UNDERSTANDING NMR SPECTROSCOPY:

PRINCIPLES, INSTRUMENTATIONS AND APPLICATIONS

-TATHAGATA CHAKRABORTY

B.Pharm , 4th Year , 1st

Semester

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.

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.

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.

The NMR Spectrometer

The NMR Graph

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.

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.

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.

Advantages of FT overCW SpectrometersIf 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.

APPLICATIONS OF NMRNuclear 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 sampleThe relative amounts of atoms present in a sampleThe specific environments of atoms within a moleculeThe purity and composition of a sampleStructural information about a molecule, including

constitutional and conformational isomerisation

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 dynamicsProtein foldingIonization stateProtein hydration 

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.

DISCUSSIONRecently 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.

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