Nuclear Magnetic Resonance

Nuclear Magnetic Resonance

Principle
Nuclear Spin

Principle
Effect of External Magnetic Field

Principle
Zeeman Effect

Principle
Absorption of Radiation
Resonant Frequency
is the gyromagnetic ratio, characteristic of each nuclei:
B0 is the magnitude of the external magnetic field.
Chemical Shift
Resonant frequency for all nuclei of same element in a molecule need not be the same.
Depends on the electronic environment of the nucleus considered.
Electrons have magnetic moment of their own, which acts opposite to the external field and
reduces the energy gap between the spin states of the nucleus.
Reduced energy gap gives rise to lower resonant (NMR) frequency.
This change in NMR frequency is called chemical shift.

Working
NMR Spectrometer
Continuous Wave NMR

Spectroscopy
(CWNMR)
Fourier Transform NMR

Spectroscopy
(FTNMR)

Working
An NMR Spectrometer consists of three main parts:
NMR Powerful Magnet
NMR Spectrometer Console
Radio frequency detector
Radio frequency generator
Signal Amplifier
Computer Workstation

Working

Working
The process:
1. The magnetic field is held constant.
2. The sample is irradiated with a short pulse (approximately 10-5s) of radio-frequency energy.
3. The radio-frequency energy flips the spins of all susceptible nuclei simultaneously.
4.When the pulse is discontinued, the excited nuclei begin to lose their excitation energy and return to their
original spin state, or relax. Since the molecule contains many different nuclei, many different frequencies of
EM radiation are emitted simultaneously. This emission is calledfree-induction decayorFIDsignal as
shown below:
5. The intensity of the FID decays with time and falls to zero as nuclei
return to their equilibrium state.
6. A computer records the intensity-versus-time information (time
domain) and then uses aFourier Transformto convert the information
to intensity-versus-frequency information (frequency domain).

Working
Why do we see peaks?
Why are the peaks at different

position?
Doublet Triplet and n+1 rule

Applications
Protein Structure
Determinations
Magnetic Resonance
Imaging (MRI)
Pharmaceuticals
Food Sciences

Proteins Structure Determination
Kurth Wthrich
Nobel Prize 2002

Applications
Applications

Protein Solution
NMR Spectroscopy
STEPS
Sequential resonance
Assignment
Collection of
Conformational Constraints
Calculation of 3D structure
Applications

Sample Preparations
Proteins
Physiological Considerations
pH
Ionic Strength
Temperature
Purity
Molecular
Weight
Concentration
Applications

Spectroscopy
The chemical shift is very sensitive

to the micro environment of a
particular nucleus.

Applications
Can the amino acid residues be connected by NMR?
If so, one can get the primary structure

Applications
Resonance Assignment
Use to connect nuclei of amino acid

residues which are neighbors in the
sequence

Applications
Determining the arrangement of amino acids by NMR
TALOS+. Predicts
protein backbone torsion
angles from chemical shift
data. Frequently used to
generate further restraints
applied to a structure
model during refinement.

Applications
Detecting the secondary structure of protein by NMR

Applications
PROSESS
Protein Structure Evaluation

Suite & Server
GeNMR
GEnerate NMR
structure
Assessment of protein structural models by NMR
chemical shifts as well as NOEs, geometrical, and
knowledge-based parameters.

Applications

Applications
Magnetic Resonance Imaging (MRI)
Overview

Applications
History

Applications
Comparison with CT
Advantages
Safety
Contrast
Disadvantages
Time for Processing
Cost of Processing

Applications
Pharmaceuticals
Drug Discovery
Conformation of Impurity Profile Composition of

Drug Target
Drug
Analysis
Clinical Diagnostics
Dissolution of Investigating the

metabolites of the
Tablet
drug in the body

Applications
Pharmaceuticals
Drug And Purity Detection
Case Study : Heparin
Applications

Pharmaceuticals
Low Molecular Weight Heparins (LMWHs v/s Unfractioned Heparin (UFH)
Limitation of UFH
Unpredictable
Anticoagulant
Effect
Platelets
Activation
Advantage of LMWH
Risk Of Hit More Predictable

Anti Coagulant
response
Reduced Protein Less Risk Of Hit

Binding
Applications

Pharmaceuticals
NMR concept involved
Overall characterization can be done by 1H & 13C NMR.

All LMW heparin varies in the Disaccharide and Sulphur content.
Molecular weight of LMW Heparin & its structural information can be
determined by 13C NMR (provide high resolution).
1H NMR is used to determine the ratio of Iduronic acid and Glucouronic
acid and the position of sulfation in the chain.
Due to presence of different reduced end anomeric carbon and all other
anomeric carbon impurities can be easily determined.
Applications

Food Science
Nuclear Magnetic Resonance (NMR) spectroscopic techniques, based on
selective nucleus such as 1H, 13C, 31P and 19F have been utilized for the study
of the food analysis because they found in abundance in food and show high
sensitivity for NMR spectroscopy.
Parameters
Food analysis for solids and multiphase systems, parameters are isotope
ratio measurements and magnetic resonance imaging.
Applications

Food Science
Milk Analysis by NMR
31P NMR work in food science has been covered as part of general NMR reviews for
milk, meat, and lipids or olive oil analysis.
The application of 31P NMR spectroscopy in the study of milk by identifying the
signals of inorganic phosphates Pi, phosphoserine (SerP) residues in casein in
spectra of milk and skim milk in bulk.
Distribution of phosphorus compounds in milk from various species, concluding
that the technique may be of value in milk authentication, and also showed that
31P NMR can be used to measure changes during lactation.
Applications

Food Science
Advantage over Chromatography
In general, NMR spectroscopic techniques present several advantages

compared to the chromatographic ones such as
(a) they are very fast while chromatographic techniques are time consuming
(b) they require less solvents thus are cheaper, since one has bought the
equipment
(c) they do not destroy the sample.
Applications

Food Science
Disadvantage of using NMR
The big disadvantage of NMR is its low sensitivity.

But this is partially compensated by the high resolution of the NMR spectra.
In the aspect of the development of novel fast NMR methods of enhanced

sensitivity, scientist has synthesized new fluorine derivatives as probes for
the food analysis.
THANK YOU

Nuclear Magnetic Resonance

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