OBJECTIVE

To study thin layer chromatography (TLC) technique for the qualitative analysis.

To identify the components of a mixture.

PROBLEM STATEMENT

How does the component work on the chromatography paper?

What are the components of mixture identified?

INTRODUCTION

Chromatography is the separation of two or more compounds or ions by the distribution

between two phases, one which is moving and the other which is stationary. These two phases
can be solid-liquid, liquid-liquid or gas-liquid. Although there are many different variations of
chromatography, the principles are essentially the same.

Thin-layer chromatography or TLC, is a solid-liquid form of chromatography where the

stationary phase is normally a polar absorbent and the mobile phase can be a single solvent or
combination of solvents. TLC is a quick, inexpensive micro scale technique that can be used to:

• determine the number of components in a mixture

• verify a substance’s identity

• monitor the progress of a reaction

• determine appropriate conditions for column chromatography

• analyze the fractions obtained from column chromatography

In paper chromatography, the stationary phase is a specially manufactured porous paper.

The samples are added to one end of the sheet of paper and dipped into the liquid or mobile
phase. The solvent is drawn through the paper by capillary action and the molecules are
distributed by partition between the mobile and stationary phase. The partition coefficient, k,
similar to the distribution coefficient for extraction, is the equilibrium constant for the
distribution of molecules between the mobile phase and the stationary phase. It is this
equilibrium that separates the components. Different inks and dyes, depending on their molecular

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structures and interactions with the paper and mobile phase, will adhere to the paper more or less
than the other compounds allowing a quick and efficient separation.

TLC works on the same principles. In thin-layer chromatography, the stationary phase is
a polar absorbent, usually finely ground alumina or silica particles. This absorbent is coated on a
glass slide or plastic sheet creating a thin layer of the particular stationary phase. Almost all
mixtures of solvents can be used as the mobile phase. By manipulating the mobile phase, organic
compounds can be separated.

A mixture to be separated is first applied to an immovable porous solid (like

paper, or alumina, or fine silica sand) called the stationary phase. The components
of the mixture then get “washed” along the porous solid by the flow of a solvent
called the mobile phase. The mobile phase can be liquid (as in column, paper, or
thin-layer chromatography) or it can be a gas (as in gas chromatography).

RETENTION FACTOR

After a separation is complete, individual compounds appear as spots separated

vertically. Each spot has a retention factor (Rf) which is equal to the distance migrated over the
total distance covered by the solvent. The Rf formula is

Rf=distance traveled by sample distance traveled by solvent

The Rf value can be used to identify compounds due to their uniqueness to each
compound. When comparing two different compounds under the same conditions, the
compound with the larger Rf value is less polar because it does not stick to the stationary phase
as long as the polar compound, which would have a lower Rf value.

Rf values and reproducibility can be affected by a number of different factors such as

layer thickness, moisture on the TLC plate, vessel saturation, temperature, depth of mobile
phase, nature of the TLC plate, sample size, and solvent parameters. These effects normally
cause an increase in Rf values. However, in the case of layer thickness, the Rf value would
decrease because the mobile phase moves slower up the plate.

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If it is desired to express positions relative to the position of another substance, x, the Rx (relative
retention value) can be calculated:

Rx=distance of compound from origin distance of compound x from origin

Rx can be greater than 1.

PROCEDURE

1. A TLC plate obtained. The plate was handled using forceps as to avoid contamination. A
light pencil line was drawn using a straight ruler about 1cm from one end of the plate.
2. A capillary micropipette was used to make a small spot of the pure dye solution on the
plate several times. The spot placed 1cm from the left edge along the pencil drawn. Also
spotted the TLC plate with each of the solution prepared. Spots labeled lightly in pencil
below the line.
3. TLC plate was developed by placing it in a beaker that has been filled with developing
solvent (5% acetic acid in ethyl acetate) to a level of less than 1cm high (the spot on the
TLC plate should be above the level of solvent)
4. The beaker was then covered with aluminum foil immediately after the TLC plate was
immersed. The solvents are allowed to migrate up the TLC plate until it is about 1cm
from the top. The solvents were not allowed to reach the top of the plate.
5. TLC plate was removed then and the level which the solvent rose was marked with a
pencil. The solvents were allowed to evaporate off of the plate in the hood and then
visualized it under UV light. The outlines were outlined with pencil.
6. The distance the solvent moved is measured as well as the distance of all the spots. TLC
plate was carefully sketched in the space provided on the report sheet.

7. The procedures for the other two compounds were repeated.

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DIAGRAM

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APPARATUS AND MATERIALS

 Tissue rolls. Thicker ones will work better.

 Scissors
 Pencil
 Beaker
 Test tubes
 Water
 Dropper
 Clean plate
 Red, green and blue food coloring liquids
 Test tube rack
 TLC plate (chromatography paper)
 Solvent (5% acetic acid in ethyl acetate)

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OBSERVATION

SPOT SPOT COLOR DISTANCE SOLVENT DISTANCE SPOT Rf

TRAVELLED TRAVELLED

SPOT 1 BLUE

SPOT 2 PURPLE
(RED + BLUE)

SPOT 3 GREEN
(BLUE +
YELLOW)

IDENTIFICATION OF SPOT COMPONENT

SPOT 1 : BLUE – BRILLIANT BLUE

SPOT 2 : PURPLE – ALLURE RED AND BRILLIANT BLUE

SPOT 3 : GREEN – TATRAZINE AND BRILLIANT BLUE

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STRUCTURE OF CAFFEINE

STRUCTURE OF WATER

STRUCTURE OF DICHLOROMETHANE

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CONCLUSION

When observing the spots created by the known dyes we can apply these known patterns to the
unknown mixtures to determine their components.

For SPOT 1 it appears to have one components to the mixture. The only known dye I could
guess as to be one of the components would be brilliant blue.

For SPOT 2 it appears to have 2 components to the mixture. The component it contains appears
to be purple which is the combination of blue and red.

For SPOT 3 it appears to have 2 components to the mixture. The component it contains appears
to be green which is the combination of blue and yellow.

So in summary Spot 1 contains one component though Spot 2 and 3 contains two components
each composed of purple and green.

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REFERENCES

 http://chemwiki.ucdavis.edu/Reference/Lab_Techniques/Thin_Lay
er_Chromatography
 http://courses.chem.psu.edu/chem36/Experiments/PDF's_for_techn
iques/TLC.pdf
 http://faculty.chemeketa.edu/jcammack/ch104/CH104%20Lab/5%
20CH104%20%20Paper%20and%20TLC.pdf
 https://www.apsu.edu/files/chemistry/Paper_Chromotography_of_
Food_Dyes_and_Colors_RF8.pdf