DISCUSSION

The photosynthetic pigments are responsible for absorbing and trapping light energy uses its radiant
energy to power up the process of photosynthesis. The main photosynthetic pigments are chlorophyll a
and b. There are accessory pigments like carotene and xanthophyll which transmit absorbed light energy
to chlorophyll a. Chlorophyll absorbs certain wavelengths of light within the visible light spectrum.Plant
appear green because chlorophyll reflect yellow and green wavelength of light,while red and blue
wavelength are absorbed and provide energy used for photosynthesis.

In order to identify the pigments that are present in leaves, they need to be separated and
characterized. Separation of the pigments can be done by paper chromatography. This separates
pigments of a spinach leaf .In order to separate the pigments in spinach leaves and carrot extract, grind
them separately in acetone medium. In a chromatography paper, draw a mark line at around 2 cm from
the tip of the paper as well as a dot in the middle of that line. With a capillary tube, place a spot of the
pigment on the pencil mark of spinach in middle paper and of carrot in the other paper. Let the
pigments dry. Now place each paper in separate arrangements of chromatography solvents and keep
each closed. Let the solvent front rise to around 3 - 6 cm and then remove the papers. Allow them to
dry. Once dried, measure the distances of the pigments moved and then calculate the Rf values.. The
separation is due to the solubility of the pigment in the chromatography solvent (acetone) and the
affinity of the pigment to the paper surface In paper chromatography, a paper strip has its tip dipped in
a non-polar solvent that contains the pigments to be separated. The water that is bound to the cellulose
fiber of the paper acts as the polar medium that attracts polar molecules. When the solvent moves
through the paper, more polar pigments are bound easily to the water in the cellulose of the paper and
stops moving.The pigments dissolve and move along with it. Less polar or non-polar pigments have
more affinity to the solvent rather than the paper. Thus they move farther from the pigment origin
compared to the distance travelled by the different pigments in the extract. The distance travel by
solvent and pigments are measured to calculate the Rf value (Retention Factor).

Rf = Distance moved by pigment/ Distance moved by solvent

To determine distance traveled by the pigment you must measure from the middle of the pigment band
to the markline and to determine the distance traveled by the solvent you must measure from the
solvent line to the mark line.A small RF value indicate a strong affinity of the compound for the
chromatography paper, move very slowly and crossed a small distance from the mark line.Those
pigments that moved the farthest are non-polar while those that stayed near are polar in
nature.Carotene is a non polar as well as the highest Rf value because it travel the furthest from the
mark line and move fastly.

In case of the spinach leaf extract, the chromatography paper showed a first band of chlorophyll b, then
xanthophyll and finally carotene at the top. This shows the polarity of the pigments. We can see that
chlorophylls are more polar than xanthophyll or carotene.For the carrot extract,only xanthophyll and
carotene appear on the chromatograhphy paper. Carotene is responsible for the orange colour
of carrots . Carotene is the most soluble and travels furthest, followed by Xanthophyll and finally
chlorophyll b.

In conclusion,we can separate different kind of pigment by using the chromatography paper and
calculate the Rf value by measuring the distance travel by solvent and pigment from the mark
line.Carotene travelled furthest than any other pigment because it is a non polar,most soluble and has
the largest value of Rf.