Separation of a Dye Mixture
Introduction
The entire palette of artificial food colors is derived from just seven dyes certified by the FDA
for use in food, drugs, and cosmetics. How can these FD&C dyes be identified in a mixture?
How do the molecular structures of the dye molecules influence their properties, solubility, or
affinity for different solvents?
Like all chemicals, food dyes have different physical properties like boiling points, melting
points, and color. These properties also can include the affinity of a dye to a particular
chemical. If a chemical “sticks” to another, it has a high affinity. If a chemical doesn’t want to
stick to another, it has a low affinity. If we have several dyes with different affinities to a single
liquid, we can separate them using a technique called paper chromatography. In this lab, we
will be separating colored food dyes using salt water and comparing them to dye standards.
Paper chromatography is a method chemists use to separate one chemical from another using
differences in physical properties. Paper chromatography is a simple separation technique that
has several components:
a. Stationary Phase—To separate chemicals, we have to have a platform on which to separate
them. In our case, the platform is paper. The paper does not move in the process of
chromatography, therefore, we call it the stationary phase.
b. Mobile Phase—a mobile phase is the solvent that carries the chemicals through the
stationary phase. Chemicals don’t move on paper alone, but if we add a solvent, it can draw
the chemicals up through the paper using capillary action.
c. Point of Origin—the spot where you put your chemicals on the stationary phase.
d. Developing—after putting chemicals on the point of origin, the chromatogram can be
“developed”, that is, the mobile phase can be pulled through the stationary phase.
In paper chromatography, organic molecules that are “spotted” onto the paper strip separate
as they are carried with the solvent at different rates. Those molecules that have a polarity
closest to the polarity of the solvent, will move up the strip the fastest.
The choice of solvent is the most difficult task in chromatography. Choosing the right polarity is
critical because determines the level of separation that will be achieved. Through different
interactions, samples will move different distances along the paper. The distance a sample
moves along the chromatography paper is compared to the overall distance the solvent travels
– this ratio is called the Rf value or rate of flow.
Pre-Lab Questions:
1. Figure 1 is a sample chromatogram for three samples A, B, and C. Label the drawing with
the following items: stationary phase, mobile phase, and the solvent front.
2. Calculate the Rf value for the spot in sample B using sample A as an example.
3. Sampe C gave two spots on the chromatogram. What does this tell you about the
composition of the sample?
4. Based on the Rf values of A and B, what can you conclude about the intermolecular
attractions that both samples have for the paper and for the solvent?
Activity I: Create Rf Standards for Dyes
It should be no surprise to you that we use a large number of dyes in our everyday life.
Plastics, resins, paints, and clothing are full of pigment from a variety of dyes. Synthetic
chemical dyes are used to color the powders and crèmes we use as makeup and the coatings
on pills we take when we are sick. Another place we use dyes is in food production. Kool-Aid,
popsicles, hard candies, and sodas are good examples of food products that contain dyes.
Believe it or not, that sour apple gummy worm has to be dyed that bright neon green color!
Our government regulates the types of dyes that are used for food, drugs, and cosmetics.
They are tested extensively for short term and long term safety before they are approved for
use. If you have ever looked on a list of ingredients, you have no doubt seen numbered colors
near the end, like red #3. You may have even seen FD&C listed before these dyes. This
stands for food, drug, and cosmetic dyes. These are pure colors, only made of one colored
chemical. In contrast, most of the uses of these dyes are in mixing them together to create new
colors and shades. Food coloring, for example, may look red, but in actuality, it is a mixture of
several different FD&C dyes that give it just the right shade of red.
Many foods, drinks, and candies have several FD&C dyes that are combined in different ratios
to give different colors. The purpose of this lab is to experimentally determine which FD&C
dyes are in food coloring and then candy samples. You will determine this by making a
chromatograph of the FD&C dyes, then making other chromatographs of the candy samples.
You will be able to compare the Rf values of the FD&C dyes to those in the candy samples to
determine which dyes were used to color the products.
Activity I: Procedure
1. Please circle the assigned Chromatography Solvent used either:
Isopropyl Alcohol Solution OR Sodium Chloride Solution
2. Please record the dyes assigned: ______________ and ________________
3. Limit handling the chromatography paper to the edges on as to not contaminate or compact
the analysis area.
4. GRAB a pair of scissors and cut two strips of chromatography paper to fit inside the tube
with the cap closed.
5. Using a ruler and a pencil, draw a faint line 15 mm from the bottom of each paper across the
width of the strip. Measure 9.5 mm from the edge and place a pencil dot. This is the starting
point for the sample.
6. Locate the dye mixtures. Be sure to locate your assigned dyes.
7. Using a clean toothpick, spot the strip by placing the toothpick into the dye mixture solution
and then touching the tip of the toothpick gently onto the designated pencil dot. Allow the
sample to air dry. Repeat the procedure 2-3 more times enough to increase the concentration
of the sample but not to allow the size of the spot to increase. The sample spots must remain
above the level of the solvent so be sure that the placement of the spots is above ~ 5 mL.
8. Repeat step 7 for the second assigned dye. Be sure to keep each strip separate. Do not
allow them to touch. Do not confuse the strips. Keep separate. Set aside to dry.
10. Obtain the assigned solvents. Add ~ 5 mL of solvent to the tube, enough to fill the bottom
well. Place the lid on the tube at all times to avoid spillage.
11. Once the chromatography strips are dry from initial spotting, gently slide the strips into their
respective solvent tubes, close the lid tightly, and LABEL the tubes with tape to keep track of
each dye/solvent combination. Keep the tubes upright. DO NOT tip tubes over.
12. The sample spots must remain above the level of the solvent at all times.
13. Allow the chromatogram strips to develop.
14. When the solvent is within 1-2 cm of the top of the strip, stop the run by removing the strip
and placing it flat on a paper towel.
15. Upon removal, using a pencil, lightly draw a line to mark the distance the solvent traveled.
This is called the solvent front.
16. Measure the distance from the pencil line at the bottom of the strip to the solvent front.
Record this distance in millimeters.
17. Using a pencil, trace the shape of each dye band or spot to mark its location on the strip.
This should be done immediately because the color and brightness of some spots may fade
over time.
18. Measure and record the distance in millimeters that each dye band or spot traveled.
Measure from the line at the bottom of the paper to the center of each band or spot.
Calculate the Rf Values for the dyes you separated using the sample from Pre-Lab.
Report your data in the appropriate column on the front board and record all class data.
Activity I Data Table – Compiled Class Data
Dye Name
Solvent
Spot
Distance Spot
Distance Solvent Average
Used
Color
Traveled (mm)
Front Traveled
Rf
(mm)
Values
Blue No. 1
NaCl
Blue No. 2
NaCl
Green No. 3
NaCl
Red No. 3
NaCl
Red No. 40
NaCl
Yellow No. 5
NaCl
Yellow No. 6
NaCl
Blue No. 1
Isopropyl
Blue No. 2
Isopropyl
Green No. 3
Isopropyl
Red No. 3
Isopropyl
Red No. 40
Isopropyl
Yellow No. 5 Isopropyl
Yellow No. 6 Isopropyl
Form a working group of 4 students and discuss the following questions:
1. Examine the structures of the Red No. 40, Blue No. 1 and Yellow No. 5 dyes from the
provided molecule samples.
What is similar?
What is different?
Which is the largest? ________________
Which is the smallest? _______________
2. View the two molecular diagrams of both solvents used, sodium chloride in water and
isopropyl alcohol in water. Identify the intermolecular attractions within each diagram.
Sodium Chloride in water has ________________________ interactions.
Isopropyl alcohol in water has ________________________ interactions.
3. Which dye and solvent combination seemed to have similar polarities? How did you know?
Activity II: Identify the dye content of candy shells
In this part of the lab, the dye content of the shell coating of M&M candies will be analyzed
using chromatography. The colors of the shells of M & M candies contain food coloring dye
that are FDA approved (5 commonly used of the 7 dyes) organic compounds and therefore
display characteristic Rf values. Some M & M’s use yellow #5 (tartrazine) as color. This dye
causes allergic reactions in some people and must therefore be made aware of the presence.
Devise a procedure to test for the presence of the allowed dyes in M&M candy. Collect
adequate data, in an organized fashion to determine the specific dyes present in colored
M&Ms. You may or may not observe all 5 spots. The data table below is a guide.
To help, view the following weblinks.
http://www.youtube.com/watch?v=F267mlsvqek
http://www.teachertube.com/viewVideo.php?video_id=244061
Activity II Data Table
M&M
Spot
Distance Spot
Color
Color
Traveled (mm)
Distance
Solvent Front
Traveled (mm)
Rf
Values
Identity Determined
by Rf Comparison
Spot #1
Spot #2
Spot #3
Spot #4
Spot #5
You will be using this data to complete a CER conclusion so be purpose driven and clear in
your understanding of the underlying scientific principles guiding this exercise.