Page 1 of 7 (Too Much??) Learning Objectives: Caffeine in Energy Drinks Preparation of energy drink sample for testing Separation of caffeine from other components in energy drinks using HPLC (high performance liquid chromatography) Determination of the amount of caffeine in the sample Calculation of the quantity of caffeine in the whole serving of energy drink The Problem Caffeine is a common ingredient in drinks people use to wake them up or provide energy. Caffeine occurs naturally in coffee but is added to drinks like cola, Red Bull, Monster, and other energy drinks. While some energy drinks tell you how much caffeine is present, others (including coffee) don t. Our job is to determine the quantity of caffeine in several common drinks. Background Caffeine is not the only component present in coffee, Red Bull, or other energy drinks. In order to determine the amount of caffeine present, we must first separate it from all the other components in the drink. We do this by using HPLC (high performance liquid chromatography). During the experiment, the instructor will explain how HPLC separates components of a mixture. The structure of caffeine is shown below: Caffeine Molecular Formula = C 8 H 10 N 4 O 2 Molecular Mass = 194.19
Page 2 of 7 Equipment and Materials: Each partner station needs: Listing of sample for prep and dilution scheme above station (see attached sheet for dilution scheme of all samples) Appropriate air displacement pipet and tips (instructions for electronic versions are in drawer #61 opposite LCs) 2 appropriate volumetric flasks and stoppers Minimum 6 HPLC autosample vials and tops (in drawer #61 opposite LCs) Water squeeze bottle Erlenmeyer flask with water from the carboy over the sink, labeled WATER Plastic transfer pipets, minimum 10 Waste container Pencil/marker (for labeling HPLC vial) Reserve caffeine standard (~38 ppm) is in cabinet Experimental Procedure Preparing the Sample 1. Using an air displacement pipet, transfer the sample to a volumetric flask. 2. Carefully fill the flask to the mark with water from the squeeze bottle. Use a plastic transfer pipet to add the final amount. 3. Seal the flask with a stopper. 4. Place your thumb on the stopper, invert the flask and gently swirl. Repeat this 5 times. 5. Use a new plastic transfer pipet to fill an HPLC vial ½ full. 6. Record the information for you sample in the table below: Sample Name Sample Amount (in ml) Dilution Volume (flask size in ml) Inputting Data into the HPLC Data System 7. Place the HPLC vial into the autosampler, noting the vial location number 8. Input sample name, sample amount, and dilution volume into the run sequence file. 9. Place your vial in the autosampler in the position listed in the sequence file.
Page 3 of 7 Once all the samples are in the autosampler and the sample information is added to the run sequence, the HPLC will be started and the samples will be analyzed. Calculating the Amount of Caffeine in a Serving 10. As each sample is analyzed a report will be printed that provides the amount of caffeine (in mg) in 1 ml of that energy drink. 11. This data will be entered into a spreadsheet to determine the mg of caffeine in a serving using the following equation. mg caffeine in 1 ml X number of oz in serving X 29.57 = mg caffeine in serving Applying Your Knowledge 1. Which sample had the most caffeine per ml? Which has he most per serving? 2. For sample with listed amounts of caffeine, how do your results compare to the label amount? 3. Why do different energy drinks have different serving sizes? If a can of energy drink says 2 servings, is mg per serving really appropriate? 4. Which samples appear to contain other components that the HPLC separates? If we did not separate out these components from caffeine, would our results be accurate? Why? Instructor Notes: The experiment requires an instructor with a minimum one assistant for 3 or 4 partners, and 2 assistants for 5 or 6 partners. NOTE: Make sure the night-light is on over the sink. If not, reset the GFI outlet so the sink drainage pump functions properly. Setting up the HPLC System: The following are the HPLC conditions using HPLC 35: Detector (switch on left rear of unit) at 275 nm, 0.05 RSP HPLC pump (switch on right side front) using Method #03 (1.95 ml/min, 100% mobile phase A). If not on Method #03, press F6 key (DIR) and arrow up or down to Method #03. Press F4 key (RCL) and recall Method #03. (NOTE: Method #06 is a copy of Method #03)
Page 4 of 7 Autosampler using Method #01 (vials 1-8, 15 ul, 3.0 min run time). If not Method #01, use similar procedure as described for the pump. (NOTE: Method #06 is a copy of Method #01) Vacuum degasser (switch on ride side the rear of the unit) Mobile phase A = 31/69/0.2 methanol/water/phosphoric acid Syringe wash = 30/70 methanol/water Caffeine standard (position 1 in autosampler) Before starting up the pump, open the front left door on the pump, place a syringe on the fitting, open the valve by turning CCW. Use the Purge key and set flow rate to 3 ml/min. Purge for 1-2 min. Press STOP. Close valve, remove and empty syringe and press key F8 (STRT). Watch to see that the pressure only varies by ~50 70 psi after a minute or so. If the fluctuation is higher, purge the pump again to remove trapped air. This start up procedure only needs to be done at the beginning of the day. NOTE: if the screen becomes unfamiliar, the QUIT key will bring it back to the beginning condition. At the beginning of the day, press the FLUSH key on the autosampler to remove air from the syringes. Setting up TurboChrom Data Acquisition: The computer should be set up and running. If so, pressing the admin key gets you to the TurboChrom program screen. If the computer has shut off, follow the procedure below: Turn on computer Go to the TCNav icon Login with a manager logon and managerx password ( where X is the current number listed near the keyboard) Status may start with Port Comm Error in red, will become No Method in red after 2-5 min. If not, shut off computer and start again The data acquisition system uses Sequences and Methods. The naming convention for sequences is as follows: Today s date color of team as in 0425green So 0425green is April 25 th and green team. Using this as an example, there will be 4 sequences for April 25 th, 0425red, 0425yellow, 0425green, and 0425blue. The data files in each of these sequences will also follow the same convention of date and team color. To find the sequences (and to save them correctly), open the Sequence Editor (Build Sequence) and go up one directory level. It is in the folder marked Caffeine Data. The entire hierarchy from the C-drive is as follows: C:PenExe/TcCS/Ver6.3.2/Caffeine data
Page 5 of 7 Copies of all the caffeine files (caffeine.seq, caffeine.mth, and caffeine.rpt) are found in the Templates directory: C:PenExe/TcCS/Ver6.3.2/Templates All the working caffeine files must reside in the Caffeine Data directory. Introducing the Experiment: The instructor will bring all the students to the back wall monitor and present the caffeine PowerPoint and the web link chromatography separation animation. Once this is complete (3 4 minutes maximum), the team partners are positioned at the six workstations. NOTE ON TIMING: With a full group, there are 8 injections (six team samples, a standard and a blank) that run ~3.4 minutes from injection to injection. That means that this takes ~28 min for the run once the sequence is filled out and the samples are loaded. To be complete within a 45 min timeframe, the introduction and ALL sample prep and data input must be done in 18 min. It can easily be done, but any discussions need to occur AFTER the start of the HPLC run. Running the Experiment: Set up with one assistant covering two separate team partners, each preparing one sample. The assistant will show the team partners how the air displacement pipet works, how to actually pipet the sample, and how to dilute to volume. This is done initially using the squirt bottle with the last few drops added with the plastic transfer pipet. The prepared sample will be loaded into an HPLC autosampler vial (only fill 2/3 of the way) and then taken to the HPLC to input the sample information (sample name, sample volume, vol flask volume) into the sequence. Once this is done, the vial will be placed into the autosampler in the appropriate position. Once all the samples are entered into the sequence file, it is saved and the Sequence Editor MUST be closed prior to initiating Instrument Setup on the screen. (This also goes if either the Method Editor or the Report Editor is opened. They must be closed for the data system to initiate setup.) The autosampler is set for running 8 injections. If you are running less, then the autosampler must be reset for the lower value. Press the F2 key (METH). Using the arrow keys, move to the LAST column and enter the number for the last injection. And the yellow ENTER key. Once the value is accepted, press the F6 key (STOR) and the unit will give you a new method number. Input 1 and when the unit asks if you want to overwrite the method press yes (the #1 key) and when it asks for a new name press no (the #0 key). Click Instrument Setup and browse the Caffeine Data directory for the correct date and team color sequence. Load it, and be sure that lines 1 through 8 are listed for loading (or 1
Page 6 of 7 through the number of rows filled out in the sequence. Approximately 30 sec or less after the Instrument Setup is closed, the status screen will change color to grey-blue and say System Ready. If this does not happen, confirm that the sequence, method, and report editors are closed. Once closed, the Instrument Setup must be re-initiated as above. Once the system is ready, press Start on the autosampler to begin the run. Click on Real Time Plot to open a screen that allows viewing of the chromatogram as it is generated. Open the Sequence Editor and the current file as Read Only to be able to see what the upcoming sample is. NOTE: If you miss inputting the correct number of injection during Instrument Setup, after the last autosampler injection, go to Run on the toolbar and Clear Setup. Go to the instrument PE-LC under Lab to clear and return to the red No Method status. Discussing the Experiment: Once the run is ongoing, the following points can be discussed: What the void volume is How the blank is the water used to dilute the samples and the need to prove it is free of caffeine The differences in serving size, i.e. both sizes of Red Bull are listed as 1 serving, but the Monster can is 2 servings (and how can that be? Would you drink half a can and then share it with someone else?) How chromatography is needed to separate all the unpronounceable ingredients in the drinks, while UV spectrophotometry can be used with the single component analgesics in the disso experiment How some of the drinks list an Energy Blend concentration that contains several ingredients, but don t list specifically how much caffeine is present How coffee has more caffeine than the other drinks, but some people find the drinks have more of an action. May be due to the other ingredients present. How the students results compare to the label claim for Red Bull How the energy drinks have different peaks that are due to the different ingredients they contain. How if we obtained standards for each ingredient, we could find out what the other peaks we see are The fact that all these drink manufacturers have a QC department and the job of testing every batch of drink made How this is similar to drug testing by pharmaceutical companies Anything else that seems appropriate.
Page 7 of 7 Sample Dilution for Caffeine Analysis Sample Name Sample Amount (in ml) Dilution Volume (flask size in ml) Red Bull 1 10 5 Hour Extra Strength 0.2 25 Monster Energy 2 25 WORX Extra Strength 0.5 50 N.O.-Xplode 0.5 25 Starbucks Regular Coffee 1 20