Chromatography of painkiller drugs

AIM

To analyse the components of painkiller tablets by comparison to known standards

YOU WILL NEED

●​ Fluorescent silica gel TLC plates

●​ 100 mL glass beaker and watch glass
●​ 6 glass vials or small beakers
●​ Filter paper
●​ Ethanol
●​ v
●​ Micro-pipettes
●​ Caffeine tablet
●​ Paracetamol tablet
●​ Aspirin tablet
●​ Ibuprofen tablet
●​ Mixed painkiller tablet X
●​ UV TLC lamp (short (254 nm) wave)
●​ Spatula
●​ Paper towels

PROCEDURE

Making your solutions: Crush up 1 aspirin tablet with a spatula, and add 2-3 mL of ethanol, in a vial
or small beaker, and label the beaker 'ethanolic aspirin solution'. Repeat this with a caffeine,
paracetamol and ibuprofen tablet, to make up your 4 known standard solutions - if the drug is in a
capsule, you can open the capsule and tip the powder into the ethanol. Stir the solutions for several
minutes to try to dissolve as much as possible (you can shake them if they are in vials with lids).
Finally, take a tablet/powder of 2 unknown mixtures of painkillers, and repeat the procedure above to
make a solution labelled 'ethanolic solution X', and 'ethanolic solution Y'.

Making the TLC tank:

Now you need to make yourself a TLC tank, with either a 100 mL beaker and a watch glass, or a
wide jam jar with a screw-top lid. Alternatively foil can be used to make a lid. Line the tank with
filter paper (this helps to saturate the air in the tank with solvent), and pour a small amount
(approximately 10 mL) of ethyl acetate into the tank - if you are doing this outside of a fumehood, do
not leave the tank without a lid on.

Allow the ethyl acetate to soak up into the filter paper (you can swirl the tank to do this), and either
add or remove some ethyl acetate so that the solvent level is roughly 0.5 cm high.

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Loading the TLC plate:
To load your TLC plate, first draw a line on the plate in pencil (not ink) 1.0 cm from the bottom of the
plate; this marks the starting point. Mark 6 dashes along this line, in pencil, as markers for your
samples: aspirin, paracetamol, caffeine, ibuprofen, X and Y. Be careful to mark the silica gently,
without scoring it.

Then dip a micro-pipette into a solution of your sample (just take the liquid from your premade
solutions, leave the solid to settle to the bottom), and make a tight spot on the plate by lightly
touching the plate on the baseline at a marked point. Make sure that you number/label these so that
you know which lane is which drug. Now check the plate before you develop it under the UV TLC
lamp, to make sure the sample is strong enough to see - you should be able to see a dark spot in
each lane along the baseline. Make sure that the solvent has evaporated from the spots before you
develop the plate. You can reuse the same micropipette, as long as you draw up some ethanol and
spot it onto some paper towel, between samples.
Caffeine, paracetamol, aspirin, caffeine, and ibuprofen all absorb UV light, and can be identified on
the fluorescent silica plates under short wave (254 nm) UV radiation; When viewed under short
wave UV the zinc sulfide in the silica plates fluoresces green, except where an eluted substance
quenches this fluorescence – these stand out as dark spots on the plate.
If the sample is too weak, allow the spot to dry and reapply. This can be done several times, but
allow the spot to dry each time to keep the spot size small.

Developing the TLC plate:

Place the loaded TLC plate into the tank carefully, making sure the baseline is at the bottom, the
back of the plate leans against the tank wall at a slight angle, and the baseline is above the level of
the eluent. Do not move the tank during the plate development. Place the lid (watch glass) on the
tank and allow the eluent to rise up the plate, until it is about 1 cm from the top. Carefully remove
the plate, mark the solvent front with a pencil, and allow the plate to dry (preferentially in a
fumehood).
Look at the TLC plate under 254 nm UV light, and use a pencil to circle the dark spots that you
can see on the plate. Measure the Rf (retention factor) of each of the standards, and draw a copy
of your TLC. Determine by comparison which drugs are in X and Y.
To measure the retention factor, Rf: this is the relationship between the distance moved by a given
TLC spot up the plate, as a fraction of the total distance travelled by the eluent. This ratio is always
constant, regardless how long the TLC plate is.

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To measure distance ‘b’, measure from the baseline to the middle of the TLC spot.
If you are having problems with understanding your TLC plate, take a look at the ‘TLC
troubleshooting page at
https://chembam.com/experiments/painkiller-chromatography/tlc-troubleshooting

This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit
http://creativecommons.org/licenses/by/4.0/ or send a letter to Creative Commons, PO Box 1866, Mountain View, CA 94042, USA.
QUESTIONS
1.​ What is the retention factor (Rf) for each of the 4 standard drugs, measured from the UV
visualisation?

Aspirina(Rf): 0,813
Cafeina (Rf): 0,346
Paracetamol (Rf): 0,64
Ibuprofen (Rf): 0,88

2.​ Looking at the structures of the 4 different drugs below, what do they all have in common,
and why do you think they absorb UV light?
4 de elles tenen un anell de benzè. Absorveixen lum ultravioleta per aquest anells cosa que
aquests anells tenen més afinitat a la lum ultra violada.

3.​ What are the stationary and mobile phases of this chromatographic technique?
Quan tenim de posar la nostra placa de cromatografia en acetat de etil, el acetat etil es la
fase estaciónaria, no tendria de moures. La fase mobil es quan injectem els principis actius i
amb el acetat etil comença a pujar.

4.​ Why is it important to use a pencil, and not pen, for marking TLC plates?
Per que si nos queden marqués en la cromatografía que fan dificultar el resultat. Per aixó
s’útlitza llapis.

5.​ What combination of drugs is in the unknown mixture XY? Fill in the table below to help
assign these:

Painkiller Rf
Aspirin 0,813
Paracetamol 0,64
Ibuprofen 0,88
Caffeine 0,346
Unknown X 0.84
Unknown Y 0,746
Unknown X= Caffeine, Paracetamol and Ibuprofen
Unknown Y= Caffeine and Paracetamol

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 6.​ When recording the Rf of a compound, what factors are important to know about the TLC
run? What factors can change the Rf?
Els factors que afecten la reporductivitat de Rf són: Fase estacionària: qualitat de l'adsorbent, gruix
de la capa, activació de la placa, distribució i grandària de les partícules. Fase mòbil: qualitat i
puresa dels solvents.

Factors que poden canviar la Rf: el tipus de fase estacionària, la polaritat del dissolvent, la
temperatura i la concentració dels components en la mescla .

This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit
http://creativecommons.org/licenses/by/4.0/ or send a letter to Creative Commons, PO Box 1866, Mountain View, CA 94042, USA.