Materials/Equipment:

2 oz. each of 43%, 70%, and 100% chocolate bars

Petroleum Ether                                                          Freezer

Mortar & pestle                                                           Electronic analytical balance

15 mL centrifuge tubes                                               50 & 250 mL Erlenmeyer flasks

Centrifuge                                                                   Dropper

Deionized water                                                          Conical funnel

Water Bath                                                                  Gilson PIPETMAN P20 pipette

1000 mL beaker                                                          0.45 mm filter paper

Hot plate                                                                     Caffeine stock, powdered

Chromatography C₁₈ column packed with silica (SiO₂)

Thermo Scientific UHPLC coupled to TSQ Quantum Vantage triple-stage quadrupole mass spectrometer (Thermo Fisher Scientific, San Jose, CA).

Cold Bruer Drip Coffee Maker                                  Folgers Classic Roast Instant Coffee

Mr. Coffee Single Serve Coffee Maker                      Folgers Regular Classic Roast Ground Coffee

 

Procedure

Caffeine Extraction and Quantitation from Chocolate

1. Obtain 2 oz portion of chocolate and slice into small pieces.  Place sliced chocolate into mortar and place in freezer along with pestle until cold.

   2.  Obtain and weigh a 15 mL centrifuge tube on an electronic balance.  Record weight then add frozen chocolate to tube.

   3.  Add 10mL petroleum ether to tube then close cap.  Shake tube vigorously for 30 seconds in order to dissolve fat from the chocolate into the solvent.

   4.  Place tube in centrifuge and spin for 1 minute at 8,000 rpm.  Decant supernatant.  Repeat Steps 3 & 4 two more times to ensure fat is completely removed from the chocolate.

   5.  Add 500 mL of water to a 1000 mL beaker and bring to a boil using a hot plate.  Place tube with chocolate in boiling water for 10 minutes in order to remove any residual solvent in the chocolate.

   6.  Calculate the mass of the chocolate residue by weighing tube with chocolate residue and subtracting the weight of the empty centrifuge tube recorded in Step 2.  Record result.

   7.  Add 3 mL of deionized water to tube with chocolate. Heat tube by placing in boiling water to soften chocolate.  Begin stirring contents to suspend as much chocolate as possible.

   8.  Obtain and weigh a 50 mL Erlenmeyer flask.  Record weight.

   9.  Pour suspension into a 50 mL Erlenmeyer flask.  Repeat Steps 7 & 8 until all chocolate from centrifuge tube has been transferred to the flask.

   10.  Add deionized water to flask to bring the volume to 30 mL.  Place flask into a boiling water bath for X minutes to extract all the caffeine from the chocolate into the water.

   11. Place the flask onto an electronic balance.  Using a dropper filled with deionized water, add water drop by drop until the weight of water equals 33.3 g.

   12.  To remove all chocolate particles from the solution, aliquot contents into centrifuge tubes and centrifuge for 1 minute at 8,000 rpm.

   13.  Place conical funnel lined with 0.45 mm filter paper into a clean flask.  Filter suspensions from centrifuge tubes by pouring contents into funnel.

   14.  Repeat Steps 12 & 13 five more times to obtain a clean sample.

2. Prepare the following standards by dissolving powdered caffeine stock into hot water:
   – 0.5 mg/mL concentration = 5 mg caffeine into 10 mL deionized water
   – 1.0 mg/mL concentration = 10 mg caffeine into 10 mL deionized water
   – 1.5 mg/mL concentration = 15 mg caffeine into 10 mL deionized water

–  2.0 mg/mL concentration = 20 mg caffeine into 10 mL deionized water

16.   Inject 20 ml of 0.1 mg/mL standard into HPLC for analysis.  Repeat for each standard to construct calibration curve.

17.  Inject 20 ml of sample into HPLC for analysis.

18.  Repeat process for each type of chocolate to be analyzed, with the exception of Steps 15 & 16 as calibration curve will be used for all chocolate samples.

19. Using chromatogram results, quantitate the amount of caffeine per each 2 oz. serving of chocolate in mg

Caffeine Extraction and Quantitation from Coffee

1. Prepare each sample by using 5 g of coffee with 8 oz water. Using regular ground coffee, follow manufacturer’s instructions for drip and brewed coffee makers. For instant coffee, simply dissolve instant coffee grinds into hot water and stir.
2. Filter drip coffee sample using a filter funnel lined with 0.45 um filter paper into a clean 250 mL Erlenmeyer flask.
3. Inject 20 ml of sample into HPLC for analysis.
4. Repeat Steps 2 & 3 for brewed and instant coffee samples.
5. Using chromatogram results, quantitate the amount of caffeine per each 8 oz. serving of coffee in mg