Vivian Nguyen
Student ID: 5005053
Group C2 Tuesday
Lab Partners: David Hirsch, Jarrett Mansergh, Emily Erhart
Instructor: Dr. Nicholas Frost
Procedure:
The purpose of this lab was to determine the mass fraction of caffeine and theobromine in
the companys Special Dark chocolate product in mg/kg.
A. Materials and Equipment used:
 Agilent 1100 Series HPLC machine, HPLC Water and Methanol
 Centrifuge machine, plastic centrifuge tube
 Vacuum Filtration kit
 Petroleum Ether
 Verified Caffeine 1.00mM standard solution
 Verified Theobromine 1.25mM standard solution
 Dark Chocolate bar
 Standard Reference Material by NIST Certificate of Analysis
B. Procedure:
In order to determine mass fraction of caffeine and theobromine in the chocolate bar, the
sample needs to be run through HPLC machine to construct a calibration curve. Prior to running
the HPLC, it is essential to defat the chocolate by extraction using petroleum ether, because the
extra materials might interfere with HPLC readings or damage the machine.
Sample preparation:
Due to many transfers and extractions, some of the original materials will be lost during
sample preparation. By adding a known quantity of analytes to the sample, a spike recovery
percentage will be calculated to give more accurate results of how much theobromine and caffeine
there initially was. To accomplish this, three samples were prepared for analysis: control
(unspiked), spiked and NIST standard reference sample.
The given frozen chocolate was ground with a mortar and pestle then placed in three preweighed plastic centrifuge tube of the following masses:
 Control: 0.5093g of chocolate, 0g of theobromine and caffeine
 Spiked: 0.5033g of chocolate, 0.0016g of theobromine, 0.001g of caffeine.
 NIST: 0.6283g, 0g of theobromine and caffeine.
*The amount of theobromine and caffeine added to the spiked tube to account for materials
loss was suggested by TA.
To defat the chocolate, each tube was added with 10mL of petroleum ether and shaken
vigorously until all remaining chocolate pieces were dissolved and not observable. The tubes were
then centrifuged for a minute; two layers, one of solid and one of liquid were observed. The liquid
part (petroleum ether) was taken out from the tube. This defat process was carried out three times
per tube until the petroleum ether fluid was clear, which would be the indication that all fats were
removed from samples.
To each tube 10mL of HPLC water was added. HPLC water was used instead of regular
water in order to minimize contaminations that could possibly interfere with the signals and to
avoid destroying HPLC machine. After shaking the tubes until all solids were in water, the contents
were poured into Erlenmeyer flasks and heated to boiling point with a Bunsen burner, to remove
any remaining petroleum ether. Vacuum filtration was performed to filter out the chocolate solids.
Prior to injecting the samples, HPLC water was added to the remaining fluids to the 100mL mark;
1mL of which was taken out and placed in labeled HPLC test tubes, ready for analysis.
HPLC Analysis:
The HPLC conditions were set at 274nm for wavelength, 80% HPLC water and 20% HPLC
methanol (1% TFA) for mobile phase composition and run at 2.000mL/min. After consulting with
TA, a total time of 5.0 min per run was chosen because that was enough time for both analytes to
be completely eluted. These conditions were discussed with TA, however, in reality, multiple trials
would need to be tested before finding the conditions at which give the best results. In this
experiment, it was learned that 30% was the maximum allowed percentage of methanol because it
might damage the HPLC internal structure if go over. A percentage of 10% methanol would take
too long (approx. 8 minutes per run) because only 5 minutes were needed for both theobromine
and caffeine to be eluted. To be safe, 20% of methanol as an average was chosen. Due to strong
radiation absorption at 274nm, peak signals of theobromine and caffeine will be observed in the
chromatogram. Since theobromine is less soluble than caffeine in silica gel, it will be eluted faster
resulting in a shorter retention time. Thus, when analyzing the chromatogram, theobromine will
be the first peak and caffeine the second. The area (width x height) of each peak is proportional to
the compound concentration. However, because only peak heights could be used to find standard
deviation of HPLC background, concentration of compound was plotted against peak heights,
instead of peak areas. This correlation will be used to find concentration of compound accordingly,
then mass and mass fraction.
Making Calibration Curve for Analytes
A calibration curve would be needed to determine the concentration of theobromine and
caffeine. Since theobromine and caffeine are immiscible, adding both analytes together in HPLC
test tubes would not interfere with the signals. Another reason for adding both analytes was time
efficiency. Using the given verified standard solutions of 1.00mM Caffeine and 1.25 mM
Theobromine, five solutions with different concentrations of each were prepared and recorded in
the following table. A reason for choosing these ratios was taking 1.25mM/1mM = 5/4. This
suggested that concentrations of each analytes should be differed by a factor of 0.25 per time,
starting from 0.00mM.
Theobromine Caffeine
Concentration Concentration
(mM)
(mM)
A
0.00
1.00
B
0.25
0.75
C
0.50
0.50
D
0.75
0.25
E
1.25
0.00
Table 1. Initial Calibration Standards
Theobromine
Volume (L)
Caffeine
Volume (L)
Water Volume
(L)
0
80
160
480
200
200
300
200
200
0
0
20
90
120
0
The calibration standard solutions were injected to HPLC. To find out the approximate
retention time of caffeine (the order of choosing which analyte was completely random), solution
A which was only consisted of caffeine, was injected first. Only one very tall peak showed up at
around 4 minutes after injection. The HPLC report also demonstrated that only one peak was
present, thus, it was concluded that 4 minutes was the approximate retention time of caffeine.
Solution B was run and two peaks were observed, one at around 1.4 minutes and the other at 4
minutes. Comparing two peak areas from chromatogram, it was clear that the 4minute peak area
was quite bigger than the 1.4minute peak, which agreed with our concentration table where
caffeine concentration was 3 times smaller than that of theobromine. This helped conclude that
peak showing up at 1.4 minutes represented theobromine. This result matched with our
prediction that theobromine was eluted faster, thus produced a shorter retention time. The same
trend was observed for the remaining C, D, E solutions. Using Excel, the calibration curve of
theobromine and that of caffeine were constructed.
A slight modification was made to calibration standards. After injecting the Control tube
to HPLC, it did not quite fit the calibration curve due to insufficient minimum peak area of each
analyte. Thus, solution C was diluted with HPLC water to a 10:1 ratio and run again to modify
the calibration curve. This change would be demonstrated in Table 2 and 3.
Final Analysis
Each sample (control, spiked and NIST standard reference) were run three trials. The
calibration curve from the standards would give concentration of theobromine and caffeine.
Percent recovery was then determined and used to calculate the original mass of each analyte in
chocolate sample, and finally the mass fraction.
Ftest, Ttest and confidence interval will be used to compare our sample to the given
standard and conclude about the methods accuracy and precision.
Data Analysis:
As explained in HPLC Analysis part, peak height will be used to plotted against
concentration. Peak area could not be used because standard deviation of HPLC background was
found in peak height unit (mAU).
Theobromine Data
The following table summarized the standard concentration used for Theobromine and
peak height found from HPLC.
Theobromine
Concentration (mM)
Peak Height
(mAU)
0.005
7.1556
0.25
335.69189
0.5
568.61804
0.75
851.49292
1.25
1192.69958
Using Excel, a calibration curve was constructed for Theobromine, with R2 and standard error
for slope and yintercept.
1400
1200
y = 945.27x + 70.288
R = 0.98227
1000
800
600
400
200
0
0
0.2
0.4
0.6
0.8
1.2
Concentration (mM)
1.4
Sample
Mass (mg)
Initial mass
(mg)
Mass fraction
(mg/g)
Mass
fraction
(mg/kg)
0.663
2.630
5.164
Control
0.705
2.798
5.494
0.743
2.948
5.788
1.109
4.399
8.740
Spiked
1.128
4.474
8.890
1.087
4.312
8.568
4.588
18.193
28.956 28956.88
Reference
4.881
19.355
30.805 30805.62
4.831
19.158
30.492 30492.89
Table 4. Theobromine Mass, Initial Mass, Mass Fraction Data
Mean of
Mass
Fraction
STDEV
5.482
0.312
8.733
0.161
30.085
0.989
Caffeine Data
The following table summarized the standard concentration used for Caffeine and peak
height found from HPLC.
Caffeine
Concentration
(mM)
Caffeine Peak
Height (mAU)
0.005
2.61711
0.25
123.99193
0.5
240.81856
0.75
346.75644
1
430.20663
Table 5. Caffeine Concentration and corresponding Peak Height from Standard solutions
Using Excel, a calibration curve was constructed for Theobromine, with R2 and standard error
for slope and yintercept.
500
450
400
350
300
250
200
150
100
50
0
y = 445.40x +2.617
R = 0.99471
0.2
0.4
0.6
0.8
1.2
Concentration (mM)
Mean of
Concentration (mM)
0.00358
0.0234
0.0169
Sample
Mass
(mg)
Initial mass
(mg)
Mass fraction
(mg/g)
Mass
fraction
(mg/kg)
Mean of
Mass
Fraction
0.0960
0.249
0.489
Control
0.0550
0.143
0.280
0.0576
0.149
0.294
0.458
1.191
2.367
Spiked
0.458
1.191
2.367
0.446
1.159
2.305
0.317
0.824
1.313
1312.72
Reference
0.336
0.873
1.389
1389.81
0.334
0.868
1.382
1382.05
Table 7. Caffeine Mass, Initial Mass, Mass Fraction Data
The standard error of yaxis was calculated using formula: =
STDEV
0.354
0.117
2.346
0.0363
1.361
0.0424
( )2
2
, n = 5.
= 25.319
= 6.959
3
++

( )2
2 ( )2
Sample Calculation
Calibration curve equation of Theobromine is y = 945.27x + 70.288
y is the peak height, x is the concentration
X = (Y70.288)/ 945.27
Using this equation, concentrations of Theobromine in Table 4 were found.
A spiked sample was made to account for materials loss.
% recovery =
100
0.0016
1
= (
)
1000
=(
)
1000
0.1
180.164
= 0.0888
= ( 5) = 0.0615
= ( 5) = 0.0391
% =
0.0615 0.0391
100 = 25.22%
0.0888
() =
= 0.1, = 180.164 )
()
()
Method of Validation
Linearity
Excel was used to calculate the linear least square regression. A fit calibration curve to
the predicted equation would be determined by how close R2 is to 1.00, the closer the fitter. From
Graph 1 and 2, R2 = 0.995 for Caffeine and 0.983 for Theobromine. R2 of these two calibration
curve are both higher than 0.98, and very close to 1.00 for Caffeine curve. Thus, it could be
concluded that the calibration curve fits well with the predicted equation and is reliable enough
to use for calculation of concentration.
Limit of Detection and Limit of Quantitation
= 0.0000159
= 0.0000338
= 0.000053
= 0.000113
Referring to the data tables, concentration of theobromine in all samples were much
higher than LOD and LOQ. The same trend is observed for caffeine and its LOD, LOQ as well.
Thus, all our concentration results are acceptable.
Accuracy:
Calculating the 95% confidence interval was needed to conclude whether or not the
standard reference mean could be the true mean of the method data from this experiment.
95% confidence interval =
( , = 4.303 ( , = 2, 95%),
, = 3)
Caffeine:
% Recovery = 38.5%
Mass fraction of Control sample: 0.354 0.117 (mg/g of chocolate)
95% confidence interval = 0.354 0.290 (mg/ g of chocolate)
Hersheys mass fraction: 0.488 mg/g of chocolate
Hersheys mass fraction 0.488 falls within the 95% range, thus we could conclude that 0.488
caffeine/ g chocolate could be the true mean of method data from this experiment.
Theobromine:
% Recovery = 25.2 %
Mass fraction of Control sample: 5.482 0.312 (mg/g of chocolate)
95% confidence interval = 5.482 0.775 (mg/ g of chocolate)
Hersheys mass fraction: 4.24 mg/g of chocolate
Hersheys mass fraction 4.24 does not fall within the 95% range, thus we could conclude that
4.24 mg theobromine/g chocolate could not be the true mean of method data from this
experiment.
For Caffeine:
The standard mass fraction given by SRM certification was 1060 50 mg/kg = 1.06 0.05mg
caffeine/ g chocolate.
1 = 1.06 mg/g
S1 = 0.05 mg/g
n1 = 8
DOF 1 = 7
2 = 1.361 mg/g
S2 = 0.042 mg/g
N2 = 3
DOF 2 = 2
F calc = (s1/s2)2= 1.42 < 19.4 (F table), the precision in two methods are not significantly
different.
=
=
(0.05)2 (8 1) + (1.361)2 (3 1)
1 2 (1 1) + 2 2 (2 1)
=
= 0.64
1 + 2 2
8+32

1
2
1 2
1 +2
1.061.361 
0.64
83
8+3

1
2
1 2
1 +2
11.630.085
17.48
83
8+3
Discussion
Since our all of data passed the Ftest and Ttest, if the given standard method was accurate, our
method would also be accurate. However, some of the following errors might have affected our
results and could be avoided for better data in the future.
 We could do many trials to see which standards would give the most fit calibration curve.
Collecting more data could also enhance the calibration curve. The closer R2 to 1.00, the
fitter the curve. At the moment, our R2=0.995 for Caffeine was already very close to
1.00. If we could find better concentrations/dilutions ratio that could bring R2 of
theobromine closer to 1.00, we would have a fitter new equation. Though it might not be
significantly different, since concentrations are needed to calculate all the other mass
data, it would eventually make a big change to our data.
 Our % recovery was very low. This was due to many transferring of solutions between
glassware which resulted in loss of materials. Vacuum filtration could have not been done
properly done, which possibly affected the concentration of sample. Another
modification that could be applied to getting a more accurate % recovery would be
measuring the peak height/peak area of spiked sample before adding any amount of
analyte.
In conclusion, our method showed accurate results but could definitely be improved to obtain
better data. However, based on our calculations, the mass fraction of theobromine and caffeine in
the chocolate found in this experiment was reliable.
Precision
Using the following equation, the precision was calculated:
S=
( )
1
= 3
For caffeine:
Our method: 1.361 0.042 mg/ g of chocolate, thus 3.08 %
Standard: 1.06 0.05 mg/ g of chocolate, thus 4.72 %
For theobromine:
Our method: 30.085 0.989 mg/ g of chocolate, thus 3.29 %
Standard: 11.6 1.1 mg/ g of chocolate, thus 9.48 %
For both caffeine and theobromine, since the relative standard deviation of our method was both
lower than that of the given standard, it can be confirmed that our method was more precise.
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