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Chemistry 315

Experiment 7: Determination of Caffeine and Vitamin B6 in Energy Drinks using High-Performance Liquid Chromatography*
In this experiment you will calculate the amount of caffeine and vitamin B6 (pyridoxine hydrochloride) in an energy drink using the standard addition method by high performance liquid chromatography (HPLC). Required Reading 1. B. L. Karger, HPLC: Early and Recent Perspectives, J. Chem. Educ. 1997, 74, 45. DOI: 10.1021/ed074p45 2. Chapter 26, An Introduction to Chromatographic Separations, Skoog, et al. 3. Chapter 28, High Performance Liquid Chromatography, Skoog, et al. 4. Section 1D-3, Standard Addition Methods, Skoog et al. or 5. Section 5.1, Fundamentals of Chromatographic Separations, pp. 159-170, et al. 6. Section 5.3, Liquid Chromatography, pp. 185-208, Kellner, et al. 7. L. R. Snyder, Modern Practice of Liquid Chromatography, Before and After 1971, J. Chem. Educ. 1997, 74, 37. 8. Section 12.2.7, pp. 743-744 (standard addition method), Kellner et al.
*Based on the procedure outlined in: Leacock, R. E.; Stankus, J. J.; Davis, J. M. Simultaneous Determination of Caffeine and Vitamin B6 in Energy Drinks by High-Performance Liquid Chromatography (HPLC). J. Chem. Educ. 2010, 88, 232-234.

Kellner

Introduction Energy drink consumption has increased exponentially since the beverages were first introduced in the 1960s. Compared to a traditional cup of coffee, which contains between 77 and 150 mg of caffeine, energy drinks can have as much as 505 mg of caffeine per can. The United States Food and Drug Administration (FDA) set a limit to the amount of caffeine a standard soda can contain of 200 parts per million (ppm) for a standard soda.1,2 In addition to providing the standard nutritional information on the label of a food or beverage product, it is important to be able to quantify the amounts of toxic substances, such as caffeine3 present in consumer products prior to marketing. Highperformance liquid chromatography (HPLC) will be used to simultaneously determine the amount of caffeine and vitamin B6 (pyridoxine hydrochloride) present in an energy drink and compare the results to the amounts printed on the label. Basic Chromatographic Concepts Chromatography allows for the separation of analytes from a complex sample matrix based upon their adsorption affinities to a support. There are many different methods of performing chromatographic separations, but all depend upon the partition of an analyte between the mobile phase and stationary phase. The classification of chromatographic techniques is often divided into two major groups: gas chromatography and liquid chromatography. For this experiment, the main focus will be on liquid chromatography, but the same general principles apply to gas chromatography. In standard column chromatography, a liquid solution is poured through a column containing silica gel. The solvent acts as the mobile phase and carries the analytes of interest down the column due to gravity while the silica gel acts as the stationary phase. Because silica gel contains alcohol groups, polar components have higher affinities for adsorbing to the stationary phase and elute from the column later than those components that are nonpolar. The time required for a component to elute is termed the retention time. Thin layer chromatography follows the same principles as column chromatography, but the mobile phase is drawn up the side of the film by capillary action, rather than downward due to gravity. The use of a polar stationary phase is known as normal-phase chromatography, but reverse-phase chromatography, containing a non-polar stationary phase, is much more common. Size exclusion and ion exchange supports can also be used to separate chemical compounds in column chromatography. The ability for a given column to separate peaks is known as the column efficiency and can be quantified as the height equivalent of a theoretical plate (HETP). This is often related to the length of the column according to the equation: where N is the number of theoretical plates, L is the length of the column in cm, and H is the height of the theoretical plates in cm. This provides a useful number for comparing different columns when considering which one to use for a given separation. Experimentally, this can be calculated according to the following equation: (

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where tR is retention time in seconds and W1/2 is the width of the peak at half of the height of the peak in cm. An additional parameter useful for characterizing separations is resolution, or the distance between two peaks relative to the widths of the peaks. This may be calculated as [( ) ( ) ] where A and B are two different components, tR is their retention times in s, and W is the width of the peaks in cm. Components that elute at very similar times or that have overly broad peaks will exhibit very poor resolution. Optimization experiments are often performed to increase the resolution of a separation. Such experiments include changes in solvent viscosity, changes to flow rate, and even changes in temperature over the duration of the elution. Chromatography, in itself, is not a detection technique. It is often coupled to some type of detector, such as a mass spectrometer or, in the case of this experiment, a UVvisible spectrometer. High-Performance Liquid Chromatography (HPLC) Due to the dependence on gravity for flow, column chromatography requires a significant amount of time to complete a separation and can exhibit poor resolution between components as the effects of longitudinal diffusion become more prominent. In high-performance liquid chromatography, we take advantage of smaller stationary phase particle sizes which provide more surface area for components to adsorb. A high-pressure pump is required to draw the eluting solvent through the smaller pores, however, increasing the cost of the instrumentation. The expense one must consider is offset by the vast information one is able to obtain from HPLC, though, since it has the ability to resolve different components from a very complex sample matrix, such as an energy drink which contains high fructose corn syrup, food coloring, and various other additives. The HPLC column material in this experiment is nonpolar (reverse-phase) porous silica with covalently bonded organic side-chains (C18). Differences in binding affinity of the vitamin B6 and caffeine to the C18 groups located on the column allow for higher resolution between the two components in this experiment. For this experiment, an isocratic elution will be performed using a mobile phase that consists of a 1:1 water:methanol + 1% acetic acid solution. The HPLC system in the lab is set up so that a UV-visible detector, set at 292 nm, monitors the elution of the analytes. Standard Addition Because the sample matrix of the energy drink is so complex, the use of internal standards cannot be used since there is no guarantee that certain peaks will not overlap with each other. We will therefore explore the standard addition method in order to determine the concentration of caffeine and vitamin B6 in the energy drink. In the method of standard additions, one spikes the unknown sample with known concentrations of a standard solution. By plotting the added concentration versus peak area or peak height, one can extrapolate the best-fit line determined through linear regression analysis to the x-axis. The absolute value of the x-intercept is the actual concentration of the unknown. 3

Example:
70000 60000 50000 Peak Area 40000 y = 1000x + 20000 R = 1

Unknown Conc 20000 = 20 ppm


10000 0

30000

-30

-20

-10

10

20

30

40

50

Standard Concentration (ppm)

References (1) Nutrition, C. for F. S. and A. FDA Basics - Why isnt the amount of caffeine a product contains required on a food label? (2) Reissig, C. J.; Strain, E. C.; Griffiths, R. R. Caffeinated Energy Drinks -- A Growing Problem. Drug Alcohol Depend 2009, 99, 1-10. (3) Peters, J. M. Factors Affecting Caffeine Toxicity. The Journal of Clinical Pharmacology 1967, 7, 131 -141.

Prelab assignment 1. Look up the MSDS for caffeine, pyridoxine hydrochloride, and methanol. What are the hazards associated these chemicals? What actions should be taken should these come into contact with your body (e.g. your skin/eyes)? 2. Calculate the required amounts in grams of caffeine and pyridoxine hydrochloride to create the stock solutions. What are the concentrations in mg/L and molarity? 3. Complete the following table according to the experimental procedures: Sample Volume Caffeine Stock (mL) Volume Pyridoxine HCL Stock (mL) Total Volume (mL) Standard Standard Caffeine Pyridoxine Concentration HCl (ppm) Concentration (ppm)

1 2 3 4 5 4. Draw a block (functional) diagram of the HPLC apparatus. 5. Draw the structure of the exterior of a silica gel particle for the column used in this procedure. 6. Explain three reasons for band broadening in a chromatogram. Be sure to discuss the van Deemter equation. 7. Which of the compounds (caffeine or pyridoxine HCl) do you expect to elute first? Why?

Materials Equipment: Column Agilent ZORBAX Eclipse XDB-C18 Guard Column Agilent ZORBAX Eclipse XDB-C18 Pump PerkinElmer Flexar Isocratic LC Pump Detector PerkinElmer Flexar UV-Vis LC Detector 7 100 mL volumetric flasks (for sample solutions) 1 5 mL volumetric pipet 1 1 mL volumetric pipets 1 2 mL volumetric pipets 1 3 mL volumetric pipets 1 4 mL volumetric pipets 2 100 mL Erlenmeyer flasks (for mixing standards) 1 stirring rod (for quantitative transfer between flasks) 1 50 mL beaker (for pipet rinse) 1 50 L blunt-tipped syringe Chemicals: Caffeine Pyridoxine hydrochloride Methanol (in 20 mL scintillation vial for column rinse) Distilled water Miscellaneous: Energy Drink Nitrile gloves Pasteur pipettes/bulbs (for topping off volumetric flasks)

Procedure Special Notes: Make sure to clean ALL glassware between each sample injection or dilution. Everything except methanol may go down the drain. There is a waste container provided for methanol waste. Turn on the instrument and start the LC Pump when you first enter to allow for proper equilibration

Standard Addition Preparation: 1. Prepare two stock solutions, one of approximately 650 ppm caffeine and the other of approximately 500 ppm pyridoxine hydrochloride, in 100 mL volumetric flasks. The weighed amounts do not have to be exact since you can calculate the actual concentration of your final dilutions using the mass you record. Bring to volume with de-ionized water. Ensure adequate mixing by inverting the volumetric flasks after capping the top with the provided ground glass stoppers. 2. Pipet 5 mL of the energy drink to each of five 100 mL volumetric flasks. Pipet the appropriate amount of stock solution to each flask according to the table below: Sample Caffeine (mL) Pyridoxine HCL (mL) 1 0 0 2 1 1 3 2 2 4 3 3 5 4 4 Bring to volume with de-ionized water. Ensure adequate mixing by inverting the volumetric flasks after capping the top with the provided ground glass stoppers. Instrument Setup 1. Turn on the UV-Vis detector and the LC Pump by clicking the buttons on the front of the instrument (the detector needs to be held down until it turns on). Open the Chromera Manager software. Double-click Honeybadger under labuser in the Configuration window. Wait for Chromera to load. 2. Under Control Mode on the left of the screen, make sure Sequence is selected. 7

3. Go to File > Open Sequence. Click the plus sign next to Sequence Group in the window that opens. Select Experiment 7 and then click Open. 4. Verify the Method and Sequence being used: a. Click on Method on the bottom-left of the screen. Go to File > Open Method. Click the plus sign next to Method Group in the window that opens. Select Experiment 7 and then click Open. Print the Method using the default options. b. Click on Sequence on the bottom-left of the screen. Go to File > Open Sequence. Click the plus sign next to Sequence Group in the window that opens. Select Experiment 7 and then click Open. Print the sequence using the default options. c. Compare the edited dates on the printed sheets to those taped to the desk. If they do not match, inform your TA. DO NOT continue the experiment until these dates match. 5. Press the Play button ( ) under Sequence and wait for the Instrument window to

open. If the pump status is listed as Shut down, click Start LC Pump in the Control Panel on the right of the screen. Wait for the pump to equilibrate if necessary. Once you get the Waiting for manual injection message and you have verified that the pump pressure is stable around 2600-3000 psi, you are ready to continue. You will not have to pause or stop the sequence at any point unless you make a mistake. 6. Load the blunt-tipped syringe with the caffeine stock solution (slightly more than 20 L) and remove any air bubbles by dipping the syringe into the sample solution and quickly pumping the syringe by moving the plunger up and down until all bubbles are removed. 7. Make sure that the valve on the left side of the detector is in the 8 oclock LOAD position and insert the syringe into the small, white circular opening on the front of the valve. Push the needle in until the glass part of the syringe touches the white opening. There may be some resistance toward the end. 8. Push in the plunger on the syringe and remove the syringe from the valve. This LOADS the sample into the valves sample loop.

9. Quickly turn the valve clockwise toward the INJECT position. This injects the sample into the HPLC column, zeroes the detector, and starts the sequence on the computer. The sequence will take 10 minutes to complete. When the run is finished and the chromatogram resets, turn the valve toward the 8 oclock LOAD position.

Loop injector valve switching patterns. The load position allows the loop to be filled with sample. The inject position injects the contents of the loop onto the column.

10. The pump may need to equilibrate between runs. Check the status window on the bottom right of the screen to ensure that the Sequence Status is Running, the Detector Status is Ready, and the Pump Status is Ready before continuing. 11. Repeat steps 6-10 with the Vitamin B6 stock solution, the standard addition samples, and methanol rinse according to the order below. For the methanol steps, only inject 20 L of methanol (provided in the scintillation vial) in order to clean the column. 1. Caffeine 2. Vitamin B6 3. Sample 1 4. Methanol 5. Sample 2 6. Methanol 7. Sample 3 8. Methanol 9. Sample 4 10. Methanol 11. Sample 5 12. Methanol

Make sure to rinse the syringe with the sample to be injected between each run to prevent contamination. The order is very important because the sequence automatically names each sample. You can check which sample the program is waiting for by simply looking at the list on the screen. If you make an error during an injection, allow 9

the run to finish. Then, stop the sequence by pressing the Stop button ( ). Select the sample you would like to repeat by clicking on the row number to the left of the sample (it is recommended to select the Column Rinse step right before the erroneous sample to clean the column first). Once selected, click the Start Row button ( ). All of the samples above the selected one should turn grey. Then, ) and continue with the sample you selected.

click on the Play button (

12. The reports for your runs have automatically been saved in the HPLC Reports folder on the desktop. Ensure that they have been saved and that each file contains a summary of the data collected as well as an image of the chromatogram. You may print the PDFs or save the Excel files to a flash drive to work with later. You must attach each of your standard addition sample chromatograms and the final column rinse chromatogram to the back of your report. 13. Click Stop LC Pump in the Control Panel and exit Chromera. Right click on the Chromera icon on the bottom right of the screen, and close the Chromera Manager. Turn off the pump module and the detector by holding down the power buttons on the front of the instruments. 14. Clean your work station. Only solutions containing energy drink, caffeine, and pyridoxine hydrochloride may go down the drain. Methanol must go in the waste container. Rinse all glassware with distilled water. For your Laboratory Report: Results: a) Identify the retention times of caffeine and pyridoxine hydrochloride. b) Prepare plots of the areas for the caffeine peaks and the peak heights of the caffeine peaks versus their concentrations in each sample. c) Prepare a plot of the peak areas of the pyridoxine hydrochloride peaks versus their concentrations in each sample. d) Perform a linear least squares analysis on the data to find the equations of the best fit lines. Also, include the R2 correlation values. e) Find the concentrations of caffeine and pyridoxine hydrochloride in the energy drink in ppm, mg/L, and molarity.

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f) Compare your findings to the reported values on the can. (WolframAlpha [http://www.wolframalpha.com] is able to calculate percent daily values on the can to a useful number) g) Calculate the number and height of the theoretical plates using your data and the column information provided on Compass for caffeine. h) Using the vitamin B6 peak and the closest neighboring peak in Sample 5, determine the resolution between the peaks. Where tR is the retention time and W is the width of the peak at the base, where B is the second species eluted. Use the ruler to determine the width of the peaks if necessary. Questions: 1) Why are peaks visible when a component elutes, rather than just vertical lines? How does the use of peak heights instead of peak areas for the determination of the caffeine concentration affect your results? Does this introduce any errors? If so, what were they? 2) Calculate the error between your findings and the concentrations listed on the can. Discuss possible reasons for any differences. Is HPLC a suitable platform for this type of measurement? 3) What are the implications of using a UV-visible detector to monitor the elution of analytes? In other words, what are some errors that may be introduced into the measurement? What are some benefits? 4) Additional Chemical Engineering Question: How might you improve this experiment to obtain higher resolution between peaks? Be sure to discuss such factors as column construction and mobile phase composition.

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