THIN LAYER CHROMATOGRAPHY

1.1. Aim
To determine the composition of given over-the-counter analgesics (pain medications) and to
select the solvent best suitable for given components using thin layer chromatography method.
1.2. Theory
The reference compounds are: Acetaminophen, Aspirin, Caffeine and Ibuprofen.
O
H
N

OH
O

HO
O

Aspirin (acetylsalicylic acid)

Acetaminophen

O
OH
O

Ibuprofen

N
O

Caffeine

a) Principle of operation
TLC is the separation of moderately volatile or nonvolatile substances based upon differential
adsorption on an inert solid (the stationary phase) immersed in an organic solvent or solvent
mixture (the mobile phase). The components are distributed between the stationary phase
(usually silica gel or alumina) and the solvent depending upon the polarities of the compound
and solvent. The compounds are carried up the plate (ascending chromatography) at a rate
dependent upon the nature of the compounds and the solvent.
Compounds are separated by adsorption chromatography based upon differential attachment of
molecules to the adsorbent and the polarity of the solvent used for the separation. Polar
compounds are strongly attracted to and held by a polar adsorbent. Nonpolar compounds are held
weakly. When a nonpolar solvent is passed through the adsorbent, nonpolar compounds are
released easily, but polar compounds are retained. When a moderately polar solvent is passed
through the adsorbent, both polar and nonpolar compounds are released, but the nonploar move

faster because there is still an attraction between the polar compounds and polar adsorbent. In
general, nonpolar compounds move faster than polar compounds for TLC on silica gel or
alumina.
Solvents are rated according to their polarities. Common solvents are listed below from most
polar to least polar.
Most Polar

Methanol (CH3OH)
Ethanol (CH3CH2OH)
Acetone (CH3COCH3)
Ethyl acetate (CH3 CO2 H2CH3)
Methylene Chloride (CH2Cl2)
Diethyl ether (CH3 CH2OCH2CH3)
Toluene (C6H5CH3)
Cyclohexane (C6H12)

Least Polar

Hexanes (C6H14 isomers)

Rf Value Calculation: The distance traveled by each component is expressed as a rate or

retardation factor (Rf). Rf values are calculated by dividing the distance traveled by a component
by the distance between the origin and the solvent front (distance traveled by the solvent).

Rf =

Rf values are measured from the origin where the initial spot was applied to the center of the
spot.
b) Description of apparatus
TLC plates and micro capillary: to spot the samples
UV-lamp: to visualize the spots
Developing Chamber (Beaker covered by a watch glass)
Materials:

Acetaminophen, aspirin, caffeine, ibuprofen, standard reference mixture, unknown over-thecounter analgesic tablets, 99/1 mixture of Ethyl Aceate/ Glacial Acetic acid, 50/50 Ethanol/ethyl
acetate solution, TLC chamber, TLC plates, micro capillaries, , ruler, 2 test tubes, spatula, small
beaker, pencil
Safety Precautions:
Solvents should be used in the fume hoods (inhalation of vapors can cause nausea or
drowsiness). Avoid spills; it can also pass through both latex and nitrile gloves. Do not look
directly at the UV lamp, the UV light can damage your eyes.
Procedures:
1. Obtain half a tablet of the analgesic to be analyzed, crush it and transferred it to a small,
labeled test tube.
2. Prepare a solution of 50/50 Ethanol/Ethyl acetate. Dissolve the unknown powdered sample
in 5 mL of the solvent (if not dissolve, heat each of them gently for a few minutes in a warm
water bath). Not all the tablet will dissolve, since the analgesics usually contain an insoluble
binder. In addition, many of them contain inorganic buffering agents or coatings that are
insoluble in this solvent mixture.
3. Obtain a TLC plates and micro capillaries. Using a lead pencil (not a pen) lightly draw a
line across the plates (short dimension) about 1 cm from the bottom. On this plate, lightly
mark off intervals on the line. These are the points at which the samples will be spotted.
4. Use a capillary tube to right spot the plate on marked points. It is important that the spots be
made as small as possible (about 1-2 mm in diameter) and that the plates not be overloaded.
5. Prepare a developing chamber using a beaker as the chamber. Pour the solvent, a 99/1
mixture of Ethyl acetate/Glacial Acetic Acid, into a beaker to a depth of about 0.5 cm. Place
the spotted plate in a developing chamber, put the top on, and allow the plate to develop.
6. When the solvent has risen to near the top of the plate (about 1 cm from the top), remove the plate
from the chamber and mark the position of the solvent front with a pencil. Let the plate dry in hood.
Examine the plate under UV light to see the components as dark spots. Lightly outline all the
observed spots with a pencil. Measure the distance that each spot has traveled relative to the solvent
front. Calculate the Rf values for each spot and record. By matching the R f values from your
reference compounds to those of the unknown drug sample, determine the identity of each spot in the
drug sample.

Observations
Table 1. Commercial Painkillers and their components
Components of Commercial Painkillers (Standards)

Co

Acetaminophen
Anacin

Aspirin

Caffeine

Ibuprofen

Painkillersmmercial

Bufferin
Excedrin (extrastrength)
Tylenol
Motrin

Molecular Structure of the

compounds

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