Abstract
The purpose of this experiment was to separate the pigments carotene,
chlorophyll a, chlorophyll b, and xanthophyll from the plant extract from
which they originated. Pigments are substances that absorb light,
and aid plants in the process of photosynthesis. These pigments were
placed on a sheet of chromatography paper, and the tip of the paper was
placed within the chromatography solvent—which was acetone. About
ten minutes were allowed for the acetone solvent to be drawn up through
the paper; this action permitted the separation of the pigments.
The soluble pigment carotene dissolved into the acetone the easiest, and
thus moved the furthest from the origin. The less soluble chlorophyll
b did not dissolve as easily into the solvent; it instead was absorbed
more readily into the fibers of the paper. Consequently, chlorophyll
b moved the least from the plant extract origin. Chlorophyll a moved
further than chlorophyll b. From this outcome one can speculate that
chlorophyll a was either slightly more soluble, or less likely to be absorbed
into the chromatography paper than chlorophyll b. Likewise, one may
suggest that xanthophyll was more soluble than both chlorophylls, yet not
as soluble as carotene, and with more of a tendency to stick or be absorbed
into the chromatography paper.
Introduction
This experiment was conducted to determine the distances the pigments,
carotene, chlorophyll a, chlorophyll b, and xanthophyll, will travel on
a chromatography paper that absorbs an acetone solvent. As the acetone
solvent is absorbed into the paper it moves through it, and separates the
pigments producing various colored stripes across the chromatography paper.
The pigments can then be identified by a color that is specific to them—the
carotenes turn a yellow color, xanthophyll also turns to a yellow color,
chlorophyll b reveals it color as light green, and chlorophyll a has a
color of dark green. Due to varying degrees of solubility the acetone
is capable of separating the pigments; for the more soluble a pigment is
the further it will be carried along the chromatography paper.
Materials and Methods
A strip of chromatography paper was needed for this experiment.
One end of the paper was then cut to make a sharpened point. Using
a pencil, a dim line was drawn across the paper approximately two centimeters
from the sharpened tip of the paper. A plant extract was then applied,
in repeated strokes, across the pencil line. Between each stroke
time was allowed for the plant extract to dry—this process was enhanced
by blowing on the paper after each application. The application of
the plant extract was stopped when an ample amount had accumulated, forming
a thin stripe. An acetone solution was placed within a test tube,
but the amount of acetone used depended on the length of the chromatography
paper because only the tip of the paper was positioned in the solution.
The chromatography paper was attached to a paper clip and fastened to a
cork that was stuck in the opening of the test tube.
Between eight and ten minutes was then allowed for the acetone solution
to travel up the length of the chromatography paper. After this allotted
time the paper was removed from the test tube and the furthest distance
that the solvent traveled was marked with a pencil, and the distance it
traveled was measured and recorded in Table 1. Then, using a completed
chromatogram the locations of the various pigments were found, and thus
the different pigments were identified. The distance of each of the
pigments from the origin of the plant extract was measured and also recorded
in Table 1. The following equation was then used to calculate the
Rf:
Rf= Distance moved by pigment
Distance from pigment
origin to solvent front
These findings were then recorded in Table 1.
Results
Table 1 shows that out of all the pigments chlorophyll b traveled the
smallest distance—2.1 centimeters—from the plant extract source.
Chlorophyll a was next having moved a distance of 3.0 centimeters, and
xanthophyll followed moving an additional centimeter with a distance of
4.0 centimeters. The pigment carotene moved the furthest from the
plant extract origin. Its distance, more than double that of xanthophyll,
was 8.6 centimeters.
The Rf represents the relationship between how far a pigment moved
in comparison to the distance traveled by the solvent. The Rf for
chlorophyll b (.227cm) is a number smaller than those for the remaining
pigments, therefore, clearly it moved the smallest distance. The
Rf of chlorophyll a was .341cm, and for xanthophyll it was .455cm, thus
xanthophyll moved further than both chlorophylls a and b. The pigment
that moved the furthest though was carotene with an Rf of .997cm.
One can also see from the results that carotene came the closet to
the solvent front—lagging behind at a distance of only 0.2cm. Xanthophyll
was 4.8cm from the solvent front. Chlorophylls a and b were 5.8cm
and 6.7cm, respectively, from the solvent front.
Table 1
Distance to Distance Moved
Pigments Solvent Front By Pigment Rf
Chlorophyll b 8.8cm 2.1cm .227cm
Chlorophyll a 8.8cm 3.0cm .341cm
Xanthophyll 8.8cm 4.0cm .455cm
Carotene 8.8cm 8.6cm .977cm
Discussion
It is important to note that determining the Rf helps to standardize
the procedure—others, by following the steps of this experiment and by
calculating the Rf, can attain similar results.
The results show how fast the pigments moved in relation to the acetone,
and in the time allowed. The pigment carotene, moved the furthest,
therefore it traveled the fastest, but at a slightly slower rate than that
of the acetone. This is evident from its distance from the solvent
front; only 0.2 cm away. The pigment that moved the slowest, and
therefore the smallest distance was chlorophyll b. The distance between
chlorophyll b and the solvent front was 6.7 cm. Chlorophyll a was
the second slowest, and xanthophyll was the second fastest pigment.
The difference in the movements of each pigment was due to the solubility
of a particular pigment and ability of that particular pigment to stick
to the cellulose fibers of the paper. The solubility of a pigment
and the size of a pigment’s molecules help to determine the rate, and thus
the distance it will travel. The more soluble a pigment is, the further,
and faster, it will travel. Conversely, the more readily a pigments
adheres to the fibers of the chromatography paper the slower its movement.
One can then state that the reason why carotene moved the furthest is because
of how soluble it is to the acetone solvent. On the other hand, one can
say that chlorophyll b traveled such a small distance from the pigment
extract origin because of its tendency to stick to the paper’s fibers.
Xanthophyll and chlorophyll a fell somewhere in between chlorophyll b and
carotene. Xanthophyll was slightly more soluble than chlorophyll
a, yet a bit less soluble than carotene.
References
Vodopich, D.S. and R. Moore. 1996. Biology Laboratory Manual,
4th ed. Wm. C. Brown Publishers, Dubuque, IA.
Purves, W.K., G. Orians, C. Heller, and D. Sadava. 1998.
Life The Science of Biology, 5th ed. Sinauer Associates, Inc., Sunderland,
MA.