Experiment 3: Paper chromatography
Aim:
To perform paper chromatography for the separation of colored compounds present in the
leaves of different plants.
Principle:
Paper chromatography is a type of partition chromatography where components of a mixture
partition between a mobile phase (solvent) and a stationary phase (water film adsorbed on
cellulose paper). Each component moves up the paper at a different rate depending on its
solubility in the mobile phase and affinity for the paper, producing separated spots. The
distance each spot travels relative to the solvent front (Rf) is characteristic under the same
conditions.
Distance moved by substance from origin
𝐑𝐟 = Eqn. 1
Distance moved by solvent front from origin
Materials:
Fresh leaves of different plants, Whatman filter paper, acetone, isopropanol, mortar and pestle,
pipette tips (10 μL), capillaries, 100 mL beaker.
Procedure:
1. Sample preparation
• Fresh leaves of different plants were collected, blotted dry, and cut into smaller pieces.
• Approx. 0.5–1 g of leaves was collected in a mortar pestle and ground with 5 mL of
acetone or 90% (v/v) ethanol until a concentrated green paste formed or the mixtures
were properly colored.
• The extract was transferred to a small vial and allowed to sediment the particulates.
2. Preparation of solvent mixture/mobile phase
• The 100 mL solvent with a mixture of acetone: isopropanol in the ratio of 80:20 was
prepared.
• A beaker was filled with the prepared solvent mixture/mobile phase such that the depth
of solvent was up to 2 cm from the base of the 100 mL beaker.
3. Preparation of paper strips:
• Chromatographic papers/ Whatman filter papers were cut into pieces with a width of 3
cm and a height of 15 cm.
• A reference horizontal line at a height of 2 cm from the base of the paper was drawn.
• A few drops of the prepared sample were placed at the middle of the reference line and
allowed it to dry.
4. Development of chromatogram
• The filter paper, loaded with the sample, was placed in the solvent-containing beaker
such that the bottom edge was in the solvent.
• The beaker was closed from the top to avoid evaporation of the solvent.
• The solvent was allowed to ascend the paper until the solvent front reached about 1–2
cm from the top.
• After the development of the chromatogram, the paper was removed, and the solvent
front was marked.
5. Visualization of spots and calculation of Rf values
• The distance of the obtained spots (at the middle of the spot) and the solvent front
(distance travelled by solvent) was measured from the baseline, and the distance was
used to calculate the Rf value using Equation 1.
Results:
• Number of obtained pigments:
• Rf values of different pigments:
Precautions:
• Do not disturb the beaker/chamber when the reaction has started.
• Do not use pen/ink jets for marking the baseline as well as centre of the obtained spots.