Discussion

Micropipette Calibration
Micropipettes were calibrated and it was found that the 0.5-10 L
pipette was accurate to 3% in neither percent error nor relative error (Table
3). As a result, this pipette will not be used and instead will be sent out for
recalibration. The 10-100 L pipette was found to be accurate to 0.6% at its
maximum volume and 1.3% at its minimum volume. This indicates that it is
accurate, especially for larger volumes. The 1.3% error at the 10 L volume
setting is a little large though, so it might be a good idea to use a
micropipette with a smaller volume setting that has already been calibrated
when working with volumes around 10 L.

Calibration Curve
The calibration curve was constructed using exact concentrations of
aspartame in water. The R2 value was 0.9997 after forcing the line through
the origin, indicating that our value for was extremely precise and that
error derived from the calibration curve is very small. As was mentioned, the
line of best fit also had an intercept of 0, as the p-value for the intercept
assigned to it by Excel was 0.09. This is in agreement with Beers law, which
does not include an intercept since absorbance is 0 for concentrations of 0.

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The molar absorptivity after eliminating the intercept was found to be 167
11 M-1cm-1.

Analysis of Safeway and Equal Sweetener Packets

Both Equal and Safeway brand sweeteners are purported to contain 1
g of sweetener per packet. It was determined that the average mass of
sweetener in a packet of Safeway sweetener was 1.09 g, which is higher
than expected. Equal, on the other hand, had an average mass of 0.99 g per
packet.
It was expected that both Safeway and Equal brand sweeteners would
contain 36.7 mg of aspartame per gram of sweetener. It was found that
Safeway brand sweetener had 42.8 1.0 mg of aspartame per gram of
sweetener while Equal only had 40.1 2.3 mg of aspartame per gram (Table
5). So not only do Safeway packets contain more sweetener overall, but
possibly also a higher concentration of aspartame.
Performing a two tailed t-test between the aspartame content in
Safeway and Equal brand sweeteners returns a p-value of 0.0725, which
means that the null hypothesis is accepted and that the two sweeteners are
from the same population. This can be interpreted to mean that the
differences observed in aspartamed content between the Safeway and Equal
brand sweeteners are not statistically significant. Calculating tcalc gives a

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value of 1.256, which is smaller than tcrit even at a 90% confidence interval,
meaning it would be very difficult to make the claim that the difference in
the amount of aspartame between Safeway and Equal brand sweeteners is
statistically meaningful considering the experiment conducted.
Using Equation 1, it can be seen that there is a bias of 3.4 mg/g for
aspartame content in Equal. Using equation with a tcalc of 3.02, which is just
slightly lower than the 95% tcrit of 3.18. Statistically, this bias is insignificant
at a 95% confidence interval, but at a 90% confidence interval it becomes
significant. Whether Equal actually does contain more aspartame than it is
supposed to, or there was some kind of systematic error with
instrumentation or lab technique cannot be confidently determined with the
amount of measurements taken, but the bias is worth noting nonetheless as
it is greater than a standard deviation (2.3 mg/g) from the measured
aspartame content.

Sweetness Taste Test

Collected data from the sweetness taste test yielded Kd and Smax values
that were significantly different when determined from double reciprocal and
hyperbolic saturation curve fittings. The double reciprocal plot gave a Kd of
4.8 2.4 mM and an Smax of 7.0 3.4. The hyperbolic saturation curve gave
a Kd of 5.9 2.7 mM and an Smax of 8.1 2.1. Both sets of values were also
very different from the literature value of Kd for aspartame, which is 0.75
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mM.2 The literature value measured by Masuda is also probably more

accurate, as his aspartame binding was measured in a petri dish in a more
controlled setting, with much less saliva and other contaminants present.
In theory, the hyperbolic saturation curved should give more accurate
values for Kd and Smax. However, the values obtained from the double
reciprocal plot were very slightly closer to literature values than those
obtained from the hyperbolic saturation curve, which should be more
accurate due to the fact that as the reciprocal of a small value is taken, error
increases very quickly and proportionally with the value. On the other hand,
the Kd and Smax values obtained from the hyperbolic saturation curve had
somewhat smaller errors associated with them, which suggests they were at
least slightly more precise. It can also be seen in Table A2 that the intercept
for the double reciprocal plot, or in this case 1/Kd, had a p-value of 0.07,
meaning that at a 95% confidence interval it is not even statistically
significant. This further suggests that our data does not give a good
approximation of Kd and Smax. It is difficult to say which values are more
reliable, but considering collected data and associated uncertainty, this
experiment would not give a good indication anyway.
Referring back to Table 7, the fact that these findings are so
inconsistent is not surprising considering our data from the taste test was so
varied. This is not unexpected, as a test based on individual perception is
going to have a scale that is different for each person contributing data.

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Overall, our data was very poor and few conclusions can be drawn from it
besides the fact that it is difficult to measure taste when everyone perceives
it so differently.

Appendix
Table A1 Linear regression data for calibration curve before intercept was forced
through 0.
Coefficien

Standard

ts
0.0115354

Error

t Stat
2.157743

P-value
0.09713448

Intercept
X Variable

29
0.1652770

0.005346062

019
168.5068

38

0.000980833

806

7.4401E-09

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Table A2 Full statistical report for linear regression of double reciprocal plot in
Excel.

Sample calculation locations:

SC-A pg. 035
SC-B pg. 035
SC-C pg. 035
SC-D pg. 035

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SC-E pg. 036

SC-F pg. 036

References:
1) Weast, R. C.; Astle, M. J. CRC Handbook of Chemistry and Physics 63 rd Edition.
CRC Press Inc.: Boca Raton, 1982. p. F-5.

2) Masuda, K. et al. Characterization of the Modes of Binding between

Human Sweet Taste Receptor and Low-Molecular-Weight Sweet
Compounds. PLoS ONE 2012, 7, e35380.

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