Introduction Chromatography is a process of separating mixtures based on a differential distribution of solutes as they pass through a stationary and mobile

phase []. Liquid chromatography is a form of chromatography employing a liquid as the mobile phase and a solid or a as the stationary phase. In particular, one example of liquid chromatography is High Performance Liquid Chromatography (HPLC). In this method the separation is conducted with the aid of high pressure. The pressure forces a solvent through a closed column containing fine particles. This method is very efficient and produces high resolution statistics for analyses. As a result this technique it is applicable in the food industry as well as biochemical fields. Coffee, Coca-Cola, Pepsi and Energy drinks are some of the most ingested beverages on a daily basis. They are consumed due to the energy boost they provide to the human body. The source of this boost is chemical called caffeine. Caffeine is a white xanthine alkaloid crystalline powder with the chemical formula C8H10N4O2. Caffeine causes changes in the chemicals of your brain, mainly in 2 ways. It mimics adenosine, and binds to all the adenosine receptors in your brain. This prevents the real adenosine from doing its job, which happens to be the slowing down of nerve impulses and the causing of drowsiness. As a result of this effect, it has become very popular among university students who need to stay awake for long hours in order to study. The caffeine content of these energy drinks can be determined by employing HPLC analysis. Problem A significant number of the university student population consume various energy drinks to prolong their study periods. This is due to the contents of the drink; of which caffeine is included. Caffeine is the main stimulant which is responsible for the expected surge of energy experienced by the consumer. However, the specific amount of caffeine contained in the energy drink is not readily known as it often not printed on the container. The recommended daily intake of caffeine for adults is 300mg. However, a problem may arise as one is not completely aware of the amount of caffeine being ingesting before consumption. Hence the consumer is ignorant to how much caffeine he/she is having with respect to the recommended daily caffeine intake.

Hypothesis The amount of caffeine contained in Full Throttle Energy Drink is not similar to the recommended daily intake of 300mg. Objectives Determine the concentration of caffeine in Full Throttle Energy Drink using both the calibration curve and standard addition techniques. The separation will be carried out under isocratic conditions. This means that the elution will be at a constant solvent composition. Determine the extraction efficiencies by spiking the sample with appropriate volumes of standard caffeine prior to extraction. Determine the detection limit and the linear operating range of the analytical machine.

Methodologies i. The gross sample of Full Throttle to be analyzed is 473 ml of energy drink which contains approximately 100mg of caffeine according to the product website. ii. iii. From this approximately 25 ml will be taken out from which our sample will be prepared. The 25ml of sample will then be prepared as follows: a. The sample will be degassed for the removal of all gases. b. The sample will then be filtered through a 0.45m syringe filter by a 10ml filter with the initial 1ml being dispensed as waste. c. The syringe will then be rinsed with 2-3ml aliquots of a 75:25 DI water/methanol solution. d. The sample will then be stored in a 250ml pre-cleaned (soaked in 1M HCl over a week) plastic bottle. iv. The following caffeine standards will be prepared from a ppm stock solution stored in a pre-cleaned plastic bottle. The final [standard] will be reached by dilution with the 75/25 methanol/solution.

Standard # 1 2 3 4 5 v.

Mass of Caffeine/g

Pipette Size

Vol. Flask

Dilution [Standard]/ppm Factor

vi.

Each standard solution will be filtered through a 0.45m syringe filter with a 10ml syringe filter. The initial 1ml will be dispensed into waste. The filtrate will be then stored in volumetric flasks. The syringe should be cleaned with the water/methanol solution. Analysis of Standards and Samples The flow rate of the HPLC system should be set at 1.5ml/min The solvent composition should be the same as that in the sample and standard 75/25 water/methanol solutions. The sample loop should be cleaned before injection a. The syringe should be rinsed with several aliquots of the solvent Flush about 250l of mobile phase though to clean the loading passages twice. The syringe should be rinsed with several aliquots of the sample to be injected, before Injection there should be no bubbles in the syringe. The samples and standards will then be injected as per the HPLC operating manual.

vii. viii. ix. x. xi. xii.

Results 1. The caffeine standards will be used to identify the caffeine peak. 2. Record the retention time and area of the caffeine peak for each of the standards. The peak area will increase from the lowest standard to the highest. 3. The retention time of the caffeine peak will be used to determine if caffeine is present in the beverage samples. 4. If the caffeine is present, record the retention time and area of the caffeine peak. Analysis of Data Use the data obtained for the standard solutions to prepare a calibration curve The calibration curve will be used to determine the concentration of caffeine in the sample. The spike samples will be used to prepare a standard addition graph from which the concentration of caffeine in the sample will be determined. The errors in the method will be analysed and reported.

1. 2. 3. 4.

References 1. Oswegu University Website, www.oswegu.edu/kadina/CHE425L 2. Westminster College Website 3. Petracco, M. 2005. Our everyday cup of coffee: The chemistry behind the magic. Journal of Chemical Education, 82, 1161-1167 4. http://coffeetea.about.com/cs/caffeine/a/caffeinefacts.htm 5. www.drinkfullthrottle.com 6. Harris, D.C. 2007. Quantitative Chemical Analysis, 7th Edition. W.H. Freeman and Company, New York. Chapter 25.

Activities Plan for the Semester Week 3 - Prepare the Sample, Degas and Filter and Store. Week 4 Prepare the caffeine standards; prepare stock solution and the aforementioned standards. Prepare sample to fit in calibration curve. Week 5 Run initial HPLC analysis of first set of prepared standards and sample and use the data for the determination of caffeine concentration via the calibration curve analysis. Week 6 Run second HPLC analysis of second set of spiked samples for the standard addition method for the determination of the caffeine concentration. Week 7 The data compiled in Weeks 6 & 7 be analyzed and a draft copy of the final written report worked on, planning for the oral presentation will begin. Week 8 The written report will be finalized and prepared for submission. The plan for the Oral Presentation will be finalized and rehearsed. Week 9 The Oral Presentation of the findings of the experiment

Activities Plan for Week 3 Degassing of the 473mL Sample: Sanjo Edwards Dornicqah Goulbourne This will be carried out by measuring 5 portions of approximately 95mL and shaking them in 250mL volumetric flasks.

Filtering of the Sample: Inkiru Bernard Sanjo Edwards Dornicqah Goulbourne This will be done with the aid of 10mL syringes.