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Exercise 6: Paper Chromatography
Introduction
In last weeks experiment you used the physical properties of density, phase, and
solubility to determine what unknowns you had been assigned. Today we will again use the
physical property of solubility to separate a mixture of substances. Once separated, it will be
possible to identify the individual components of the mixture.
There are two parts to the experiment today. In the first part of the experiment, you will
be working with food dyes (food coloring). A typical set of food coloring contains four base
colors: red, blue, yellow and green. It is possible, however, to make many more colors by
mixing the different food colorings together in various relative proportions.
Sometimes food dyes are made up of only one type of pigment (coloring agent).
Sometimes, however, food dyes are made up of two or more pigments mixed together. If we
want to know what pigment(s) are present in a certain dye, we must have a procedure capable of
separating the individual pigments. Your first task is practice the separation technique on food
coloring.
You should also know that there is no single pigment for any given color. There are
thousands of red, green, yellow, purple, etc., pigments. For example, there are many pigments
that produce the color “red”.
In the second part of the experiment you will separate dyes used in the candy coating of
M&M’s. By comparing the chromatogram with the names of the dyes shown on the list of
ingredients you should be able to identify which dyes correspond to the spots on the
chromatogram.
The separation technique you are going to use in lab this week is called paper
chromatography. You already know the solubility of a substance depends on the solvent being
used. Suppose we had two substances, lets be creative and call them substance 1 and substance
2. Let’s suppose substance 1 is soluble in water, and substance 2 is insoluble in water. Suppose
we place a drop of substance 1 on a paper towel close to the edge and place the paper towel in a
cup of water so that the level of the water is just below the spot of substance 1. You probably
know that as the paper towel sits in the water, capillary action will cause the water to be drawn
up the paper towel. What do you suppose will happen to the spot of substance 1 that we put on
the paper towel? Since it is soluble in water, as the water passes over it, substance 1 dissolves in
the water and gets carried up the paper towel as high as the water rises. Let’s perform the same
experiment with substance 2. We place a drop of substance 2 on a paper towel close to the edge
and place the paper towel in a cup of water so that the level of the water is just below the spot of
substance 2. As water moves up the paper towel, what do you suppose happens to the spot of
substance 2 we placed on the paper towel? Since substance 2 is insoluble in water, will it be
dissolved in the water and carried up the paper towel? No. The spot of substance 2 does not
move at all. Therefore, if we had a mixture of substance 1 and substance 2, we could separate
them by this technique. The water is called the mobile phase (it moves). The paper towel is
called the stationary phase (it does not move). Paper chromatography can be adjusted to
separate a vast array of mixtures. Virtually any mobile phase can be used: water, acid, alcohol,
etc. depending on the substances we are trying to separate. The stationary phase can also be
selected (paper towels are rarely used). We can adjust the attraction that different substances
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have to the stationary phase, making it either easier or more difficult for the substances to
separate as the mobile phase passes over them.
In this experiment you will use a common mobile phase (distilled water) and a common
stationary phase (filter paper) to perform your separations and identifications.
Procedure
Part A. Food Coloring
1.
2.
3.
4.
5.
Obtain one filter paper strip from the front table.
Use a pencil (not a pen) to a draw a light line about 1 cm from the bottom of the paper.
Obtain a set of food dyes and a spot plate from the front table.
Place one drop of each food coloring into separate wells on the spot place.
Dip an end of a capillary tube into the red dye. Lightly touch the end of the dipped capillary
tube onto the pencil line on the filter paper. Leave enough spacing for four more spots to be
applied.
6. Repeat step 5 with fresh capillary tubes for each of the remaining 3 dyes.
7. Obtain, from your instructor, an unknown food dye mixture. Using a fresh capillary tube and
your spot plate, apply your unknown to your filter paper.
8. Add 2.5 mL of water and 7.5 mL of isopropyl alcohol to a 125 mL Erlenmeyer flask. Insert
your filter paper strip into the Erlenmeyer flask with the pencil line at the bottom of the flask.
The level of the water MUST BE BELOW the pencil line. If it is not, remove the filter paper
strip and pour off a little of the water. Reinsert your filter paper strip.
9. Cover the Erlenmeyer flask with plastic wrap and allow the flask to sit, undisturbed until the
mobile phase has almost reached the top of the filter paper strip (about 30 min.).
10. Allow the chromatogram to dry.
11. When dry, circle the separated spots on the chromatogram.
12. Calculate the Rf value for each spot on the chromatogram.
13. Fill in and answer the questions for Part A on the Data Sheet.
Part B. M&M Analysis
1. Place a red, blue, yellow, and green M&M into separate wells on your spot plate.
2. Using distilled water and an eyedropper place one drop of water onto each M&M. Allow the
M&M to sit in the water for several minutes while turning the M&M over from time to time
(this will dissolve some of the dye in the candy coating).
3. Obtain one filter paper strip from the front table.
4. Use a pencil (not a pen) to a draw a light line about 1 cm from the bottom of the paper.
5. Dip an end of a capillary tube into the colored solution made from the red M&M. Lightly
touch the end of the dipped capillary tube onto the pencil line on the filter paper. Leave
enough spacing for three more spots to be applied.
6. Repeat step 5 with fresh capillary tubes for each of the remaining 3 colored M&M solutions.
7. Add 2.5 mL of water and 7.5 mL of isopropyl alcohol to a 125 mL Erlenmeyer flask. Insert
your filter paper strip into the Erlenmeyer flask with the pencil line at the bottom of the flask.
The level of the water MUST BE BELOW the pencil line. If it is not, remove the filter paper
strip and pour off a little of the water. Reinsert your filter paper strip.
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8. Cover the Erlenmeyer flask with plastic wrap and allow the flask to sit, undisturbed until the
mobile phase has almost reached the top of the filter paper strip (about 30 min.).
9. Allow the chromatogram to dry.
10. When dry, circle the separated spots on the chromatogram.
11. Calculate the Rf value for each spot on the chromatogram.
12. Fill in and answer the questions for Part B on the Data Sheet
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Data Sheet for Exercise 6:
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Paper Chromatography
Part A. Food Coloring
1. Sketch your chromatogram below:
To calculate the Rf value of a particular spot,
you must make two measurements:
1. The distance from the initial point of
application (the pencil line) to the
midpoint of the particular spot
and
2. The distance the solvent front traveled
(starting at the pencil line).
Rf 
distance the spot trave led (1)
distance the solvent tr aveled (2)
2. Calculate the Rf values for all the spots on your chromatogram (start at the bottom for every
color).
Red
Blue
Green
Yellow
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Part B. M&M Analysis
1. Sketch your chromatogram below:
To calculate the Rf value of a particular spot,
you must make two measurements:
1. The distance from the initial point of
application (the pencil line) to the
midpoint of the particular spot
and
2. The distance the solvent front traveled
(starting at the pencil line).
Rf 
distance the spot trave led (1)
distance the solvent tr aveled (2)
3. Calculate the Rf values for all the spots on your chromatogram (start at the bottom for every
color).
Red
Blue
Green
Yellow
4. Do any of the M&M dyes appear to be the same as any of the dyes from Part A? If so, list
them.