1. Team 7 Terence ChoyGurinderpal Doad Sheetal GandhiNicole Hair Patricia LiPulsar Li Caitlin OksienikBryan Shilowich Michael SwitaDaniel Wunderlich Advisors: Jean Barmash and Amy Katz Sweet Mysteries: An HPLC-Analysis of the Active Ingredients in Chocolate

2. History Aztecs began eating chocolate Spaniards took the idea and sweetened it with sugar Chocolate spread throughout Europe Variations of chocolate were created In 1895 Hershey perfected the mass production of chocolate

3. Active Ingredients in Chocolate Caffeine C 8 H 10 N 4 O 2 (trimethylxanthine) Stimulant Increases Heart Rate Increases Mental Awareness

4. Caffeine Dancing

5. Active Ingredients in Chocolate Theobromine C 7 H 8 N 4 O 2 (3,7-dihydro-3,7- dimethyl-1H-purine-2,6-dione) Stimulant Increases Heart Rate Less active than caffeine

6. HPLC Chemistry

7. Hypothesis More theobromine than caffeine will be found in all types of chocolate Dark chocolate will have more of both active ingredients than milk chocolate, which will have more of both ingredients than white chocolate

8. Extraction Procedures Pulverizing using liquid nitrogen Dissolving in methylene chloride (1 g in 5 mL) Extraction using needle Re-dissolving in ethanol  Cocoa butter  Less Corrosive Transfer into HPLC-vial (500 µL)

9. HPLC Data Analysis The detection apparatus uses UV rays to analyze the substances within a chocolate sample. The substances are output as a chromatogram. The chromatogram must be manually integrated and analyzed to discover relative concentrations of the active components.

10. Chromatogram Information Important data gleaned from the chromatogram are:  Retention Time  Integrated Area  Area Ratio All are important for identifying and evaluating the components in question.

11. This is what we call… THE MEAT

14. Oh Yeah… IMPORTANT: NOT ALL CHROMATOGRAMS ARE NICE AND SIMPLE. THIS IS HOW NASTY THEY CAN GET!

16. NOISY!!!! This kind of chromatogram is designated as unreadable and is not used in final analysis.

17. OOPS!

18. This is what most relatively normal chromatograms look like. They must be toyed with so that the areas are as accurate as possible.

19. HPLC Analysis

21. Subjective Study Purpose: Correlation between chocolate components and alertness levels. Chocolate preference’s affect on ratings.

22. Procedure 50 volunteers 3 samples in 5 minute intervals Blindfolding/Random groups Questions:  Gender & Caffeine Consumption  Taste Preference  Alertness level after each sample

23. Pictures!!

24. More Pictures…

25. Analysis Preference does affect rating! Note: The lower the number, the higher the preference

26. More Analysis…

27. Look!

28. Subjective Study II 10 volunteers (small size) 6 samples/ 10 minute intervals Results  Darker chocolate - more alertness  Darker (more bitter) chocolate/ lower rating

29. Conclusion Were we right? Data analysis Future Research and Improvement PProcedure to extract chocolate SSubjective Tests

30. Acknowledgements We would like to thank Dr. James Fukunaga and Carrie Williams for their assistance, PerkinElmer Corporation and Waters Corporation for donated HPLC equipment, and, William-Sonoma for donated chocolate.