Determining Concentrations of

Red Dye No. 40 in Powerade

CHEM 101
Sakhaya T

November 14, 2023

Date Performed: October 30, 2023

Partners: Kaan C
Clara P
Instructor: Professor R

1 Results and Discussion

In order to determine how much Red Dye #40 is in a given solution, we used spec-
troscopy to measure the absorbance of different concentrations of the dye. Then
using that data in conjunction with spectrometric absorbance readings for our tar-
get solutions, Fruit Punch and Orange flavored Powerade, we were able to calculate
those mystery concentrations of Red Dye #40. The results are discussed in what
follows.

1.1 Determination of Molar Absorptivity based on Absorbance

measurements
Starting out with a pure sample of the dye, we first aim to calculate the Molar Ab-
sorptivity (which should remain constant) The first step of our experiment involved
doing a series of dilutions, for each of which we measured the peak absorbance
( _;0F ) of the dye. After calculating the concentrations of dye for each dilution, we
were able to calculate the Molar Absorptivity using Beer’s Law:

_
Y_ =
:⇠

A table of these findings is shown below.

1
 Dilution (Dye to 2 $) Dye Concentration (M) Absorbence ( _;0F )* Molar Absorptivity (Y_ )
0.25 6.058 ⇥ 10 5 1.185 ± 0.002 19560.911
0.125 3.029 ⇥ 10 5 0.679 ± 0.002 22416.639
.0625 1.515 ⇥ 10 5 0.362 ± 0.002 23894.389
0.0313 7.573 ⇥ 10 6 0.176 ± 0.002 23240.460
0.0157 3.786 ⇥ 10 6 0.094 ± 0.002 24916.359
*error shown is standard deviation calculated from a sample of 6 peak absorbance readings

Table 1: Spectrometer Readings for a series of Red Dye #40 Dilutions

This table shows the individual values of Molar Absorptivity, however we can
calculate an average by using Beer’s law with the averaged concentration and aver-
aged _;0F values:

Mean Concentration Mean _;0F Resulting Y_

2.34758 ⇥ 10 5 0.4992 21264.451

Table 2: Averaged Concentration and Absorbence and the Resulting Molar Absorp-
tivity

As we can see in Table 2, the average Y_ value is 21264.451. This constant

will later be used to determine Concentrations in our sports drink samples. Below
we see the Concentration and Maximum absorbance values plotted in the form of a
Standard curve.

2
Figure 1: Standard curve: Maximum Absorbance as a Function of Dye Concentra-
tion.

In generating this table we included the point (0,0) to show that the baseline
absorbance of a 0M concentration of dye was 0. Error bars are included for Ab-
sorbance measurements, showing a standard deviation of 0.002 which is too small
to see. For this plot, '2 = 0.994 , which shows that Absorbance has strong linear
dependance on dye concentration.
Something worth noting about our method in collecting data for our standard
curve is that rather than measuring the absorbance at a specific fixed wavelength,
we at each reading recorded the peak absorbance, regardless of the wavelength. We
reasoned that this would give us a better representation of the absorbances of our
dye, as it was not dependent on one initial reading to determine a _ ;0F
The next step in our process was to find the wavelength of maximum ab-
sorbance, which would subsequently be used to determine the amount of Red Dye
#40 in our Powerade samples. This is important because without specifying a wave-
length for Red Dye #40 during the measurement of our samples, it would be unclear
whether other dyes or substances in the drink are interfering with our maximum
Absorbance measurements.

3
1.2 Determining Concentrations of Dye in Powerade using Ab-
sorbance at a fixed wavelength

0.8

0.6
Absorbance

0.4

0.2

0
360

380

400

420

440

460

480

500

520

540

560

580

600
Wavelength [ nm]

Plot

Figure 2: Absorbance Spectrum of a 1:4 water dilution of Red 40

Figure 2 shows that when a spectrometer was used to record Absorbances and
Wavelengths for a 1:4 dilution of Red 40, we saw that peak Absorbance was at a
wavelength of roughly 500 nm. This is our _ ;0F value at which to measure the
absorbances of each Powerade sample.

1.2.1 Method 1: Beer’s Law

Measuring a 1/2 dilution of red Powerade three times at _ = 500<; yielded a mean
Absorbance of 1.3707, While measuring a 1/2 dilution of orange Powerade three
times yielded a mean Absorbance of 1.381.

4
 Using these values, we were able to use Beer’s law in the form:

0D6,500<;
⇠@43 40 =
Y_

to calculate the Concentrations of Red Dye #40. These results are shown in Table
3. Once again the variable of length can be disregarded because the the length is a
constant 1cm.

0D6,500<; ⇠@43 40 ( " )

Red Powerade 2.741 1.289 ⇥ 10 4
Orange Powerade 2.762 1.299 ⇥ 10 4

Table 3: Absorbances and Concentrations of Red Dye#40 in our Powerade samples

1.2.2 Method 2: Using Linear Regression

How we originally planned to calculate our Concentrations was to simply use the
line equation generated by Google Sheets in Figure 1: 0D6,_;0F = 19557⇠ + 0.0334.
Inputting our average absorbance values for the two Powerades gives us the con-
centrations:
2.741 0.0334
⇠ '43 ( " ) = = 1.384 ⇥ 10 4
19557
2.762 0.0334
⇠$@0<64 ( " ) = = 1.395 ⇥ 10 4
19557
While these concentration values are slightly higher than our results for Method
1, they are very close, (within 0.1 ⇥ 10 4 of each other). Because it gave such close
values, it can be said that Beer’s law does indeed hold in this case.

1.3 Comparison to Literature Values

During my search of the internet, I was unable to find any credible sources giving
the exact concentrations of Red 40 in Powerade, however I came across a study
including a data sheet for various drinks with masses of total AFCs (Artificial Food
Colorings) per 240mL serving, showing that there are 17.4mg per 240mL 1 . Since
Red Dye #40 is listed as the only coloring agent in the nutrition facts for "Fruit
Punch" flavored Powerade, it should be possible to calculate the concentration from
this. I used stoichiometry to convert:

17.4;6 6 1;=: ;=:

= 0.725 ⇥ = 14.54 ⇥ 10 4
240;! ! 498.426 !
2
This concentration is greater than our calculated concentrations by roughly a fac-
tor of 10. This could mean that there is in fact another dye present in Fruit Punch
1 Stevens LJ, Burgess JR, Stochelski MA, Kuczek T. Amounts of Artificial Food Colors in Commonly

Consumed Beverages and Potential Behavioral Implications for Consumption in Children. Clinical Pedi-
atrics. 2014;53(2):133-140.
2 Molarity of Red 40 is 498 6
;=:

5
Powerade (as it was noted that they used many wavelengths to test Absorbance, for
multiple dyes) It is also possible that a calculation error was made either by us or
them.

1.3.1 How much Powerade is too much?

The ADI (Acceptable Daily Intake) listed for Red Powerade is 7mg/kg/day. When
we calculated the ADI for a 140lb person, using our Concentration values for Red
Powerade from Table 3, the result was 6.92L/day. This means that drinking more
than 6.92 liters of "Fruit Punch" Powerade is not acceptable and could be harmful.

2 Sources of Error
The main sources of error in this experiment came from the slight glassware un-
certainty in our volume measurements during our initial dye dilutions (affecting
the concentrations data), and the fluctuations in Absorbance measurements on the
spectrometer. Based on the fact that the spectrometer constantly fluctuates and is
inherently less accurate than precision volumetric glassware, I would infer that the
Absorbance data is a larger source of error.
The dilutions were made solely using two kinds of glassware: 10mL Volumet-
ric flasks (uncertainty 0.02mL) and 5mL Volumetric pipettes (uncertainty 0.01mL).
For Volume a simple addition gives us an uncertainty of 0.03mL. The relative Error
calculated for Absorbance is much larger: 0.313 (See Appendix for calculation),
which confirms my inference.

6
3 Appendix
3.1 Initial Procedure

Figure 3: Our initial procedure as written out on Oct. 30, 2023 and approved by
Prof. Rawle

7
3.2 Sample Calculations

Figure 4: Calculations for determining ADI

8
Figure 5: Relative Error of Absorbance Measurements