Analysis of Caffeine Content in Red Bull Energy Drink Using High-

Performance Liquid Chromatography

Dennis Wrin
Department of Chemistry, Coastal Carolina University, Conway, SC
Experiment designed and performed in collaboration with lab partner Cameron Hance

November 19, 2013
2
Abstract
The purpose of this experiment was to make an accurate comparison between the
experimental and manufacturer provided caffeine contents in the Red Bull energy drink by
measuring its concentration. The design of the experiment implemented three trials of spiked
sample solutions prepared using a constant volume standard addition method. Test samples were
then processed through a reversed-phase HPLC instrument application. The experiment resulted
in a suggested caffeine content of 72.5 mg per 250ml can of the energy drink. Through
appropriate data analysis it was determined that the experimental and known value, printed on
the tested can size as 80 mg, statistically agree with each other. Accepted results suggest that the
manufacturer of the Red Bull energy drink has accurately provided this ingredients content
information to their consumers.
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Introduction
Many of peoples daily lives are spent interacting in an ever demanding society where
efficiency and production are highly valued. In result this concept has created a market where
any food or drug that contains active components focused on boosting the bodys performance
are readily produced, sold, and consumed.
1
A majority of the products filling this demand
contain the natural compound of caffeine, which acts on the body as mild stimulant. This
abundance in consumed goods is related to its naturally occurring status in many plant species
found across the world. All of these factors combined with its low risk in moderation have led to
caffeine being the most popular and used drug in the world. The amount contained however
varies widely from product to product and is not required by the FDA to be listed on nutrition
panels.
2
Methods used to determine low level concentrations in complex mixtures, such as those
produced for human consumption, require analytical techniques that employ separation and
accurate measurement. The method used for preparation of the solutions used for testing must
also take into consideration the complex nature of the sample.
One of the most common analytical technique used in a lab for separating, identifying,
and measuring components in a liquid state sample is High-Performance Liquid Chromatography
(HPLC). The instrument used combines separation and analysis of sample components into a
single process, this is not only efficient but also leads to less error from interference. The HPLC
method can vary, but is determined based on the component of interest. For caffeine separation is
achieved by the use of reversed-phase Chromatography, a technique that employs the use of a
non-polar stationary phase and a more polar mobile phase.
3
The HPLC instrument operates by
injecting a small amount of the sample into the appropriately prepared column and then forcing it
through the column under a constant high-pressure flow. The difference in polarities causes each
component to pass through at a different rate, allowing for the measured retention time to be
used as an identifier when compared to a known standard. As each component passes the
detector absorbency is measured and recorded as a peak with a given height and area
corresponding to its concentration in the sample.
4
The standard addition method best used when
composition of the sample is complex and/or unknown. Known quantities of the desired analyte
are added to spike concentrations in a fixed quantity of the unknown and then the increase in
instrument response is measured. Preparing all samples to a constant volume is necessary when
the analytical method being used consumes part of each sample, as in HPLC. The series of
sample solutions should be prepared so that their measurements bracket the unknown in a linear
range.
5

The purpose of this experiment was to measure the amount of caffeine contained in the
Red Bull energy drink using a series of solutions prepared by the method of standard addition
that were then processed through a reversed-phase HPLC instrument application. Because the
product provides a value listed on the can, results were then used to determine if the
experimental caffeine content in a Red Bull energy drink is in agreement with the amount
reported by the manufacturer. The experiment was designed so that the concentration is
approximated and the results are analyzed by statistical comparison to the theoretical value. It
was hypothesized that the experimental content found will be in agreement with the
manufacturers listed amount.




4
Experimental
Preparation of Standard Solution. A standard solution of 0.05M of aqueous caffeine was
determined appropriate for creating the linear range of samples required to measure the
concentration of the unknown present. This was prepared by accurately weighing out 0.972 g of
solid caffeine (analytical balance) that was then dissolved into 100.0 ml of deionized water in a
volumetric flask. Results provided 100.0 ml of a 0.05M aqueous caffeine solution for use as a
standard in the experiment.
Preparation of Sample. To create a linear range for construction of the calibration curve
five sample solutions were required using different dilutions of the standard solution and
constant a constant volume of the unknown measured using volumetric pipets. All five sample
solutions were prepared to a consistent volume of 10.0 ml in volumetric flasks. Prior to creating
testable samples the Red Bull drink had to be prepared for experimental use by removing as
much carbonation from it as possible. This was done by boiling it in a beaker on a hot plate to
expel the dissolved carbon dioxide gas. The desired samples prepared for this unknown
contained a constant volume of 2.00 ml of the decarbonized Red Bull, added to this was the
increasing amounts of standard solution; 0.00 ml, 2.00 ml, 4.00 ml, 6.00ml, and 8.00 ml.
Resulting samples contained the calculated concentrations 0.00M, 0.01M, 0.02M, 0.03M, and
0.04M of standard respectively. This series of five sample solutions was prepared three times to
allow for three trials of HPLC measurements.
Sample Data Measurement. A small amount from each prepared sample was filtered into
appropriately marked autosampler vials. The resulting fifteen sample vials, three trials for each
of the five samples, were then given to the instructor for measurement in the HPLC instrument.
Chromatographic results were then returned to the group providing measured peak information
of caffeine content for each of the fifteen provided vials.
Data Analysis. The resulting data was then subjected to appropriate statistical analysis.
This consisted of calculating the average peak areas, along with the standard deviations, for all
three trials. This allowed for the construction of a single calibration curve which was then used
to determine the initial concentration, including error, of the Red Bull. This value was then
applied to a t-test designed for verifying agreement with the known value posted on the product
packaging.

Results and Discussion
Raw data for the experiment was returned to the group in the form of individual
chromatograms for each autosampler vial that was provided to the instructor. From this
information data analysis began by determining an average and standard deviation, calculated in
Microsoft Excel,
S1
for each all three trials performed. The calculated averages and deviations for
the peak areas reported in the chromatograms are shown below in Table 1.

5

(Table 1)
From this data an equation for a line of best fit was determined using linear regression.
This was produced using the LINEST function
S1
and it returned the following results displayed
in Table 2.


(Table 2)
Consideration was given to the linearity of the calculated line, indicated by the R
2
value,
to assess how well it fits the measurement data from the experiment. Its value should be very
close to 1, which it is in this case. Given values for the slope and y-intercept a standard line
equation is easily derived from this information. Equation 1 shows the linear regressions
corresponding equation in standard form.


(Equation 1)
The construction of the calibration curve for this standard addition procedure was
generated by comparing the concentration of the known standard added versus the area of the
peak in the instruments response for each sample. The calibration line was then extrapolated to
include the x-intercept, which has an absolute value that is equal to the unknowns concentration
of analyte in the spiked solutions. This was calculated by setting the y-value in Equation 1 equal
to zero. The resulting value of the x-intercept is -0.00031, indicating a 0.00031M caffeine
concentration in the diluted test samples.
To determine the uncertainty associated with this result the standard deviation of the x-
intercept (

) was calculated using Equation 2.

||

(Equation 2)
The calculation
S1
returns a value of

equal to +/- 0.001M. Graph 1 shows the final

extrapolated calibration curve with each measurements associated error
Solution Concentration Concentration Average Std dev
# of Known (M) of Known (mg/ml) Retention Area of Area (+/-)
1 0.00 0.000 426500 8000
2 0.01 1.944 6045000 60000
3 0.02 3.888 11500000 100000
4 0.03 5.832 16800000 300000
5 0.04 7.776 24000000 300000
Standard Addition Calibration Curve Data
m 578269927 177605 b
s
m
19330121 473489 s
b
R
2
0.997 611272 s
y
LINEST Function Output:
6

(Graph 1)
To determine the initial concentration of Red Bull ([]

) the prepared samples dilution

factor, given by Equation 3, was applied to the determined diluted analyte concentration ([]

).

[]

) []

(Equation 3)
Where []

is the concentration in the sample (x-intercept), is the total volume of the

sample, and

is the volume of unknown added to the sample. Plugging in measured

experimental values and data results in the value of the initial caffeine concentration that is given
in Figure 1.


(Figure 1)

To compare the experimental value to the theoretical value a t-test was performed by
calculating the 95% confidence interval and checking to see if the theoretical value falls within
it. Equation 4 shows the equation used to determine this interval.

(Equation 4)
The following Figure 2 displays the resulting 95% confidence interval.

{ }
(Figure 2)
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It can easily be suggested that the experimental measurement produces an interval that
does include the manufacturers supplied value on the packaging. It can be stated that the values
statistically agree with each other.
Data analysis was concluded with simple calculations to convert all of the reported
concentrations from molarity units to milligrams per milliliter units (mg/mL). This was done
only with the intention of making the final data and results more relatable to real world
applications. The final results of the experiment, along with the theoretical values, are displayed
in Figure 3.


(Figure 3)

Conclusions
The results from this experiments design provided measurements that agree with the
initial hypothesis. There is a significant agreement between the experimental and theoretical
caffeine amounts in the energy drink. From this it can be determined that the manufacturer of
Red Bull has accurately reported their products caffeine content on its packaging. With this
accurate information consumers can readily know the amount of caffeine entering their body
when consuming a can of this energy drink. It can also be concluded that the design of this
experiment was well suited for testing the hypothesis and producing appropriate data for
analysis.
Supporting Information
Workbook S1. A majority of calculations used in this experiment were done using Microsoft
Excel. All supporting calculations for data analysis are included in this file,
Analysis_of_Caffeine_Report.xlsx
















Experimental Theoretical
0.0015M 0.0016M
0.29 mg/ml 0.32 mg/ml
72.5 mg per
250 mL serving
80.0 mg per
250 mL serving
Summary of Results
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References
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Scientific. [Online]. http://www.jenway.com.
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University: Northridge, CA, 2012; p 32.
4. Department of Chemistry, MNState. Chemistry 380 Laboratory Manual; Determination of
Caffeine by Solid Phase Extraction and High Performance Liquid Chromatography;
Minnesota State University: Moorhead, MN. http://web.mnstate.edu.
5. Harris, D.C. Quality Assurance and Calibration Methods. Quantitative Chemical Analysis,
8
th
ed.; W.H. Freeman: New York, 2010; p 106.