TEACHER RESOURCES

1. ANALYSIS OF FOOD DYES IN SCPORTS DRINKS

Introduction
If you have ever added a powdered drink mix to water, you will realize that the more concentrated the drink, the
deeper the color of the solution. Analytical chemists routinely use a quantitative approach called spectroscopy that
uses a photometer to measure light intensity to determine the concentration of solute in a solution. A colorimeter is
an instrument that measures light at specific wavelengths in the color spectrum. In this lab, you will investigate and
design an experimental procedure that utilizes spectroscopy and quantitative analysis to determine the concentration
of FD&C food dyes in sports drinks.

Pacing and Length of the Lab

Initial Investigation Advanced Investigation

Preparation 30 min Experiment Design 15 min
Initial Investigation 45 min Experiment 45 min
Data Analysis 30 min

The time required for planning, preparing and executing advanced and extended inquiry labs will vary depending on
the type of activity selected by your students. Please provide them the opportunity to compare data and discuss
outcomes of their selected activities.

AP* Connections
The concepts covered in this lab align to the “AP Chemistry® Course Framework.”

Learning Objective SAP-8.A: Explain the relationship between a region of the

Primary Learning Objective
Secondary Learning Objective
Science Practices
electromagnetic spectrum and the types of molecular or electronic transitions
associated with that region.
Learning Objective SAP-8.C: Explain the amount of light absorbed by a solution of
molecules or ions in relation to the concentration, path length and molar absorptivity.
SP 2.C, 5.A, 5.D, 6.A, 6.B

Next Generation Science Standards (NGSS)**

Disciplinary Core Ideas Crosscutting Concepts Science and Engineering Practices

Structure and Properties of Matter (HS- Patterns Asking questions and defining
PS1) Structure and Function problems
Planning and carrying out
investigations
Analyzing and interpreting data
Constructing explanations
Engaging in argument from evidence

*
AP is a registered trademark of the College Board, which was not involved in the production of, and does not endorse, this product.
**NGSS is a registered trademark of Achieve. Neither Achieve nor the lead states and partners that developed the Next Generation Science
Standards were involved in the production of this product, and do not endorse it.

PASCO / PS-2828A 1
 ¡ERROR! UTILICE LA PESTAÑA INICIO PARA APLICAR HEADING 1 AL TEXTO QUE DESEA QUE APAREZCA AQUÍ. /
TEACHER RESOURCES

Concepts
Students should be familiar with the following concepts:
 Electromagnetic spectrum
 Spectroscopy or colorimetry
 Transmission and absorbance
 Wavelength
 Solution concentration
 Molarity
 Beer's law

Materials and Equipment

For Each Student Station
 Data collection system  Test tubes and racks
 Wireless colorimeter  FD&C Blue # 1 food dye stock solution1
 Plastic cuvettes  Distilled water in wash bottle
 Plastic transfer pipettes or eyedroppers  Sport drink or Kool-Aid® with FD&C Blue # 1
 10 mL graduated cylinder food dye
1
Refer to the Lab Preparation section.

Safety
Follow these important safety precautions in addition to your regular classroom procedures:
 Wear safety goggles and gloves at all times.
 Avoid direct contact of FD&C dyes with skin, eyes and clothing.
 Wash hands thoroughly with soap and water before leaving laboratory

Lab Preparation
These are the materials and equipment to set up prior to the lab. Adjust volumes as needed for your class size.

1. FD&C Blue # 1 stock solution: Prepare 1000 mL of Blue 1 concentrate by adding 0.12 g of powdered dye to
100 mL distilled water in a 1000 mL volumetric flask. Swirl flask to dissolve dye powder completely. Add
enough distilled water to the 1000 mL mark. To make the FD&C Blue 1 stock for your students, mix 50 mL of
your concentrate with 950 mL distilled water (1:20 dilution).

2. FD&C Red # 40 stock solution: Prepare 1000 mL of Red 40 concentrate by adding 0.38 g of powdered dye to
100 mL distilled water in a 1000 mL volumetric flask. Swirl flask to dissolve dye powder completely. Add
enough distilled water to the 1000 mL mark. To make the FD&C Red 40 stock for your students, mix 50 mL of
your concentrate with 950 mL distilled water (1:20 dilution).

3. FD&C Yellow # 5 stock solution: Prepare 1000 mL of Yellow 5 concentrate by adding 0.39 g of powdered
dye to 100 mL distilled water in a 1000 mL volumetric flask. Swirl flask to dissolve dye powder completely.
Add enough distilled water to the 1000 mL mark. To make the FD&C Yellow 5 stock for your students, mix 50
mL of your concentrate with 950 mL distilled water (1:20 dilution).

4. Absorbance of the final stock solutions must have a maximum absorbance of 1.0 absorbance units at the
following wavelengths:

2 PASCO / PS-2828A
 ¡ERROR! UTILICE LA PESTAÑA INICIO PARA APLICAR HEADING 1 AL TEXTO QUE
DESEA QUE APAREZCA AQUÍ. / TEACHER RESOURCES

FD&C Blue # 1 = 600 nm

FD&C Red # 40 = 500 nm
FD&C Yellow # 5 = 450 nm

5. Many commercially available sports drinks contain FD&C Blue 1: Powerade®, Gatorade®, Mixed Berry® or
Kool-Aid®. Choose only drinks that are clear to avoid any interference with light transmission in the
colorimeter.

6. FD&C Blue # 1 dye is also available as a liquid concentrate in the baking aisle of most grocery stores and
supermarkets. A comparable stock solution can be prepared by adding 6 drops of liquid Blue 1 dye to 1000 mL
distilled water.
Note: Liquid formulations may vary slightly depending on the brand. It is important to test which colorimeter
wavelength (600 or 650 nm) is optimal to use for your experiment.

7. Calculate the molar concentration of the FD&C Blue # 1 stock solution by measuring absorbance at 600 nm.
Absorbance must be within the 0.7-1.0 absorbance units. Absorbance must be divided by 1.3 x 105, the molar
absorptivity of FD&C Blue # 1 at 600 nm. Thus, for a solution with an absorbance of 1.0:
1.0/1.3 x 105 M-1cm-1 = 7.7 x 10-6 M

8. Follow the same process for each of the Extension Inquiry investigations. Molar absorptivity values for FD&C
Red # 40 and Yellow # 5 at specific wavelengths are given below:

FD&C Red # 40 at 500 nm = 2.9 x 103 M-1 cm-1

FD&C Yellow # 5 at 450 nm = 2.73 x 104 M-1 cm-1

Teacher Tips
Tip 1 – Sport drink too concentrated
If the sports drink chosen for analysis is too concentrated, dilute it and keep track of the dilution factor for
calculations of the final concentration of FD&C Blue # 1 dye.

Tip 2 – Interference with colorimeter

Other sport drink ingredients may interfere with colorimetric readings. Thus, it is critical to select a commercial
product that is clear or transparent

PASCO / PS-2828A 3
 ¡ERROR! UTILICE LA PESTAÑA INICIO PARA APLICAR HEADING 1 AL TEXTO QUE DESEA QUE APAREZCA AQUÍ. /
TEACHER RESOURCES

Pre-Lab Questions
The visible absorbance spectrum of a 0.1 M Co(NO3)2 solution is shown in the figure below.

1. What wavelength would be optimal for measuring the absorbance versus concentration of a series of solutions
of Co(NO3)2? Explain your answer.
The wavelength for maximum absorbance of Co(NO3)2 is approximately 520-525 nm. The range of wavelengths is in the “green” region of
the color spectrum which is consistent with the fact that it is the complimentary color that is absorbed. Green is complimentary to red (see
color wheel below).

2. A series of solutions of Co(NO3)2 were prepared to construct a calibration curve. Using the dilution information
below from a 0.1 M Co(NO3)2 stock solution, determine the concentration of each of the dilutions. Hint: M1V1
= M2V2

Dye Stock Solution B C D E F G H

(A)
Concentration 0.1 M 0.08M 0.06M 0.04M 0.03M 0.02M 0.01M 0.00M
Water (mL) 0 2 4 6 7 8 9 10
Stock Solution (mL) 10 8 6 4 3 2 1 0

4 PASCO / PS-2828A
 ¡ERROR! UTILICE LA PESTAÑA INICIO PARA APLICAR HEADING 1 AL TEXTO QUE
DESEA QUE APAREZCA AQUÍ. / TEACHER RESOURCES

Initial Investigation
The Initial Investigation is designed to familiarize students with the equipment and techniques necessary to design
their own experiment. If students are comfortable with probeware, and have a strong understanding of the concepts,
this section may be removed, placing more responsibility on the students for developing an experiment.

Determination of Optimal Wavelength for Measuring Absorbance of FD&C Blue # 1

1. Using a sample of the stock solution of FD&C Blue # 1 food dye provided to you by your instructor, determine
the optimal wavelength for measuring absorbance of FD&C Blue 1 food dye.

2. Start a new experiment on the data collection system from your Chromebook, computer or mobile device.

3. Connect the colorimeter to the data collection system. SPARKvue was used for this analysis.

4. Select “Absorbance and Transmittance” from the Quick Start Lab menu or open lab file 01 Analysis of Food
Dyes in Sports Drinks. Calibrate the colorimeter using distilled water. You should read zero for absorbance and
100% for transmittance.

5. Measure absorbance of your dye sample at the different wavelengths. Determine the wavelength that gives the
maximum absorbance.

Maximal absorbance of FD&C Blue 1 food dye is achieved at a wavelength of 600 nm.

Calibration Curve of FD&C Blue # 1 Food Dye

1. Prepare a series of dilutions of FD&C Blue 1 food dye from a stock solution of known concentration prepared
for you by your instructor, and distilled water. Prepare 10 mL of each dilution, calculate and record the
concentration of each sample in the table below. Using a colorimeter, you will measure absorbance of each
solution in the Initial Investigation.

PASCO / PS-2828A 5
 ¡ERROR! UTILICE LA PESTAÑA INICIO PARA APLICAR HEADING 1 AL TEXTO QUE DESEA QUE APAREZCA AQUÍ. /
TEACHER RESOURCES

Table 1: Calibration Curve of Stock FD&C Blue Dye # 1 (Absorbance at 600 nm)
Blue #1 Sample Stock Solution Distilled Water Concentration2 Absorbance %Transmittance
(mL) (mL) (M) (-log T)
1 10 0 6.78 x 10-6 0.882 13.1
2 8 2 5.42 x 10-6 0.722 19.0
3 6 4 4.07 x 10-6 0.571 26.8
4 4 6 2.71 x 10-6 0.401 39.7
5 3 7 2.03 x 10-6 0.306 49.4
6 2 8 1.36 x 10 -6
0.189 64.7
7 1 9 6.78 x 10-7 0.092 81.0
8 0 10 0.00 0.00 101.2
2
Calculations based on the concentration of FD&C Blue 1 stock solution = 6.78 x 10 M. (0.882/1.3 x 10 M cm )
-6 5 -1 -1

6. Start a new experiment on the data collection system on your Chromebook, laptop or mobile device.

7. Connect the colorimeter to the data collection system.

8. Select “Absorbance and Transmittance” from the Quick Start Lab menu or open to page 2 of lab file. Calibrate
the colorimeter using distilled water. You should read zero for absorbance and 100% for transmittance.

9. Transfer prepared dilutions to cuvettes, filling to three-fourths full. Using the most concentrated solution, insert
into the cuvette port of the colorimeter and record the absorbance data. Remember that the cuvettes must be
handled by the ribbed sides.

10. Of the given wavelengths of light, determine the optimum wavelength that demonstrates your sample’s
maximal absorbance. What do you notice about the wavelength of highest absorbance?
Hint: Consider the color wheel of complementary colors.

11. Save your data and start a new experiment by selecting Beer’s Law from the Quick Start Menu. Adjust the
absorbance wavelength if needed.

12. Measure and record the absorbance of your first dilution sample at the optimal wavelength. Enter the calculated
dye concentration in moles/L (M). Press the green “check” mark to record the reading.

13. Remove the cuvette from the colorimeter and replace with a second sample. Repeat the necessary steps to test
the remaining dye dilution samples.

14.When completed, stop data collection and save your experiment. Use SparkVue’s graphing tools to determine
the linearity of your calibration curve.

15. Record all absorbance data in the prepared table above.

16. Convert y-axis in data table to Transmittance at 600 nm and record the corresponding % Transmittance (%T) in
the prepared table.

6 PASCO / PS-2828A
 ¡ERROR! UTILICE LA PESTAÑA INICIO PARA APLICAR HEADING 1 AL TEXTO QUE
DESEA QUE APAREZCA AQUÍ. / TEACHER RESOURCES

Advanced Investigation
To further support your understanding of the graphical relationships between absorbance and transmittance,
complete Table 2 using the data set from Table 1.

Table 2: Graphical Analysis of Absorbance and Transmittance of FD&C Blue Dye # 1 (600 nm)
Blue Dye # 1 Concentration (M) %T Transmittance (T) log T Absorbance (-
Sample log T)
1 6.78 x 10-6 13.1 0.131 -0.882 0.882
2 5.42 x 10 -6
19.0 0.190 -0.722 0.722
3 4.07 x 10-6 26.8 0.268 -0.571 0.571
4 2.71 x 10-6 39.7 0.397 -0.401 0.401
5 2.03 x 10-6 49.4 0.494 -0.306 0.306
6 1.36 x 10-6 64.7 0.647 -0.189 0.189
7 6.78 x 10 -7
81.0 0.810 -0.092 0.092
8 0.00 101.2 1.01 0.00 0.000

1. Prepare separate graphs of (a) %T, (b) T, (c) log T and (d) -log T as y-axis values and molar concentrations of
food dye as the x-values. Remember that the value for transmittance (T) is percent transmittance (%T) written
in decimal form. Using SPARKvue’s analytical tools, determine which curve has the best linear fit. Which
graph has the most linear and positive slope?
As is indicated by the graphs below, the calibration curve that has a positive slope and produces the best linear fit is -log T vs. Blue 1
concentration.

PASCO / PS-2828A 7
 ¡ERROR! UTILICE LA PESTAÑA INICIO PARA APLICAR HEADING 1 AL TEXTO QUE DESEA QUE APAREZCA AQUÍ. /
TEACHER RESOURCES

8 PASCO / PS-2828A
 ¡ERROR! UTILICE LA PESTAÑA INICIO PARA APLICAR HEADING 1 AL TEXTO QUE
DESEA QUE APAREZCA AQUÍ. / TEACHER RESOURCES

2. Obtain a sports drink containing FD&C Blue # 1 dye. You may choose a ready-to-drink product or prepare one
from a powdered mix according to manufacturer’s instructions. Using the procedure from the Initial
Investigation and graphs produced in the graphing challenge above, develop an experimental protocol to
measure the concentration of FD&C Blue # 1 dye in your chosen drink using the optimal calibration method
that gives the best linear correspondence for quantitative analysis.
The experimental protocol must use a calibration curve of Absorbance @ 600 nm (-log T) versus the concentration of FD&C Bue # 1 dye.

3. Unlike your dye dilution samples which contained only dye and water, the typical sport drink formulation
contains other ingredients which may include sugar and electrolytes. What effect will the presence of additional
ingredients potentially have on the quantitative determination of the food dye content?
Many sports drinks contain ingredients (perhaps some ionic compounds) that may not be completely soluble in water thus making the
drinks cloudy. This will interfere with the colorimetric analysis. White sugar, in and of itself, should not interfere with colorimetry, if the
concentration is low. Very high concentrations of sugar, or colored sugars may affect colorimetric analysis and should be avoided for this
analysis.

4. Measure the absorbance and % transmittance of your sports drink. Do the numbers fall within the range of your
calibration curve? If not, what should you do to fix this?
The final concentration of the sports drinks tested are all within the range of the calibration curve. If a drink is out of range, it must be
diluted until it falls within the linear range of the calibration curve. Special attention, however, must be given to the dilution factor as this
will be used in the calculation of the final concentration of FD&C dye.

5. Determine the concentration of the dye in your beverage and calculate the mass of dye (mg/L) of the sports
drink. FD&C Blue # 1 dye has a molar mass of 793 g/mole.
Sample Calculations for Sport Drink
Data obtained using Lower Sugar Gatorade® Thirst Quencher Cool Blue sports drink:
Absorbance = 0.335 %T = 46.3
y = mx + b
A = m[concentration] + b
Calculation: 0.335 (A) = 125,000 (m) X + 0.0456 (b). (Data from absorbance graph above)
[FD&C Blue # 1] in Gatorade® Lower Sugar Cool Blue Drink = 2.3 X 10-6 M
Mass of FD&C Blue #1 in Gatorade® Cool Blue Lower Sugar Sport Drink:
2.3 X 10-6 moles/L x 793 grams/mole x 1000 mg/gram = 1.8 mg/L

PASCO / PS-2828A 9
 ¡ERROR! UTILICE LA PESTAÑA INICIO PARA APLICAR HEADING 1 AL TEXTO QUE DESEA QUE APAREZCA AQUÍ. /
TEACHER RESOURCES

Extended Inquiry Investigation

Following the procedures used above, similar investigations can be conducted to investigate the dye content of other
food dyes in other drinks. Two common dyes are FD&C Red # 40 and FD&C Yellow # 5. The structures and
molar masses of these dyes are shown below.

Measure the visible spectra of FD&C Red # 40 and FD&C Yellow # 5 and determine the wavelength for each dye
that gives maximal absorbance. Compare your absorbance data with that observed for FD&C Blue # 1. What
trend(s) do you notice? Formulate a possible explanation for your observations that correlates the molecular
structure of the food dye and absorbance wavelength. Determine the concentration of the food dye in the red and/or
yellow sports drinks using Beer’s law.

10 PASCO / PS-2828A
 ¡ERROR! UTILICE LA PESTAÑA INICIO PARA APLICAR HEADING 1 AL TEXTO QUE
DESEA QUE APAREZCA AQUÍ. / TEACHER RESOURCES

Synthesis Questions
1. In a copper(II) sulfate solution, copper(II) ions cause the blue color. Sulfate ions have no color. Sodium sulfate
solution has no color because neither ion has color. Could you use Beer's law to find the concentration of a
sodium sulfate solution?
No. The absorbance would not change with the concentration of sodium sulfate.

2. Hypothetically, how would you modify the experiment if the measured absorbance of the unknown was too low
to measure?
Increasing the path length would increase the absorbance. Therefore, using a cell with a longer path length would work.

2. How would you modify your experiment if the measured absorbance of the unknown was too high?
Making a proportional dilution of the solution would decrease the absorbance.

AP® Chemistry Review Questions

1. Which of the following variables affects the absorbance of light in a solution?

A. The distance the light must travel through the solution (path length)
B. The amount of solute in each volume (concentration)
C. The wavelength of the light that is interacting with the solution
D. All of the above

2. A sample of 0.10 M copper(II) chloride is placed into a cuvette with a 1.00-cm path length. The solution has a
measured absorbance of 2.000. What would you expect the absorbance of a 0.05 M copper(II) chloride solution
to be?
A. 4.000
B. 2.000
C. 1.000
D. Not enough information

PASCO / PS-2828A 11