Analysis of Caffeine Content in Red Bull Energy Drink Using HighPerformance Liquid Chromatography 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
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 people’s 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 body’s performance 1 are readily produced, sold, and consumed. 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 spe cies 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 2 panels. 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 anal ysis 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 3 non-polar stationary phase and a more polar mobile phase. 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 4 corresponding to its concentration in the sample. 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 5 range. 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 manufacturer’s listed amount.
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Experimental Preparation of Standard Solution. A standard solution of 0.05M of aqueous caffeine was determined appropriate for creating the linear range o f 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 p roviding 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 pe ak 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 p rovided 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.
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Standard Addition Calibration Curve Data Solution
Concentration
Concentration
Average
Std dev
# 1 2
of Known (M) 0.00 0.01
of Known (mg/ml) 0.000 1.944
Retention Area 426500 6045000
of Area (+/-) 8000 60000
3 4 5
0.02 0.03 0.04
3.888 5.832 7.776
11500000 16800000 24000000
100000 300000 300000 (Table 1)
From this data an equation for a line of best fit was determined using linear regression. S1 This was produced using the LINEST function and it returned the following results displayed in Table 2. LINEST Function Output: m
578269927
177605 b
sm
19330121
473489 s b
0.997
611272 s y
2
R
(Table 2) 2
Consideration was given to the linearity of the cal culated line, indicated by the R 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 regression’s 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 instrument’s 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 unknown’s 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 xintercept ( ) was calculated using Equation 2.
S1
|| √ ∑ ̅
(Equation 2)
The calculation returns a value of equal to +/- 0.001M. Graph 1 shows the final extrapolated calibration curve with each measurement’s associated error 5
[ ]
(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 b y 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.
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(Figure 2)
It can easily be suggested that the experimental measurement produces an interval that does include the manuf acturer’s 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. Summary of Results Experimental 0.0015M 0.29 mg/ml 72.5 mg per 250 mL serving
Theoretical 0.0016M 0.32 mg/ml 80.0 mg per 250 mL serving (Figure 3)
Conclusions The results from this experiment’s design provided measurements that agree with the initial hypothesis. There is a significant agreement between the ex perimental 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 ca ffeine 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 p roducing 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
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References 1. Reid, T.R. Caffeine-What's the Buzz?. National Geographic. [Online] 2005, January, Excerpt. http://science.nationalgeographic.com. (accessed October 11, 2013). 2. Jenway Scientific Equipment. The quantitative determination of caffeine in beverages and soft drinks using UV wavelength spectroscopy; Technical R eport for Bibby Scientific. [Online]. http://www.jenway.com. 3. Department of Chemistry, CSUN. Chemistry 321 Laboratory Manual; California State 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 Anal ysis, th 8 ed.; W.H. Freeman: New York, 2010; p 106.
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