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SBL 1023




MATRIC NUMBER E 20161015941

Date and time of practical class

19th December 2017 2.00 p.m. – 4.00 p.m.


A pigment is a molecule that absorbs light. White light contains all of the different colours of
the visual spectrum. This can be observed in a sample rainbow during rain storm or by using
a prism that splits white light into its various colours.

In plants, there are two categories of pigments used for photosynthesis such as
primary pigments and accessory pigments. The chlorophylls are the primary pigments of
photosynthesis, with two types called chlorophyll a and chlorophyll b. The chlorophyll is
green pigment molecules that absorbs blue, red, yellow, orange, etc but reflects green light.
However, in accessory pigments are red, yellow or orange. They are absorbing the other
entire colour.

In this experiment, we will use paper chromatography to separate the plant pigments
from a plant using hydrophobic solvents. Chromatography is an analytical chemical
technique that allows separating different molecules from a mixture based on difference in
solubility. Some compounds do not dissolve in water. These are called hydrophobic
compounds. On the other hand, some molecules are hydrophilic, meaning it is like to dissolve
in water.


To determine the RF value


1. The chloroplast extract have been dropped on the chromatography paper.

2. The extract has been drop at about 1.0 cm from the pointing end of the paper.
3. The drop was dry with a hair dryer and the process was repeated for 3-4 times until a
small dot of thick pigment available.
4. The paper strip was attached at the cork stopper using a pin. The strip was placed
vertically and straight into the test tube which contains solvent.
5. The solvent was let to move and remove the paper before the solvent front reaches the top
of your chromatography paper.
6. The last range of the solvent was mark with pencil.

𝒅𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒕𝒓𝒂𝒗𝒆𝒍𝒍𝒆𝒅 𝒃𝒚 𝒄𝒐𝒎𝒑𝒐𝒖𝒏𝒅

𝑹𝒇 =
𝒅𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒕𝒓𝒂𝒗𝒆𝒍𝒍𝒆𝒅 𝒃𝒚 𝒔𝒐𝒍𝒗𝒆𝒏𝒕

Green spinach

Colour of pigment Distance travel by Distance travel by Rf value

compound (cm) solvent (cm)
Red 2.5 cm 6.4 cm 2.5 𝑐𝑚
𝑅𝑓 = = 0.39
6.4 𝑐𝑚
Pale yellow 6.2 cm 6.4 cm 6.2 𝑐𝑚
𝑅𝑓 = = 0.97
6.4 𝑐𝑚
Red spinach

Colour of pigment Distance travel by Distance travel by Rf value

compound (cm) solvent (cm)
Yellow green 3.0 cm 7.2 cm 3.0 𝑐𝑚
𝑅𝑓 = = 0.42
7.2 𝑐𝑚
Green 4.9 cm 7.2 cm 4.9 𝑐𝑚
𝑅𝑓 = = 0.68
7.2 𝑐𝑚
Pale yellow 6.8 cm 7.2 cm 6.8 𝑐𝑚
𝑅𝑓 = = 0.94
7.2 𝑐𝑚


Paper chromatography is a form of liquid chromatography where the components of a

mixture of organic compounds get separated as unique spots by unidirectional flow of the
developing liquid mobile phase solvent mixture over the filter paper to which a spot of the
sample is applied. The distance travelled by each component is specific under the given set of
operational conditions.

1. Why the developing solvent mixture is prepared fresh before use?

The developing liquid phase comprises of a pure solvent but more often it is a
mixture of two or more solvents in specified proportions. In case solvents are
mixed and stored for long periods there could be loss of volatile component which
will alter the mixing proportions.
2. Why is it important to keep the dye spots (leaf extract) above the solvent level?
It is important to keep the dye spots above the solvent level because if the dye
spots of submerged in the solvent, then the spots would dissolve into the solvent
preventing them from separating out and no measurements or observations could
be made.

3. Why is it necessary to cover the test tube during the paper development?
The entire reservoir of solvent will evaporate prior to the completion of the
chromatography if the beaker isn't properly covered. Thus, it will cause the
solvent mixture to more quickly diverge from the intended mixture for the
chromatography, which will detract from reproducibility and likely harm the
usefulness of the separation.

4. Why it is important to stop the chromatogram before the solvent front leaches the
top of your chromatography paper?
If did not stop the chromatograph before the solvent reached the top of the paper,
then the solution will travel to the top edge and couldn’t calculate the Rf value
that is to compare the distance that a component "travelled" in the stationary phase
with the distance reached by the solvent

5. Why is it important to mark the solvent level on the chromatography paper when
you remove it from the test tube?
It is important to mark the solvent level on the chromatography paper when
remove it from the test tube because the point at which the solvent stopped could
be noted in case the solvent kept advancing when removed.

6. Explain what would happen to your chromatogram if you let it run too long?
Too high a solubility will lead to transfer of the component along with the solvent
front and on the other hand if the solubility is too low the component will not be
carried by the solvent mixture and will remain close to the initial applied spot.

The experiment was carried out and it was proven that the green spinach contains Carotene
and Chlorophyll B as pigment in the leaf. While red spinach contains Carotene, Chlorophyll
A and Chlorophyll B. This was shown when the paper chromatography was done, the read
spinach trials had many different coloured pigments over the paper.


Deepak Bhanot. (2014). 10 Common Interview Questions on Paper Chromatography.

Retrieved from

St. Rosemary Institution. (2016). Chromatography Lab Answers. Retrieved from