DEPARTMENT OF BIOLOGY

FACULTY OF SCIENCE AND MATHEMATICS

SULTAN IDRIS EDUCATION UNIVERSITY

SBL 1023

TECHNIQUE IN BIOLOGY AND BIOCHEMISTRY LABORATORY

EXPERIMENT 6: PLANT PHYSIOLOGY

BY

BIL NAME STUDENT ID LECTURER

GROUP

1. NOR AIYNA SYERA BT MOHD ASRI E20161015981 B

Introduction

The purpose of this lab experiment was to separate plant pigments using paper
chromatography, and to measure the rate of photosynthesis in isolated chloroplasts. Because
of capillary action the solvent moves up the paper causing the pigments to become visible at
certain distances.

The substances visible on the paper are called pigments. Chlorophyll a is the main
pigment that makes up about 75% of the pigmentation in plants. Chlorophyll b makes up
about 25% of the pigmentation. And carotenes and xanthophylls are accessory pigments that
make up the rest of the pigmentation. Carotene is the most soluble of the pigments and as a
result will be carried the farthest by the solvent. The paper will display a spectrum of the
pigments found in the spinach leaves. Using the formula Rf one can determine the
relationship between the distance the solvent travelled to the distance the pigment travelled.

What is pigment? 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 simple rainbow
during a 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: primary
pigments and accessory pigments. The chlorophylls are the primary pigments of
photosynthesis, with two types called chlorophyll a and chlorophyll b. The chlorophylls are
green pigment molecules that absorb blue, red, orange, yellow, etc., but reflects green light.
On the other hand, the accessory pigments are red, yellow or orange – they absorb all of the
other colours.

In this experiment, we used paper chromatography to separate the plant pigments

from a plant using a hydrophobic solvent. Chromatography (“colour” “measure”) is a
technique that allows us to separate different molecules from a mixture based on differences
in solubility. Some compounds do not like to dissolve in water.
Objective

- To separate the plant pigments from a plant using a hydrophobic solvent.

Equipment/ materials

- Cork stopper
- Strips of filter paper
- Test tube
- Pin
- Leaf extract ( green and red spinach)
- Sample substance (red and blue ink)
- Solvent (petroleum ether)
- Mortar

Procedure

1. Using the microbiurette/ pin head as the dropper, the chloroplast extract of green
spinach was dropped on the prepared chromatography paper.
2. The extract was dropped at about 1.0 cm from the pointing end of the paper. The
drop was dried with a hair dryer and the process was repeated for 3-4 times until one
small dot of thick pigment available.
3. The paper strip was attached at the cork stopper using a pin. The strip was placed
vertically and straight into the test tube which contained solvent.
4. The solvent was let moves and the paper was removed before the solvent front
reaches the top of the chromatography paper.
5. The last range of the solvent was marked with pencil.
Result

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
Discussion

In our discussions, we have discussed why:

1. 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. 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
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. Is it necessary to cover the test tube during the paper development?

During the chromatogram development chamber is covered. This is essential as the
environment inside the chamber should remain saturated with the solvent vapour.
Development times can vary from about an hour to several hours and a saturated
environment prevents losses due to evaporation

4. It is important to stop the chromatogram before the solvent front reaches the
top of your chromatography paper?
It is important to stop it because you cannot determine the Rf value unless you can
measure the distance from the start to the front of solvent, you have to be able to see
where it stops even if you let it go as high as you want

5. 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 you
remove it because so the point at which the solvent stopped could be noted in case
the solvent kept advancing when removed and so that you will be able to measure
how far it travelled.
 6. Which of the pigments migrated the farthest and why the separation of
pigments occur as it did? How does paper chromatography work?
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.

7. Explain what would happen to your chromatogram if you let it run too long?
If we allowed the chromatogram to develop too long the solutes and the solvent
would travel too far and possibly run of the plate. Therefore we would not be able to
calculate the Rf without a measured solvent front.

Conclusion

Paper chromatography proved to be an accurate method of separating and observing

the various colours of plant pigments. The pigments dissolved in the solvent and migrated
upward. The colours were observed and their migration distances measured & recorded. The
Rf value of each pigment was determined by dividing its migration by the migration of the
solvent. It was determined that 4 pigments were present in the original spot — carotene,
xanthophyll, chlorophyll a, and chlorophyll b. Carotene was the most soluble, while
chlorophyll b was the least soluble.

Reflection

From this experiment, we have learned on how to separate the plant pigments from a plant
using a hydrophobic solvent. The objective was received. Apart from that, we also have
learned on how to determine the retention factor of plant extract by separating the extracts
based on their pigments by paper chromatography technique. Lastly, we also able to calculate
the value of Rf from this experiment. Overall the experience was amazing.
Reference

1. Retrieved from, http://lab-training.com/2014/12/05/10-common-interview-questions-

paper-chromatography/ on 18 January 2018.
2. Retrieved from, https://en.wikipedia.org/wiki/Plant_physiology on 18 January 2018.
3. Retrieved from, lab manual lab 4: plant physiology on 18 January 2018.