Paper Chromatography

Purpose:
You will separate the colors out of the black ink mark from a marking pen line on a coffee filter. As water seeps up the filter paper, the molecules of color are carried with them. They can be separated because they are in a mixture rather than being chemically combined. They will attach themselves to the cellulose in the paper, but with differing affinities depending on their chemical nature. Some cling hard, others are only weakly held. Those that are weakly attached to the cellulose travel further up the paper than those with the stronger bond, and they will spread further. Components in similar chemical tests, such as DNA segments in gel electrophoresis, can be identified by how far they are carried.

Supplies:
water and rubbing alcohol
coffee filter (or filter paper)
water soluble food colors, water-soluble marking pens,
and/or Skittles™ candy and Q-Tips™
clear glasses or other containers

Supplies
Cut the coffee filters into strips about 1 “ wide.

Fill one glass about 1” full of water, the other about 1” full of rubbing alcohol.

Toward the end of the filter strips, draw a line with a black watercolor marking pen, or 2 or 3 primary colors of food coloring, or use a Q-Tip™ to rub off color from candies and rub onto the filter paper. (May take several times to get a dense spot of color.) Let them dry.

Place that end of the papers in the glasses (don’t let the dot of color touch the water or alcohol), and watch the water soak in and travel up the papers (this is called capillary action, which is how water goes up a tree trunk into the branches). As it does, it will dissolve the colors, which are carried up the paper. The lighter ones will be carried higher than the others.
 Smudges
Close-up
After the dots have been completely dissolved, remove the papers from the liquid and allow them to dry. Observe the results, and compare the differences between the two liquids.

Try variations, using different kinds of paper, different markers with different colors, and adding vinegar to the water. Compare results.


Results, Questions:
1. What did you see? What colors were actually in the black ink?

2. Which colors were carried furthest? (the lighter colors) Which remained lowest? (the darker colors)

3. Which color is the lightest in weight? (those lightest in color) The heaviest in weight? (the darker colors)

4. What pattern was there to the change?

5. What is happening when the colors move up the paper? (the molecules of color are being dissolved by the water and carried with the water up the paper)

6. What causes the colors to separate? (the different colors have different affinities for clinging to the paper, and those that cling hardest to the cellulose in the paper will stop first, and those that cling the weakest will travel further up the filter paper before stopping)

7. Predict what might happen with different colors.
Try it again. Do you get the same results?

Applications:
1. This is the same principle used in gel electrophoresis, which is a type of DNA fingerprinting. A DNA sample is placed in a gel bed, current is run through it, and the DNA fragments move across the bed, ending up in different spots. Identical samples of DNA will always have the same results. The banding seen in these results is used to compare DNA samples. See sln.fi.edu/qa98/biology/biopoint6.html