Separation by Chromatography

Chromatography is a physical method used to separate mixtures and has many applications in science research, the
medical field, and forensics. In chromatography, a mixture is separated by distributing it through two phases; a
stationary phase and a mobile phase. The mobile phase moves through the stationary phase, carrying the
different components of the mixture with it. Each component in the mixture will have a different affinity
(attraction) for the stationary phase. A high affinity (attraction) for the stationary phase means a component in the
mixture will not travel far. Less affinity (attraction) to the stationary phase causes a component to travel further.
Each component in a mixture will travel different a distance based on how attracted it is to the stationary phase.
The components of the mixture are eventually separated into bands and the result is called a chromatogram.
There are four major types of chromatography methods: paper chromatography, column chromatography, thin
layer (TLC) chromatography, and gas-liquid chromatography. The method you are using today is paper
Scientists can identify molecules by calculating their relative rate of migration, or Rf value. Any molecule separated
by chromatography in a given solvent has a uniquely consistent Rf value. If you know the Rf value, you know the
You can calculate the Rf values for each of the pigments viewed today by dividing the distance the substance
(pigment) traveled by the distance the solvent traveled from the original starting line.
Rf =
Distance substance (pigment) traveled
Distance solvent traveled
Skittles, M&M candies, filter paper, test tubes, 1% salt water solution, pencil, toothpicks, wax paper
1. Cut two stips of filter paper strip in half (horizontally) so you have four pieces of paper total.
2. Using a pencil (ink from a pen will run, so pencil is better), draw a line 1 cm from the top and bottom edges
of each piece of paper.
3. Make a pencil dot along one of the lines on each paper. Underneath the dots, label the color of the candy
you will test on that spot. Since you will not have space to write the whole color name, put Bl for blue, G
for green, etc.
4. Space 4 drops of water equally distant on the piece of wax paper. Position one candy of each color on the
drops. Give the color about a minute to come off into the water. Pick up the candy and eat it or throw it
5. Dip a toothpick into a color and dab the color onto the pencil dot for that color. Use a clean toothpick for
each color. Try to keep each color dot as small as possible. Allow the filter paper to dry, then go back and
add more color to each dot, a total of three times, so you have lots of pigment in each sample.
6. When the paper is dry, fold it in half vertically with the color sample dots on the bottom. Ultimately, you are
going to stand this paper up in a salt solution (with the liquid level lower than the dots) and capillary action
is going to draw the liquid up the paper, through the dots, and toward the upper edge of the paper. If the
candy color contains multiple pigments (dyes) they will become separated as the liquid moves up the paper.
7. Prepare the salt solution by mixing 1/8 teaspoon of salt and three cups of water (or 1 cm3 of salt and 1 liter
of water) in a clean pitcher or 2-liter bottle. Stir or shake the solution until it is dissolved. This will produce a
1% salt solution.
8. Pour the salt solution into a clean test tube so that the liquid level is 0.5 cm. You want the level to be below
the sample dots. You can check this by holding the paper up against the outside of the glass. Pour out a little
salt solution if the level is too high. Once the level is correct, stand the filter paper inside the glass, with the
dot side down and the edge of the paper wetted by the salt solution.
9. Capillary action will draw the salt solution up the paper. As it passes through the dots, it will begin to
separate the dyes. You will notice some candy colors contain more than one dye. The dyes separate because
some dyes are more likely to stick to the paper, while other dyes have a higher affinity for the salt water. In
paper chromatography, the paper is called the 'stationary phase' and the liquid (salt water) is called the
'mobile phase'.
10. When the salt water (solvent front) reaches the pencil line drawn 1cm from the top edge of the paper,
remove it from the glass and place it on a clean, flat surface to dry.
11. When the coffee filter is dry, compare the results of chromatography for the different candy colors.
Construct a data table to record the distances traveled by the solvent (salt water) and each pigment, as well
as the Rf values for each of the pigments discovered in your four chromatograms.
12. With a ruler, measure the distance from the bottom and top lines on each chromatogram. Record in your
data table. Measure the distance from the starting line to the middle of each pigment in each of the
chromatograms and record in the data table. Finally, calculate the Rf values for each pigment in the mixture.
Record these in your data table.
13. Finally, compare your chromatograms of the same colors with another group’s data. Did the M&Ms
contain the same pigments as the Skittles of the same color?
Analysis Questions
1. Which candy colors contained the same pigments?
2. Based on your results, do you think the pigments used in M&Ms and Skittles are the same? Explain why/why
3. Paper is composed of cellulose (plant cell walls). Cellulose is an organic polymer (made of many repeating
units) of molecules which are much less polar than water molecules.
(a) According to the information above and your calculated Rf values, which of the pigments you separated is
the most polar? The most nonpolar?
(b) Why do water molecules exhibit ‘capillary action’ (move up a piece of paper against the force of gravity)?
4. Go to the “AP Chemistry Topics” page on the wikispace and click on “Column Chromatography Tutorial”.
Briefly compare and contrast paper chromatography with column chromatography.