Introduction to chromatography and spectroscopy
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Transcript of Introduction to chromatography and spectroscopy
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• Introduction to chromatography and spectroscopy
• Using chromatography and spectroscopy to solve a mystery
• Seeing in 3D
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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
Thin-layer chromatography (TLC)
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Assessing purity
Pure compound 1Impure compound 1
impurity
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Identifying unknown samples
Compound A Compound B Unknown
How would you confirm that the unknown was compound B?
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Identifying unknown samples
Compound AMixed
UnknownCompound B
MixedUnknown
Consistent with unknown being compound A
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How does spectroscopy work?
• Light is a mixture of different colours (wavelengths/frequencies)
Ultra-violet
Infra-red
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How does spectroscopy work?• Different materials absorb different
parts of the spectrum
Ultra-violet
Infra-red
Ultra-violet
Infra-red
Absorbs everything but blue and red
This reflected light tells us something about the material
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Infra-Red (IR) spectroscopy
• In IR spectra, regions of light are absorbed by specific parts of the molecule.
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
IR light C
O
For example
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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
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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?
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SafetySafety glasses and gloves to be worn throughout
PainkillersAspirin and paracetamol are active medicines. Do not ingest.
TLC tank solvent: diethyl etherdichloromethanetetrahydrofuran
Solvent for dissolving the painkillers:ethanol
Solvent for washing the IR sodium chloride platesAcetone
Use of UV lamp to observe TLC plates
} Do not inhale the solvent or allow it to contact your skinDiethyl ether and tetrahydrofuran are highly flammable
} Ethanol is highly flammable
Acetone is highly flammable
UV light is harmful to eyes and skin. Do not look directly at the light or place your hands under the light
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Which one could have killed the victim?
Aspirin Paracetamol
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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
• We are going to use visible spectroscopy to reach a conclusion
http://www.le.ac.uk/spectraschool/sias/UV%20poster.pdf
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Solving a mystery – Part 2
• How much aspirin was present in the victim’s blood?
• What was the likely cause of death?
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Colour filters and 3D visualisation
• In IR spectra, regions of light are absorbed by specific parts of the molecule.
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
IR light C
O
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• A colour filter on a camera works in a similar way
white light
It absorbs all the colours in white light apart from red, making everything look red
As a result, red/orange objects are difficult to see with a red filter
Colour filters and 3D visualisation
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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
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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?
Colour filters and 3D visualisation
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We draw them flat but they do have 3D shape
Aspirin Paracetamol
http://www.3dchem.com/molecules.asp?ID=157 http://www.3dchem.com/moremolecules.asp?ID=9&othername=Paracetamol
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Aspirin
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Paracetamol