Quantitative Analysis of Caffeine in Energy Drinks by High Performance Liquid Chromatography CHEM 329 Professor Vogt TA: Fahad Hasan Allison Poget Date Performed: April 5, 2016 Date Submitted: April 12, 2016 ABSTRACT In this experimental, techniques of high performance liquid chromatography (HPLC) were introduced through the quantitative analysis of caffeine in Red Bull energy drink. Four standard solutions of varying concentrations are prepared from a pre- prepared stock solution of 0.1667 mg/ml. When the components of the analytical sample has passed through the column and reached the detector, a spectra will result with peak height and peak area information that can be correlated to the concentration of the respective component. In this experimental, the component caffeine is observed and a calibration curve is generated showing the relationship between the concentration (mg/ml) of caffeine in the prepared stock solutions to the resulting caffeine peak areas. From there, a linear trendline and a correlation coefficient is added to the data. Using the trendline equation, the unknown concentration of caffeine in the Red Bull is calculated using the resulting peak area for the caffeine peak of the respective spectra. The caffeine content was calculated to be 0.215 mg/ml in this laboratory and the expected value was 0.313 mg/ml resulting in a 31.3% error.
Quantitative Analysis of Caffeine in Energy Drinks Poget 2 Introduction: In analytical chemistry high performance liquid chromatography, or HPLC, is utilized to measure, identify, and quantify components within a liquid mixture by passing the liquid sample through a column. Each column is packed with a stationary phase through which the analyte sample components will pass at differing rates. Within the HPLC instrument, there is also a liquid mobile phase that will push the analyte sample through the column so that its components will reach the detector to generate a spectra. The varying rates at which the components of the analyte sample elude are a direct result of the partitioning between the packing and liquid mobile phase. This will cause the various components of the analyte sample to elude at various speeds, causing each component to possess a specific retention time on the spectra. This type of analysis will also quantify the amount of each component within the sample by correlating the amount of a component to the peak area on the spectra of that respective component. Advantages to using HPLC for analysis of a chemical include that it provides high resolution, sensitivity, accuracy, and speed. Because of these and many other advantages, HPLC has been utilized in the field of pharmacology and pharmaceuticals to identify drug components and measure the concentration of the respective components [1]. This analytical instrumentation is being used as a means of quality assurance in this field. Additionally HPLC has been utilized in the fields of water quality testing. Pollutants in water can be detected using HPLC, and once identified and quantified, they can be eliminated with respect to the type of pollutant identified in the water sample source [2]. Farmers, city officials, and commercial fish farmers are among the many who utilize HPLC technology as a means of water quality testing because of the speed and accuracy of the instrumentation [2]. HPLC has also been used by food scientists working for companies such as the Food and Drug Association (FDA) to analyze the concentration of water- soluble vitamins and minerals within a food sample. Other components like cholesterol, sugars, and flavanoids can be identified and quantified using this method of chemical analysis [1]. Ultimately, HPLC is a widely used chemical analysis technique that has applications in a plethora of real world situations. The necessity of these quality assurance proceedings is that the producer of a good must ensure that their customers are getting exactly the product promised by the product label. These quality assurance testing method are utilized to check a random sample of the products produced in order to ensure that the concentration of the components that are listed on the label are in fact contained within the sample in the package. One type of product that is highly regulated by the FDA because of its potential negative effects to the consumer is the realm of energy drinks [1]. Because energy drinks contain such a high level of stimulant chemicals, there are increased potential risks for the consumers of such a product [2]. In this experiment, the concentration of caffeine within a can of Red Bull energy drink is quantified using HPLC instrumentation. First, a calibration curve is developed using prepared standards of decreasing concentration. All standards are prepared from the same stock solution containing caffeine. The peak areas will be analyzed as they relate to the concentration of caffeine in the respective samples. This experimental will introduce HPLC techniques as well as refine skills of utilizing a calibration curve in analytical chemistry.
Quantitative Analysis of Caffeine in Energy Drinks Poget 3 Experimental: The HPLC solvent is prepared with 70% methanol and 30% water (HPLC grade). By placing the solution in a sonicator, it is degasses to remove the air bubbles that may exist in the solution. Additionally a 0.2 micron filter is used to remove the larger particles that could exist within the solution. In order to prepare the standards of the unknown solution, ~0.016 g of pure caffeine is added to a 100- ml volumetric flask and then diluted to the line with HPLC grade water to generate the stock solution (0.16 mg/ml) which will be used in the rest of the experimental. Labeling five 10- ml volumetric flasks for the respective standard value, and using a micropipette, four calibration standards of known concentration and one unknown energy drink solution were prepared with information from the following table of dilutions. Sample Type Volume of Stock Solution (ml) Volume of HPLC grade water (ml) Standard 1 10 0 Standard 2 7.5 2.5 Standard 3 5 5 Standard 4 2.5 7.5 Unknown (Red Bull) 1.5 (Raw Energy Drink) 8.5 Table 1: This table contains the information for dilutions of the solutions generated for the standards as well as the unknown. Using five new, clean 10- ml volumetric flasks and a syringe, the prepared standards were filtered through a 0.2 micron filter. Additionally, each prepared sample was degassed in the sonicator for 5 minutes. These steps must be performed prior to running the HPLC column in order to eliminate potential sources of error and to make the column run more accurately and precisely so that more definite conclusions may be made from the data and the results. In order to conserve time, the teaching assistant performed all of the aforementioned proceedings. After all the samples were prepared for analysis by HPLC, the instrument was initialized under the instruction of the teaching assistant. The initialization will take care of any gas bubbles that could occur in any of the instrumentation lines and will pass an HPLC solvent through the column for 15 minutes starting at 1.5 ml/min and regressing to 1.25 ml/min. The reverse phase C18 column (5 micrometer) is used as the column in this HPLC experimental. The UV/Visible detector was monitored during this initialization process to ensure that there was a steady absorbance response appearing at 254 nm. Following the initialization, 100 microliters of Standard 1 was injected into the column slowly. This action was performed twice, each time carefully ensuring no air bubbles were in the syringe prior to injection into the column to remove potential sources of error. The standard was allowed to pass through the column for 10 minutes, and the resulting spectrum contained a major caffeine peak around 1.9 minutes. A window of retention time from 1.85-2.15 minutes was set to record all peaks within a region around 1.9 minutes in order to ensure that the major caffeine peak was recorded. In this, the resulting spectra indicate that there were multiple caffeine peaks because it recorded any peak within the set window to represent caffeine when in fact, the only peak truly
Quantitative Analysis of Caffeine in Energy Drinks Poget 4 representing caffeine is the major peak within this window. The spectra file was saved, and this process was repeated for all of the standards prepared as well as the prepared unknown solution. The spectra were then analyzed by generating a table containing peak area for each sample and using this information to prepare a calibration curve in excel of peak area on the y- axis and caffeine concentration (mg/ml) on the x- axis. This data should generate a linear calibration curve whose line of best fit can be used to identify the concentration of the unknown solution. The line of best fit and R 2 values are also to be reported for the plot. The information about the caffeine concentration listed on the can of Red Bull is used to calculate the proposed concentration of caffeine in the energy drink, and the resulting value is compared to the experimental value calculated. Data: Conc (mg/ml) Peak Area Standard 1 0.1667 detection limit Standard 2 0.125025 1982.4021 Standard 3 0.08335 1480.8521 Standard 4 0.041675 1036.4020 Unknown 0.032193286 919.2838 Table 2: This table contains the concentrations (mg/ml) of the standards as well as the calculated unknown concentration and the corresponding peak areas from the HPLC spectra. Peak Area vs. Concentration (mg/ml) 2500.0000 2000.0000 y = 11350x + 553.89 R² = 0.99879 Peak Area 1500.0000 1000.0000 500.0000 0.0000 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 Concentration (mg/ml) Figure 1: This is the graphical depiction of the relationship between the concentration (mg/ml) and the respective peak areas from the resulting HPLC spectra. Intercept: 553.89; Slope: 11350; R 2 : 0.99879
Quantitative Analysis of Caffeine in Energy Drinks Poget 5 Figure 2: This spectrum results from running the Standard 2 solution through high performance liquid chromatography. Figure 3: This spectrum results from running the Standard 3 solution through high performance liquid chromatography.
Quantitative Analysis of Caffeine in Energy Drinks Poget 6 Figure 4: This spectrum results from running the Standard 4 solution through high performance liquid chromatography. Figure 5: This spectrum results from running the Red Bull solution of unknown concentration through high performance liquid chromatography.
Quantitative Analysis of Caffeine in Energy Drinks Poget 7 Calculations: Concentration of caffeine in diluted energy drink Equation of line from the plot: y = 11350x + 553.89 Peak Area from the spectra of the Unknown Sample: 919.2838 919.2838 = 11350x + 553.89 x = (919.2838-553.89)/11350 = 0.0322 mg/ml Concentration of caffeine in energy drink Concentration of caffeine in diluted energy drink: 0.0322 mg/ml C1 =? V1 = 1.5 ml C2 = 0.0322 mg/ml V2 = 10 ml Expected concentration of caffeine in energy drink from label Information from label of one can of Red Bull: 111 mg/12 fl oz. (12 fl oz)*(29.574 ml/1 fl oz.) = 354.888 ml 111 mg/354.88 ml = 0.313 mg/ml C1V1 = C2V2 (C1)*(1.5 ml) = (0.0322 mg/ml)*(10 ml) C1 = 0.215 mg/ml Comparison of expected and experimental caffeine concentrations in the unknown Red Bull solution Experimental caffeine concentration in the unknown solution: 0.215 mg/ml Expected caffeine concentration in the unknown solution: 0.313 mg/ml Percent Error = [(accepted- experimental)/(accepted)]*100 = [(0.313-0.215)/(0.313)]*100 = 31.3% error Results and Discussion: In this experimental, a pre- prepared stock solution containing caffeine was prepared by the TA of concentration 0.1667 mg/ml. From this the four standards prepared of concentrations 0.1667 mg/ml, 0.125025 mg/ml, 0.08335 mg/ml, and 0.041675 mg/ml for Standards 1, 2, 3, and 4 respectively. Standard 1 resulted in a peak area that exceeded the detection limit, so this value was omitted from further use and analysis in the calibration curve. This is because since the peak was greater than the detection limit allowed, the peak area listed on the resulting spectra was not representative of the whole peak area. If this value were not omitted from the calibration curve, there would be a great deal of error in the correlation and the resulting linear trendline. This would yield more error in the calculation of the concentration of the unknown. Standard 2, 3, and 4 resulted in peak areas of 1982.4021, 1480.8521, and 919.2838 respectively. These peak areas were plotted against the values reported for the concentration in mg/ml. The trend of this plotted data was linear, so a linear trendline was fit for the data. The equation of the linear trendline was y = 11350x + 553.89 with a slope of
Quantitative Analysis of Caffeine in Energy Drinks Poget 8 11350 and an intercept of 553.89. The correlation coefficient was R² = 0.99879. This correlation coefficient indicates the closeness of relation of the data points. The closer to unity this value is, the higher degree of relation the data points have to one another and the better representation the trendline is for the data. In this case, the correlation coefficient is extremely close to unity, and thus is a great representation of the data. This is ideal for the line of best fit for a calibration curve because this line will be used to calculate an unknown value, so the more accurate it is initially, the less error one will incur. Using the generated line equation, the peak area of the unknown solution s caffeine peak, which was 919.2838, was plugged into the equation in order to solve for the concentration of the caffeine in the unknown Red Bull solution. It was calculated that 0.0322 mg/ml was the concentration of the diluted Red Bull solution. Utilizing a simple reverse dilution calculation, it was calculated that the original concentration of caffeine in the solution of Red Bull was 0.215 mg/ml. When this experimental value is compared to the expected value of 0.313 mg/ml calculated from the information on the nutritional label, the resulting percent error is found to be 31.3% error. While this is a relatively high percent error value, it is still lower than it could have been. Our experimental was successful because it generated a reasonable value that fell close to the expected value. The difference between the expected and observed data could be explained by a variety of possibilities. One reason could be that the label listed what should be in the can of Red Bull, but the contents contained a drink with a lower caffeine content. Another reason could result from the choice to omit one of the standards. Perhaps if more standards were included in the development of the plot of peak area versus the concentration, the resulting line equation would be that much more representative of the actual trend between the data. This would result in a better line equation and thus a better or more accurate value calculated for the unknown caffeine contents. Additionally, calculations could contain error if too much or not enough rounding is done within computation. If one uses the wrong equations for the wrong parts of the laboratory, this could also negatively impact the accuracy of the lab and contribute to the error and discrepancies between the expected and observed data. If the solutions were not filtered and degassed properly or if they were injected with a syringe containing an air bubble, more error could be added to the experimental negatively impacting the results and increasing the error. Questions: 1. Discuss the reliability of this experiment and possible sources of error. How can you improve this method to overcome these errors and increase the reliability? Possible sources of error include calculation and mathematical error, instrumentation usage error, or error in degassing or filtering. If errors are made at any point during calculation whether the wrong equation is used, the calculation is performed wrong, or the wrong values are used in the wrong parts of the calculation, the resulting calculation values could be off and would negatively impact the validity of the laboratory results. This can be avoided by carefully labeling calculations performed and results achieved. This includes adding units to all numerical values to understand what measurement each value is representing. Instrumentation error could include injecting the sample too fast, choosing an improper mobile or stationary phase for the respective analyte sample, or not preparing the system for analysis prior to adding any solutions. Proper education must be received by machine or instrument user in order to eliminate improper
Quantitative Analysis of Caffeine in Energy Drinks Poget 9 machine usage as a form of error. Additionally, it is vital to the success, accuracy, and precision of this experimental that there be no large particles or air within the solution, so the importance of the sonicator and the filter are priceless. The only way to avoid error that could result from forgetting to use these tools or using them improperly is to make sure the process is fully understood before instrument usages. It is also necessary to eyeball the sample to double check that the sonicator and the filter are performing their respective duties. There could be error in either of these machines or instrumentation that needs to be detected to preserve the accuracy and precision of the experimental at hand. If the system is not calibrated at the beginning prior to any sample analysis, there could be leftover solutions from previous analysis that would contaminate the spectra and cloud our spectra with distracting and misleading peaks. This error can be avoided by remembering to reset the machine and clear the column prior to each analysis. Lastly, it is of the utmost importance to remember to degass and filter the sample analyte to remove distracting and contaminating particles that would negatively impact the solution s motion through the column. 2. The detector is a UV/Vis detector which responds to a fixed wavelength (254 nm) only. Can you detect/identify all the components in your unknown sample at a fixed wavelength? Explain why? You should be able to identify all of the components of the unknown sample, but changing the wavelength could allow for a lower limit of detection and permit you to detect lower concentrations of components that may exist within the analyte sample. Although this is a potential benefit, if a wavelength of less that 220 nm is used, the solvent will typically display some interference here and show some absorbance introducing more noise to the experimental. To a certain extent, the wavelength selected must allow for detection of components while not allowing too much noise to impact the system. 3. Why blank (HPLC grade water) is not required for this experiment? Can we use methanol/water solution instead of HPLC grade water for the dilution step? Explain why? HPLC grade water must be used in this experimental because it has little to no UV absorbance so it will not interfere with the other experimentation, there is little mineral or background interference from this source, and can be run through a sonicator and a filter. This step is performed in order to clean out the column from the last run essentially. A methanol/water solution could be used instead of HPLC grade water because the stationary phase used in this experimental is nonpolar and the polar molecules will not heavily impact the peak areas or retention time. Additionally, mixing the caffeine containing solution with methanol could induce side reaction that would impact the peak areas and the retention times. They would also distract from the resulting spectrum validity because it would introduce more noise to the spectrum. 4. Briefly explain the importance of this type of analysis for quality control. This type of chemical analysis is vital for quality control and assurance experimentation for a company to utilize. For example, a pharmaceutical company
Quantitative Analysis of Caffeine in Energy Drinks Poget 10 must ensure that all of the expected and advertised components are contained within the drug produced. Even more importantly, the company MUST ensure that the proper amount of each component is included in the pill or sample size. Here is where an analytical method like HPLC would prove most vital. Since HPLC will show representative peaks for the components of the solution and the peak area can help to quantify the component in the solution analyte, this is exactly how quality control can utilize this chemical method to keep their products honest. It is also important to the health of their consumers. 5. Explain peaks other than caffeine in the chromatogram of energy drink solution? (Bonus question) The large initial peak likely represents the sugar within the energy drink that may not have been added to the prepared standard solutions, which would explain its absence from those spectra, as seen above. Additional small peaks would likely represent vitamins and minerals included in the solution, one more than likely being Vitamin B6. The vitamins will elude before the caffeine in the spectra because they will have a shorter retention time. The peaks for vitamins including Vitamin B6 are short in height and broad in width. Conclusion: In this experimental, four standards were separately run through a column of high performance liquid chromatography analysis and the resulting peak area values for the caffeine peak were plotted against the concentration of caffeine. The Standard 1 value was omitted because it fell outside of the detection limits of the instrument. The plot displayed a linear relationship represented by y = 11350x + 553.89 with R 2 = 0.99879 indicating great correlation value of the data described by the linear trendline. This trendline is used along with the peak area for the caffeine peak in the diluted sample of Red Bull to calculate the experimental observed value of concentration of caffeine in the Red Bull Sample to be 0.215 mg/ml. When compared to the calculated expected caffeine concentration value of 0.313 mg/ml, a percent error of 31.3% was obtained. This is not as ideal as one would like, but it is more accurate than inaccurate in which case we can assume there must have been experimental errors and latent variables that impacted the experimental determination of the concentration. This type of instrumentation is vital in the realms of commercial quality control of foods, drugs, and other products. In the field of caffeine or energy drinks, this type of instrumentation is widely used because if its speed and accuracy in measurement. This experimental was overall very successful in perfecting skills of calibration curve development and use to the linear trendline to calculate an unknown value. Additionally, this experimental introduced the instrumentation of HPLC. References: [1] Bhanot, Deepak. "Laboratory Training Courses on HPLC, GC, AAS, Lab Safety, Spectroscopy." LabTraining.com. Lab Training, 2012. Web. 11 Apr. 2016. [2] Thomas, G.P. "High Performance Liquid Chromatography Methods, Benefits and Applications." High Performance Liquid Chromatography Methods, Benefits and Applications. AZO Materials, 13 Dec. 2013. Web. 11 Apr. 2016.