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In this experiment the analgesics were purified and identified by the means of a column
chromatography packed with the silica as a stationary phase. The three analgesics were
provide that is aspirin, paracetamol and caffeine and were purified under four solvent viz.
acetone, ethyl acetate 50 : 50 hexane, ethyl acetate 75 : 25 hexane and ethyl acetate 90 : 10
hexane. The solvent or mobile phase, ethyl acetate 75: 25 hexane was chosen as the best
solvent since it gave the best separation of the analyzed compounds. The components were
separated and collected as fractions of up to 30, the first compound appeared in spot 13 that is
in spots 1 to 12 there were spots observed. The fractions 27 – 29 have the same retention
factor (Rf = 0.12) which corresponds to that of an aspirin in ethyl acetate 75: 25 hexane and
fraction 17 have the same Rf value to that of paracetamol. From these values it was
concluded that the sample contain only aspirin and paracetamol and no caffeine.


The column chromatography is a chemistry based technique used to isolate a single chemical
compound from a mixture. Chromatography is a able to separate the components in a mixture
based on different adsorption of compounds to the adsorbent; compounds move through the
column at different rates allowing them to be separated into fractions. The method of
chromatography was first discovered by a Russian-Italian Botanist, Mikhail Tsvest, as he
applied his observations with filter paper extraction to the new methods of column
fractionation that had been developed in the 1890s for separating the components of

The compounds that need to be separated in a chromatography are distributed between a

mobile phase and a stationary phase, in such a system distribution based on selective
adsorption give rise to separation. The technique can be used on scales from microorganism
to kilograms. The main advantage of column chromatography is the low cost and low
disposability of the stationary phase used in the process.

The preparation of a column involves the packing of a solid absorbent into a cylindrical glass
or plastic tube. The size will depend on the amount of a compound being isolated. The base
of tube contains a filter, either a cotton or glass wool plug. A solvent reservoir must be
attached at the top of the column. In the column chromatography the stationary phase is
usually silica or alumina placed in a vertical glass column and mobile phase is a solvent of
choice added from the top and left to flow down through the column by either gravity or
alternatively external pressure. Below is a diagram of a prepared column chromatography.

Fig 1. A diagram of a prepared column chromatography


TLC results

Rf = distance travelled by spot (mm)/ distance travelled by solvent (mm)


Distance travelled by solvent: 37 mm

Distance travelled Rf
by spot (mm)
Unknown 19 0.51
Paracetamol 16 0.43
Caffeine 24 0.65
Aspirin 29 0.78

Ethyl acetate 50: 50 hexane

Distance travelled by solvent: 39mm

Distance travelled Rf
by spot (mm)
Unknown 5 0.13
Paracetamol 4 0.10
Caffeine 3 0.08
Aspirin 24 0.62

Ethyl acetate 75: 25 hexane

Distance travelled by solvent: 42mm

Distance travelled Rf
by spot (mm)
Unknown 16 0.33
Aspirin 14 0.12
paracetamol 5 0.33
Caffeine 28 0.67

Ethyl acetate 90: 10 hexane

Distance travelled by solvent: 37mm

Distance travelled Rf
by spot (mm)
Unknown 11 0.23
Caffeine 4 0.11
Paracetamol 12 0.32
Aspirin 25 0.68


Using the TLC plates, the solvent that was best for separation of the components of the
mixture is ethyl acetate 75: 25 hexane because it gave the good separation of the analyzed
components that is aspirin, paracetamol and caffeine over acetone, ethyl acetate 50: 50
hexane and ethyl acetate 90: 10 hexane.

The components were separated from the mixture using the column packed with silica, the
first twelve fractions contained no compound which means that the compound was still at the
bottom of the column. The compound started to elute at fraction 13, and this compound was
of less polarity since the less polar components elutes first compared to components of higher
polarity. The compound appeared up to fraction 30 and from there no compound showed in
other fractions. This means that all the compound was now eluted and ready to be identified.

The Rf values of the known and unknown compounds were compared. The fractions 27 to 29
had the Rf value of 0.12 which is the same for that of aspirin eluted by ethyl acetate 75:25
hexane and fraction 17 has Rf value of 0.68 same for that of paracetamol. There was no
significant evidence that shows the present of caffeine since it had the lowest Rf value
compared to other fractions. From the results it is deduced that the analgesics that were given
to be separated in a sample are aspirin and paracetamol.


The objection of this experiment was successful since the analgesics in a mixture was
separated and identified to be aspirin and paracetamol according to their similarity in Rf
values. There was no sufficient evidence that shows the presence of caffeine in a mixture
since it Rf value differs from that obtained in 30 fractions. Column chromatography method
is good and efficient for this experiment.


1. Lehman. J.W. Operational Organic Chemistry. A Problem-‐-Solving

Approach to the Laboratory Course, 3rd ed. Prentice Hall: Upper Saddle River,
NJ, 1999; pg 117-‐-122.
2. Revell, K. D. J. Chem. Educ. 2011, 88, 1413–1415.