Chromatography of Food | Custom PHD Thesis

Dyes
Hands-On Labs, Inc.
Version 42-0148-00-02

Review the safety materials and wear goggles when
working with chemicals. Read the entire exercise
before you begin. Take time to organize the materials
you will need and set aside a safe work space in
which to complete the exercise.
Experiment Summary:
You will learn how chemical compounds can be
separated into their constituent components
based on size and/or polarity. You will explore
how mixtures of compounds can be separated
using paper chromatography, and through paper
chromatography, determine what food dyes are
found in certain foods.

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Experiment

Chromatography of Food Dyes

Learning Objectives
Upon completion of this laboratory, you will be able to:
●●

Define the term chromatography.

●●

Explain the terms mobile phase and stationary phase.

●●

Describe the process by which different compounds are separated from each other in paper
chromatography, including the relevant physical properties.

●●

Discuss why polarity of a molecule is an important characteristic in chromatography analysis.

●●

Define Rf (Retention factor) values.

●●

Perform paper chromatography analysis to analyze food dyes.

●●

Measure and utilize Rf values to identify the food dyes in different food resources.

Time Allocation: 2.5 hours

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Experiment

Chromatography of Food Dyes

Materials
Student Supplied Materials
Quantity
1
1
1
1
1
1
1
1
1
1
1
1
16

Item Description
Bottle of distilled water
Digital Camera
Kool-Aid® drink mix: strawberry
Kool-Aid® drink mix: grape
Pair of scissors
Pencil
Ruler
Salt
Set of food coloring (usually boxed in tubes of red, yellow, green,
and blue)
Small bag of M&M® candy
Stapler
Tape
Toothpicks

HOL Supplied Materials
Quantity
1
3
1
1
1
1
1

Item Description
Beaker, 50 mL, plastic
Filter paper, 14 x 7 cm
Pair of gloves
Pair of safety goggles
Petri dish, 60 mm
Well plate, 24 wells

Experiment Bag: Chromatography of Food Dyes:
1- FDC Blue Dye #-1 – 0.5 mL
1- FDC Blue Dye #-2 – 0.5 mL
1- FDC Red Dye #-3 – 0.5 mL
1- FDC Red Dye #-40 – 0.5 mL
1- FDC Yellow Dye #-5 – 0.5 mL
1- FDC Yellow Dye #-6 – 0.5 mL
1- Unknown – 0.5 mL

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Experiment

Chromatography of Food Dyes

Note: To fully and accurately complete all lab exercises, you will need access to:
1.   A computer to upload digital camera images.
2.   Basic photo editing software such as Microsoft Word® or PowerPoint®, to add labels, leader
lines, or text to digital photos.
3.   Subject-specific textbook or appropriate reference resources from lecture content or other
suggested resources.
Note: The packaging and/or materials in this LabPaq kit may differ slightly from that which is listed
above. For an exact listing of materials, refer to the Contents List included in your LabPaq kit.

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Experiment

Chromatography of Food Dyes

Background
Chromatography Basics
Chromatography is a technique commonly used to separate mixtures of chemical compounds
into their constituent components. This is useful for determining the presence of a substance in a
mixture (for example, identifying unknown substances in a mixture) or for separating components
in a mixture. Separation is based on a physical characteristic of the compounds, such as polarity.
Chromatography is based upon two phases – a mobile phase (eluent) composed of a liquid or
gas solvent, and a stationary phase, composed of a type of specialized solid, called an adsorbent
that has an affinity for the solvent and the chemical components of the mixture. The compounds
involved must have different solubilities in the eluent (mobile phase) and different affinities for
the solid (stationary phase) for them to be separated by the procedure. The compounds that
have the strongest affinities for the solid phase will move the least during the chromatographic
procedure while those that have the highest solubility for the mobile phase will move the most.
The interaction of the compounds with the two phases makes it possible to separate many
different substances from one mixture. Scientists can identify components by comparing the
chromatogram (the chromatography results) of the unknown mixture to chromatograms of
known substances carried out using the same two phases.
There are different types of chromatography that are specialized for different applications:
Liquid and Gas chromatography are also called column chromatography due to the equipment
design. Liquid chromatography separates liquid samples in a liquid solvent (the mobile phase)
through a column of solid beads (stationary phase); Gas chromatography separates vaporized
samples in a gas mobile phase through a column of solid beads; Thin-layer chromatography uses
a thin layer of particles spread on a glass plate as the stationary phase, with a liquid solvent
applied to dried liquid samples. Paper chromatography is the simplest form of chromatography.
It uses specialized paper samples (often polar cellulose paper) as the stationary phase, across
which a liquid solvent applied to dried samples moves up the paper.
Paper chromatography is a simple yet extremely effective method for determining the presence
of a substance in a mixture or for separating components from a mixture. A strip of paper (special
chromatography paper or simple filter paper) serves as the stationary phase. A small spot of the
sample mixture to be separated is placed near one end of the paper strip and a solvent (or solvent
mixture) called the mobile phase (or eluent) is passed over the spot. The solvent moves up the
chromatography paper due to capillary action – the same way that water will move up a paper
towel – and carries with it the various components. The leading edge of the solvent, as it moves
up the chromatography paper, is called the solvent-front.
The choice of a solvent in paper chromatography is crucial. The solvent must dissolve the various
components in the mixture and there must be at least slight differences in solubility of each
component. If the components are insoluble they will remain where originally placed and not
move with the solvent. If all the compounds are extremely soluble, then each component of the
mixture will travel along with the leading edge of the solvent and not separate. One solvent does
not normally work equally well for all compounds, thus it is common for solvent mixtures to be
used.

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Experiment

Chromatography of Food Dyes

The different components will move up the chromatography paper at different rates depending
on their affinities for the stationary and mobile phases. If the components are colorful, their final
positions (spots) on the chromatogram are easily viewed. If they are not colorful, they may be
visible under UV light or a chemical reaction may be used to make them visible.

Retention Factor and FD&C Dyes
Paper chromatography can be used to indicate whether or not a sample contains one or more
components. The ending positions of each spot can be compared with those from known
compounds (called standards) to identify a compound, or confirm its absence. The location of the
spot is indicated by an Rf – Retention factor – value. The Rf value is calculated by comparing the
distance from the origin to the center of the spot to the distance the solvent traveled during the
chromatography. See Figure 1.

Figure 1. Schematic of paper chromatogram and Rf calculation.

The resulting Rf factor, which has no units, is dependent upon the specific stationary and mobile
phase used. Chromatogram results are considered to have better resolution if the Rf values of the
components being separated are farther apart (e.g. 0.3 and 0.7) rather than closer together (e.g.
0.4 and 0.5). The distance the compound moves relative to the distance the solvent (or mobile
phase or eluent) moves is a characteristic of that compound.

Rf =

Center of compound distance from base line
Solvent-front distance

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Experiment

Chromatography of Food Dyes

In this activity, you will be investigating the presence of FD&C dyes, which are specific dyes
approved for human consumption under the Food, Drugs and Cosmetics Act, in food coloring and
on colorful candy coatings. Common FDA color additives are shown in Table 1. You will absorb the
dyes onto chromatography paper (stationary phase) and allow a dilute salt-water solution (mobile
phase) to move up the paper. The dyes will travel up the paper at a rate that is determined by
their relative affinities to the two phases. You will be able to identify the dyes in the food coloring
and candy coating by comparing the R f values to those of the FD&C dyes.
Table 1. FDA Color Additives.

In addition to detecting food dyes as you’ll do in
this experiment, chromatography can be used to:
Analyze pigments found in different plant leaf colors
Determine the chemicals found in different
pharmaceutical products
Detect blood or alcohol levels in a bloodstream
Examine levels of pollutants in water supplies
Assist in analyzing specific types of crime scene samples

Can you think of other examples of how
chromatography is used?

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Experiment

Chromatography of Food Dyes

Exercise 1: Paper Chromatography
In this experiment, you will analyze food dyes from a variety of sources using paper chromatography
technique. The components in the food dyes are polar and, therefore, soluble in water. Both the
stationary phase (chromatography paper) and the mobile phase (dilute salt water) are polar. The
components will be separated by their relative affinities (attractions to the two phases). Some
dyes will be more strongly attracted to the paper and will move slowly up the paper while others
will spend more time dissolved in the mobile phase and will travel more rapidly up the paper.
Note: Completely read all instructions and assemble all equipment and supplies before beginning
work on this experiment.

1.   Using the 50-mL beaker, dissolve a pinch of salt into 50 mL of warm (room temperature)
distilled water. Stir until completely dissolved and set aside. This will be the salt water (0.1%
NaCl).
2.   Gather 2 sheets of the filter paper – 14 x 7 cm from your kit.
a.   Create a base line by drawing a thin horizontal line with a pencil (NOT a pen!) across the
paper, 10 mm (1 cm) from the bottom. The line should barely be visible.
b.   With a pencil, draw 9 small cross lines along the horizontal line, 1.5 cm apart, as shown in
Figure 2.

Figure 2. Preparing the filter paper.

3. Repeat step 2 for the second piece of filter paper.
4. On Sheet 1, from left to right, use a pencil to lightly label each section between the cross lines
with the abbreviation for the six FD& C food colors from your experiment bag that will be
tested: B1, B2, R3, R40, Y5, and Y6.

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Experiment

Chromatography of Food Dyes

5.   Also on Sheet 1, after Y6, place the labels KG and KS to represent the Kool-Aid® Grape and
Kool-Aid® Strawberry drink mixes to be tested.
6.   On Sheet 2 from left to right, use a pencil to lightly label each section between the cross lines
with the abbreviation for the grocery store food coloring that will be tested: SR, SY, SG, and SB.
7.   Also on Sheet 2, after SB, place the label “UK” for unknown and then “M” plus a letter for the
color of 4 different candies from your bag of M&Ms®: such as; M-R (for M&M® red), M-Y, M-B,
M-G.
8.   Set Sheet 2 aside and perform the following for Sheet 1. After you have completed the
experiment for Sheet 1, repeat for Sheet 2.
9.   Put on your safety gloves and goggles.
10.   For each dye on the sheet to be tested:
a.   Use the scissors to cut open the pipets and place them in the 24-well plate, bulb side
down, using the well plate as a pipet holder.
b.   Place a few drops of the dye in a well of the 24-well plate, as shown in Figure 3.

Figure 3. Dye drops and pipets in well plate.

c. Dip a clean toothpick tip into the dye and then briefly touch the toothpick tip to the
CENTER of the appropriately marked section on the horizontal line on the filter paper. See
Figure 4.

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Experiment

Chromatography of Food Dyes

Figure 4. Dye on filter paper.

Note 1: A small drop of water on a little bit of Kool-Aid® powder will be sufficient to make a sample
to spot the paper. A small drop of water on the coating of an M&M® will dissolve enough dye for
the toothpick to pick up. See Figure 5.The coating of blue and brown M&Ms® is made up of very
interesting dye combinations!

Figure 5. Drops of water on M&Ms®.

Note 2: Apply only a small drop of each prepared dye (Kool-Aid® or M&M®) on the paper. Big
drops may spread over a greater area and overlap with other dyes. This is called band broadening.
Although a larger dot will produce a more vivid color, a smaller dot will show the most distinct
break between colors. The smaller and more intensely-colored the dot is, the better your final
chromatogram will be.
11. Once the paper is spotted with all the dyes, allow the spots to dry for a few minutes.

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Experiment

Chromatography of Food Dyes

12. Form the spotted filter paper into a cylinder (colored spots on the inside) with the edges
touching, but NOT overlapped, and staple at the top and bottom as shown in Figure 6. (You
may want to use small pieces of tape on the outside to lightly hold the cylinder together
while you securely staple it. If so, remove the tape after stapling.) Set the cylinder aside for a
moment.

Figure 6. Placing the staple in the paper.

13.   The salt water (~0.1% NaCl) you previously prepared will be your eluting solvent and a Petri
dish will be your elution chamber. Pour salt water into the clean and empty Petri dish to a
depth of about 1/4 cm. This is just enough to cover the bottom of the elution chamber.
14.   Set your paper cylinder next to (not in) the Petri dish, with the spots at the bottom, and look
to make sure the solvent level is below the base line. If it is not, pour out a little solvent until
it is. The base line (and spots at the bottom of the chamber) must be above the solvent level
for this experiment to work.
15.   Carefully place the paper cylinder into the eluting chamber, making sure not to touch the Petri
dish sides. See Figure 7. The solvent-front will travel up the paper rapidly at first and then will
slow down. Let the solvent-front rise, monitoring every few minutes. Immediately remove the
cylinder if any dye or the solvent front moves up higher than 2 cm from the top of the paper.
If all the solvent is soaked up before the front has time to move toward the top of the paper
carefully add a little more solvent to the Petri dish.

Figure 7. Filter paper in Petri dish.

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Experiment

Chromatography of Food Dyes

16. When the solvent-front has traveled up the filter paper, 2 cm from the top, remove the filter
paper cylinder from the chamber and immediately mark the top the solvent-front with a
pencil. See Figure 8.

Figure 8. Solvent-front is marked with a pencil.

17.   Allow the paper to dry for several minutes, carefully remove the staples, and draw an outline
around each spot using a pencil.
18.   Use the digital camera to take a photograph of your chromatogram after you have circled the
spots. Resize and insert the photograph of your chromatogram in Data Table 1 of your Lab
Report Assistant.
Note: Refer to the appendix entitled, “Resizing an Image” for guidance with resizing an image.
19.   Use the ruler to measure the distance of the solvent front, in millimeters. Use Figure 1 in the
Background section as needed. Record the distance of the solvent front in Data Table 2 of
your Lab Report Assistant.
Note: The distance of the solvent front will be the same for the entire filter paper chromatogram.
20.   For each of the dye spots on the filter paper chromatogram, measure and record to the
nearest millimeter the distance each of the spots has traveled up the solvent front. Start from
the original horizontal pencil line (base line) and measure to the top center of where the dye
stops in each column on the paper. See Figure 1 in the Background section as needed.
Note: There is room for each of the samples to separate into 3 spots. This may not happen for all of
the samples. Therefore, some of the samples will not have data recorded for Spot 2.
21.   Calculate the Rf value for each spot and record in Data Table 2.
22.   Repeat steps 9 through 21 for the second sheet of filter paper. Record all data for this chromatogram in Data Table 3 of your Lab

Report Assistant.

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Experiment

Chromatography of Food Dyes

23.   Compare the Rf values for the spots of the Kool-Aid®, unknown sample, store food colorings,
and M&Ms®, to those of the known FD&C food dyes, to determine which FD&C dyes are used
in the Kool-Aid®, the grocery store food colorings, and in the M&Ms®.
24.   Record the FD&C dyes present in each of the samples in Data Table 4 of your Lab Report Assistant.
25.   Clean up all equipment and return to kit for future use.
26.   When you are finished uploading photos and data into your Lab Report Assistant, save your
file correctly and zip the file you can send it to your instructor as a smaller file. Refer to the
appendix entitled, “Saving Correctly,” and the appendix entitled, “Zipping Files” for guidance
with saving the Lab Report Assistant correctly and zipping the file.

Questions
A.   What might happen you used a pen to mark the baseline on the chromatography paper?
B.   What would happen if you did not stop the chromatograph before the solvent reached the
top of the paper?
C.   What would happen if the solvent level was above that of the dye spots on the paper at the
start of the chromatography?
D.   Why is it important to mark the solvent level on the chromatography paper as soon as you
remove it from the Petri dish?

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Experiment

Chromatography of Food Dyes

Chromatography of Food Dyes
Hands-On Labs, Inc.
Version 42-0148-00-02

Lab Report Assistant
This document is not meant to be a substitute for a formal laboratory report. The Lab Report
Assistant is simply a summary of the experiment’s questions, diagrams if needed, and data tables
that should be addressed in a formal lab report. The intent is to facilitate students’ writing of lab
reports by providing this information in an editable file which can be sent to an instructor.

Exercise 1: Paper Chromatography
Data Table 1. Photographs of Chromatograms

Photograph of Chromatogram #1

Photograph of Chromatogram #2

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Experiment

Chromatography of Food Dyes

Data Table 2. Chromatogram Sheet 1 Data
Sample

Solvent
front (mm)

Spot 1
mm

Spot 2
Rf

Spot 3
Rf

Blue 1
Blue 2
Red 3
Red 40
Yellow 5
Yellow 6
KG
KS
Data Table 3. Chromatogram Sheet 2 Data
Sample

Solvent
front (mm)

Spot 1
mm

Store Red
Store Yellow
Store Green
Store Blue
Unknown
M&M: M -_
M&M: M -_
M&M: M -_
M&M: M -_

Spot 2
Rf

Spot 3
Rf

Data Table 4. FD&C Dyes in Samples
Sample:
Kool-Aid Grape ®
Kool-Aid Strawberry ®
Store Red
Store Yellow
Store Green
Store Blue
Unknown Sample
M&M®__________
M&M®__________
M&M®__________
M&M®__________

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FD&C Dyes in Sample

Experiment

Chromatography of Food Dyes

Questions
A. What might happen you used a pen to mark the baseline on the chromatography paper?

B. What would happen if you did not stop the chromatograph before the solvent reached the
top of the paper?

C. What would happen if the solvent level was above that of the dye spots on the paper at the
start of the chromatography?

D. Why is it important to mark the solvent level on the chromatography paper as soon as you
remove it from the Petri dish?