1. Analysis of Caffeine in Coffee Kate Penrod, Nikki DeLuca, Christina Robinson, and Jess Bases

2. Purpose Quantify caffeine in brewed coffee – Varying strengths Extract caffeine from grounds Maxwell House: Regular and Decaf.

3. Background Caffeine in food/drink CNS stimulant Schedule V drug High levels – Restlessness – Irritability – Headaches – Heart complications

4. Methods HPLC – Liquid Separation – Liquid IR – Solid and liquid SFE – Solid UV/Vis – Disabled

5. HPLC Hypothesis Samples – 10-50ppm caffeine standards – Strong caffeinated/decaffeinated – Regular caffeinated/decaffeinated – Weak caffeinated/decaffeinated Dilution of coffee samples

7. SampleConc [ppm]Retention TimeArea Standard102.3461026491 Standard202.3231360973 Standard302.3141612710 Standard402.3051915989 Standard502.3022330806 Weak DC 1:246.553691532.2661436697 Reg DC 1:577.881659332.2931193080 Str DC 1:5227.3816352.2832139010 Weak C 1:5180.64694982.2941843306 Reg C 1:10321.80749682.3131718385 Str C 1:25548.24560022.3101394082

8. Organic Separation Sample Preparation (Dr. Peterman’s Coffeemaker) – Regular Caffeinated/ Decaffeinated 1 Tbsp. grounds, ¾ cup water – Strong Caffeinated/ Decaffeinated 3/2 Tbsp. grounds, ¾ cup water – Weak Caffeinated/ Decaffeinated ½ Tbsp. grounds, ¾ cup water

9. Organic Separation First Method – Adapted from Dr. Halligan’s Organic I Lab Manual – Extraction of Caffeine using Dichloromethane – Large emulsion layer, not a clear separation

10. Organic Separation Revised Method – Added Na 2 CO 3 which increased caffeine’s solubility in aqueous layer – Separation layer more pronounced – Dried with Anhydrous MgSO 4 – Vacuum under reduced pressure

11. Mass Quantification AppearanceMass of Product Regular CaffeinatedYellow/brown liquid0.2753g Weak CaffeinatedLight brown liquid0.2731g Strong CaffeinatedYellow/ brown solid, black spots 0.2892 g Regular DecaffeinatedYellow/brown liquid0.2006 g Weak DecaffeinatedLight brown liquid0.2001g Strong DecaffeinatedYellow/ brown liquid0.2296 g

12. Extracted Caffeine IR Characterization Substituted Alkene

13. Extracted Caffeine IR Characterization Strong Decaff Regular Decaff Weak Decaff C = N C = O O – H stretch C = N C = O Substituted Alkene C = O C = N O – H stretch CH3 C - N Substituted Alkene

14. Extracted Caffeine IR Characterization Strong Caffeinated Regular Caffeinated C = O C = N O – H stretch CH3 C - N Substituted Alkene C = O C = N O – H stretch CH3 C - N Substituted Alkene C - N CH3 C = N O – H stretch Weak Caffeinated

15. The Devil’s Instrument a.k.a. SFE

16. 4 Samples – “Pure” coffee grounds – Post brewing coffee grounds 1 gram of each sample 10 minute runs End mass indications IR results

17. SFE Data SamplePercent Yield Pure Caffeinated5.186 % Pure Decaf.6.286 % Brewed Caffeinated1.185 % Brewed Decaf.3.576 %

18. SFE Extraction IR Analysis

19. UV/Vis Attempted to use the DRA-600 – Solid Analysis Missing part! Call EVERYONE – Manufacturer – Distributor – Delivery Guy Piece was backordered until May 20 th Put on hold INDEFINITELY.

20. Sources of Error Organic Method – Impure caffeine for mass quanitification – Transfer between different glassware – Incomplete drying in extraction (O-H stretch peak) HPLC – Day 1: Low pressure, purge lines – Day 2: Invalid date/time error, inconsistent – Day 3: Consistent results, ran all samples

21. Sources of Error (Part 2) SFE – Leaks in the line – Issues with the amount of sample – Needed constant supervision UV/Vis – Missing part – Phone tag – Wasted 2 whole lab periods

22. Sources of Error (Part 3) Jerry Sainsbury – Sales Representative Thermo Fisher Scientific

23. Conclusions HPLC: Strong decaf. was comparable to weak caf. Weak and regular brews – 4:1 caf. vs. decaf. Strong brew – 5:2 caf. vs. decaf. Organic Method: Qualitative data Erroneous quantitative data Masses very similar SFE: Low percent yields Higher yield from decaf Did not extract pure caffeine May be caffeine plus side products If UV/Vis hadn’t been in the initial method, further research would have been conducted

24. Further Research Organic Method – Develop a new or revised method to extract pure caffeine in its solid form as was done in Dr. Halligan’s Organic I Lab IR – Analyze liquid only – Create calibration curve HPLC – Compare different coffee makers and brands

25. Further Research (Part 2) SFE – Brew remaining grounds, analyze with HPLC – Determine other components of extracted grounds UV/Vis – Obtain calibration disc and sample holder – Develop SOP for DRA-600