Separation of Plant Pigments Using Chromatography
By: Kasey Brent
Statement of the Problem
From prior knowledge, one knows that chromatography paper is a useful technique for
identifying unknown compounds. According to The Biology Corner, chromatography paper is
often used in crime scene investigations to match ink, lipstick, or colored fibers. By determining
and comparing Rf value of unknown compounds, crime scene investigators are able to identify
the compound.
Materials
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Strips of Chromatography paper (the number of strips depend on the desired number of
test subjects)
Pencil
Beaker or flask
English Ivy leaf
Purple Plum tree leaf
Acetone (fingernail polish remover)
Popsicle sticks ( the number of popsicle sticks depend on the desired number of test
subjects)
Penny
Tape
Scissors
Procedure
In this lab, plants are rubbed onto chromatography paper and placed in acetone to
determine the plant pigments in the plants. When the chromatography paper comes into contact
with the acetone, the acetone begins to climb up the chromatography paper spreading the plant
pigments up the chromatography paper. The following are the steps taken during the procedure:
1. Use the scissors to cut a strip of the chromatography paper.
2. Draw a horizontal line of one end of the paper with a pencil approximately ½ inch from
the bottom.
3. Place a leaf on the line drawn during step 1 and roll the penny over the plant to create a
line of pigment on the filter.
4. Repeat step three until the line is fairly dark.
5. Pour approximately one inch of acetone (fingernail polish remover) in a beaker.
6. Tape the top of the chromatography strip to a popsicle stick across the top of the beaker.
7. The bottom of the chromatography paper strip needs to be in the acetone, but the green
line does not need to be in the liquid (one may need to adjust the length of the
chromatography paper accordingly).
8. As the pigments move up the paper, take pictures to document the experiment.
9. Set the beaker aside and observe as the acetone moves up the chromatography paper and
deposits the pigment components along the way.
10. Leave the chromatography paper in the acetone for ten to fifteen minutes.
11. Once the chromatography paper is taken out of the acetone, draw a line where the
pigment ends (solute) and where the acetone stopped being absorbed by the
chromatography paper (solvent).
12. Find the Rf value of the plants.
13. Repeat all steps for each subject being tested
Results (Data)
Table 1
Results of Experiment
Materials Tested:
English Ivy
Band Color:
Yellow
Yellow Green to
Olive Green
Purple Plum Tree Yellow
Bright Green to
Blue Green
Yellow Green to
Olive Green
Pink/Magenta
Pigment:
Distance (mm):
Xanthophyll
Chlorophyll b
Solute- 17
Solvent- 30
Xanthophyll
Chlorophyll a
Solute- 30
Solvent- 30
Chlorophyll b
Anthocyanin
Rf Formula
(mm):
0.57
1
Figure 1 Jordan Greer checks to make sure she is making a dark line on the chromatography paper.
Figure 2 The Chromatography Paper is placed in the acetone.
Figure 3 As the acetone travels up the chromatography paper, so does the plant pigments.
Conclusions
After drawing lines where the solute and solvent ended on the chromatography paper, one
measured the distance from the starting point to the solute and from the starting point to the
distance traveled by the solute from the original line
solvent. By using the Rf formula distance traveled by the solvent from the original line, one found the Rf
formula of the English Ivy and the Purple Plum tree. This resulted in the Purple Plum tree having
a slightly large Rf then the English Ivy. One also found the Purple Plum tree to have more
pigments then the English Ivy.
During the experiment one learned human error does occur when conducting science
experiments. The experiment had to be conducted twice due during the first trial the group did
not mark the distance traveled by both the solvent and solute, which is essential in order to figure
the Rf value of the plants. A second trial was then conducted.
After studying the results of the second trial, the group noticed that although the majority
of the pigments of the English Ivy ended at seventeen millimeters, there was still a little bit of
pigment that had ended at the solvent line. This may have occurred due to an error unobserved
by the group during the experiment.
Also during the duration of the experiment, one learned plants all vary one from another
in pigment color. Some plants have pigment colors that others do not and sometimes they share
similar colors with just a slight difference in shade. Furthermore, it was interesting to know that
plants have many pigments combined to create their color besides the colors seen by the naked
eye.
References
The Biology Corner. (2001). Separation of plant pigments using chromatography (demo lab).
Retrieved from http://biologycorner.com/worksheets/plant_pigments.html
on September 29, 2013