Name: _________________________________________Date: ______ Period: _______
Separation of FD&C Food Dyes in
M&M’s Using Paper Chromatography
Background
Paper chromatography is an important separation technique that
depends on differences in both absorption and solubility. A small amount
of the mixture to be separated is placed close to the edge of a piece of
chromatography paper. The edge of the paper is then immersed in a
developing solution. As the developing solution ascends up the paper by
capillary action, the components of the sample are carried along at
different rates. To prevent evaporation of the developing solution, this
process is carried out in a closed container.
Each component of the mixture will move a definite distance on the paper
in proportion to the distance that the solvent moves. This ratio,
distance component moves
Rf (retention factor) = __________________________
distance solution moves
can be calculated for each component, to aid in identification. Retention
factor values are dependent upon the paper, developing solution, and
sample size.
Candies, such as M & M's, contain FD&C (Food, Drug, and Cosmetic Act)
dyes, sugars, and other organic and inorganic substances in their coatings.
Color additives are available for use in food as either "dyes" or "lakes".
Dyes dissolve in water, lakes are the water insoluble form of the dye, are
more stable than dyes, and are ideal for coloring products containing fats
and oils. There are nine certified colors approved for use in food in the
United States. Four dyes can be found in M & M ® candies: Red #40,
Blue #1, Yellow #5, and Yellow #6.
If the coating of an M&M candy is dissolved in an acidic solution (white
household vinegar), the FD&C dyes will be released from the candy. The
FD&C dyes can be absorbed from the solution containing vinegar, dye, and
sugar by wool. The dyes can then be released from the wool by placing it
in an alkaline environment. This simplified equation represents the
process involved:
WOOL
+
DYE
+
H+ →
DYED WOOL+
A solution of household ammonia can be used to supply sufficient OH─ to
reduce the concentration of the H+ and cause the dye to be released from
the wool.
Purpose
In this experiment, you will extract the dyes in candy coatings using wool
and then separate the dyes using the technique of paper chromatography.
If samples containing known FD&C dyes are run as standards, it is
possible to identify the dyes used in the candy coatings.
A sample containing a few micrograms of the dye is placed near one end
of a strip of filter paper. That end of the paper is then immersed
vertically in a solvent. As the solvent rises up the paper by capillary
action it tends to carry the sample along with it, to a degree that depends
on the solubility of the sample in the solvent and its tendency to adsorb
on the paper. When the solvent has risen a distance of L centimeters,
the solute, now spread into a somewhat diffuse zone or band, will have
risen a smaller distance, say D centimeters. It is found that D/L is a
constant independent of the relative amount of that substance or other
substances present. D/L is called the Rf value for that substance under
the experimental conditions.
Rf = D = distance solute moves
L
distance solvent moves
The Rf value is a characteristic of the substance in a given
chromatography experiment, and it can be used to test for the presence
of a particular substance in a mixture of substances with different Rf
values.
The first part of the experiment will involve the determination of
the Rf values for five common acid-base indicators. These substances
have colors that will allow you to establish the positions of their bands at
the conclusion of the experiment. When you have found the Rf value for
each substance by studying it by itself, you will use these Rf values to
analyze an unknown mixture.
Procedural Notes
1. Don't let the chromatography paper touch the sides of the beakers.
2. Handle chromatography paper carefully. Always handle the paper by
the edges because their surfaces can easily be contaminated by your
fingers.
Materials
Equipment:
2 pieces of chromatography paper (10 cm x 8 cm)
2 600 mL beakers
6 test tubes
Test tube rack
Glass stirring rod
Forceps
Beaker tongs
2 evaporating dishes
400 mL beaker
Hot plate
toothpicks
2 15 cm pieces of wool yarn
2 beral pipets
Substances:
2 M&Ms of each of your assigned color of candy
1 set of standards FD&C dyes
Vinegar
Ammonia
Pre-Lab Questions
1. What is the mobile phase in a paper chromatography investigation?
2. What is the stationary phase in a paper chromatography investigation?
Number
1
Standard
McCormick Red Food Coloring
2
Archer Farms Red Food
Coloring
McCormick Green Food
Coloring
McCormick Yellow Food
Coloring
Wilton Orange Food Coloring
McCormick Blue Food Coloring
3
4
5
6
Components
Red #40
Red #3
Red #40
Blue #1
Yellow #5
Blue #1
Yellow #5
Red #3
Yellow #6
Blue #1
Red #3
3. The table above lists the standards you will be given in this laboratory
experiment. Which standard(s) would you need to use to determine
the Rf values for the following FD&C food dyes? In some cases you
might need to compare two standards to determine the Rf value for a
specific dye.
a) Red #40
b) Red #3
c) Yellow #5
d) Yellow #6
e) Blue #1
Procedure
Part 1 – Extraction of Dye From M&M Candies
1. Place a 200 mL of water in a 400 mL beaker. Heat the water in the
beaker until it is boiling. Turn off the hot plate once the water is
boiling.
2. Add vinegar to the test tube containing M&Ms of your assigned color.
You should add just enough vinegar to cover the candy in the test
tube.
3. One at a time, hold a tube with a test tube holder and dip it into
boiling water to heat the vinegar and dissolve the color coating, so
that the candies turn white. The color comes off very fast, usually
within a few seconds, so it is usually not necessary to keep it in there
for longer than 10 seconds. Be careful to dissolve as little as
possible of the white layer (sugar) under the colored layer and do
not dissolve any of the chocolate.
4. Immediately pour the colored solution into a clean test tube, trying
not to transfer any of the sediment. Repeat with the remaining test
tube and M&M color.
5. Now you need to extract the dye from the solution onto the strips of
yarn. To do this, place one piece of yarn into each test tube of colored
solution and top off the level of vinegar so that there is at least 4mL
(4cm). Heat the tubes in the boiling water bath for 5 minutes. You can
remove the tubes from the water when the solution is milky-white and
the yarn is the color of the dye, in other words, when all of the dye
has been extracted from the solution.
6. Remove the dyed yarn from the solution and rinse it with distilled
water.
7. Now it is necessary to extract the dye from the yarn and get it back
into solution. Into 2 clean test tubes place a piece of yarn and 3mL
of 1M ammonia. Make sure that the yarn is immersed and mix with a
stirring rod, rinsing the stirring rod before placing it in each tube so
as not to mix any of the colors.
8. Heat the tubes containing the yarn and ammonia in the water bath
for 5 minutes or until the color of the yarn has faded. Remove the
yarn.
9. You now may need to concentrate each color. Gently pour the
ammonia solution into an evaporating dish. Take your evaporating
dish to the fume hood and heat the dish on a hot plate until the
solution is almost entirely evaporated.
10. Carefully remove the evaporating dish from the hot plate. You now
have dye from M&Ms to separate using paper chromatography.
Part 2 – Chromatography of Standards and Candy Dyes
1. Obtain chromatography paper and samples of each FD&C food dye
from the front supply counter.
2. Place the chromatography paper on a clean dry sheet of paper.
3. Make a pencil mark about 2 cm from the end of the chromatography
paper. Mark five equally spaced positions along the pencil line (the first
and last positions should be at least 0.5 cm from the edge of the paper).
4. Use a toothpick to place a small spot (no larger than 5 mm in diameter)
of each of the concentrated FD&C dyes on the marked positions. Allow
the spots to dry completely.
5. Label the chromatography paper at the top (opposite of where you
spotted the sample) so that you can identify which standard you
applied.
7. Repeat steps 4 and 5 for the remaining standards and the
concentrated M&M dyes, using a clean toothpick for each sample.
8. Cut the corners off the bottom of the paper (below the baseline).
9. Repeat steps 2-8 using the concentrated dye from the M&M samples.
10. Add 15 ml of vinegar to 2 different 600 ml beakers.
11. Carefully hand the piece of chromatography paper from a paper clip
suspended over a glass stirring rod.
12.
Place the paper in the beaker with the spots at the bottom of the
cylinder. Be careful that the paper does not touch the sides of the
beaker.
Cover the beaker with plastic wrap and allow the
chromatogram to develop until the developing solution is about 1 cm
from the top of the paper.
13. Remove the paper from the beaker, open it out, and lay it on a piece of
paper towel to dry. Mark the solvent line with a pencil, all the way
across the paper. It is important you do this NOW. After the paper
has dried, you won't be able to see the solvent line!
14. Let the strips air dry for completely.
15. Measure the distance from the center of the band to the point where
the indicator was applied. Record this information in your data
table.
16. For each sample (including the M&M dyes), also measure the
distance from the solvent front to the point of application.
17. Clean and return all supplies to the front supply counter.
18. Wash your hands before leaving the lab today.
Data Analysis and Conclusion
1. Which FD&C food color has the longest retention time? Which has the
shortest?
2. Calculate the Rf (retention factor) ratios for each of the FD&C dyes used
as standards.
3. Calculate the Rf ratio(s) per dye for the six M&M colors you tested.
4.
Using the FD&C chromatogram and the calculated Rf values as
standards, list the FD&C dyes used in your M&M experimental groups.
5. Some people are allergic to yellow #5 food dye. What colors of M&Ms
should a person avoid if they are allergic to yellow #5?
Example Data Table
Table 1 – Paper Chromatography of FD&C Food Dyes
Sample
Distance
solvent
moved
(cm)
Distance
sample
moved
(cm)
Rf
Color in
NH3
vapor
Color
in HCl
vapor