ACTIVITY 3.

SEPARATION AND PURIFICATION OF SUBSTANCES

CHROMATOGRAPHY
Chromatography is one of the most useful methods of separating minute amounts of
complex mixture into the pure substances contained therein. It was originally concerned with the
separation of natural pigments which form color zones or bands on a column, but is now
employed with also in the separation of colorless substances. Techniques are available for
separating mixtures of gases, liquids and solids in solution.

Chromatographic separations are effected by allowing the components of the mixture to be

distributed in 2 phases, one being stationary and the other, mobile. In paper chromatography, the
stationary phase of this system is a filter paper saturated with water, along which moves an
immiscible solvent carrying the solute. The mobile solvent moving over the paper allows the
separation of the solutes of different partitions coefficients, hence the formation of a
chromatogram

Each component has a definite and characteristic rate of travel on the paper when using
specific mobile solvent. This characteristic of a substance can be used for its identification. The
rate of migration of solute on the paper will depend on the mobile solvent used. The will migrate
at a definite relative rate as compared to the rate of travel of the mobile phase.

The ration of these two rates of travel is called the Rf value and can be defined as the ratio
of the distance traversed on the paper by the substance to the distance traversed by the mobile
solvent from the same starting point.

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Rf =
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Reagents and Materials:

4.0 ml 95% Ethyl alcohol (C2H5OH) Marking Pen (Any color)

Distilled water Aluminum foil

Procedure:

1. Preparation of the Developing Chamber

Mix 4.0 ml of 95% ethyl alcohol and 4.0 ml of distilled water in a dry 250 ml beaker.
(Avoid splashing the liquid on the sides of the beaker.) Cover with a piece of aluminium
foil and let stand for 10 minutes for the atmosphere inside to become saturated with
solvent vapour.
 2. Preparation of the Paper Chromatogram
a. Cut a piece of Whatman filter paper no.1, 16.50 cm long and 8.00 cm wide. With a
pencil draw a line 6 mm from the lengthwise edge of the paper and 1 cm from each
crosswise edge for handling
b. Mark lightly with pencil 4 equidistant spots along the lengthwise line of the filter
paper.
c. Gently and quickly touch the first mark with the point of black – colored marking pen.
(The area touched should not be more than 2 mm in diameter)
d. Repeat step c on the other marks using different color marking pens for each mark.
Apply the same color on the same mark 4 to 6 times, allowing the spot to dry before
each application.

3. Development of the Spot

a. With careful handling at the crosswise edges, staple the paper into a cylindrical form.
Do not overlap the edges.
b. Put the cylindrical paper upright into the equilibrated beaker with the spotted edge at
the bottom. The solvent should wet the lower edge of the paper without reaching the
spots. Put the aluminum foil cover in place and let it stand for 30 – 45 minutes or
until the solvent front is 1 cm away from the upper edge.
c. Remove the paper from the beaker. Mark the position of the solvent front before it
dries up. Open up and dry completely.

4. Calculation of the Rf value

a. Calculate the Rf value for each of the colored dyes used.

Questions:
1. What is Chromatography?
2. What is the principle involved in paper chromatography?
3. What factors determine the rate of migration of the solute?
Name Date

Group no.

Activity 3.3
SEPARATION AND PURIFICATION OF SUBSTANCES
Chromatography

I. Results and Observation

A. The Paper Chromatogram
(Attach resulting paper chromatogram)

B. Calculations Involved: