Chromatography Lab
Objective:
To separate and identify the colors of the plant extracted pigments by using the paper
chromatography technique, indicate the distance of each pigment from the starting line, and
discuss about the result and the fluorescence emitted.
Introduction:
In photosynthesis process, light reaction occurs to produce ATP and NADPH which later
be converted carbohydrate in Calvin cycle reaction. The production cannot exist without energy
input. The main source of energy comes in a type of solar energy. The energy is captured by
three major pigments in plant. Those are chlorophyll A, chlorophyll B, and carotenoid. These
pigments absorb different wavelength, which calls the absorption spectrum. These pigments
absorb visible light, and the ones that do not get absorbed will reflect back. That is why we see
different colors.
Hypothesis:
Three different colors are expected to appear on a watercolor paper. Those are yellow
from carotenoid, green and light green from chlorophyll A and chlorophyll B. The color which will
move furthest is yellow. It is because of the non-polarity of the chlorophyll. Three pigments have
different polarity, which causes them to move in various distances.
Materials:
- 1 x 250 mL Beaker
- A strip of watercolor paper
- Glass pipette
- Scissors
- Ruler
- Pencil
- 9:1 petroleum ether-acetone solvent
Procedure:
1.
Cut the watercolor paper paper into 2.5cm x 8cm.
2.
3.
4.
5.
6.
7.
Draw one line 1.5cm away from the end of the paper and another one 0.5cm from the
other side.
Draw a dot in the middle of the 1.5cm away line.
drop one drop of the extracted pigment at that dot and wait until it dries, repeat this
method for 5 times (concentrating the dot).
Pour 9:1 petroleum ether-acetone solvent into the beaker, makes sure that it is not high
enough to touch the spot.
Put the paper and leave it until the solvent is absorbed to the 0.5cm away line.
Count the number of separate pigments and measure the distance from the starting line.
Result:
Identify pigments table
Color
Distance from the starting line
Pigment
Orange
5 cm
Carotenoid
Blue-greenish
3 cm
Chlorophyll A
Green
1.7 cm
Chlorophyll B
Analysis:
1.
How many substances are on the Chromatogram?
4 which are:
- The extracted chlorophyll from plant’s leaves
- Solution (9:1 petroleum ether:acetone solvent)
- The separated colours of pigment (Carotenoids, Chlorophyll A and Chlorophyll B)
- pencil line
2.
What colours are they?
There are yellow from the carotenoid, blue color from the chlorophyll A and green from
the chlorophyll B.
3.
Which color moved furthest?
The yellow color which is carotenoids moved furthest.
Conclusion:
From this experiment, we were able to prove that plants have more than one type of
pigments due to the presence of more than one color on the testing paper and the result has
supported the hypothesis. Each of the color on the testing paper represents 1 type of pigment in
plants. The blue-greenish one represents chlorophyll a, green color represents chlorophyll b,
and the orange-yellowish one represents carotenoids. In addition, more different pigments help
plants to get more energy from different wavelength of light. For instance, Carotenoid is
responsible for absorbing sunlight with wavelength that Chlorophyll a cannot.
Fluorescence is a kind of radiation that emitted by an electron when it falls back to its
ground state. We see Fluorescence in the extracted pigment because when we shine light
through them, the electron gets to the excited state. After that it falls back to the ground state
and releasing fluorescence. Unlike the extracted pigment, In the leaves electron doesn't fall
back to the ground state because there is an acceptor that catches electron and take them
through electron transport chain. So, that is why we cannot see fluorescence ,when we shine
light through them.
If this experiment is to be repeated, we might use chromatography paper instead of
watercolor paper. Because it is better in extracting colors than drawing paper. If we had more
time, we would also be able to define other possible pigments that could be extracted and do
the experiment more about how they are different that make some of them move further except
from their polarity. We would also repeat the experiment at least three times for data accuracy.