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Lauren Dattilo

Class: AP Biology

Teacher: Mrs. Schulte

Experiment #: 4

Date of Experiment: 1/30/2018 and 1/31/2018

Title: The effect of a isopropyl alcohol on the pigmentation of spinach leaves.


All organisms rely on energy to perform their basic biological processes. Plants in
specific use the sun and process of photosynthesis to create their energy. During photosynthesis
carbon dioxide (CO2), water, and sunlight is absorbed by an autotroph were it is converted into
sugar (in specific C6H12O6) and oxygen. In plants, pigments are required in order to absorb the
sunlight required in order to perform this process. Each pigment gains its color based on the
color it reflects, for example if the pigment is green that means the pigment reflects green light
and can absorb all colors of light except for green. The most common pigment in plants are
chlorophylls which reflect green light, this is how plants get their color. Most green plants
contain both chlorophyll ​a​ and chlorophyll ​b​. Chlorophyll ​a​ is the main pigment and collects
blue-violet light as well as red-orange light. Chlorophyll ​b​ is more of an accessory pigment and
absorbs green light which is later passed on to Chlorophyll ​a​. Leaves of plants may also contain
carotenes which are orange pigments and xanthophylls which are yellow in color. Both are
however outnumbered by the amount of Chlorophylls.

Pigments in plant cells of leaves can be separated from one another in a technique called
chromatography. Chromatography is used to for separating and identifying substances in a
mixture based upon their solubility in a solvent. This is commonly used by scientists to analyze
environmental pollutants, identify drugs in urine, and even separate proteins that can identify
evolutionary relationships. In this lab chromatography is used to separate the different pigments
found in a spinach leaf. When you place some of the spinach leaves pigments on
chromatography paper and place it in a solvent, the individual substances will migrate up the
paper at different rates. The rate of migration is based upon the adsorption capacity of the
chromatography paper as well as the solubility of the sample in the leaf pigment. Substances that
are more attracted to the chromatography paper than to the solvent stop moving and form bands
or spots along the paper strip. During the lab you will see the different pigments represented on
as lines in there correct color on the chromatography paper.


This, on the left, was our first trial of using the chromatography
paper to separate the pigments found in a spinach leaf. The
paper itself was not chromatography paper but in fact filter
paper. This paper was very thin and the pigments though they
did show up were very hard to measure and see due to bleeding.
On the left we have our second trial. This time we used a
significantly thicker form of filter paper. As you can see in the
picture, The pigmentation was a lot clearer to read and measure.
However, unlike the first trial, not as many pigments moved up
the paper.

Total distance the Total distance the Rf Value

Solute solvent traveled pigment traveled (D unknown/ D

Trial 1 Blue: 11 cm Blue: 10 cm Blue: 0.909 cm

Coffee Filter Green: 11 cm Green: 8.5 cm Green: 0.773
(Picture 1) Orange: N/A Orange: N/A Orange: N/A
Yellow: N/A Yellow: N/A Yellow: N/A

Trial 2 Blue: 15.5 cm Blue: 13.5 cm Blue: 0.871

Thick Filter Paper Green: 15.5 cm Green: 12 cm Green: 0.774
(Picture 2) Orange: N/A Orange: N/A Orange: N/A
Yellow: N/A Yellow: N/A Yellow: N/A

In this table you are able to see the exact measurements of each pigment and the Rf value. As
shown in the table, the blue pigment moved further up the paper in both trials. Also. we were not
able to see an orange or a green pigment in either trial.

After the completion of both trails we were clearly able to see a Blue and a Green
pigment on both of the papers. The green pigment can be identified as chlorophyll ​a​ and the blue
pigment can be identified as chlorophyll ​b​. The absence of carotene (orange lines) and
xanthophylls (yellow lines) can be understood based on the lack of these pigments normally
found in leaves. Chlorophyll ​a​ and ​b​ are more plentiful and common within leaves and therefore
would be more likely to show up on the paper.

There were many human errors involved in this experiment that could have contributed to
the lack of orange and yellow pigments seen on our paper. First, this experiment was conducted
without actual chromatography paper. Therefore, the paper may not have shown the correct
movement and the correct pigment. Another human error was the use of spinach leaves that are
bought from a store in winter. Due to the fact we bought it from a store, there may have been
pesticides and other chemicals placed on the spinach in order to maintain its freshness for longer
than average. These chemicals may have directly influenced the results and lack of certain
pigments. Also since the leaves were bought during the winter, it guarantees that chemicals were
used due to the fact that it is not the correct growing season for spinach. The last human error
that may have affected our results was the coloring of the isopropyl alcohol. We used green
isopropyl alcohol and that may have altered the coloration of the pigments that did show up the
on paper.

For further testing I recommend that the experiment is performed using the correct paper.
This will give you definite results and could change the data provided in this experiment. I would
also recommend to test different types of leaves. For example the use of a flowering plant and
you could compare that to spinach, an edible plant. It would also be very interesting to test the
difference in pigmentations in different growing seasons. There may be more pigmentation
shown in the summer and less in the winter.