Anna Roman

AP Biology

Mrs. Schulte

Experiment 4

January 30-31

The use of chromatography to separate and identify plant pigments

Abstract:(1)
In this lab we used chromatography to separate the individual pigments involved in
photosynthesis. There were four reasons for completing this lab: to understand the use of
chromatography in identifying compounds, perform chromatography to identify the pigments of
green leaves, calculate the R​f​ values of individual pigments in green plants, and study the use
and relation of plant pigments in photosynthesis. We were able to fulfill this purpose by using
spinach as our green plant; we understood that it was green because it reflected green light and
absorbed all other colors, especially red and blue. The pigments in plant cells can be separated
from one another by chromatography which is a technique used for separating and identifying
substances in a mixture based on their solubility in a solvent.
The most important pigments in photosynthesis are the “chlorophylls”. Green plants
mostly contain chlorophyll a and chlorophyll b but also have “carotenes” and “xanthophylls”
which are orange and yellow; they are what turns the leaves colors in the fall but are most of the
time masked by chlorophyll. Chromatography works by adding a dye mixture on a strop of
chromatography paper and placing it into a solution, the substances in the mixture will migrate
up the chromatography paper at different rates. The different rates are based upon the
absorption capacity of the paper and solubility of the solvent.
In our experiment we tested three different types of paper as a replacement to
chromatography paper, isopropyl alcohol, vegetable oil, and distilled water as our solutions, and
rubbed spinach onto the paper with a coin to make our mixture. To complete the experiment we
poured our solutions into test tubes, cut the paper, rubbed on the spinach with a coin, and let
the paper soak in the solutions for a while. We tested all three solutions on coffee filters and
found the oil did not work and the isopropyl alcohol worked the best; therefore, we decided to
test the alcohol on filter paper to see what the results would be. We found the isopropyl alcohol
on the coffee paper gave us the best results as we were able to see orange, green, yellow, and
blue lines on the paper. We were then able to calculate the relative mobility factor (R​f​) which is
the ratio of the distance a substance (solutions) travels to the distance the solvent (spinach)
travels.

Results:

Coffee filter:​ (because the oil, second from the left, did not produce any effect we left it out of
our report as there was no data to collect)
Water:(furthest on the left)
 Paper length 18 cm

Distance water traveled 18 cm

Green/yellow line 6 cm

Green line 8 cm
This chart shows the data we collected from putting our solute onto a strip of coffee filter paper
and letting it soak in water. We found the solution (water) traveled all the way up the paper and
the solute (spinach) separated into a green/yellow line and a distinct green line. We measured
the distance the pigments traveled from the original solute line so that we could calculate the R​f
values for both:
Green/yellow R​f​ value:
6 cm (distance pigment traveled) / 18 cm (distance solution traveled) = ​.33

Green R​f​ value:

8 cm (distance pigment traveled) / 18 cm (distance solution traveled) = ​.44

Isopropyl Alcohol:(third from the left)

Paper length 11.5 cm

Length water traveled 11.5 cm

Orange line 4.25 cm

Green line 5 cm

Yellow line 5.5 cm

Blue line 7 cm
This chart shows the data we collected from putting our solute onto a strip of coffee filter paper
and letting it soak in isopropyl alcohol. We found the solution (the alcohol) traveled all the way
up the paper and the solute (spinach) split into orange, green, yellow, and blue pigments. This
combination gave us the best results out of all of our trials. We measured the distances of each
line to be able to calculate the R​f​ values:

Orange R​f​ value:

4.25 cm (distance pigment traveled) / 11.5 cm (distance solution traveled) = ​.37

Green R​f​ value:

5 cm (distance pigment traveled) / 11.5 cm (distance solution traveled) =​ .43

Yellow R​f ​ value:

5.5 cm (distance pigment traveled) / 11.5 cm (distance solution traveled) = ​.45

Blue R​f ​ value:

7 cm (distance pigment traveled) / 11.5 cm (distance solution traveled) = ​.61

Filter paper:​(second from the right)

Isopropyl alcohol:

Paper length 10.75 cm

Distance water traveled 10.75 cm

Yellow line 3 cm

Green line 5 cm
This chart shows the data we collected from placing the solute on a regular filter paper and
letting it soak in isopropyl alcohol. We only put this paper into the alcohol because it is what
gave us the best results prior and we wanted to see if we could get even greater results with
different paper. We found the solution (the alcohol) traveled all the way up the paper and the
solute (spinach) separated into yellow and green pigments. We measured the distance of the
lines of these pigments from the original solute line so we could calculate the R​f​ values of both.

Yellow R​f​ value:

3 cm (distance pigment traveled) / 10.75 cm (distance solution traveled) = ​.28

Green R​f ​ value:

5 cm (distance pigment traveled) / 10.75 cm (distance solution traveled) = ​.47

Thick filter paper:​ (furthest on the right)

Isopropyl Alcohol:

Paper length 14.5 cm

Distance water traveled 14.5 cm

Green line 10 cm
This chart shows the data we collected from placing the solvent onto a thicker filter paper and
letting it soak in isopropyl alcohol again. We used the alcohol for the same reasons as before;
we found that is gave us the best results prior and wanted to test it on different types of paper.
We found that the solution (the alcohol) traveled all the way up the paper and the solute
(spinach) only separated into a green pigment. We measured its distance from the original
solvnet line so we could find the R​f ​ value:
Green R​f​ value:
10 cm (distance pigment traveled) / 14.5 cm (distance solution traveled) = ​.69
Conclusion:
Our hypothesis for this experiment was that we would see the spinach separate into
multiple, distinct colors and that the isopropyl alcohol would cause the greatest results. We
found that our hypothesis was true and the alcohol formed the best results while the oil did
nothing and the water formed little results. Because of this we decided to test the alcohol and
the spinach on different papers thinking thicker filter paper would be the best for this and we
evidently found this not to be true as the thinnest coffee filter paper showed us the greatest
results.
We were able to fulfill our purpose and were able to identify the different compound in
the spinach. On the filter paper with the alcohol, from the bottom up the compounds in the
spinach that separated are chlorophyll b, chlorophyll a, xanthophyll, and carotene. We
successfully used chromatography to separate the pigments in the green plant and were able to
see green, yellow, orange, and a blue line. After performing chromatography we collected
measurements that enabled us to calculate the R​f​ value for each of the individual pigments in
the plant. With our results we were then able to better understand the relationship and use of
pigments in photosynthesis.
Some sources of error in this lab would include the fact that we did not use actual
chromatography paper instead we used coffee filter paper and then decided to test regular filter
papers of different thicknesses. The chromatography paper would have given us the greatest
results because it is made specifically for this kind of experiment; however, we made due with
what we had. This spinach we used was not fresh spinach as it came from a package and was
most likely packed with preservative chemicals. It was also grown in the winter making it less
“colorful”. If we had used fresh spinach grown in the summer we would have seen greater
results. Another possible source of error could be the fact that we touched the filter paper that
we used for the chromatography and the oils on our fingers could have skewed the data in one
direction or another.
I think it would be interesting to do this lab again but with the proper materials so that we
could get the best possible results and get a greater range of colors/compounds. We could also
test this experiment with different kinds of plants: other green plants and plants of other colors.
For example, would a flower produce the same results?

Background Research: (1)

All life on earth depends on the process of photosynthesis. Photosynthesis is a process
that plants, cyanobacteria, and algae use to convert CO​2​ and water, in the presence of light
energy, into sugar. Photosynthesis requires the presence of special pigments that can absorb
the energy of light. A pigment is a substance that absorbs light of a particular wavelength. Its
color depends upon the color of light that it reflects. For example, a green substance appears
green because it reflects green light and absorbs all other colors of light, especially red and
blue.
The most important plant pigments in photosynthesis are the “chlorophylls”. Green plants
contain both chlorophyll a and chlorophyll b. In addition to chlorophyll, the leaves of many green
plants also contain one or more other pigments, including “carotenes”, which are orange, and
“xanthophylls”, which are yellow. The presence of these other pigments is masked by the
abundance of chlorophyll during most of the year.
The pigments in plant cells can be separated from one another by a technique known as
chromatography. Chromatography is a technique for separating and identifying substances in a
mixture, based upon their solubility in a solvent. It is one of the most valuable techniques
chemists and biochemists use to determine ingredients that give flavor or scent, analyze
environmental pollutants, identify drugs in urine, and even separate proteins that can identify
evolutionary relationships.
The name chromatography is derived from the Greek words “chroma” and “graph”, which
mean “color writing”. Chromatography was invented in 1910 by a Russian botanist, Mikhail
Tswett, who used it to separate plant pigments. When a dye mixture is placed on a strip of
chromatography paper and placed into a solvent solution, the individual substances in the
mixture will migrate up the chromatography paper at different rates. The rate of migration is
based upon the capacity of the chromatography paper and the solubility of the sample in the
solvent. As the solvent moves up the chromatography paper stip substances in the mixture that
are soluble in the solvent are carried along up the strip. On the other hand, the substances that
are more attracted to the chromatography paper than to the solvent stop moving and from
bands or spots along the paper strip. After chromatography, each band or spot on the
chromatogram can be assigned a Relative Mobility Factor (R​f​) which is characteristic of the
specific substance that is associated with it. The R​f​ is a ratio of the distance a substance travels
to the distance the solvent travels. It is calculated as follows:
R​f​ = ​Distance solute traveled (D​unknown​)
Distance solvent traveled (D​solvent​)
The R​f​ value for a particular substance is constant with a consistent set of
chromatography conditions. Therefore, substances can be identified according to their R​f​ value
when compared to a known standard. In this investigation we separated the individual pigments
involved in photosynthesis.

Works cited:
1) “Separation of Plant Pigments Using Chromatography.” ​Separation of Plant
Pigments (Chromatography)​,
www.biologycorner.com/worksheets/plant_pigments.html.