Introduction History Principle Assembly Solvents Chemical shift Factors affecting chemical shift 2D NMR NOE effect NOESY COSY Application Conclusion References

1. NMR
By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )

2. CONTENTS
• Introduction
• History
• Principle
• Assembly
• Solvents
• Chemical shift
• Factors affecting chemical shift
• 2D NMR
• NOE effect
• NOESY
• COSY
• Application
• Conclusion
• References

3. INTRODUCTION
• Nuclear magnetic resonance spectroscopy,most commonly known as NMR
spectroscopy or Magnetic resonance spectroscopy is a spectroscopic technique to
observe local magnetic fields around the atomic nuclei sample with the radio waves
into the nuclear magnetic resonance which is detected with the sensitive radio
receivers.
• The intramolecular magnetic field around an atom changes the resonance frequency.
• thus giving the details of the electronic structure of a molecule and its individual functional
group.
• It is used to study the wide variety of nuclei:
1H 15N
19F 13C
31P

4. HISTORY OF NMR
• In 1938 I.I.Rabi of columbia university sucessfully made accurate
measurement of nuclear magnetic resonance absorbstion of a molecular
beam and was awarded nobel prize in 1944 in physics
• In 1946,F.Block and E.M.Purcell sucessfully demonstrated nuclear magnetic
resonance (NMR) for condensed matter and shared nobel prize in the year
1952,marking the start of NMR.

5. THEORY
• Spin Quantum Number (I) is related to the atomic and mass number of the
nucleus
• Elements with either odd mass or odd atomic number have the property of
nuclear spin
• If the external magnetic field is applied the number of possible orientation
calculated by (2I+1).
• Hydrogen has spin quantum number I=1/2 and possible orientation is
(2*1/2+1=2) two +1/2 and -1/2

6. PRINCIPLE
• The theory behind the NMR comes from the spin of the nucleus and it generates
a magnetic field.without an external applied magnetic field,the nuclear spin are
random in the direction
• But when an external magnetic field (Bnot) is present the nuclei align themselves
in the either with or against the field of external magnet.
• If external magnetic field is applied ,the energy transfer (ΔE) is possible between
ground state to excited state.
• When the spin return to its ground state level,the absorbed radiofrequency
energy is emitted at the same frequency level.
• The emitted radiofrequency signal that gives the NMR spectrum of the
concerned nucleus.
• The emitted radiofrequency is directly proportional to the strength of the applied
field

7. • V=YB0/2 pie
• Where:
Bᴼ=external magnetic field experienced by the Proton
ϒ=Magnetogyric ratio(the ratio between the nuclear magnetic moment and
angular momentum.

8. ASSEMBLY
• sample holder:It is a glass tube,the
prescribed diameter of that glass
tube is approximately 8.5com long
and 0.3cm diameter
• Permanent magnet:this
permanent magnet is used to
provide homogenous magnetic
field that is 60-100MHz
• Magnetic coils:these coils are used
to induce magnetic field when
current flows throught them.

9. • Sweep generator: to produce equal amount of magnetic field pass though
the sample.
• Radiofrequency transmitter: a radio frequency transmitter is is a unit that
produces powerful pulse of radio waves.
• Radio frequency:A radio receiver coil that detects Receiver radio frequencies
emitted as nuclei relax to a lower energy level.
• Readout system:a computer that analyses and record the data. Usually a
high processing power computer is required specially designed for this
purpose only.

10. SOLVENTS USED IN NMR
The following solvents are normally used in the hydrogen is replaced by the
deuterium
• CCl4:Carbon Tetrachloride
• CS2:Carbon Disulfide
• CDCL3:Deuteriochloroform
• C6D6:Hexa Deuteriobenzene
• D2O:Deuterium Oxide

11. CHEMICAL SHIFT
• A chemical shift is defined as the difference in parts per million(ppm)
between the resonance frequency of the observed proton and tetramethyl
silane hydrogens(TMS)
• TMS is the most common reference compound in NMR,it is set at δ=0 ppm.

12. • Sheilding of Protons:high density of electron around the
a nucleus shields the nucleus from the external magnetic
field and the signals are upfeild in the NMR spectrum.
• Desheilding of protons:lower electron density around
the nucleus deshields the nucleus from the external
magnetic field and the signals are downfield in the NMR
septrum.

13. FACTORS AFFECTING CHEMICAL SHIFT
• Electronegative groups:
Electronegative groups attached to the C-H system decrease the electron
density around the protons,and there is less shielding (i,e.desheilding) and
chemical shift increases.
CH2I 2.16
CH3Br 2.65
CH3Cl 3.10
CH3F 4.26

14. • Magnetic anisotrophy of π-systems
The word anisotropic means non-uniform.So magnetic
anisotropy means that there is a non uniform magnetic
field
The electrons in π systems
(e.g.aromiatics,alkenes,alkynes,carbonyl,Etc.)intract with
the applied field which induces a magnetic field that
causes the anisotrophy.
It causes both shielding and desheilding of proton
Example:Benzene

15. • Hydrogen bonding:
Protons that are involved in hydrogen bonding are
typically change the chemical shift values.
The more hydrogen bonding the more proton is
desheilding and chemical shift value is higher.

16. SPECTRUM OBTAINED BY NMR
• The peaks of energy absorption
is observed during the NMR
spectroscopy in a graph like
projection in X and Y axis
mostly.these obtained peak is
of two types i,e
• Higher(deshielded):it means
that higher amount of enery is
required to turn the element to
opposite direction of the
applied magnetic field.i,e
minimal electron cloud is
around the element and it may
be singlet or doublet.

17. • Lower(Shielded):it means lower
amount of energy is required to
turn the element to the
opposite direction of applied
magnetic field.it means that
many many electrons are
surrounding the element of
intrest.having electronegative
impact.
• This above data is analysed by
computer and give a particular
imaging.usually every molecule
or compound have its own
unique type of graph which is
nanlysed by computer to give
structural details of the
compound or the element.

18. 2D NMR
• In this type of spectrum the one graph is correlated with the graph of
another spectrum.
• It may be same for the different nuclei.
• It correlate the spectrum differently through:
1.COSY-through bonding nature.
2.TOCSY-through extended system.
3.NOESY-through space around the element.
2-D NMR signal is recorded as a fuction of two time variables,T1 and T2
Rf pulse are generally applied during the preparation and mixing period

19. APPLICATION OF 2D NMR
• Simplifies analysis of more complex or ambiguous cases
such as proteins.
• Obtained structural information is not accessible by one
dimensional NMR method.

20. TECHNIQUES OF 2D NMR INCLUDES
• Correlation spectroscopy(COSY)
• Heteronuclear correlation spectroscopy(HETCOR)
• Heteronuclear multiple-quantum coherence(HMQC)
• Nuclear overhauser effect spectroscopy(NOESY)
• Incredible natural abundance double quantum
transfer experiment (INADEQUATE)…Etc…Etc

21. COSY
• homonuclear correlated spectroscopy.correlation between proton that are
coupled to each other.
• This is commonly used for regiochemical assignment.
• Cross peak appear if spin coupling is present
• Protons that are separated by 2 or 3 bonds are usually detected.

22. NUCLEAR HAUSER EFFECT
• NOE is caused by the dipolar coupling between the nuclei.
• The local field at one nucleus is effected by the presence of another
nucleus.the result is the mutual modulation of resonance.
• The intensity of the interaction is a function of of the following distance
between the nuclei according to the following equation
I=A(1/r6)
Where,
I-intensity.
A-scaling constant.
r-internuclear distance.

23. Arrow denote the cross relaxation pathways
R1=distance between the proton 1 and 2
R2=distance between the proton 2 and 3
R3=distance between the proton 3 and 1
• The NOE provides a link between an experimentally
measurable quantity,I,and internuclear distance.
• NOE is only observed upto 5Ǻ

24. GENERAL APPLICATION OF NMR
• NMR is used in biology to study the Biofluids,cells,Per fused organs and
biomacromolecules such as nucleic acid (DNA,RNA),carbohydtrates proteins and
peptides and also labeling studies in biochemistry.
• NMR is used in physics and physical chemistry to study high pressure diffusion
,liquid crystals,liquid crystals solution,membranes,rigid solids.
• NMR is used in food science
• NMR is used in pharmaceutical science to study pharmaceuticals and drug
metabolism.
• NMR is used in chemistry to:
1. Determine the enatiomeric purity.
2. Elucidate chemical structure of organic and inorganic compounds.
3. Macromolecules-ligand interaction.

25. • H1 NMR spectroscopy is widely used for structure
elucidation.
Inorganic solids: inorganic compounds are investigated by
solid state 1H-NMR.eg CaSO4.H2O.
Organic solids:solid state 1H-NMR constitutes a powerful
approach to investigate the hydrogen-bonding and
ionization states of small organic compounds.
Direct correlation with the hydrogen bonding lenth could
be demonstrated e.g for amino acid carboxyl groups.

26. • For polymer and rubber
• Peptides proteins
• Clinical and scientific research
• Invivo NMR studies
Concerned with the 1H-NMR
• Spectroscopy of human brain
• Many studies are concerned with the altered levels of
metabolites in brain diseases
• To determine spatial distribution of any gven metabolite
detected spectroscopically

27. • Application in medicine:
MRI is specialist application of multi dimentional
fourier transformation NMR which includes:
1.Anatomical imaging
2.Measuring physiological function
3.Flow measurement
4.Tissue perfusion studies
5.Tumors

28. REFERENCES
• Organic Spectroscopy: By William kemp
• Principle and techniques of biotechnology and
Molecular biotechnology:By K.Wilson and J.Walker
• Instrumental methods of chemical analysis:By Willard
• WiKipedia article on NMR
• Shomu Biology Youtube Channel
• Kimball Biology pages