AP Chemistry – Laboratory Investigation
What is the Concentration of Food Coloring Dyes in Gatorade and other
beverages?
Purpose: To spectrophotometrically determine the concentration of either blue dye #1 or yellow dye #5
in Gatorade, Powerade, and another beverage, and compare the concentrations.
Introduction: In this experiment, you will be assigned a Gatorade, Powerade, and another beverage of a
particular color (yellow or blue). You will first prepare a set of standard solutions using FD&C yellow dye
#5 or blue dye #1, and then you will determine their absorbance values using a colorimeter. A Beer’s
Law graph of absorbance values vs. concentration will be generated. You will then use the graph of your
data to determine the concentration of the dye in the beverages.
Background Information: White light is composed of many different wavelengths of light combined
together. A spectrophotometer (or colorimeter) is an instrument that shines a single wavelength of light
of a known intensity into a solution and then measures the intensity of the light exiting the solution (see
figure 1 below). If a solution contains any compound that absorbs light, the intensity of light exiting the
solution will be less than what entered the solution. On the other hand, if none of the compounds in the
solution absorb at that wavelength of light, then the intensity of the light exiting the solution will be the
same as that entering.
Figure 1
The intensity of light that passes through an absorbing solution is dependent on the concentration of
the absorbing compound in solution. The greater the concentration of the absorbing compound, the
less light will get through. This relationship is expressed as Beer’s Law:
A = abc
A is the absorbance. a is the molar absorptivity constant (units are M-1cm-1). It is specific for the
substance that is absorbing light at a given wavelength. It represents how much light is absorbed at a
particular wavelength. b is the path length of the light through the solution. For most standard cuvets, the
path length is 1.00cm. c is the concentration of the solution (M).
If we were to prepare several solutions of various concentrations of a colored solute, measure the
absorbance of each solution and then plot absorbance versus concentration, we should get a
straight line with a y-intercept of zero and a slope of a∙b. With this graph (known as a standard
graph or standard curve), we would be able to determine the concentration of the solute in any
solution just by measuring its absorbance (see figure 2 below).
Figure 2
How do we know which wavelength to use? Our colorimeters can be set to 1 of 4 wavelengths: 430nm,
470nm, 565nm, or 635nm. You will want to take your absorbance measurements at the wavelength at
which the solute is absorbing the strongest. This would be λmax. To find λmax, a plot of absorbance vs.
wavelength for a given substance is collected. This plot is called an absorption spectrum. Below are the
absorption spectra for the 2 dyes used in this lab (as well as for red dye #40):
Food coloring dyes have been used for centuries. We have come to expect that our food will look a
certain way. We expect that orange flavored drinks will be orange. They also help us identify the flavor
of a food. For instance, lime and orange sherbets would be nearly indistinguishable based on
appearance if not for the green and orange colors. In addition, coloring agents add a festive appearance
to foods – M&M candies would all be the same color (probably gray) if no food dyes were used.
Historically, coloring agents have also been added to foods to cover up inferior quality foods by making
them look different. Since the passage of the Federal Food, Drug, and Cosmetic (FD&C) Act of 1938,
color additives in the U.S. have been the responsibility of the Food and Drug Administration (FDA). The
structures of the 2 food dyes tested in this lab are as follows:
Pre-lab questions:
1.
Which wavelength (430, 470, 565, 635) should you set the colorimeter to for your experiment?
Determine this by looking at the Food Dyes Absorption Spectra. Why should you use that
wavelength?
2. Why can’t we test red dye #40 with our colorimeters?
3. You will be preparing a set of standard solutions of your food coloring dye from a stock solution
of known molarity (Blue #1 = 7.0µM, yellow #5 = 27.8µM). From the concentration of the stock
solution, determine the concentration of each of the following dilutions that you will be making
when you do the lab. (µM of diluted sample = µM of stock soln X (mL stock soln/mL total).
Record the concentrations in the table below.
4. Using the Beer’s Law equation from the background information, predict the absorbance of
each standard. The molar absorptivities (a) of blue #1 and yellow #5 are 130,000 M-1cm-1 and
27000 M-1cm-1 respectfully. The path length (b) is 1cm. Record your absorbance predictions in
the table below.
Standard #
1
2
3
4
5
6
7
mL stock soln
1.0
2.0
4.0
6.0
8.0
9.0
10.0
mL water
9.0
8.0
6.0
4.0
2.0
1.0
0
Concentration (µ)
Predicted absorbance
5. Beer’s Law only holds true at low absorbance values. The colorimeter you are using will be
sensitive to about an absorbance of 0.8. Above an absorbance of 0.8, the instrument becomes
saturated and the readings no longer maintain a linear relationship. Do your predicted
absorbances for your standards fall within the linear range of the colorimeter?
6. When testing your beverage samples, the absorbance you get for each one must fall within the
aborbances of your standards. The beverages you are using may be too concentrated, so you
will need to prepare dilutions of them. You will probably have to try a few dilutions to find the
one that will fall within your standard curve. Some example dilutions are:
 1:2 – this means that the original sample has been diluted to twice its original volume
(add equal volumes of sample and water)
 1:5 – this means that the original sample has been diluted to 5 times its original volume
(add 1 part sample and 4 parts water)
For these dilutions, we are assuming that volume is additive (a valid assumption since the
original solutions are aqueous and we are diluting them with water)
What dilutions are you planning to make of your samples? (pick at least 2 dilutions, and also run
the undiluted sample)
7.
Make a data table for the lab. You will need to record the concentration and absorbance
readings for each of your standards, and the dilution and absorbance readings for each of your
beverages.
Materials:
LabQuest
Vernier Colorimeter
one cuvette
16 medium test tubes
5 10-mL serological pipets
two 100 mL beakers
Beverage samples
100 ml volumetric flask
FD&C dye from Flinn Scientific
pipet filler
distilled water
test tube rack
stirring rod
Lint-free tissues
Magnetic stirrer and stir bar
1 - 5ml serological pipet
Procedure:
1.
Prepare your stock solution (7µM for blue #1 and 27.8µM for yellow #5). Using a 5ml
serological pipet, accurately transfer 4.0ml of the FD&C dye from Flinn Scientific to a 100ml
volumetric flask that you have previously rinsed with distilled H2O. Dilute to the mark with
distilled H2O. Mix thoroughly by putting a magnetic stir bar in the volumetric flask (after you’ve
filled it to the mark) and stir using the magnetic stirrer.
2. Label your test tubes #1-7. Using two 10ml serological pipets (1 for the stock solution and 1 for
the distilled water), prepare your standards in the test tubes using the dilutions from your
prelab chart. Mix each standard thoroughly with a stirring rod. Wipe off the stirring rod
between each use.
3. Set up the data-collection system.
a. Connect the Colorimeter to the labquest.
b. Turn on the labquest and open the labquest app.
c. On the Meter screen, tap Mode. Change the mode to Events with Entry. Enter the name
(concentration) and the units (µmol/L).
4. Calibrate the Colorimeter as follows:
a. Prepare a blank by filling an empty cuvette 3/4 full with standard #1 (the distilled
water). Be careful to only handle the cuvette on the ridged sides, wipe the outside of
the cuvette with a lint-free tissue before use, and rinse a couple times with distilled
water.
b. Place the blank in the cuvette slot of the Colorimeter and close the lid. Make sure the
light will pass through the smooth side of the cuvette.
c. Press the < or > buttons on the Colorimeter to set the wavelength to your chosen
wavelength. Then calibrate by pressing and holding down the CAL button on the
Colorimeter. When the LED stops flashing, the calibration is complete.
5. You are now ready to collect absorbance-concentration data for the five standard solutions.
a. Start data collection (press the green arrow).
b. Remove the cuvette from your Colorimeter and pour out the water. Using the solution
in Test Tube 2, rinse the cuvette twice with ~1 mL amounts, and then fill it 3/4 full. Wipe
the outside with a tissue, place it in the Colorimeter, and close the lid.
c. When the absorbance readings have stabilized, select Keep and enter the concentration
you calculated in the prelab. Select OK. The absorbance and concentration values have
now been saved for the first solution.
d. Repeat Parts b and c of this step for Test Tubes 3 – 7
e. Stop data collection.
f. To examine the data pairs on the displayed graph, tap any data point. As you tap each
data point, the absorbance and concentration values are displayed to the right of the
graph. Write down the absorbance values and concentrations for each of the data
points in your data table.
6. Determine the absorbance value of the unknown beverage solutions.
a. Using 10ml serological pipets and medium test tubes, make the dilutions of your
beverages that you decided upon in your prelab.
b. Rinse the cuvette twice with each of your beverage dilutions (starting with the most
dilute solution) and fill it about 3/4 full. Wipe the outside of the cuvette, place it into the
Colorimeter, and close the lid.
c. Monitor the absorbance value. When this value has stabilized, record it in your data
table.
d. If the absorbance readings of all the dilutions are greater than 0.8, make more dilutions
until the absorbance reading is below 0.8.
7. Discard the solutions as directed by your instructor.
8. Display a graph of absorbance vs. concentration with a linear regression curve as follows:
a. Choose Graph Options from the Graph menu.
b. Select Autoscale from 0 and select OK.
c. Choose Curve Fit from the Analyze menu.
d. Select Linear as the Fit Equation. Select OK. The best-fit linear regression line will be shown
on the graph for your five data points.
9. On the Graph screen, choose Interpolate from the Analyze menu. Find the absorbance value
closest to the absorbance reading you recorded in Step 6. The corresponding concentration
value is the concentration of the food dye in your diluted samples, in µM.
10. Save the graph of absorbance vs. concentration on a flashdrive as your period # and your names
or initials.
Calculations / Conclusions:
1.
What is the concentration of your dye in each of the 3 beverages you tested? Remember you
diluted your sample, so you need to calculate the concentration in the undiluted beverage
2. Which beverage had the greater concentration of the dye?
3. Using one of your data points for your standards, use Beer’s Law to calculate the molar
absorptivity of your dye. (remember that the concentrations in the lab are in µM. Change to M
to use in the Beer’s Law formula). The path length of the sample is 1cm. Does this value come
close to the value given in pre-lab question #4? Do a percent error with the value given in the
pre-lab as the accepted value.
4. Are the absorbance values you got for your standards higher or lower than what you predicted
in pre-lab question #4? What might be the cause of the difference?