NMR Is a technique used to determining the structure of complex biochemical molecules. It can only
be used with molecules that contain atomic nuclei that have a ‘spin’. Overall spin can only be
found in nuclei with have either an odd number of protons, or an odd number of neutrons or an
odd number of both protons and neutrons. This is because if all the nucleons spins were paired
they would cancel out.
Electrons, protons and neutrons can be thought as either spinning up or down.
A charged particle with spin creates a magnetic field along its axis (a magnetic moment). When a
external magnetic field is applied the nucleus can be aligned with (lower energy) or against the
field (higher energy)and theses orientations have different energies (slightly more nucleons will
be in the lower state aligned with the external field). The energy difference between the higher
and lower states (ΔE = hν) is so small that electromagnetic waves with low frequencies (i.e.
Radio waves) can cause a nucleus to flip its orientation and absorb the wave, this region is called
the radio region.
This difference in energy of the two spin states depends on which kind of nucleus it is (i.e. Different
isotopes have different nucleuses) and the chemical environment that surrounds the nucleus.
A nucleon can absorb a quantum of energy in the radio region and be put into the
higher spin state. When it relaxes it returns to the lower level and will emit the
same radio frequency (see image)
The magnetic field experienced by the nucleus is a result of the electrons around it
spinning and having a associated magnetic field . This electron generated field
ensures that the field experienced by the nucleus is not the external magnetic
field. This is known as Nuclear shielding and extent of it is determined by the
other atoms surrounding the nucleus as the magnetic field of neighbouring atoms
modifies the external magnetic field experienced by the atom within a molecule. .
The amount of shielding also changes the required energy for the nucleus to
change its spin. For example the frequency absorption would be minutely
different for the hydrogen in –CH3 than the hydrogen –CH2-.
J-coupling (also called indirect dipole dipole coupling) is the connection between two
nuclear spins due to the influence of bonding electrons on the magnetic field
running between the two nuclei. J-coupling contains information about dihedral
angles, which can be estimated using the Karplus equation. It is an important
observable effect in 1D nuclear magnetic resonance spectroscopy.
A basic NMR spectrometer includes:
•
Very strong magnets because NMR signals are more spread out when a more intense
magnetic field is used (for interpreting).
•
A radio transmitter coil
•
The sample (if it is a solid it is dissolved in a solvent that will not emit a signal such as
CD2CL2 as Deuterium [D OR 2H], 16O and 12C have paired protons and neutrons
which means they lack overall spin)
•
A radio receiver coil so that when relaxing nuclei move down to a lower energy level
and emit a radio frequency it can be detected.
•
A computer which both analyses and records the data.
NMR is used in a wide variety of scientific disciplines for a range of purposes.
The most well known use of NMR is when used in medicine for magnetic resonance
imaging (MRI).MRI works by measuring the amount of time it takes for exited
protons in the human body to relax back down to the lower energy state. This time
is different for all the different tissues in the body which means that a tumour
can be identified as it also has a different response . In this case a computer
analyses the data (in quantitative form) and gives off a coloured image
(qualitative data) of the persons body that is easy for doctors to interpret.
Another very common application of NMR is in a chemical analysis. Theses can be
carried out by a great number of different scientists such as forensic scientists
(to identify toxins, etc) and analytical chemists (to discover/learn about/identify
the structure of a specific molecule).The data comes in the form of peaks on a
spectrum (quantitative data)
Other uses include non-destructive testing, In the petroleum industry to find pore size
distribution and identify pore fluids (water, oil and gas) in boreholes, In quantum
computing using the spin states of molecules as qubits (a unit of quantum
information) as well as many other uses in a large variety of disciplines .
It is very hard to compare frequencies from one NMR to another but, it is necessary
for reliability. So, in order to standardise the measurements of different machines
and under different experimental conditions, a ‘control’ sample is used as a
reference. This is almost always tetramethylsilane (TMS), (CH3)4Si (see image)
The reason for using TMS is because its structure ensures that all the H atoms in the
molecule are in an identical environment. It is inert so it can be added to the
sample without causing a reaction. Also, when looking at the data of a NMR the
TMS peak is well away from the ones the experimenter would be interested in.
T he difference in energy needed to change the spin state in the sample is compared
to the TMS and is called ‘chemical shift’. Chemical shift is measured in ppm and
its symbol is δ. The chemical shift of TMS is defined as zero.
For large molecules (for example protein molecules containing thousand of atoms ) it
is likely that NMR signals will overlap, making it harder to use NMR to learn about
its structure. However, some of the different forms of NMR such as solid state
NMR can be used with proteins ,polymers, etc (refer to future slide).
NMR is very useful because it can give many types of information. It is similar to
infrared spectroscopy to identify functional groups. analysis of a NMR spectrum
provides information on the number and type of chemical entities in a molecule.
However, NMR provides much more information than IR. However, much more
information can be obtained from using NMR.
The data is fairly easy to understand for the trained eye.
Largely non destructive so it is good for expensive (DNA/RNA) or dangerous samples
(as long as they are not ferromagnetic)
It is considerably slower than other techniques
The atomic nucleus of an isotope in the molecule needs to have overall atomic spin.
Other limitations on sensitivity arise because NMR requires an majority of the
population to be higher in the lower energy state and also that particles goes from
the low energy state to the high energy state. It has been discovered that radiation
and temperature can affect the atoms ability to move from energy states. However,
ways have been found to decrease the sensitivity of NMR such as changing the
temperature which have made them hardier.
MRI
low resolution NMR- The chemical shifts give information about the sort of environment the
hydrogen atoms are in, the ratio of the areas under the peaks gives the ratio of the numbers of
hydrogen atoms in each of these environments; the number of peaks gives the number of different
environments the hydrogen atoms are in.
-
The ratio of the number of hydrogen in the various environments can be found in the
ratio of the areas under the peaks. In the case of methyl propanoate, the areas were in
the ratio of 3:2:3, which is exactly what is expected for the two differently placed CH 3
groups and the CH2 group.
-
The position of the peaks tells you useful things about what groups the various
hydrogen atoms are in.. The important shifts for the groups present in methyl
propanoate are:
High resolution NMR – Very similar to low resolution except what were peaks on low
resolution are split up into what look like many peaks in clusters. These should be
treated the same way as a low resolution spectrum, just treat each cluster as a
separate peak.
-The type of NMR that uses the differences in the magnetic properties of 1H atoms in
compounds in order to identify the number of chemically distinct hydrogen
‘environments’ there are ,is known as proton NMR or 1H NMR.
-When looking at the data the number of peaks is equal to the number of different
bonding environments experienced by all the hydrogen nuclei in the molecule.
-For example in the case of the ethyl benzene all the hydrogen in the same
environment are in the same peak on the spectrum. For example all of the single
hydrogen atoms that are bonded to a single carbon are together in one peak.

In the previous graph (because it was a high resolution NMR spectrum) the
various peak s due to protons in the same environment were split into finer
peaks. The amount of splitting provides information about the number of
hydrogen atoms attached to adjacent carbon atoms. This interaction is known
as spin-spin coupling.

The clusters are known as singlets , doublets, triplets and Quartets.
The number of peaks caused by splitting equals n+1, n is the number of H atoms on
the neighbouring atom. So, A Singlet is next to carbon with no hydrogen attached
•
A doublet next to a CH group
•
A triplet next to a CH2 group
•
A quartet next to a CH3 group
On the graph depicting ethanol(CH3CH2OH) on the next page:
CH splits the signal from hydrogen attached to adjacent atoms into two peaks,
CH2 splits the signal from hydrogen attached to adjacent atoms into three
peaks and CH3 splits the signal from hydrogen attached to adjacent atoms into
four peaks.

Also, the peak areas give us information about the amount of equivalent H
atoms ( i.e. CH3 has the largest area under the peak because it has more
hydrogen than the other s).

H and OH groups do not split the peaks . The reason for this is oxygen shielding
. Effects of shielding

For H1 Intensity of the lines is equal to the Patterns present.

-Spectra fine structure due to spin-spin coupling is when the hydrogen atoms
are on adjacent atoms ( for example H-C-C-H) and not when separated by one
or more atoms (H-C-O-C-H).
To find the compound chemical shift data is also used to help to identify the
environment each group was in.
The second most common base isotope for NMR is Carbon-13 as
most biomolecules contain carbon. 13C is naturally occurring and has
nuclear spin. It is used to determine the chemical environment of
carbon atoms within a molecule.
In the spectra the chemical shift from the TMS is depends on the
carbon atoms environment within a molecule. The shifts range from
0ppm to 200ppm (see graph below). There is only one peak produced
for each carbon atom environment (see Buckyball).
The proton NMR and carbon-13 NMR’s graphs are very different and
should not be confused with one another.
Electrical Hazards-The NMR spectrometers in the lab operate on either 240 volts AC or 120
volts AC and have several high-voltage DC components, all of which can be
hazardous or fatal if the equipment is used improperly and accidental electrocution occurs.
To prevent such accidents, safety precautions are put in place such as:
-No person may access the instruments without proper training and
Without proper authorisation.
-Extreme caution should be used whenever the instruments are being used in a way that
makes it necessary to be near the console
or magnet.
-No one can access the instrument panels or spectrometer consoles unless
under the observation and guidance of the a NMR supervisor.
-Any accumulation of water around or near the instruments should be
reported to a NMR supervisor and the wet areas should be avoided
to prevent electrocution.
-Any accidental exposure to electricity must be reported to the NMR
Supervisor.
Cryogenic Liquids-Cryogenic liquids such as Liquid Nitrogen and liquid Helium are used in the NMR laboratory and
both
Can be extremely dangerous. In order to prevent accidental exposure to liquid
cryogens, and to avoid asphyxiation (suffocation) in the event of a magnet quench safety precautions are put in
place such:
-No person may use cryogenic liquids in the NMR laboratory without first
having been trained in the safe handling of cryogens and other dangerous substances.
-. Before using cryogenic liquids in the NMR laboratory, a NMR Facility
Supervisor (of some description)must be notified. Cryogenic liquids stored in the NMR laboratory
are not for general use.
-Protective clothing including lab coats, gloves and eye-protection will be
worn by all individuals whenever handling cryogenic liquids in the NMR
laboratory. Individuals near the vicinity of the NMR equipment when cryogens are being handled may also be
required to wear
protective clothing.
-In the event of a magnet quench (the sudden evaporation of
cryogenic liquids in the magnet), all present must immediately and orderly
exit the NMR laboratory. A magnet quench is usually obvious from the
loud rushing sound of the evaporated gases escaping the magnet and may
displace sufficient Oxygen to cause asphyxiation. Since Helium is less
dense than air, exiting the laboratory by crawling on the floor is
recommended. 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 supervisor in order to get the appropriate medical attention.
-
Magnetic Fields and Electromagnetic Radiation
Strong magnetic fields and several sources of electromagnetic radiation are
present in the NMR laboratory that may be dangerous. To prevent such accidents,
safety precautions are put in place such as:
-Users of the NMR equipment are subjected to exposure limits to static magnetic
fields. Exposure limits are the concentration of static in the workplace to which
most people can be exposed without experiencing harmful effects.
-No person may enter the NMR laboratory without authorization from the
NMR supervisor.
-People with pacemakers, defibrillators, metal surgical implants or
prosthetics must stay at least 6 feet away from the magnets at all times.
-Personal articles such as hairpins or jewellery must be kept away from the
magnets at all times.
-Metal tools, carts, and gas cylinders must be kept away from the magnet
at all times.
Glass Tubes and Evacuated Storage Dewars – Glass tubes (to keep the sample in) and storage
dewars (Dewar's are a container with an evacuated space between two walls that are highly
reflective, capable of maintaining its contents at a near-constant temperature over relatively
long periods of time-) . Like all equipment these could become quite dangerous if used
incorrectly. To prevent such accidents, safety precautions are put in place such as:
-NMR Tubes must be handled with extreme caution(They are thin-walled
glass and can cause dangerous wounds. If broken)
- Never force an NMR tube into the
NMR spinner holder and never force the cap on or off an NMR tube.
-Evacuated storage dewars are in the probes on all the
spectrometers, and are sometimes used around spectrometers.
These can be very dangerous when broken as the vacuum can cause implosion.
-Always make connections to storage dewars carefully and without force.
-External storage dewars should always be wrapped in plastic mesh or tape
to prevent flying glass if they are broken.
-Broken glass should be cleaned up under the supervision of the NMR
Supervisor and should be disposed of in approved glass waste
containers.
-All injuries must be reported to the NMR supervisor.
NMR Dewar
Chemical Hazards- NMR uses various hazardous chemicals (such as solvents) which can
be harmful if used incorrectly, To prevent such accidents, safety precautions are put in
place such as:
-NMR Solvents must be handled as specified in the Material Data Safety Sheets .
. Because there are no fume hoods in the NMR
laboratory, samples requiring a hood for safe handling must be prepared
outside of the NMR laboratory.
-Chemical spills or accidental exposure to NMR solvents must be reported
to the NMR supervisor.
-Material Data Safety Sheets should be available for all standard and calibration
samples used in the NMR laboratory.
Physical Hazards – There are many potential physical hazards in a NMR lab that can happen if the
equipment is used improperly. To prevent such accidents, safety precautions are put in place
such as:
-Stairways to magnets must be used with extreme caution.
-Care must be taken to avoid overturning of the magnets. No one should
ever lean on the magnets or pull on the magnets when getting up or down
from the floor (as when tuning the magnets). They are not stable and can
be overturned easily.
-Cryogenic storage dewars can also be overturned quite easily. They
should never be pulled from the top, but rather from the handles provided.
-All injuries in the NMR laboratory must be reported to the NMR supervisor.
The 1H NMR spectrum of methyl methanoate , HCOOCH3 , consists of two apparent
singlets at 8.07ppm and 3.76ppm, with relative areas of 1:3. It is difficult to detect
any fine structure due to spin-spin coupling because
a)
The presence of oxygen eliminates the spin- spin coupling.
b)
A single hydrogen atom has no interaction with a –CH3 group.
c)
The hydrogen atoms are not on adjacent atoms.
d)
Spin-spin coupling doe not occur in esters (Esters are derived from carboxylic
acids which contains the -COOH group, and in an ester the hydrogen in this group
is replaced by a hydrocarbon group of some kind).
1. NMR spectra can also be observed for other atoms besides from 1H. For which
one of the following atoms could an NMR spectrum be observed?
a)12C
b)16O
c) 19F
d) 32S
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