Overview of Nuclear Magnetic Resonance

Bella Anbar, Camellia Elvi S, Miranti, Veryco Budianto

Group 8
Biomanufacturing Training Program, 2016
Outline:
1.
2.
3.
4.
5.
6.
7.

Introduction
Basic principal
Safety
Sample preparation and general steps of operation
Data analysis
Application
Conclusion

1. Introduction
In practical life, we may encounter many reactions that involved numerous compunds
as reactants or products. We often need to identify the stucture of those compounds. Until the
mid-twentieth century, the determination of the structure of a compound was difficult and
time-consuming. But now as the modern instrumental techniques has been greatly developed,
there are many tools that can be used to determine the structure of a compound. One of them
is NMR spectroscopy. NMR spectroscopy has been the most important tool in the
determination of organic structures. The particular value of NMR spectroscopy is that it gives
us information about the number and types of atoms in a molecule and also provide
information on how they are connected. Two common types of NMR spectroscopy usually
used to characterize organic structure are 1H NMR and 13C NMR.
2. Basic Principles
NMR spectroscopy basically exploits the idea of spinning electron and nuclei. We are
already familiar with the concept that an electron has a spin and also a charge. When a
charged particle spins on its axis, it creates an associated magnetic field. In effect, an electron
behaves as if it is a tiny bar magnet. An atomic nucleus that has an odd mass or an odd atomic
number also has a spin (nuclear spin) and also behaves as if it is a tiny bar magnet. The
examples are 1H and

13

C isotopes, which are the two most common elements in organic

compunds.
Basically, the spins of 1H or

13

C nuclei are completely random in orientation. But

when we place them in a powerful magnetic field, however, interactions between their
nuclear spins and the applied magnetic field are quantised, and only two orientations are

allowed. They are oriented aligned with or against the direction of the magnetic field, as
illustrated in Figure 1. But majority of their nuclear spins are aligned with the applied field
because this state has lower energy.

Figure 1. Nuclear spins orientation (a) in the absence of applied magnetic field, (b) in the presence of applied
magnetic field (Blackman, 2005)

When external energy source (electromagnetic radiation in radiowaves frequency) of

the appropriate energy is applied, some of those nuclei in the lower energy spin state absorb
the energy and results in their nuclear spins flipping from the lower energy state to the higher
energy state, as illustrated in Figure 2. In this context, resonance is defined as the absorption
(or emission when the nuclei return to equilibrium) of electromagnetic radiation by a
spinning nucleus and the resulting flip of its nuclear spin state.

Figure 2. An example of resonance for nuclei of spin +1/2 and -1/2

For 1H NMR, hydrogen atoms in organic molecules are surrounded by electrons and
by other atoms. The electrons that surround a nucleus also have spin so they create local
magnetic fields that oppose the applied field. Consequently, the magnetic field experienced
by a hydrogen atom in a molecule is slightly less than that of the applied magnetic field of the
instrument. The result of these local magnetic fields is that hydrogen atoms are shielded from
the applied field, as illustrated in Figure 3. The greater the shielding of a particular hydrogen

atom by local magnetic fields, the lower is the energy (frequency of radiowaves) required to
bring that hydrogen atom into resonance.

Figure 3. Shielding and deshielding effects (Smith, 2008)

The power of the shielding depends on electron density around the hydrogen atoms.
That electron density can be influenced by the atoms that surround the nucleus. For example,
the electron density around the hydrogen atoms in fluoromethane is less than that around the
hydrogen atoms in chloromethane, due to the greater electronegativity of fluorine relative to
chlorine. Thus, we can say that the hydrogen atoms in chloromethane are more shielded than
the hydrogen atoms in fluoromethane. Conversely, we can say the hydrogen atoms in
fluoromethane are more deshielded (reduced electron density around each hydrogen atom)
than those in chloromethane. So, different molecules will give different strength of shielding.
Then, the different shielding will also give differences in resonance frequencies received in
the NMR spectroscopy.
Modern NMR spectrometers (1H NMR), work by irradiating a sample with a short
pulse of radiation that covers the entire range of relevant radiowave frequencies. All
hydrogen atoms are simultaneously excited and then begin to return (or decay) to their
original spin state. As each atom decays, it releases energy of a particular frequency that is
detected as an electrical impulse in the receiver coils. Those electrical impulses can be
recorded every 12 seconds, and many are collected and then averaged to generate a
spectrum using a mathematical technique.
3. Safety
There are some important safety considerations which one should be familiar with
before using a NMR spectrometer. The concern of hazards associated with work in NMR

spectrometer comes from the high magnetic fields, the handling of cryogens and high
electrical voltages/RF sources.

Magnetic Fields

People with medical implants such as, surgical clips, cardiac implants pacemakers or
prostheses should check with the NMR Facility Manager before entering the NMR
room. No person may enter the NMR laboratory without authorization from the NMR

Facility Manager.
All magnetic objects, electronics, credit cards and magnetic storage media should be
kept outside the NMR magnet room. This includes keys, wallets, mechanical watches,

tools, etc.
Use only non-magnetic tools inside the NMR magnet room.
In case a metallic object strikes the magnet, do not attempt to pull the object off
yourself, get NMR Facility Staff immediately.

Cryogens Liquids

The NMR facility manager must be notified before using cryogenic liquids in the

NMR laboratory.
Before using cryogenic liquids in the NMR laboratory , the person must be trained
first in the safe handling of such substances. No person may use cryogenic liquids in
the NMR laboratory without first having been trained in the safe handling of such

substances.
Protective clothing, including lab coats, gloves and eye protection will be worn by all
individuals who handle, or are in close proximity when cryogenic liquids are being

transferred.
In the event of a magnet quench (the sudden evaporation of cryogenic liquids in the

magnet), the NMR lab must be immediately evacuated.

Doors to the laboratory should be left open to aid in the dispersal of helium and

nitrogen gases.
Any accidental exposure to cryogenic liquids must be reported to the NMR facility
manager and the Director of Environmental Health & Safety.

Electrical Hazards
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No person may operate the equipment without proper training and authorization from

the NMR manager.

Only authorized and qualified personnel shall access electrical panels or instrument

consoles.
Report any accumulation of water on the floor of the NMR laboratory to the NMR

Facillity Manager. Wet areas should be avoided to prevent electrocution.

Extreme caution should be used whenever the instruments are being tuned or
otherwise used in a way that makes it necessary to be near the console or magnet.

4. Sample Preparation and General Step for Operating The Equipment

Sample Preparation
1. Samples should be dissolved in 0.6-0.7 ml of a deuteriated solvent.
2. Filtered through a Pasteur pipette, equipped with a cotton wool, that discharges into an
NMR tube. The purpose of filtration is to remove any undissolved sample, such as dust,
hair, etc. from the solution, any of which may adversely affect the resolution and
lineshape of your NMR spectrum.
3. Use the ruler and ensure that the height of the solution is at least 50 mm from the bottom
of the NMR tube. If it is not, add additional deuterated solvent until the height is 50 mm.
4. Place the cap firmly onto the NMR tube.
Analysis of Sample with The NMR Spectrometer
1. Open Bruker ICONNMR Software.
2. Put the sample into NMR Spectrometer.
3. Analyze the sample.
4. Get the spectrum.

5. Data Analysis
The output NMR method is a series of peaks known as NMR spectrum. The typical
NMR spectrum is shown in Figure 4. The horizontal axis of the graph is called chemical shift
(, in part per million, ppm) scale runs from right to the left. By interpreting the signal from
NMR spectrum, we can know the structure of the molecule.

Figure 4. An NMR spectrum showing peaks result of molecule NMR analysis

The area under the peak gives information about how many protons are in each
environment. The area is given as an integration curve and the height of the curve can be
measured. By comparing the heights of the integration curves we can determine the ratio of
protons in each environment.
NMR analysis can be used as a qualitative analysis, usually done as an identification
of molecule structure. There are some steps from the guidelines of how to interpret NMR
spectrum. Which are:
1. Molecular formula and number of elements of unsaturation
The first step is to determine the type of bonding in the molecule by determine the
number elements of unsaturation. This number represent the number of double bonds
plus rings in the molecules. This is given by the formula:
EOU =1+ Carbons

( hydrogens+ halogens)
2

2. Number of signal
If two or more protons are in an equivalent environment, then they will appear as one
signal. So, the number of signals shows how many different kinds of protons are
present.
3. Integration of signal area
Area under each peak of signal is represent the number of hydrogen that are giving
rise to that peak. A convenient way to analyze this peak is by convert the area into a

distance by electronically integration. The result of the distance of each peak than can
be compared and from this we can find the ratio of the number of hydrogen.
4. Pattern of chemical shift
To make it easier to analyze the NMR data, we have been provided by an approximate
of chemical shift trends, as shown in Figure 5. We can determine the type of proton of
a peak by finding the closest match of the chemical shift value from the spectrum with
the data/table.

Figure 5. Proton Chemical Shift Ranges (Organic chemistry, MSU, 2016)

5. Splitting patern
Nonequivalent protons on adjacent carbons have magnetic fields that may align with
or oppose the external field. This phenomena can cause splitting of signal. Signal
splitting shows the number of protons on adjacent atoms, following (n+1) rules.
6. Structural formula
If possible, consider the present of barrier group. After obtaining all the data needed,
try to write all of the fragment of the molecule that may occur. We should be able to
assemble all of our fragments into a complete structure.
6.

Application of 1HNMR spectroscopy

According to Vasavi (2014), The NMR spectroscopy is very widely used for the

detailed investigation of an unknown organic compound by providing these informations:

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a) Identification of structural isomer.

b) Detection of hydrogen bonding: Hydrogen bonding affects the value (chemical
shift). As hydrogen bonding involves electron cloud transfer from hydrogen to
neighbouring electronegative atom like O, N, S etc. So the hydrogen experiences
a net deshielding effect and higher value. With increasing in concentrations the
hydrogen bonding increases, in turn the value of increases and with increases in
temperature the hydrogen bonding falls, in turn the value of decreases.
c) Detection of aromaticity: Protons attached to the benzoyl, polynuclear and
heterocyclic compounds whose electrons follow Huckel rule (i.e. no. of
electrons = (4n+2) where n=0, 1 ,2 etc.) are extremely deshielded due to the
circulating sextet (ring current) of electrons. As a result of this, the signal for
the aromatic protons appears at a very low field than that observed even for
benzene. From this, the aromatic character of the compound under investigation
can be predicted.
d) Distinction between Cis-trans isomers and conformers: The cis and trans isomers
of a compound can easily distinguished as the concerned protons have different
values of the as well as the coupling constants (J).
e) Detection of electronegative group or atom: It is known that the presence of
electronegative atom or group in the neighbourhood of the proton cause
deshielding and the signal is shifted downfield. Greater the electronegativity of
the adjacent atom, smaller is the tau value of absorption for the concerned proton.
f)

Detection of some double bond character due to resonance.

The NMR spectroscopy could aslo provide informations about:

g) Quantitative analysis: The NMR is used to determine the molar ratio of
components in a mixture.
h) Structure determination of SOF4; ClF3; HF2; polyethylene as well as metal
complex as WF6L.

6.1.

NMR application in Biosimilar field

Biosimilar drug products must have a demonstrated similarity with respect to the

reference products molecules in order to ensure both the effectiveness of the drug and the
patients safety. Japelj et. al. (2016), used NMR approach to estimate the degree of similarity
between the biosimilar and the reference product. The similarity study was performed on
protein filgrastim (G-CSF, granulocyte colony-stimulating factor, reference product Amgen
trade name Neupogen and Sandoz trade name Zarxio, which is the first biosimilar approved
in the US). The similarity was evaluated using qualitative NMR spectral overlays.
Japelj et. al. (2016), used the 1H-15N Heteronuclear Single Quantum Correlation
(HSQC) experiment on Heteronuclear NMR Spectroscopy to estimate the degree of similarity
between the biosimilar and the reference product. The 1H-15N HSQC spectra were acquired
for the biosimilar and originator filgrastim products to obtain the amide fingerprints and the
through space dipolar correlations. A complete cross-peak overlay of the 1H-15N HSQC
spectra indicated the highest level of similarity for the compared proteins and their threedimensional structures (Figure 7).

Figure 7. Overlay of the amide fingerprint spectra Japelj et. al. (2016)

7.

Conclusion
NMR spectroscopy used relaxation from an atom to produce resonance. Using
resonance, NMR is used to detect an unknown organic compound. NMR can be applied in a
wide area from food science to biosimilar. Sample preparation is very simple too. In
Biosimilar, NMR is used to compare the structure of CGSF.
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References
Blackman, A., Bottle, S., Schmid, S., Mocerino, M., Wille, U. 2012. Chemistry. 2nd edition.
John Wiley & Sons.
Connecticut College. NMR Safety. https://www.conncoll.edu/offices/environmental-healthand-safety/laboratory-safety/nmr-safety/. Accessed on November 14, 2016.
Japelj, B., Ilc, G., Marusic, J., Sencar, J., Kuzman, D., & Plavec, J., 2016. Biosimilar
structural comparability assessment by NMR: from small proteins to monoclonal
antibodies. Scientific Report. Nature.
J.B. Stothers NMR Facility Department of Chemistry Western University London, Ontario,
Canada.

NMR

Sample

Preparation.

http://publish.uwo.ca/~chemnmr

usingthefacility/NMR_Sample_Preparation_2.0.pdf. Accessed on November 8, 2016.

Smith, J. G. 2008. Organic Chemistry. 2nd edition. New York: McGraw-Hill.
Vasavi, N. 2014. 1HNMR spectrometry in structural elucidation of organic compounds.
Journal of Chemical and Pharmaceutical Sciences 5: 26-29.

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