Separation of Pigments from the Extract of
Spinach Leaves by Paper Chromatography Paper chromatography is an analytical method
used to separate coloured substances, especially pigments. In paper chromatography, the sample
is distributed between a stationary phase and a mobile phase. The stationary phase is
usually a piece of high-quality filter paper. The mobile phase is a developing solution
that travels up the stationary phase, carrying the samples with it. When the mobile phase
is moved over the sample on the stationary phase, the components of the sample get gradually
separated from one another. Our aim here is to separate the pigments from
an extract of spinach leaves by paper chromatography. Materials required: Chromatographic chamber with lid, extract
of spinach leaves, isopropyl alcohol- water mixture, Whatman filter paper strip, fine
capillary tube, thread, scale and a pencil. Procedure: Take a Whatman filter paper strip and draw
a line with a pencil above 2cm from one end. Draw another line lengthwise from the centre
of the paper. Name the point at which the two lines intersect
as P. Take the extract of spinach leaves using a
capillary tube. Put a drop of the leaf extract at point P.
Allow it to dry in open air. Put another drop on the same spot and dry
again, so that the spot is rich with the mixture. Take a piece of thread and tie the filter
paper with it. Suspend the filter paper vertically in the
chromatographic chamber containing solvent, which is a mixture of isopropyl alcohol and
distilled water. Make sure that the pencil line remains about
1cm above the solvent level. Keep the chamber undisturbed for some time.
Notice the rising solvent along with the leaf extract.
As the solvent has risen, you will notice different spots of the pigments, such as:
carotene, xanthophyll, chlorophyll a and chlorophyll b, of spinach leaf extract on the filter paper.
Take the filter paper out of the chamber and mark the distance that the solvent has risen
on the paper with a pencil. This is called the solvent front.
Dry the filter paper and put pencil marks on the centre of the pigment spots.
Measure the distance of the solvent from the original line and the distance of the spots
from the original line. The Rf values of the pigments can be calculated
using the formula, Rf value of carotene is 0.94, xanthophyll
is 0.89, chlorophyll a is 0.46 and chlorophyll b is 0.22. Precautions: Always make use of a fine capillary tube.
Keep the chamber undisturbed and covered during the experiment.
The spot should be rich with the sample.