Mary Ward

AP Biology

Schulte

Experiment #3

1/30/18 - 1/31/18

Title
The Usage of Chromatography Methods to Identify, Separate, and Classify Plant Pigments based
on movement of solvents
 Abstract

In this experiment students used coffee filter paper and thick paper to witness how color
pigments and solvent can travel up the papers. Students drew lines on each strip of paper two
centimeters above the bottom of the paper, then used a quarter to roll pigments from spinach
leaves on the pencil line. The strips of paper were then placed into different test tubes with
various solutions (distilled water, isopropyl alcohol, and vegetable oil). Each test tube was filled
with two milliliters of a certain solution. Coffee filter paper was used for each solution and the
thick paper was used only for the isopropyl alcohol solution. When the pigment had reached
close to the top of the strips of paper they were taken out of the solution to get measured. The
pigment and solvent were then measured to see how far they had traveled. The solvents were
measured starting at the very bottom of the strip of paper and the pigments were measured
starting at the pencil line.

The purpose of this experiment was to witness chromatography, which is the separation
of of pigments in plant cells. It is a technique that can separate and identify substances in a
mixture based on their solubility in a solvent. It can be used to determine ingredients, analyze
environmental pollutants, identify drugs in urine, and separate proteins. In terms of this
experiment the purpose of the use of chromatography was to identify the pigments of green
leaves. Which shows how photosynthesis requires the presence of different pigments in order to
absorb energy from light. Different pigments absorb the light of a particular wavelength. The
main pigments in plants are chlorophylls. The hypothesis of this experiment is that the isopropyl
alcohol would have more of an effect than any of the other solutions and that the pigments would
be more present and travel further on the thicker paper rather than the coffee filter paper.
 Results

Table #1: Various Solvents traveling up Coffee Filter Paper and Thick Paper
1​st​ day in Solvent 2​nd​ day in Solvent

This table shows how various solvents travel up coffee filter paper and thick paper. The
strip of paper on the far right in these pictures are the thick paper with isopropyl alcohol. The
remaining three strips of paper towards the left are coffee filter paper with the solvents in the
order left to right: isopropyl alcohol, distilled water, and vegetable oil. The first picture shows
the solvent and pigments after the first day and the second pictures shows the solvents and
pigments after the second day. The independent and dependent variables are the same for each
graph. The independent variable is the type of solvent and the dependent variable is how much
the solvent and pigment moved up the strips of paper.

Table #2: Isopropyl Alcohol on Coffee Filter Paper

Blue 11 cm 10 cm .909

Green 11 cm 8.5 cm .773

This table shows how the solvent isopropyl alcohol traveled up coffee filter paper and
shows how far the pigment traveled. The two main colors seen on this paper was green and blue.
As seen in the graph the blue pigment traveled farther than the green pigment. As mentioned in
the previous graph summary the independent variable is solvent and the dependent variable is
how much the solvent and pigment moved up the strips of paper.

Table #3: Isopropyl Alcohol on Thick Paper

Blue 15.5 cm 13.5 cm .871

Green 15.5 cm 12 cm .774

Both Colors N/A

This table shows how the solvent isopropyl solvent traveled up the thick paper and how
far the pigment moved. Similarly to the coffee filter paper, the thicker paper main two pigments
were blue and green. However, the pigments were much easier to see on this paper. Just like the
past two graphs the independent variable is the solvent of isopropyl alcohol and the dependent
variable is how much the solvent and pigment moved up the strips of paper.

Analysis/Conclusion

In this experiment the solutions isopropyl alcohol, distilled water, and vegetable were
placed in test tubes then strips of paper were placed in the test tubes with the solutions. Coffee
filter paper was used for all three solutions and the thick paper was only used for the isopropyl
alcohol. The purpose of this experiment was fulfilled, students used chromatography to identify
different pigments from the spinach leaves. The hypothesis was also validated because the thick
paper showed the pigment clearer than the coffee filter paper and the pigment traveled further on
this strip on paper.

As seen in table #1 only the isopropyl alcohol worked to separate the pigments. The
distilled water and vegetable oil had no effect on the separation of pigments. This is why only the
thick paper was used in the isopropyl alcohol because since the coffee filter paper had been
placed in the solution the day before students saw that it was the only one that produced an
effect. Therefore, in tables #2 and #3 only the movement of solvents and pigments caused by
isopropyl alcohol were measured. The R​f​ value was then determined using the distances
measured. The formula for the R​f​ value is the distance solute (pigment) traveled divided by the
distance solvent traveled. A substance can be identified by the R​f​ value. It is the ratio of the
distance a substance travels to the distance the solvent travels.

There were many sources of error for this lab. The first one being an equipment error that
the isopropyl alcohol was green. Usually this solution is just clear but there was only access to
the green isopropyl alcohol solution which could have affected the presence of the pigment.
Since the solution was green the pigment could have been instensed because both are green.
Another equipment error would be that there was no access to the thick paper that first day of the
experiment which was when the majority of the lab was set up. Only coffee filter paper was
available on the first day which did not show the pigments as vividly as the thick paper. This also
means that these strips of coffee filter paper had an extra day to show how the pigment moved.
For further investigation of this experiment scientists should have access to the chromatography
paper in order to see the pigments more vividly and get results faster. There could also been a
wider variety of solutions used as the solvents.
 Background Research and Bibliography

It’s no secret that photosynthesis is needed for all life on Earth. This lab helped students
to better understand photosynthesis and how important pigments are. Photosynthesis requires the
presence of special pigments that can absorb energy from light. Certain pigments can absorb
different kinds of light energy through particular wavelengths. The most important pigment in
plants is chlorophyll. There are two types of chlorophyll which are chlorophyll a and chlorophyll
b. Chlorophyll a is the primary pigment that absorbs energy from wavelengths that are
blue-violet and orange-red light while chlorophyll b is the accessory pigment that absorbs energy
from wavelengths that are green light. These pigments can be separated using chromatography
which separates and identifies substances in a mixture based on their solubility in a solvent.

Spinach Chromatography Lab. ​Adapted from Colors of Autumn Lab by Neo/Sci.