Chawalnrath Wongdeshanan Kat 11-01

Chanunchida Nimyesuin
Ging 11-01
Karnsinee
Yotsakulsate
Jenny 11-01
Pitchaya
Areekarnlert
Petch 11-01

Lab Reort - Chromatography of Plant Pigments

Introduction
We do chromatography to separate chlorophyll a, chlorophyll b, xanthophyll, and
carotenoids. They are pigments in chlorophyll. We cant see them without doing
chromatography, because they will not separate if we dont put them to the pigment
concentration extract. Importantly, we cant put the chlorophyll that is solid on the
chromatography paper. So we have to mix the chlorophyll with petroleum ether and
acetone to change it to liquid. We will see four colors of pigments separating on the
chromatography paper. How far each pigment goes is depends on their polarity. Less
polarity will goes farther, because the chromatography paper is polarity, so more polarity
will spread very slow and stop and the less polarity will spread farther.
Material
1.Chromatography paper 2.5cm * 8cm
2.A pipette for dripping
3.Pigment extract
4.Solvent
5.Beaker
6. A pen and ruler for marking a line.
Procedure

Cut the Chromatography paper (2.5cm * 8cm)

Draw a line at the end of the Chromatography paper (1.5cm and 0.5cm. away from the end)
On the line 1.5cm.,draw a small dot on the middle of the line
Take a pipette, dip it briefly into the pigment extract
Apply the tip of the pipette to the pre-drawn dot
Produce a spot 0.5cm in diameter (keep the spot a circular shape)
Allow the solution to dry
Apply the pipette once again to the center of the spot
Fill up the beaker with solvent (make sure the spot above the solvent)
Leave until solvent soak up the Chromatography paper (until the 0.5 marked line)
Count the number of separated pigments and indicate the distance

Result
For this experiment the result is show in colors a length. On the chromatography
paper show that the orange color move the furthest from the start point, and light orange
come after. The orange colors we predict that it is a carotenoid pigment in the
chlorophyll. The third one that move furthest is dark green color. The last one is light
green color. Both green color we predict that it is a chlorophyll a and b respectively. As
the picture below show our result for the experiment.

Conclusion
We used the chromatography to separate the spectrum color in each the pigment and
the solubility. We saw the different color in the chromatography paper that the further is
orange color because of the absorption of spectrum that have capable of absorbing
various portion of visible light. Therefore, the best of moving is the most capable to
absorb the wavelength of light.
Question
1. How many substances are there in the chlorophyll?
There are four substances in chlorophyll; chlorophyll a, chlorophyll b,
xanthophyll, and carotene.
2. What colors are they?
Chlorophyll a is pale green, chlorophyll b is dark green, xanthophyll is yellow, and
carotene is orange-yellow.
3. Which color move further?
The color can move further is yellow color because of Carotenoids in chloroplast.
4. Why are they more than one pigment?
Plants have to absorb the sunlight and get the energy for photosynthetic so it have
visible of light spectrum and get more of light energy.
5. Why do you see fluorescence in the extracted pigment but not in the
leaves(when you shine light through them)? What is fluorescence?
Because fluorescence is the emission of radiation, especially of visible light, by a
substance during exposure to external radiation as light. We dont see the fluorescence
in the leaves because in the extracted pigment there is solvent to help us through.