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 Pigment Separation Using
Chromatography Paper

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.

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.

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

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.

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.