Experiment #2: Paper Chromatography of Food Dyes
Background:
Chromatography is a technique used to separate substances dissolved in a mixture.
Chromato means color and graphy means to write. Paper is the medium used to separate the
components of the mixture.
Chromatography paper is made of cellulose fibers that are pressed together. As a liquid
solvent passes over the fibers and through the pores, the paper acts as a filter and separates the
mixtures components. Each component of the mixture has a unique attraction for the liquid.
The greater the attraction for the liquid, the farther it will travel up the paper. Each different dye
will produce a unique color band on the paper. The components of the mixture that have the
strongest affinity (attraction) for the liquid solvent and the least attracted to the paper will travel
the farthest. Their band will be closest to the edge of the paper.
The FDA (Food and Drug Administration) is responsible for approving the dyes that can be
used in food products for consumption by humans. Currently, there are seven approved dyes
(FD&C dyes) for use in food products. These are red 3, red 40, blue 1, blue 2, yellow 5, yellow 6,
and green 3. In this lab, chromatography paper will be spotted with individual solutions of
each of the FD&C dyes along with the mixtures and then dried. Then the spotted
chromatography paper is placed in contact with a salt solution, and the dye molecules will be
transported through the paper.
After development each individual dye molecule may be characterized by its retention factor
(Rf). The Rf is a unitless mathematical expression for the distance traveled by the dye molecules
divided by the distance traveled by the solvent front. The R f factor will be constant for a given
dye provided all conditions in the experiment are kept constant.
Rf =
distance travelled by dye
--------------------------------------distance travelled by solvent
Dye #1
Dye #2
Dye #3
Dye #4
In this experiment, a chromatogram for each of the pure dye molecules will be developed and R f
values of each dye calculated. The components of 3 unknown mixtures of dyes will then be
identified.
Procedure:
1. Obtain a sheet of chromatography paper. Hold the paper by the edges to avoid finger
prints which could interfere with the chromatography process.
2. Draw a PENCIL line on the chromatography paper 1 cm from the bottom. This will be the
origin for the different dyes.
3. Label the marks on origin line with the names of the known FD&C dyes: B1 B2 Y5 Y6 R3
R40 G3 and the 3 mixtures.
4. Using a well plate get ONE drop of each of the FD&C food dyes and the 3 dye mixtures.
5. Use a separate wooden toothpick for each solution. Using the thinnest end, slowly dip into
the solution and then spot the paper keeping the toothpick vertical. Repeat two more
times (total of 3 spots directly on top of each other) for the FD&C dyes and 3 mixtures. See
the figure below.
___________________________________________________________
B1
B2
Y5
Y6
R3
R40
G3
Pencil Line
M1
M2
M3
(1cm)
6. Roll the paper into a cylinder, butt the ends together, but do not overlap them, and staple
the paper together.
7. Add about 25 mL of a 0.10% NaCl solution to a 600 mL beaker. Place the chromatography
paper into the beaker. Cover the beaker with plastic wrap. See figure below.
8. Allow the beaker and the chromatography paper to sit for up to 30 minutes for the dyes to
separate. Do not disturb the beaker during this time.
9. When the dyes have separated sufficiently or the liquid solvent is 2 cm from the top of the
paper, remove the chromatography paper from the beaker. Open it flat and place it on a
towel to dry.
10.With a pencil, mark the top point on the chromatogram where the developing solvent
reached. All spots can be marked either immediately upon removal of the chromatogram
or after it has been allowed to sit for one minute.
11.Pour all solutions down the sink with an excess of water. Wash all glassware with water.
Return all borrowed items to their original locations.
DATA TABLE
Distance Solvent Traveled: _____________________________
FD&C DYES
Distance Traveled by Spot
Rf
Blue 1
Blue 2
Yellow 5
Yellow 6
Red 3
Red 40
Green 3
Unknown Mixture #1:
Distance Traveled by
Rf
Identity of Spot
Spot
Spot 1
Spot 2
Spot 3
Unknown Mixture #2:
Distance Traveled by
Spot
Rf
Identity of Spot
Distance Traveled by
Spot
Rf
Identity of Spot
Spot 1
Spot 2
Spot 3
Unknown Mixture #3:
Spot 1
Spot 2
Spot 3
Pre-laboratory Questions: Paper Chromatography of Food Dyes
After reading the laboratory procedure, answer the following questions.
1. What is the liquid solvent used in this chromatography experiment?
2. Explain why you would not use an ink pen to draw the line on the chromatography paper.
3. Using the figure on the front page of the lab, determine which dye would have the strongest
affinity (attraction) for
the liquid solvent. Explain your choice.
4. Using the figure on the front page of the lab and a ruler (use centimeters), determine the
Rf value for dye #4. Show
your work. How would the R f value for dye #3 compare to the Rf value you calculated for dye
#4?
