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Experiment #3 - Extraction and Chromatography of M&M Candy Dyes

Introduction:
This experiment is one that illustrates a problem chemists face every day the separation of a mixture into its
components and the subsequent identification of those components. The mixtures used in this experiment are the dyes
used to create the colored M&M candy shells.

In the United States, the use of color additives in food is regulated by the Food and Drug Administration (FDA). The
seven color additives permitted for direct addition to human food in the US are described Table 3.1. Of these seven,
five are used in M&M candies: FD&C Green No. 3 is not one of the dyes used. The FD&C portion of the dye name
stands for Food, Drug and Cosmetic. These dyes are approved for use in products in all three of these categories.

Dye Common Name Color Common Food Uses

FD&C Brilliant Blue Beverages, dairy products, powders, jellies, confections,
Bright blue
Blue No. 1 FCF condiments, icings, syrups, extracts
FD&C Baked goods, cereals, snack foods, ice cream, confections,
Ingigotine Royal blue
Blue No. 2 cherries
FD&C Beverages, puddings, ice cream, sherbert, cherries, confections,
Fast Green FCF Sea green
Green No. 3 baked goods, dairy products
FD&C Cherries in fruit cocktail & in canned fruit for salads,
Erythrosine Cherry red
Red No. 3 confections, baked goods, dairy products, snack foods
FD&C Gelatins, puddings, dairy products, confections, beverages,
Allura Red AC Orange-red
Red No. 40 condiments
FD&C
Tartrazine Lemon yellow Custards, beverages, ice cream, confections, cereals, preserves
Yellow No 5
FD&C Cereals, baked goods, snack foods, ice cream, beverages,
Sunset Yellow Orange
Yellow No. 6 dessert powders, confections

Table 3.1 Color Additives Permitted for Direct Addition to Human Food in the United States. (1)

You have all had experience mixing colors to create new combinations, whether as a child mixing red and yellow Play-
Doh to create orange or as an aspiring artist mixing various greens to create that perfect shade of moss green. Mars
Company, the manufacturers of M&M candies, use similar color mixings to create the colors of the hard candy shells.
Your task in this experiment is to identify the individual dyes used for each M&M color.

In order to analyze the dyes that are used in the M&M candies, you must first remove the colored candy shells from
the M&Ms. The chocolate, sugar and other ingredients that make up the rest of the candy will interfere with our
tests. Paper chromatography (2) will be then be used to separate each color into the individual dyes used in its creation.

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(1) U.S. Food and Drug Administration web site, www.fda.gov, accessed September 2003.
(2) Birdwistell, K. R.; Spence, T. G. J. Chem. Educ. 2002, 79(7), 847.
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Procedure:

There are two parts to this experiment. You should begin Part II of the experiment until your instructor calls you over
to the UV Station to complete Part I.

PART I Investigation into the Fluorescence of Dyes used in M&M Candies

SAFETY CAUTION

The light used in this part of the experiment emits ultraviolet radiation. Never look directly
into the lamp. Always wear the appropriate protective eyewear.

Your instructor will help you with this part of the experiment. You should begin Part II until your instructor calls you
over to the UV Station to complete this part.

In this part of the experiment, the standard dyes are exposed to ultraviolet (UV) light. Some chemicals will fluoresce
under UV light. Others, even though they do not fluoresce, may appear to be a different color. These characteristics
will aid in determining which dyes are used in M&M Candies.

1. A strip of chromatography paper spotted with the seven dye solutions has been prepared for your use. Place this
paper under the ultraviolet (UV) light (365 nm). Turn on the light. Observe.

2. Do any of the dyes fluoresce? What color is each dye under the UV light? Record your observations on the data
sheets.

PART II Separation and Identification of M&M Candy Dyes

SAFETY CAUTION

The M&M candies used in this experiment are laboratory samples. Therefore, they are not,
under any circumstances, to be eaten. Any student who eats the M&M candy samples will be
in violation of the safety rules. This student may be asked to leave the lab, resulting in a zero
grade for the experiment.

3. Use a clean, small graduated cylinder to measure 20 mL of the mobile phase (0.10% NaCl in H2O). Pour this
solution into an 800-mL beaker. Cover the beaker with a piece of plastic wrap, securing the plastic wrap with a
rubber band. Set the beaker aside until it is needed.

4. Lay the chromatography paper onto

a clean paper towel. With a pencil,
draw a horizontal line 1.5 cm from
the bottom edge of the paper, as
shown in Figure 3.1. Be sure to
orient the paper as shown in this
diagram. Starting line
drawn
5. Place six X marks on the line (again, (in pencil)
using a pencil). Place the X marks so 1.5 cm from
that they are about 2 cm from bottom
either end and about 2 cm between
them. Label the marks with the
names of the M&M colors which you X X X X X X
are assigned. These may differ blue brown green orange red yellow

from those shown.

6. Place 4 M&M candies of the Figure 3.1 Chromatography paper labeled for use.
SAME COLOR into a 50-mL beaker.
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7. Use a clean, small graduated cylinder to measure 3 mL of the 50:50 ethanol: water mixture. Pour this measured
solution into the beaker containing the M&M candies.

8. Swirl the solution gently over the M&Ms. The colored candy coating will dissolve into the solution as you swirl.
As soon as you begin to see the chocolate parts of the M&Ms, remove the candies from the beaker. You do
NOT want the chocolate to dissolve in the solution. This process does not take very long.

9. Use a plastic transfer pipet to transfer the solution (be careful not to spill any) to the small vial provided
(labeled with the appropriate M&M color). Cap this solution and set aside.

10. Throw the M&Ms into the trash. Rinse the beaker well with water. Dry it for use again.

11. Repeat steps 6 through 10 for the other M&Ms, until all 6 M&M colors have been collected.

12. Read this entire instruction before proceeding. You must now apply the M&M colors to the paper. To do this
you use a capillary (a narrow bore glass tube). The end of the capillary is immersed in the solution, so that a small
volume of the solution is drawn up into the capillary. The capillary is then gently touched to the surface of the
paper (on the appropriate X mark). The solution in the capillary will be transferred to the paper. This process is
illustrated in Figure 3.2.

You must use a separate capillary for each solution to avoid cross contamination of the solutions. Spot each color
once on the appropriate X mark (save the capillaries). Allow the spots to dry completely (use the heat lamp, if
necessary), then reapply the colors (on top of the existing spots). You must do this at least 7 times in order to
apply enough solution to the paper.

(a) (b)

X X

(1) Immerse capillary in solution (a) (2) Gently touch capillary to X mark (3) Spot of solution covering X mark
to fill with colored solution (b). to spot paper with solution. Hold on paper.
vertically.

Figure 3.2. Process for spotting chromatography paper with solution using a capillary.

13. Once all of the spots have completely dried, carefully pick up the paper.
Touch only the edges of the paper, similar to the way you would handle a
photograph.

14. Place the paper in a cylinder form, as shown in Figure 3.3. Roll the paper staple
so that the pencil marks are on the outside of the cylinder. Staple the
edges together (2 or 3 staples are enough). Leave a small gap between
the stapled edges so that the edges do not touch.
X
X X
15. Carefully lift the plastic cover off the beaker and place the cylinder
into the beaker, so that it stands freely. The bottom edge of the paper
should be placed on the bottom of the beaker. Quickly recover the
beaker with the plastic wrap. Do not pick up the beaker while the paper Figure 3.3 Spotted paper rolled
is within. and stapled.
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16. The solution in the bottom of the beaker will begin to rise up the paper. Keep an eye on the level of the solution.
When it has risen to about 1 cm from the top of the paper, remove the paper from the beaker. This takes about 20
to 25 minutes. Be careful because the wet paper will tear easily. Open it (remove the staples) and place it on a clean
paper towel.

17. With a pencil, draw a line across the top of the paper to mark the height to which the solution rose. Carefully circle
any colored spots that you see.

18. Let the paper dry completely. You may place your paper under a heat lamp to speed up this process.

19. Once the paper is dry, circle any visible spots. For each M&M color, record the color of the observed spots in the
appropriate data tables.

20. With the lab instructors guidance, place your paper under the UV light. Do the spots fluoresce? What color are
they? For each observed spot, record this information in the appropriate data tables.

This developed paper is called a chromatogram. It is needed to answer the questions. STAPLE THE
CHROMATOGRAM TO EITHER YOUR LAB REPORT OR TO YOUR PARTNERS LAB REPORT.

CLEANUP

21. Leave the M&M dye solutions in the capped, labeled vials on the bench top at your work area. These will be collected
and used for next weeks experiment.

22. Pour out the solution in the 400-mL beaker. Clean the beakers, graduated cylinders and all other glassware that you
used. Place them on a clean paper towel and leave on the bench top at your work area.

23. Place all used capillaries in the beaker labeled USED CAPILLARIES located on the side bench near the balances.
DO NOT PUT THESE IN THE REGULAR TRASH.
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DATA TABLES and INTERPRETATION OF CHROMATOGRAM

A separate data table is provided for each M&M color (6 tables, total). The information for each column in the tables is
described below. Use this information and the sample chromatogram shown in Figure 3.4 to guide you in completing these
tables.

COLUMN A: Look at the chromatogram. Count the number of spots that you circled above each X mark for a given color.
There may be more rows in the table than you need. Ignore these.

COLUMN B: Record the color of the spots that you have observed.

COLUMN C: Record the color of the spots under the UV light.

COLUMN D: Record whether the spots fluoresced under the UV light.

COLUMN E: This is a measurement that you must make using a ruler and the chromatogram (see sample). Locate the
starting line that you drew (the one with the X marks). Locate the center of the spot under consideration. Measure the
distance from the X-mark-line to the center of the spot. Measure this distance in centimeters (cm) and record to the tenth
of a cm (for example, 5.2 cm, not rounded to 5 cm).

COLUMN F: This is a calculation. The equation for this calculation is given below. Record your answers in the data table.
Report these answers with 2 decimal places (for example, 0.55, not 0.6). The mobile phase distance measurement is shown
on the next page.

dis tan ce spot traveled

Rf =
dis tan ce mobile phase traveled
COLUMN G: Identify the individual spots by comparing the fluorescence data and the Rf values of the dyes (see Part I) to
those that you have recorded in your data tables. Write the name of the dye in this column. Be specific. For example, simply
identifying a dye as blue is incorrect. Is this Blue No. 1 or Blue No. 2? Specify!

Mobile phase line

drawn as
instructed in
step 17.
Spot 2

Distance mobile phase traveled (only

Distance spot 2 traveled
need to make this measurement
once)

Spot 1

Distance spot 1 traveled

Starting line
X X X X X X drawn
blue brown green orange red yellow (in pencil)
1.5 cm from
bottom
Figure 3.4 Sample chromatogram. For clarity, spots are shown for a blue M&M only (yours may not look like this). On
your chromatogram you will have similar spots for the other M&M colors.

Measure the distance that the mobile phase has traveled. This measurement is done once only. Locate the starting line
(with the X marks). Locate the mobile phase line (drawn after you removed the paper from the beaker). Measure this
distance in cm using the ruler provided. Report the answer to the tenth of a cm.
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Experiment #3 - Extraction and Chromatography of M&M Candy Dyes

Name: _____________________________________________________ Report (of 95): ____________

Partner(s): _________________________________________________ Clean-up (of 5): ___________

Lab Station #: ______________________________________________ REPORT GRADE: __________

PART I Investigation into the Fluorescence of Dyes used in M&M Candies

FD&C Dye Fluoresce (yes or no)? Color under UV light Rf

Red No. 3 (R3) 0.13

Red No. 40 (R40) 0.36

Yellow No. 5 (Y5) 0.57

Yellow No. 6 (Y6) 0.40

Green No. 3 (G3) 0.90

Blue No. 1 (B1) 0.86

Blue No. 2 (B2) 0.36

PART II Separation and Identification of M&M Candy Dyes

Distance mobile phase traveled: ___________ cm

M&M _______________________________ (provide color)

Column A Column B Column C Column D Column E Column F Column G

Fluoresce
Color on Color under Distance spot
Spot # under UV Rf value Dye Name
paper UV light traveled (cm)
light?

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M&M _______________________________ (provide color)

Column A Column B Column C Column D Column E Column F Column G

Fluoresce
Color on Color under Distance spot
Spot # under UV Rf value Dye
paper UV light traveled (cm)
light?

M&M _______________________________ (provide color)

Column A Column B Column C Column D Column E Column F Column G

Fluoresce
Color on Color under Distance spot
Spot # under UV Rf value Dye
paper UV light traveled (cm)
light?

M&M _______________________________ (provide color)

Column A Column B Column C Column D Column E Column F Column G

Fluoresce
Color on Color under Distance spot
Spot # under UV Rf value Dye
paper UV light traveled (cm)
light?

2
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M&M _______________________________ (provide color)

Column A Column B Column C Column D Column E Column F Column G

Fluoresce
Color on Color under Distance spot
Spot # under UV Rf value Dye
paper UV light traveled (cm)
light?

M&M _______________________________ (provide color)

Column A Column B Column C Column D Column E Column F Column G

Fluoresce
Color on Color under Distance spot
Spot # under UV Rf value Dye
paper UV light traveled (cm)
light?

IF YOU BORROWED A CALCULATOR, RETURN IT TO THE LAB INSTRUCTOR.

Q1. Staple the chromatogram to either your lab report or to your partners lab report.

My chromatogram is stapled to: my lab report my partners lab report.

Q2. Some people are allergic to FD&C Yellow No. 5, or tartrazine. These people may break out into hives if this dye is
ingested. The M&M candy label lists this dye in the ingredient list, but does not indicate the colors that actually use
it. Based on your analysis of M&M candies, which of these colored candies should a person with this allergy avoid?

Q3. If the first (closest to the origin line) spot observed with the brown M&M was purple, what two dyes (specific colors
and numbers) would make up this spot? Explain your answer.

Q4. List the five FD&C dyes used in M&Ms.