The Separation of Plant Pigments Using Paper Chromatography and the Measurement of Light
Absorption Using Spectrophotometry
Callie Mills
Florida Southern College
BIO 1500 003L
Dr. Herrick
11.27.14
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Introduction
In 1952, an experiment that dealt with the study of steroids in mammals was conducted,
using the process of paper chromatography (Bush, 1952), but how can paper chromatography be
used in relation to plants and their pigments? This laboratory report involves two labs and their
connection to each other; Laboratory 10: “The Separation of Plant Pigments” and Laboratory 11:
“Spectrophotometry.” Both involve pigments and their distinct colors, lab 10 requiring
exclusively spinach extract, and lab 11 requiring spinach extract and other kinds of pigments.
The study of pigments and their absorption of different forms of light is very important to
Biology. Understanding why leaves appear green to the human eye, or why they change color in
Autumn, or developing the ability to identify a specific compound by the light it absorbs at
different wavelengths can allow for a better understanding of biology in general. In these
experiments, paper chromatography and spectrophotometry can do just that. Paper
chromatography, often done in a tank with glass rods and larger pieces of paper and can be done
over a period of twelve hours (Toennies and Kolb, 1951), serves the purpose of separating
different pigments in a solution. Partition Chromatography demonstrates the properties of
substances that may be used for structure determination. (Chance, 1954) In the case of this
experiment, paper chromatography is done on a much smaller scale. The solvent is absorbed up
the paper from the origin through capillary action, and the pigments dissolve and move with the
solvent, forming lines and sections. The finished product is termed a “chromatogram” (Biology
Faculty, 2014). Spectrophotometry involves the use of “quanta,” also called “photons,” and their
ability to travel and be absorbed by different materials. Wavelengths are generally measured by
angstroms or nanometers, and shorter wavelengths hold greater energies. (Biology Faculty,
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2014) Choice of wavelength and elimination of interference can greatly improve the validity of
the results.
In laboratory 10, we hypothesize that there would be five pigment bands, and that the
order would be: Chlorophyll A, Chlorophyll B, Xanthophyll, and Carotene. In laboratory 11, we
hypothesized that the highest wavelength, at 640 nm, would have the most absorbance. Since
shorter wavelengths have more energy, longer wavelengths can be more easily absorbed due to
low energy levels.
Materials and Methods
In laboratory 10, the materials needed were mostly everyday items and quite easy to find.
Chromatography paper was held up with aluminum foil in a 500 mL beaker containing
petroleum ether and acetone. Much care was taken to avoid accidents, the entirety of the
experiment was done under the fume hood, due to the strong fumes of the ether/acetone solution
and its flammability. Gloves were also needed, chromatography paper tends to absorb the oils
from skin, invalidating the results. A thin line of spinach extract was introduced to the strip of
filter chromatography paper, approximately one inch long and six inches wide. An identifying
mark was made to mark the start, or the “origin,” (Biology Faculty, 2014) of the pigments. The
9:1 petroleum ether/acetone solution was carefully added under a fume hood, to a depth of .5
inches. The paper was oriented so that it would not make contact with the sides of the beaker,
and a lid of foil was made to prevent rapid evaporation. It was left for approximately seven
minutes, with frequent checks. Identifying marks were made at each band of pigment, where it
started and ended. The bands were cut and added to their corresponding vials. The Rf values for
each band was calculated and the pigments were identified. In laboratory 11, the
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spectrophotometer was used. A small cuvette was filled with a specific pigment, the
spectrophotometer was set to the correct wavelength, beginning at 380 nm and increasing by 20
each trial, a blank was set between each trial, and the absorbance and percent transmittance was
measured for each wavelength and each pigment.
Results
The results of laboratory 10 matched our hypothesis quite closely. As the bands traveled
further up the chromatography paper (See Table A), they became exponentially lighter and
yellower in color. In total, the solvent moved 46 mm from the origin, and 80 mm from the
bottom of the paper.
Table A.
Band Number
Distance (mm)
Band Color
1
8
olive green
2
17
bright green
3
30
white
4
44
yellow
5
46
yellow-orange
The Rf values, the ratio of the distance traveled by a compound to the solvent carrying it
(Biology Faculty, 2014), was calculated by dividing the distance traveled by the solvent by the
distance traveled by the compound from the origin. The values are expressed in Table B.
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Table B.
.575
=Rf yellow to yellow-orange band
.2135
=Rf bright green to blue-green band
.1
=Rf yellow-green to olive green band
.55
=Rf yellow band
.375
=Rf other pigment(s)
After analyzing the bands, how far they traveled, and their colors, it was determined that
the order in which they appeared was as follows, from bottom to top: xanthophyll; chlorophyll b;
other; chlorophyll a; carotene. The identification of the pigments to their colors can be found in
Table C.
Table C.
carotene
=yellow to yellow-orange band
chlorophyll b
=bright green to blue-green band
xanthophyll
=yellow-green to olive green band
chlorophyll a
=yellow band
other
=other pigment(s)
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The visible spectrum of light that is seen by the human eye and their color components in
relation to their wavelengths is as expressed below (Biology Faculty, 2014):
380-435 nm…….violet
435-480 nm…… blue
480-580 nm…… green
580-595 nm…… yellow
595-610 nm…… orange
610-750 nm…… red
Table D.
Pigment Color/Band #
Spinach Extract
Pigment Color/Band #
Orange
Wavelength
(nm)
Absorbance
(O.D.)
% Transmittance
Absorbance
(O.D.)
% Transmittance
380
0.851
14.1
0.524
29.9
400
1.143
7.2
0.666
21.6
420
1.254
5.6
0.765
17.5
440
1.167
6.8
0.756
17.6
460
0.979
10.5
0.670
21.4
480
0.776
16.7
0.586
26.0
500
0.612
24.4
0.519
30.3
520
0.641
24.3
0.537
29.0
540
0.502
31.5
0.396
40.2
560
0.323
47.6
0.126
75.8
580
0.390
40.7
0.020
95.5
600
0.586
25.9
0.001
99.7
620
1.130
7.3
0.004
100.9
640
1.059
8.7
0.003
100.7
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After measuring the light absorption in the different pigments, it was determined that
higher wavelengths were more likely to be absorbed. The percent transmittance rose steadily and
reached its height at 500 nm for all of the data collected, then steadily fell again. Graphs of this
data can be found in figures 1, 2, 3, and 4 (Excel, 2014) at the end of this report. The data
collected is shown in Tables D and E.
Table E.
Pigment Color
light green
Pigment Color
blue
Wavelength
(nm)
Absorbance
(O.D.)
% Transmittance
Absorbance
(O.D.)
% Transmittance
380
0.382
41.5
0.179
66.3
400
0.490
32.3
0.232
58.6
420
0.510
30.9
0.210
61.7
440
0.409
39.0
0.125
74.9
460
0.291
51.2
0.109
77.7
480
0.156
69.9
0.091
81.1
500
0.046
89.8
0.091
81.1
520
0.047
89.8
0.107
78.1
540
0.087
81.8
0.138
72.7
560
0.194
63.9
0.215
61.0
580
0.386
41.1
0.389
40.9
600
0.606
24.8
0.609
24.6
620
1.188
6.5
1.192
6.4
640
1.161
6.9
1.115
7.7
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Discussion
The hypothesis for laboratory 10 states that there would be five bands or sections of
pigments, ordered Chlorophyll A, Chlorophyll B, Xanthophyll, and then Carotene. Laboratory
11’s hypothesis said that the 640 nm wavelength would have more absorbance. Since shorter
wavelengths have more energy, longer wavelengths can be more easily absorbed due to low
energy levels. For both experiments, the hypotheses were not entirely supported.
According to the data expressed in laboratory 11, the light that is absorbed is based
entirely on the type of pigment, and therefore differs greatly. There is no common wavelength
that represents the maximum absorbance or the maximum percent transmittance for all trials. For
75% of the four pigments tested in the spectrophotometer, the percent of transmittance is highest
around the mid-range wavelengths. For the spinach extract, absorbance was highest at 1.254
O.D., with the highest percent transmittance at 40.7%. The lowest was 0.323 O.D and 5.6%. For
the orange pigment, the high was 0.765 O.D. and 100.9% and the low was 0.001 O.D. and
17.5%. The blue pigment had a high of 1.192 O.D. and 81.1%, and a low of 0.091 O.D. and
6.4%. The light green pigment had a high of 1.188 O.D. and 89.8%, with a low of 0.046 O.D.
and 6.5%. From this, we can determine that the spinach extract had the highest transmittance at
580 nm, reflecting mostly green light. The orange pigment had it’s highest transmittance at 620
nm, reflecting mostly orange and red hues. The blue pigment transmitted at 480-500 nm, making
blue and green hues more visible, and the light green pigment transmitted the highest at 500 nm,
reflecting green light. If a leaf appears green to the human eye, if is reflecting light at 380-480
nm and 580-750 nm (Biology Faculty, 2014). Every color that is not reflected is absorbed.
Organic compounds that absorb light are separated into three groups; polymethines, porphyrines,
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and polyenes. The first absorption band, which absorbs a particular component of light, is a
result of the transmission of a 𝛱-electron from a higher to lower energy level (Hans, 1949).
Possible sources of error in these experiments include human error, for example, the lack
of wiping down the spectrophotometer cuvettes between each trial and each blank setting, or
placing a line of spinach juice that was too thin, therefore making the bands less decipherable
than they could have been.
Some future studies include some interesting experiments that have been reopened, such
as light absorption in different types of phytoplankton (Morel, Bricaud, 1981), or light absorption
in F-Centres (Huang, Rhys, 1950). Light absorption can be used in the future to help create more
efficient solar panels, for a more environmentally friendly energy plan.
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Figure 1
10
Figure
2
Figure 3
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Figure 4
Figure 5
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Figure 6
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Figures 7 & 8; The chromatography paper immediately after being removed from the petroleum
ether/acetone solution.
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Chance, Britton. "Spectrophotometry of Intracellular Respiratory Pigments." Science 120.3124
(1954): 767-75. Science Mag. AAAS. Web. 27 Nov. 2014.
Biology Faculty, Florida Southern College. “BIO 1500 Biological Essentials Laboratory
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Kuhn, Hans. "A Quantum‐ Mechanical Theory of Light Absorption of Organic Dyes and Similar
Compounds." The Journal of Chemical Physics 17.12 (1949): n. pag. AIP
Scitation. 22
Dec. 2004. Web. 28 Nov. 2014.
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Huang, Kun, and Avril Rhys. "Theory of Light Absorption and Non-Radiative Transitions in
F-Centres." Proceedings A 204.1078 (1950): n. pag. The Royal Society Publihing. Web.
28 Nov. 2014.
Morel, Andre, and Annick Bricaud. "Theoretical Results concerning Light Absorption in a
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