Title: Use of chromatography to investigate the pigments isolated from the leaves of different
plants.
Introduction:
Being photoautotrophs, plants are the primary producers in the food chain since they manufacture
their own food from carbon dioxide and other raw materials. Photosynthesis is a process that plants
go through to create nutrients and add energy to the ecosystem.
Chloroplasts are the organelles in the plant cell that is responsible for photosynthesis. The light
dependent reaction and the light independent (Calvin cycle) reaction are the two reactions that
photosynthesis must go through. In a reduction process, water's electrons are transformed to
carbon dioxide. When sunlight is absorbed by chlorophyll, an electron is energised and proceeds
along the electron-transfer chain while releasing some energy. Protons (hydrogen ions) will then be
transported from the matrix to the opening between the inner and outer mitochondrial membrane
using this energy. A water molecule is prepared to donate an additional electron to the initial
chlorophyll molecule. Without the assistance of chlorophyll, this oxidation-reduction reaction
(photosynthesis) is not possible. Chlorophyll transfers light energy into chemical energy by capturing
solar energy and then transforming carbon dioxide into oxygen (sugars).
I will compare leaves from Sage plant and Kale plant in this experiment. Both of these plants are
examples of shade tolerant and shade intolerant plants. Plants that are shade tolerant can
photosynthesize even in the shadow, whereas plants that are shade intolerant cannot. Shade-
intolerant plants have thicker leaves due to the extended palisade cells, whereas shade-tolerant
plants often have bigger surface areas and thinner leaves. Shade-tolerant leaves are heavier than
shade-intolerant leaves because they have more chlorophyll and more grana in their chloroplasts.
Hypothesis:
Null Hypothesis- There is no significant effect on the pigments in the two different leaves Sage and
Kale
Alternative Hypothesis- There is a significant effect on the pigments in the two different leaves Sage
and Kale when chromatography is done.
Equipment:
Boiling tube rack
2 boiling tubes with bungs
Small glass measuring cylinder
Running solvent
Chromatography paper
Glass rod
2 leaves (spinach and birch)
Cork borer
Tile
Ruler
Pencil
Drawing pins
Marker pens
, Risk Assessment
Hazard Risk Control
Running solvent Irritating to the skin Wear safety goggles and a lab
coat; if you come in contact
with the solvent make sure to
wash it off immediately
Glassware Very sharp when broken, can Wear safety goggles and
cause cuts. handle equipment with care.
Drawing pins Can poke/cut someone Make sure to keep them in
one spot so they don’t go
missing
Method:
1. Set up boiling tubes at the start of the investigation. Add 7cm^3 of running solvent to each
of the two boiling tubes. Put a bung on top of each tube and stand them upright in a rack.
Label the tubes A and B.
2. Take a piece of chromatography paper that fits the boiling tube. Rule the pencil line 2cm
from the bottom of the filter paper. This line is called the origin. Write leaf A at the top of
the chromatography paper in pencil.
3. Cut a disc from leaf A with a cork borer. Avoid the veins and midrib of the leaf when you do
this.
4. Place the lead disc on the chromatography paper at the centre of the line marking the origin.
Crush the disc into the paper with the end of a glass rod. The crushed leaf should leave a
stain on the chromatography paper.
5. Pin the chromatography paper to the bung with a drawing pin, and then put the
chromatography paper into the labelled A. Make sure the end of the chromatography paper
is in the solvent and that the solvent doesn’t come above the origin. Put the tube carefully
back into the rack and don’t move again.
6. Let the solvent run up the chromatography paper until it almost reaches the top of the
paper. Remove the chromatography paper from the tube and immediately draw a pencil line
to show how far the solvent moved up the paper. This line marks the solvent front.
7. Replace the bung in the tube.
8. The filter paper with its coloured spots called chromatograph. Let the chromatograph dry.
Using a pencil, draw around each coloured spot on the chromatogram. Repeat two with the
second piece of paper but write B at the top of the chromatography paper.
9. Repeat steps 3-8 with leaf B.
10. Calculate the Rf value of the pigment spots on each chromatograph.
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