Paper Chromatography:
Separating Pigments found in a Plant Extract
Purpose: The purpose of this investigation is to separate the pigments in a plant extract by a process called paper chromatography. In this process, various pigments will separate along the chromatography paper depending on polarity of the pigments. The most polar will be in the lowest band on the paper, while the least polar will be in the uppermost band .
Materials and Methods: Although an extract may be obtained by grinding up spinach leaves in acetone, the instructor may prefer to obtain the extract from a biological supply house. In any case, the student will require the following materials for this experiment:
Plant extract, a clean glass slide, chromatography paper, scissors, pencil, large test tube with a cork, a pin or paper clip, and petroleum ether:acetone. (Be careful because the ether:acetone is very flammable.)
Experiment: Set up the experiment in the following manner:
| Cut a strip of chromatography paper so that it will fit in the test tube at hand. |
| Use your pencil to draw a line near one end of the paper about 1 inch from the end. |
| Now dip the end of a clean glass slide in the extract and apply the fluid at the pencil line. Allow the extract to dry, and repeat this application two or three times. |
| Pour petroleum ether:acetone into the test tube, but not enough to cover the pencil line or the extract on the chromatography strip. |
| Attach the chromatography paper to the test tube cork using a pin or paper clip. |
| Lower the paper into the test tube and insert the cork into the test tube lip. Make sure that the extract is out of the ether. |
| Allow the petroleum ether:acetone to "run" up the paper. Although this will probably take about 30 minutes, watch the movement carefully nevertheless. Stop the experiment before the ether reaches the top of the paper. |
| After the ether has moved up the paper, take the paper strip out of the test tube and examine the bands of pigments. The top band should contain the carotenes which are yellow to orange; the next band down will contain xanthophylls which are yellow; below this band, is the blue-green band of chlorophyll a pigments; and the lowest band is a yellow-green band of chlorophyll b pigments. (Sometimes yellow-brown bands may be present.) |
Conclusion: How many pigment bands do you observe on your chromatography paper?
How do you explain the fact that the pigments separated out into distinct bands?