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Analysis of Single-Origin Chocolates for Caffeine and Theobromine Composition

using High-Peformance Liquid Chromatography

1. Standard Solution Preparation
- Prepare a series of solutions before you can prepare the chocolate samples.
- All solutions will be prepared in Milli-Q water. The solids will be slow to dissolve so be
a. Internal Standard Solution.
- This solution is added to all chocolate samples and is used to prepare calibration
- Prepare 500 mL of an approximately 1000 mg/L 7-(hydroxyethyl)theophylline solution.
- Weigh 0.5 g of 7-(hydroxyethyl)theophylline and adjust to 500 mL with Milli-Q water.
b. Pure Standard Stock Solutions
- Prepare 500 mL of 500 mg/L solution of caffeine by weighing 250 mg of caffeine and
adjust to 500 mL with milli-Q water.
- Prepare 500 mL of 500 mg/L solution of theobromine using same method
c. Calibration Standards.
- Prepare following mixed calibration standards containing both theobromine and caffeine:
1, 3, 5, 10, 25, 50, 100 mg/L
- Each calibration solution should have the same concentration of the internal standard
solution (50 mg/L 7-(-hydroethyl)theophylline) Volume of theophylline stays the same
Use 5 mL of 1000 mg/L stock solution for each calibration standard solution
- All solutions are adjusted to 100 mL with Milli-Q water
100 mg/L mixed calibration standard solution
(SH 1)
50 mg/L mixed calibration standard solution
(SH 2)
25 mg/L mixed calibration standard solution
(SH 3)
10 mg/L mixed calibration standard solution
(SH 4)
5 mg/L mixed calibration standard solution
(SH 5)
3 mg/L mixed calibration standard solution
(SH 6)
1 mg/L mixed calibration standard solution
(SH 7)

Caffeine: 20 ml of 500 mg/L stock solution

Theobromine: 20 mL of 500 mg/L stock
Caffeine: 10 ml of 500 mg/L stock solution
Theobromine: 10 mL of 500 mg/L stock
Caffeine: 5 ml of 500 mg/L stock solution
Theobromine: 5 mL of 500 mg/L stock
Caffeine: 2 ml of 500 mg/L stock solution
Theobromine: 2 mL of 500 mg/L stock
Caffeine: 1 ml of 500 mg/L stock solution
Theobromine: 1 mL of 500 mg/L stock
Caffeine: 0.6 ml of 500 mg/L stock solution
Theobromine: 0.6 mL of 500 mg/L stock
Caffeine: 0.2 ml of 500 mg/L stock solution
Theobromine: 0.2 mL of 500 mg/L stock

2. HPLC Optimization Phase

- We will use two different wavelength (two HPLC machines): 274 nm and 300 nm
- The mobile phase will be a mixture of acetonitrile and 0.5% (V/V) acetic acid in Milli-Q
water. The optimum ratio of these two solvents will be determined by the lab section. The

resulting chromatograms will be evaluated to determine how well the analyte peaks are
Reservoirs for EACH HPLC machine
o 1 L of acetonitrile
o 1 L of 0.5% (V/V) acetic acid in Milli-Q water (add 5 mL of HPLC acetic acid to 1 L
bottle and adjust to the volume with milli-Q water)
Samples to run for mobile phase optimization:
a. Highest concentration mixed standard.
b. Pure solutions of each of the three compounds
HPLC parameters:
o Flow rate: 1.5 mL/min
o Detector wavelength: 274 nm (and 300 nm)
o Run time: 20 minutes
o Column temperature: 25oC
o Injection volume: 20 L
Mobile phase composition to run
100% acetic acid
95/5 acetic acid/acetonitrile
93/7 acetic acid/acetonitril
92/8 acetic acid/acetonitrile
90/10 acetic acid/acetonitrile
88/12 acetic acid/acetonitr
83/17 acetic acid/acetonitrile
85/15 acetic acid/acetonitrile
80/20 acetic acid/acetonitr
Follow the standard operating procedure instructions for the HPLC, using the parameters
listed above. Be sure to enter your assigned mobile phase percentages into the pump
parameters field.
After collecting the HPLC data at your assigned mobile phase composition, determine the
retention factor (k) for all three compounds and also calculate the selectivity factor
() and resolution (R) values between all pairs of compounds.
Record data in an excel file.
Prepare a single graph of k vs % acetonitrile for the five components. Use this graph to
determine the % acetonitrile which gives the greatest difference in capacity factor
between the peaks. This should be your optimum setting for later analysis. Set the pump
to this setting and repeat the two samples to confirm that you are getting a good
Prepare a graph of log (k) vs 1/T with temperature in Kelvin for each of the five
components. The log (k) should be a linear function of 1/T. You can do least squares trend
lines to determine the optimal temperature for separation.
Once the optimum setting is determined, you will run the calibration standards and
samples described in the lab manual using this combination. Set up a sequence file with
all of your calibration standards and chocolate samples and run it overnight.

3. Chocolate Sample Preparation

- You will be preparing seven single-origin chocolates, and three chocolate of unknown
- Accurately weigh 500 mg of each freshly crushed (use a mortar and pestle) chocolate
sample into a clean, dry disposable vial (record the mass of vial and after adding chocolate
to determine the chocolate mass).
- Tightly cap each vial and place into the 80 oC water bath to melt the chocolate.
- Once the chocolate is melted, add 20.00 mL of Milli-Q water and 5 mL of the 1000 mg/L
internal standard solution, recap and mix well.
- Next you will isolate the methylxanthines using solid phase extraction (SPE). The SPE
extraction manifold should be operated under vacuum (~5 mm Hg), maintaining a drop
wise flow rate at all times.
- SPE setup

SPE procedure
a. Conditioning SPE tube in vacuum (5 mmHg)
Activation: 1 mL methanol
Equilibration: 1 mL of Milli-Q water
Reversed phase type silicas and nonpolar adsorption media are usually conditioned with a
water-miscible organic solvent such as methanol, followed by water or an aqueous buffer.
Methanol wets the surface of the sorbent and penetrates bonded alkyl phases, allowing
water to wet the silica surface efficiently.
b. Sample loading step - make sure you added the internal standard to the
chocolate before this step!
0.5 mL of sample
Accurately transfer the sample to the tube or reservoir, using a volumetric pipette or
micropipette. The sample must be in a form that is compatible with SPE
c. Wash step
1 mL of milli-Q water
If compounds of interest are retained on the packing, wash off unwanted un-retained
materials using the same solution in which the sample was dissolved, or another solution
that will not remove the desired compounds.
d. Elution step place clean sample collection tube under each extraction column
before starting elution!
2.5 mL of ethanol
Rinse the packing with a small volume of a solution that removes compounds of interest
(typically 200 L to 2.5 mL depending on the tube size), but retain any impurities not
removed in the washing step.
Two small aliquots generally elute compounds of interest more efficiently than one larger
aliquot. Recovery of analytes is best when each aliquot remains in contact with the tube
packing for 20 seconds to 1 minute. Slow or drop wise flow rates in this step are beneficial.
Collect elutes and further prepare as appropriate.
e. Reconstitution for each sample
All samples need to be evaporated to dryness using the solvent evaporator system. If your
samples are not completely dry at the end of lab, tightly cap and place them in the
refrigerator and continue drying next week.
The dried chocolate extraction residues should be reconstituted in 2.00 mL of Milli-Q water
and filtered using 0.22 m filters into autosampler vials. When sample has been filtered,
run HPLC right away.