“spin flip” from one orientation to another.
• The energy difference between these two nuclear spin
states corresponds to the low frequency RF region of
the electromagnetic spectrum.

5. The NMR Spectrometer

6. The NMR Graph

7. Continuous-Wave (CW)
Spectrometers
 The spectrometer scans through a range of
frequencies
 The sample is interrogated with one frequency
at a time.
As the frequency range is scanned, a plot of
signal intensity vs. frequency is generated.

8. Fourier Transform (FT)
Spectrometers
 The sample is interrogated with a range of
freqencies, “all frequencies at once.”
The decay of the signal over time is observed as a
Free Induction Decay (FID)
 A Fourier transform changes the signal vs. time
plot into a signal vs. frequency plot.

9. ADVANTAGES OF FT NMR
 Dramatic increase in the sensitivity of NMR
measurements .
 Has widespread applications esp. for 13C NMR, 31P
NMR and 19F NMR giving high signal to noise
ratio facilitating rapid scanning.
 Can be obtained with less than 5 mg of the
compound.
 The signals stand out clearly with almost no
electronic background noise .
 Used in engineering, industrial quality control and
medicine.

10. Advantages of FT over
CW Spectrometers
 If a signal is weak, many scans must be averaged
to enhance the signal/noise ratio.
Signal/noise ratio rises as the square root of
number of scans.
Only FT instruments can obtain a large
number of scans in a reasonable time.
 A scan can be performed much more quickly.
CW-NMR: 5 minutes; FT-NMR: 5 seconds.

11. APPLICATIONS OF NMR
Nuclear Magnetic Resonance (NMR) Spectroscopy is a
non-destructive analytical technique that is used to
probe the nature and characteristics of molecular
structure. A simple NMR experiment produces
information in the form of a spectrum, which is able to
provide details about:
 The types of atoms present in the sample
 The relative amounts of atoms present in a sample
 The specific environments of atoms within a molecule
 The purity and composition of a sample
 Structural information about a molecule, including
constitutional and conformational isomerisation

12. APPLICATION OF NMR
SPECTROSCOPY
Today, NMR has become a sophisticated and powerful
analytical technology that has found a variety of
applications in many disciplines of scientific research,
medicine, and various industries. Some of the
applications of NMR spectroscopy are LISTED BELOW:
 Solution structure
Molecular dynamics
Protein folding
Ionization state
Protein hydration

13. APPLICATION OF NMR
SPECTROSCOPY
 Hydrogen bonding
 Drug screening and design Particularly useful for identifying
drug leads and determining the conformations of the
compounds bound to enzymes, receptors, and other proteins.
 Native membrane protein Solid state NMR has the potential
for determining atomic-resolution structures of domains of
membrane proteins in their native membrane environments,
including those with bound ligands
 Metabolite analysis A very powerful technology for metabolite
analysis.
 Chemical analysis A matured technique for chemical
identification and conformational analysis of chemicals whether
synthetic or natural.

14. DISCUSSION
 Recently 19F NMR studies of the cyan variant
of GFP conducted in a laboratory indicated
conformational flexibility in or near the
chromophore moiety with residue His148
being most likely involved in this process.
NMR has been suggested as a tool for
elucidating the dynamics of chromophore
formation, water accessibility of the
chromophore and conformational flexibility
in GFP. In contrast to many spectroscopic
techniques, NMR spectroscopy provides a
large frequency range for studying dynamical
processes from pico second to second
timescales and even longer at atomic
resolutions.