LAB 6

PLANT PHYSIOLOGY

Name Muhammad jasmi arif bin jefri

No.Matrix E20161015638
Group B
Lecturer Professor Madya Dr. Shakinaz Binti Desa
Procedure

1.Used the microbiurette/ pin head as the dropper, dropped the chloroplast
extract on the prepared chromatography paper.
2.Dropped the extract at about 1.0 cm from the pointing end of the paper. Dry
the drop with a hair dryer and repeated the process for 3-4 times until one
small dot of thick pigment available.
3.The paper strip was attached at the cork stopper used a pin, and then
placed the strip vertically and straight into the test tube which contained
solvent.
4.Let the solvent moved and remove the paper before the solvent front
reached the top of your chromatography paper.
5.The last range of the solvent has been marked with a pencil
Result

Distance Distance
Pigment Colour traveled by traveled by Rf value Observation
compound(cm) solvent(cm)

2.7 8.4 0.32 Chlorophyll b

1 Yellow
-green

orange 8.1 8.4 0.96 carotene

2

Red spinach

Distance Distance
Pigment Colour traveled by traveled by Rf value Observation
compound(cm) solvent(cm)

5.5 7.0 0.78 Chlorophyll a

1 blue -green
 orange 6.4 7.0 0.91 carotene
2

Green spinach

Discussion
While performing experiments there are some errors that directly affects the
experiment changed which we used small dot of specimen,so the colour was
not clear.To fix it we need to use large dot of specimen so we can see it very
clear and accurate.Next,there are some pigment can be obtain in experiment
which carotene,chlorophyll a,and chlorophyll b.

Carotenoids are especially useful to humans since they are broken down in
our bodies to become Vitamin A, an essential nutrient for health and survival.
They are typically yellow-orange pigments that give carrots their characteristic
color. Beta carotene is the primary carotenoid pigment found in spinach.Other
than that,When carotenoids become oxidized, or take on an oxygen molecule,
they are known as xanthophylls. The name changes since their structure has
changed. These pigments are still yellowish in color but not reddish or
orangish the way that carotenoids often are.

Chlorophyll is the green pigment in most plants that is associated with

photosynthesis. The pigment absorbs all colored light except for the green
band, which it reflects to give spinach its characteristic leaf and stem color.
"Chlorophyll a" is a strong blue-green color and primarily responsible for
photosynthesis, while "chlorophyll b" is a supporting photosynthetic pigment,
according to University of Wisconsin Chemistry.

Discussion from question

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?

- 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.Why 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 we cannot determine the Rf value unless we

can measure the distance from the start to the front of solvent,we have to be
able to see where it stops even if we let it go as high as we want.

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
you remove it form the tube because so the point at which the solvent stopped
could be noted in case the solvent kept advancing when removed

6.Which of the pigments migrated the farthest and why the separation of
pigments occur as it did? How does paper chromatography work?

-carotene ( orange) because it was the most soluble in the solvent.They all
have a stationary phase (a solid, or a liquid supported on a solid) and
a mobile phase (a liquid or a gas). The mobile phase flows through the
stationary phase and carries the components of the mixture with it. Different
components travel at different rates. We'll look at the reasons for this further
down the page.
In paper chromatography, the stationary phase is a very uniform absorbent
paper. The mobile phase is a suitable liquid solvent or mixture of solvents.

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

Conclusion

The objective have been achieve in our experiment. Paper chromatography

proved to be an accurate method of separating and observing the various
colors of plant pigments. The pigments dissolved in the solvent and migrated
upward. The colors 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 3 pigments
were present in the original spot — carotene, chlorophyll a, and chlorophyll b.
Carotene was the most soluble, while chlorophyll b was the least soluble.

Reference

1.https://www.hunker.com/12550803/plant-pigments-found-in-spinach

2.https://www.colby.edu/academics_cs/courses/BI214/upload/lab3-

pigments.pdf
3.http://www.biologyjunction.com/chromatography_of_simulated_plan.htm