Paper Chromatography Chromatography is a technique used to separate
molecules on the basis of differences in size, shape, mass, charge, solubility and adsorption
properties. In paper chromatography, the mixture is spotted on the paper, dried and the solvent
is allowed to flow along the sheet by capillary attraction. As the solvent slowly moves through
the paper, the different compounds of the mixture separate into different coloured spots.
The paper is dried and the position of the different compounds is visualized. Different
plant pigments can be separated by using the technique of paper chromatography.
Materials required: Fresh spinach leaves, filter paper strips,
chromatography chamber, mortar and pestle, ether acetone solvent (9 ether: 1 acetone),
measuring cylinder containing 5 ml acetone, capillary tube, thread, stapler, scissors,
spatula, watch glass, scale and pencil. Procedure: Take a few freshly plucked green spinach leaves.
Using scissors, cut the spinach leaves into small pieces and let them fall into the mortar.
Take a measuring cylinder that contains 5ml of acetone and pour into the mortar.
Grind the spinach leaves using the mortar and pestle.
Place the extract into a watch glass using a spatula.
Take a strip of filter paper having a narrow notch at one end of the strip.
Take a pencil and a scale and draw a horizontal line with a pencil about 2-3 cm away from
the tip of the notch. Put a drop of the pigment extract in the middle
of the line with the help of a capillary tube. Allow the drop to dry and repeat till four
or five drops are placed on the paper. Take the chromatographic chamber and pour
ether acetone solvent in it. Fold one end of the filter paper strip and
staple it. Using a thread, hang the filter paper strip
into the chromatographic chamber. The loading spot should remain about 1 cm
above the solvent level. Leave the chromatographic chamber undisturbed
for some time. We can observe, as the solvent moves through
the paper, it spreads the different pigments of the mixture in various distances.
When the solvent rises about 3/4th of the way up the strip, remove the strip carefully
and let it dry. Observations: The dried chromatographic paper strip shows
four distinct pigment bands. Different pigments can be identified by their colours. Calculations R_f Value of the each pigment spot can be
calculated by the equation; R_f=(Distance travelled by the compound)/(Distance
travelled by the solvent) Measure the distance of each pigment band
from the loading spot and also the distance travelled by the solvent. Calculate the Rf
value using the equation and record the values into a table. Result The topmost orange yellow band of pigments
in the separation corresponds to carotene. The yellowish band appearing below it indicates
the xanthophylls. The third from the above dark green band represents chlorophyll a.
The lowermost yellowish green band is that of chlorophyll b. Precautions: Spinach leaves should be fresh and green
The loading spot should be 2-3 cm away from the tip of the notch.
While hanging the strips in the chromatography chamber, the loading spot should remain about
1 cm above the solvent level.