Laraib Awan

AP Biology
Schulte
Experiment 3
1/30/18-1/31/18

The Usage of Chromatography Methods to Identify, Separate, and Classify Plant Pigments Based
on Movement of a Solvent
 Abstract
This experiment was performed in order to exhibit and prove the idea that plants are not only
composed of one singular pigment type, but instead, several different pigment molecules on a
large scale with absorbance. Although they appear green, it is not due to the absorption of green
pigment. The chlorophyll is the reason plants have the color green: they are quite attracted to
absorbing blue and red pigments and reflect green pigments; therefore, the plants physically
show the color green. This experiment allowed the participants to observe the different pigments
which were absorbed and in which combinations the colors would be observed best. In the end,
this benefits those who hope to find the optimal environment in order to speed the rate of
photosynthesis by finding which abundance of light works best.
Four test tubes were prepared with certain liquids: 2 test tubes of 2mL tinted green
isopropyl alcohols, 2 mL of tap water, and 2 mL of canola oil. This lab took place over a 3 day
period. On the first day, coffee filter paper was cut into 3 pieces which were about 11 cm long.
A spinach leave was then pressed onto a certain point of the filter paper, being compressed by a
penny which left a green line on the paper. A strip of paper was placed in one test tube of the
isopropyl alcohol, tap water, and canola oil. It was left overnight to observe the changes the next
day. The strip of paper which was held in the test tube of isopropyl alcohol was the only one
which had reacted to the experiment.
We then prepared a fourth strip of paper which was conducted the same way as the
previous strips of paper; however, this was made of a thicker filter paper and the test tube was
filled with 2 mL of isopropyl alcohol, which was the only one which was tested. The test tubes
were left over night again. Data of the different colored line and distance between certain
subjects were observed and recorded. This represented different colored pigments in the cell.
There was no change in the test tubes of water and canola oil, therefore, the data has been
discarded.
The experimenters then calculated the Rate of Flow (rf: distance the solvent travelled to
the distance of the solute). The spinach marks represent our solute. The Rf values could not be
compared in this lab due to there being different papers and solvents. However, the lab still
exhibited the idea that there are a variety of different pigments in plant cells.

Results
*Data for test tubes which contained tap water and canola oil have been discarded due to
having no effect on the lab nor the results*
Coffee filter and Isopropyl Alcohol: A pencil line was drawn 2 cm from the bottom of
the slip of coffee filter paper. The base of the slip was then placed at the bottom of the test tube
which was filled with 2 mL of isopropyl alcohol. The solvent travelled 10.4 cm from the bottom
of the slip of coffee filter paper.
Initial Point Distance Travelled Rf value
 Green line (Spinach) 2 cm from bottom 7.8 cm .75

Blue 2 cm from bottom 8.3 cm .798

Isopropyl Alcohol Bottom of slip 10.4 cm N/A

Thick filter paper and Isopropyl Alcohol: A pencil line was drawn 2 cm from the bottom
of the slip of thick filter paper. The base of the slip was then placed at the bottom of the test tube
which was filled with 2 mL of isopropyl alcohol. The solvent travelled 11 cm from the bottom
of the slip of thick filter paper.
Initial Point Distance Travelled Rf value

Dark green line 2 cm from bottom 9 cm .818

(Spinach)

Blue Line 2 cm from bottom 8.5 cm .773

Isopropyl Alcohol Bottom of slip 11 cm N/A

Conclusion and Analysis

This lab has revealed the variety of pigments which chlorophyll contains. The
experimenters compared the two different slips of paper: one coffee filter strip and one thick
filter strip. Both were placed in isopropyl alcohol. The initial points and initial colors were
observed. After leaving the coffee filter strip in for two days and the thick filter strip for one,
different lines at different points were observed. The green line which was left by a spinach
imprint on the coffee filter strip had travelled upwards about 7.8 cm and an rf value of .75. The
blue line had travelled 8.3 cm and an rf value of .798. The rf value, which is the calculated ratio
of the distance the solvent travelled and the distance the solute travelled. The solvent, which in
this case is isopropyl alcohol, travelled 10.4 cm. It is not possible to calculate the rf value of
this.
The next day, the experimenters had placed a strip of thick filter paper (the same size as
the coffee filter strip at 11 cm) in the isopropyl solution after they had made a spinach imprint
which left a dark green line 2 cm from the bottom. This line had travelled 9 cm and had an rf
value of .818 while the blue line travelled 8.5 cm and had an rf value of .773. The dark green
line from the thick filter paper strip had a greater rf value than any of the others. The blue line of
the thick filter paper strip had the least rf value than any of the others. Due to the two
extremities being on the same strip of paper, it might have something to do with the thick filter
paper as it was easier for the solvent to move up the coffee filter strip quicker.
 Although the coffee filter paper had absorbed the isopropyl alcohol quicker than the thick
filter paper strip that was dipped into the isopropyl alcohol, the thick filter paper helped the
solvent move farther by .6 cm. This may be because the thick filter paper was thicker than the
coffee filter paper.
Some human error was committed throughout this experiment. It was not handled with
gloves, therefore possibly contaminating the solvent. It also might have been better to use
freshly grown spinach rather than the spinach that had come from a supermarket bag; this is due
to the fact that the spinach would have been fresher and still had been through the process of
photosynthesis. The experimenters also tested two different filter papers, therefore, they were
unable to compare it. There was also the accidental sharing of tools, therefore possibly
contaminating the solvent with the canola oil and water. The canola oil and water had been
tested in place of the isopropyl alcohol with the coffee filter paper; however, no results were
shown and there was no charge. Therefore, it was not recorded when observed.
The bands in the experiment of the green and blue lines were most likely composed of
chlorophyll a or b. Due to the lack of actual chromatography paper, no carotene pigments were
evident nor observed, which would have shown off the orange red pigments that the chlorophyll
is composed of. If chromatography paper had been used, there is a possibility that carotene
would have been evident along with chlorophyll a and b.

Background information and Bibliography

Campbell, N. A., Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V., & Reece, J. B.

(2017). ​Campbell biology in focus​. Harlow, Essex, England: Pearson Education Limited.