• Introduction to chromatography
and spectroscopy
• Using chromatography and
spectroscopy to solve a mystery
• Seeing in 3D
Thin-layer chromatography (TLC)
Used to investigate the purity and identity of samples
– Stationary phase: silica plate
– Mobile phase: solvent
As the solvent rises up the plate, different compounds move up the plate
at different speeds
– Polar compounds move slowly
– Lipophilic compounds move quickly
UV lamp often used to visualise the compounds on the plate
Assessing purity
Pure compound 1
Impure compound 1
impurity
Identifying unknown samples
Compound A
Compound B
Unknown
How would you confirm that the unknown was compound B?
Identifying unknown samples
Compound A
Mixed
Unknown
Compound B
Mixed
Consistent with unknown being compound A
Unknown
How does spectroscopy work?
• Light is a mixture of different colours
(wavelengths/frequencies)
Infra-red
Ultra-violet
How does spectroscopy work?
• Different materials absorb different
parts of the spectrum
Infra-red
This reflected light
tells us something
about the material
Ultra-violet
Absorbs everything but blue and red
Infra-Red (IR) spectroscopy
• In IR spectra, regions of light are absorbed by
specific parts of the molecule.
For example
O
IR light
C
1715cm-1
The C=O bond of a ketone absorbs light in the region
of 1715cm-1 so that the emerging IR light has this
piece of its spectrum missing
Infra-Red (IR) spectroscopy
• Every compound has a unique IR spectrum that
tells us something about the structure
IR light
Unknown compound
http://www.le.ac.uk/spectraschool/sias/UV%20poster.pdf
Solving a mystery – Part 1
• A dead body has been found
• 2 empty bottles of 3 painkillers found at the scene
– Aspirin
– Paracetamol
• It is suspected that the victim took an overdose of
one of these
• Using the Thin-Layer Chromatography (TLC) and
Infra-Red (IR) spectroscopy, can you work-out
which painkiller is most likely to have led to the
death of the victim?
Safety
Safety glasses and gloves to be worn throughout
Painkillers
Aspirin and paracetamol are active medicines. Do not ingest.
TLC tank solvent:
diethyl ether
dichloromethane
tetrahydrofuran
}
Do not inhale the solvent or allow it to contact your skin
Diethyl ether and tetrahydrofuran are highly flammable
Solvent for dissolving the painkillers:
Ethanol is highly flammable
ethanol
}
Solvent for washing the IR sodium chloride plates
Acetone
Acetone is highly flammable
Use of UV lamp to observe TLC plates
UV light is harmful to eyes and skin.
Do not look directly at the light or place your hands under the light
Which one could have killed the
victim?
Aspirin
Paracetamol
Solving a mystery – Part 2
• We now know that aspirin was present in the
victim’s blood.
• What we don’t know is whether there was
enough in the blood to kill the victim.
• Therapeutic concentrations: <300mg/L
• Severe overdose: >750mg/L
http://www.le.ac.uk/spectraschool/sias/UV%20poster.pdf
• We are going to use visible spectroscopy to
reach a conclusion
Solving a mystery – Part 2
• How much aspirin was present in the victim’s
blood?
• What was the likely cause of death?
Colour filters and 3D visualisation
• In IR spectra, regions of light are absorbed by
specific parts of the molecule.
O
IR light
C
1715cm-1
The C=O bond of a ketone absorbs light in the region
of 1715cm-1 so that the emerging IR light has this
piece of its spectrum missing
Colour filters and 3D visualisation
• A colour filter on a camera works in a
similar way
It absorbs all the colours in white light apart
from red, making everything look red
white light
As a result, red/orange objects are difficult
to see with a red filter
Colour filters and 3D visualisation
Here is a light orange circle
and a light blue square
What do you expect to see when you look at this through a red filter?
What do you expect to see when you look at this through a blue filter?
(e.g. 3D specs, Quality Street wrappers)
What about a red filter for one eye and a blue filter for the other?
Colour filters and 3D visualisation
When you do the same thing with shapes that are
similar and are almost on top of one another,
something strange happens
What do you think is happening?
How can this help us think about the shape of molecules?
http://www.dddesign.com
We draw them flat but they do
have 3D shape
Aspirin
http://www.3dchem.com/molecules.asp?ID=157
Paracetamol
http://www.3dchem.com/moremolecules.asp?ID=9&othername=Paracetamol
Aspirin
Paracetamol