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Chem. Educator 2013, 18, 269–272 269
© 2013 The Chemical Educator, S1430-4171(13)12512-7, Published 10/18/2013, 10.1333/s00897132512a, 18130269.pdf
Determination of Artificial Dyes in Mountain Dew Products
Jason Hofstein*,†, Michael Murphy†, Nikolas Zagarella†, Kevin Rhoads†, Cynthia Woodbridge‡,
and Robert Williams†
Siena College Department of Chemistry and Biochemistry, Loudonville, NY, jhofstein@siena.edu; Baker College,
Baldwin City, KS
Received August 1, 2013. Accepted September 24, 2013.
Abstract: The concentrations of tartrazine (Yellow #5), Red #40, and Blue #1 were successfully determined for
seven flavors of Mountain Dew using the method of constant volume standard addition. These data were used to
determine the extent of possible health risks involved with drinking Mountain Dew by comparison to the
acceptable daily intake (ADI) values and other research. The range of concentrations for the six varieties of
Mountain Dew containing Yellow #5 ranged from 1.3 to 32 ppm. Of the four varieties containing Red #40, the
concentrations ranged from 2.2 to 39 ppm. For two varieties containing Blue #1, the concentrations were both
below 2.5 ppm. The experimental procedure was developed to teach students the method of standard addition and
to illustrate an example of the matrix effect. After doing this experiment, students should also have a better
appreciation of the concentrations and the potential health dangers of artificial dyes found in their diets.
Introduction
Colorants in various foods and drinks have been
commonplace for hundreds of years but it was not until
recently that synthetic color additives have been used
extensively. Tartrazine, which is also known as Yellow #5, is a
synthetic yellow azo dye used in many foods and drinks
products [1]. There are a multitude of other dyes which are
commonly used in food and drinks, the most common of which
are Yellow #6, Red #40 and Blue #1. In recent years, it has
been shown that consumption of these synthetic dyes can lead
to multiple health risks [1].
One of the health effects associated with tartrazine is the
disruption of biochemical markers in vital organs such as the
liver and kidney. A study done by K.A. Amin et al. found that
at low doses of tartrazine there were significant changes in the
hepatic and renal parameters [2]. These effects were magnified
when taken larger doses, where they induced oxidative stress
by the formation of free radicals [2]. In addition, increased
concentrations of aromatic amines, some of which are known
carcinogens, were detected in the urine of test animals
following the introduction of azo dyes [3].
Reproductive performance and sperm counts are have also
been observed to be effected in laboratory rodents that
consume substantial quantities of artificial dyes in their diet. In
a study by Saidi et al. mice were orally treated with levels of
tartrazine based on 0, 0.1, 1.0, and 2.5% of their body weight.
The reproductive performance was shown to have been
adversely affected in a group that received 2.5% tartrazine
when compared to the control group (p < 0.01). In addition, the
litter size was significantly reduced in all three experimental
groups in comparison to the control. Furthermore, sperm
counts were significantly reduced for the mice administrated
2.5% tartrazine (p < 0.01) and the sperm concentrations in the
* Address correspondence to this author.
† Siena College Dept. of Chemistry and Biochemistry, Loudonville, NY.
‡ Present address: Baker College, Baldwin City, KS.
epididymis were reduced significantly in all three experimental
groups [4].
Lastly, behavioral problems in children have also linked to
the consumption of artificial dyes such as Red 40 and Yellow
5. In a blind study done by McCann et al., parents and teachers
recorded the behavior of children who were either being
treated with mixtures of artificial dyes or part of a control
group [5]. In addition, older children between 8 and 9 years
old were given a computerized attention test. The findings
clearly demonstrated a difference in behavior between the
control and experimental groups. The children who were given
the artificial dye mixture showed a significant increase in
“hyperactivity” which included behaviors such as
inattentiveness, impulsivity, and over activity [5].
From a pedagogical standpoint, Mountain Dew is an ideal
candidate for teaching students about the uses of Beers’ Law,
standard curves, standard addition, and the concept of a sample
matrix in spectrophotometric analyses. After obtaining an
absorbance spectrum of the analyte, the analytical wavelength
(max) is determined for the analysis. At this wavelength, a
standard curve is prepared by measuring the absorbance of
samples of known concentrations. The plot of absorbance
versus concentration (mol/L or ppm) yields a straight line that
follows the equation A = bc, A is absorbance, is the (molar)
absorptivity coefficient, b is the pathlength (in cm) and c is
concentration. A solution of unknown concentration (of
analyte studied) is determined by taking an absorbance
measurement and subsequently calculating a concentration
using the line equation determined by the standards. This
procedure is followed by students to determine the amount of
tartrazine in Mountain Dew by the standard curve method.
However, this protocol assumes that the only analyte in
Mountain Dew absorbing at the analytical wavelength is
tartrazine in an aqueous medium. This is obviously not the
case, and there are several ingredients in Mountain Dew that
can be responsible for partial light absorption at max. This
effect is called the “matrix effect” and is defined as “a change
in the analytical signal caused by anything in the sample other
270 Chem. Educator, Vol. 18, 2013 Hofstein et al.
© 2013 The Chemical Educator, S1430-4171(13)12512-7, Published 10/18/2013, 10.1333/s00897132512a, 18130269.pdf
than analyte” [6]. Using the method of constant volume
standard addition, known amounts of a concentrated standard
is added to a known volume of a solution that is unknown in
analyte concentration. The “spiked” unknown solutions are
then diluted to a constant volume creating a consistent matrix.
The increased absorbance (at max) over that of the unspiked
unknown allows for the determination of analyte concentration
in the original unknown. By having the students compare the
results obtained using the standard curve method versus that of
standard addition, a direct observation of the matrix effect is
realized.
This research quantifies tartrazine, Red #40 and Blue #1 in
several varieties of Mountain Dew by the determination the
concentrations of each dye using the method of constant
volume standard addition. The developed method is used in an
analytical chemistry experiment dealing with standard
addition. While it is not within the scope of the research
project, potential health problems that could arise from
consumption have been noted.
Experimental
Both standard curve and standard addition experiments used
tartrazine standards prepared from tartrazine purchased from
ACROS Chemicals (89% pure catalog number AC19189–
1000). Degassed water was used to prepare a working solution
of 6.1 10–3 M (this is a specific concentration, but any value
close to this will work, so long as you know the concentration),
which was then used to make a diluted stock solution by
diluting the working solution 1:10. This stock solution was
then used to prepare solutions for the standard curve analysis.
The same stock solution was also used to prepare the standard
addition solutions. Several types of Mountain Dew were
purchased at a local retail store (Baja Blast was purchased
from a local Taco Bell) and used in the analysis, including
regular Mountain Dew, diet Mountain Dew, Mountain Dew
Code Red, Game Fuel – Cherry-Citrus (Halo 4 Edition), Baja
Blast, and Livewire. Each soda sample was degassed by
placing the soda in a vacuum flask under reduced pressure for
up to 60 minutes until the carbonation was removed. The “flat”
soda samples were then used without further treatment.
Absorbance measurements were performed on a Vernier
Software & Technology SpectroVis Plus and data was
collected using Logger Pro 3.8.4.
The procedure used for this work is very similar a wonderful
procedure posted online by Dr. Lawrence McGahey at The
College of St. Scholastica [7]. Changes to that procedure were
as follows: For the standard addition analysis, flat soda was
transferred by volumetric pipette to each of the six labeled 25
mL volumetric flasks. The volume of Mountain Dew added
was adjusted so the blank absorbance was between 0.03 and
0.1 at the max for the target dye, which was done so as to
reduce the amount of observed scattering in an undiluted
Mountain Dew sample – see Figure 1 and associated text).
Then additions of 0 (sample blank), 1, 2, 3, 4 and 5 mL of
standard at a concentration around 30 ppm (values can vary -
see above), and fill each to volume with distilled water. The
concentration of standard added raised the absorbance around
0.1 for each mL added.
A max of 421.6 nm was used for the tartrazine analysis,
504.3 nm for red #40, and 620.5 nm for Blue #1. The same
cuvette was used through the standard addition run, rinsing
thoroughly between readings. The procedure was repeated to
verify the results and evaluate the uncertainty in absorbance.
Additionally, this work spawned a capstone laboratory
experience, where students were asked to develop their own
procedure to solve situational problems.
Results
It was alluded to previously that the concentration of
Mountain Dew in a sample directly contributed to the amount
of observed scattering in an absorption experiment, artificially
enhancing the absorption signal at the analytical wavelength.
This would lead an experimenter performing a standard curve
analysis to think that the sample in question contained more
Yellow #5 than it really does. To show the effect of this
scattering, the absorption spectra of both diluted and undiluted
Mountain Dew were taken and superimposed on the same
wavelength scale, as can be seen in Figure 1. It is immediately
evident that there is more than just Yellow #5 absorbing at
421.6 nm. It is this scattering, or the matrix effect, that makes
standard curve analysis of a turbid solution like Mountain Dew
difficult to perform.
One then can use constant volume standard addition to
lessen the effect of the solution matrix. To illustrate the effect
of scattering caused by the turbidity of the undiluted soda
matrix, Figure 2 shows two standard addition plots on the same
scale. The blue diamonds show the averaged data for an
undiluted sample of Mountain Dew and the red boxes show
averaged data for a 2:25 dilution of Mountain Dew in degassed
water. The diluted sample data is linear, with an R2 value of
0.99985, where the undiluted sample data is not linear. This
nonlinearity can be directly attributed to the matrix effect. It is
for this reason that the amount of Mountain Dew in the
standard addition sample blank was adjusted accordingly so as
to minimize the matrix effect, but still allow for quantifiable
absorbance at the analytical wavelength.
The determination of concentration for Yellow #5, Red #40
and Blue #1 was successfully completed and shown to be quite
precise. Of the seven varieties of Mountain Dew studied, six
contained Yellow #5 in varying concentrations. The most
concentrated (in Yellow #5) was found experimentally to be
Game Fuel, which had a concentration of 32 ppm with a
standard deviation of 2.1 ppm. Furthermore, concentrations of
Livewire, Code Red, Regular Mountain Dew, Diet Mountain
Dew and Baja Blast were found to be 13, 7.7, 4.1, 4, and 1.3
ppm with standard deviations of 0.89, 1.3, 0.39, 0.51 and 0.068
ppm, respectively. These data can be found in Table 1.
Red #40 dye was present in four of the seven varieties of
Mountain Dew and was experimentally found to have the
highest concentration in Game Fuel at 38 ppm with a standard
deviation 2 ppm. The concentrations for Code Red, Livewire
and Voltage were found to be 25, 39, and 2.2 with a standard
deviation of 0.95, 1.8 and 0.14, respectively. These data can be
found in Table 2.
Blue #1 was present in both Voltage and Baja Blast and
found to have concentrations of 2.2 and 0.72 ppm with
standard deviations of 0.11 and 0.02 ppm, respectively. These
data can be found in Table 3.
Discussion
The data collected was observed to be precise as evidenced
by the low standard deviation values; however, no
Determination of Artificial Dyes in Mountain Dew Products Chem. Educator, Vol. 187, 2013 271
© 2013 The Chemical Educator, S1430-4171(13)12512-7, Published 10/18/2013, 10.1333/s00897132512a, 18130269.pdf
Table 1. Yellow-5 data for six varieties of Mountain Dew (MD)
Reg. MD Diet MD Code Red Game Fuel Baja Blast Livewire
n 36 10 10 18 10 10
Average Conc. (ppm) 4.1 3.1 7.7 32. 1.3 13
Std. dev.
(ppm)
0.4 0.5 1.3 2.1 0.068 0.89
Table 2. Red #40 data for four varieties of Mountain Dew
Code Red Game Fuel Voltage Livewire
n 10 10 10 10
Average Conc. (ppm) 25 38 2.2 39
Std. dev. (ppm) 0.95 2 0.14 1.8
Table 3. Blue #1 data for two varieties of Mountain Dew
Baja Blast Voltage
n 10 10
Average Conc. (ppm) 0.72 2.2
Std. dev. (ppm) 0.020 0.11
0
0.1
0.2
0.3
0.4
0.5
0.6
350
400
450
500
550
600
650
700
750
Absorbance
(A.U.)
Wavelength
(nanometers)
Diluted
Mt.
Dew
Undiluted
Mt.
Dew
λ
ma
x
=
421.6
nm
Figure 1. Plot of absorbance versus wavelength for diluted and
undiluted Mountain Dew samples. Note the pronounced matrix effect
at the analytical wavelength.
Figure 2. Standard addition plot for averaged diluted and undiluted
Mountain Dew samples (five runs averaged. Standard error is reported
in the error bars). The marked nonlinearity for the undiluted sample is
a direct result of a matrix effect at the analytical wavelength.
manufacturer or literature data could be found to confirm
accuracy. An experimental value from a lab experiment in
Microspectral Analysis i-LAB Academic Package where 10
mL of Mountain Dew was spiked with a Tartrazine standard
[8] resulted in a concentration of tartrazine in regular Mountain
Dew of 1.3 ppm but no standard deviation was given. The
discrepancy between our experimental value of 4.10 ppm and
this value is likely due to the matrix effect stemming from their
larger amount of Mountain Dew used (10 mL instead of the 1
mL per 25 mL used in this work). We also performed a
constant volume standard addition experiment instead of their
singularly spiked standard addition. This allowed for more
sample runs and let us compare our trials and determine the
effectiveness of our data more efficiently.
The concentrations of artificial dyes in each Mt. Dew
variety, along with the total artificial dye concentration, was
determined and plotted in Figure 3. These data can be used in
further investigation into the potential health implications of
the consumption of Mountain Dew. The highest total
concentration of dyes in was found in Game Fuel, totaling 70.0
ppm. However, this is far lower than the acceptable daily
intake (ADI) of Yellow #5 (5 mg dye/kg body weight/day) and
Red #40 (7 mg/kg bw/day).
Conclusions
This experiment may serve as a laboratory experiment for
General or Analytical Chemistry effectively illustrating the
method of standard addition and provides an example of a
matrix effect. In addition, the procedure incorporates basic
laboratory techniques including performing dilutions,
preparing standards, UV-Vis spectrometry, along with various
safety precautions. Analytical calculations such as unit
conversion, concentration determination, and linear regression
analysis are also reinforced.
Although all of the total dye concentrations found in our
work are well below the ADI for adults, more research needs
to be conducted to look into the long-term potential health
complications associated with the consumption of artificial
dyes. In addition, particular attention needs to be made about
children consuming large quantities and Mountain Dew and
potential developmental complications involved. Further work
needs to be done to explore the concentration of artificial dyes
272 Chem. Educator, Vol. 18, 2013 Hofstein et al.
© 2013 The Chemical Educator, S1430-4171(13)12512-7, Published 10/18/2013, 10.1333/s00897132512a, 18130269.pdf
Figure 3. Concentration of Yellow #5, Red #40 and Blue #1 in seven
varieties of Mountain Dew.
in other food sources. These findings could lead to stricter
regulations regarding the use of artificial dyes in food and
beverages and improve the health of millions of people.
Acknowledgement. We gratefully acknowledge financial
support from Siena College. In addition, we would like to
thank the analytical chemistry students at both Siena College
and Baker University for their patience, understanding, and
hard work.
Supporting Materials. One supporting file is available. The
Capstone Laboratory as used at Baker University
(http://dx.doi.org/10.1333/s00897132512a).
References and Notes
1. Gao, Y.; Li, C.; Shen, J.; Yin, H.; An, X.; Jin, H. Journal of Food
Science 2011; 76(6), 125–129.
2. Amin, K. A.; Hameid, H. A.; Elsttar, A. H. Food and Chemical
Toxicology 2010; 48, 2994–2999.
3. Cerniglia, C. E.; Zhuo, Z.; Manning, B. W.; Federle, T.W.; Heflich,
R. H. Mutat. Res. 1986; 175 (1), 11–16.
4. Mehedi, N.; Ainad-Tabet, S.; Mokrane, N.; Addou, S.; Zaoui, C.;
Kheroua, O.; Saidi, D. Am. J. Pharm. & Toxicol 2009; 4(4), 130–
135.
5. McCann, D; Barrett, A.; Cooper, A.; Crumpler, D.; Dalen, L.;
Grimshaw, K.; Kitchin, E.; Lok, K.; Porteous, L.; Prince, E.; Sonuga-
Barke, R.; Warner, J. O.; Stevenson, J. Lancet 2007, 370, 1560–
1567.
6. Harris, D. C. Quantitative Chemical Analysis, 7th Ed.; W. H. freeman
and Company: New York, 2007; pp 87.
7. http://faculty.css.edu/lmcgahey/web/Analytical/Lab%20Manual/Tartr
azine%20%20Analysis%20Calibration%20curve%20only.pdf
(accessed 1/3/2012).
8. http://www.microspectralanalysis.com/TechnicalDownloads/MSA%
20Academic%20Brochure-Student%20Manual.pdf (Accessed
1/30/2012).
