C 245

NAME:_______________________ BATCH:__________
ROLL NO:____________________ SEM:__________
SIGNATURE:_________________ DATE:__________

QUALITATIVE ANAYSIS OF MIXTURES USING TLC

AIM
To analyze a sample mixture qualitatively using TLC, a chromatographic technique.

THEORY
TLC, i.e., Thin Layer Chromatography, a technique that is used to analyze a small spot of the sample,
is made at one end of a glass or plastic plate that has been coated with a thin layer of silica gel. In a
process known as “development” the plate is then immersed spot-end down in a pool of solvent (the
exact solvent used depends on the sample and is determined by experimentation). The solvent is
allowed to move up the plate by capillary action (the silica gel “soaks it up”). Compounds present in
the sample are carried up the plate by the solvent. However, different compounds generally move at
different rates. Therefore, if the sample is a mixture of compounds, it will separate into a series of spots
at varying distances up the plate (fig 1). If the sample is pure (i.e., only a single compound is present)
then only one spot will result.

If the compounds in the sample are colorless, then the spots will be hard to see against the white
background of the silica gel, and a process for “visualizing” them must be used. A UV light source can
be used for this purpose if the silica gel contains a small amount of a fluorescent substance. (The
commercial TLC plates used in this experiment have silica gel to which the compound fluorescein has
been added). Under UV light the spots will show up as dark spots against a bright background.
How far a particular compound moves from the original spot depends on the rate at which the solvent
moves it. The fastest the solvent can move the compound is the same rate at which the solvent moves.
In this case, the compound forms a spot at the same distance from the original spot as the distance the
solvent was allowed to move (the spot's Rf value = 1.0; see below). At the other extreme, a compound
may be moved so slowly by the solvent that its spots remain where the original spot was placed (Rf =
0.0; see below). By measuring the distance of spot (solute) and solvent moves, we can quantify the rate
of migration of any compound using the ratio referred to as the Rf value.
Distance moved by solute front
Rf=
Distance moved by solvent front
The Rf value of a particular compound is an identifying characteristic of the compound just like
its boiling point and melting point. Therefore, spots with identical Rf values in two different samples
can reasonably be concluded to indicate the presence of the same compound in both samples. For
example, if TLC analysis of an unknown sample gave two spots with Rf values of 0.22, 0.57 and 0.77
(as in fig 1[b]) then the sample can be concluded to consist of a mixture of three compounds. Further, if
TLC analysis of a known sample of pure caffeine resulted in a spot with a Rf value of 0.23, then one
may reasonably conclude that one of the compounds present i n the unknown sample is caffeine. ( The
small difference between the two values, 0.22 and 0.23, is ascribed to unavoidable random error in the
measurement of Rf values, which at best are accurate to no more than ±0.02).

MATERIALS REQUIRED
TLC plates, diethyl ether, hexane, ethanol, dichloromethane, chloroform, o- & p- nitroaniline, TLC
chamber, ruler, capillary tube, spatula, forceps etc.
Example illustration

PROCEDURE
1. Take approximately 25 mg of the sample in a small labeled test tube and add 1.0 mL of
developing solvent to it.
2. Mix and stir vigorously to dissolve the solid.
3. Now obtain a TLC plate. Draw a light pencil line at the straight edge about 1 cm from one end
of the plate.
NOTE: The plate should be handled using forceps so as to avoid contamination.
4. Use a capillary micropipette to make a small spot of the solution (made in step 1) on the plate.
5. Place the spot at the midway point along the pencil line you drew.
6. Develop the TLC plate by placing it in a beaker that has been filled with developing solvent to a
level of less than 1 cm high (the spot on the TLC plate should be above the level of the solvent).
7. Cover the beaker with aluminum foil immediately after the TLC plate is immersed.
8. Allow the solvent to migrate up the TLC plate until it is about one centimeter from the top. Do
not allow the solvent line to reach the top of the plate.
9. Remove the TLC plate and mark the level to which the solvent rose with a pencil. Allow the
solvent to evaporate off the plate in the hood and then visualize the plate. Outline all spots with
a pencil.
10. Measure the distance the solvent moved as well as the distances of all spots. Carefully sketch
the TLC plate in the space provided on the report sheet.

Results:
TABLE, OBSERVATIONS AND CALCULATIONS
S.No. Distance Solvent Compound Spots in Spots in Sketch of developed TLC
moved sample 1 sample 2 plate

Comment on the results.