EXPERIMENT 28
TABLETOP SWEETENERS
INTRODUCTION
Worldwide consumption of tabletop sweeteners is huge, but not without controversy. It is easy, especially via
the internet, to find massive amounts of “information” about these substances, both positive and negative.
Interested persons examining such information must be very careful to “consider the source” and look for any
possible biases that might be present. The consumption of any substance, natural or “artificial”, has both positive
and negative possible consequences, and these must be weighed against each other. For example, an individual
attempting to cope with obesity and its negative health effects may be more than willing to ignore the remote
possibility of bladder cancer from the use of artificial sweeteners.
Artificial sweeteners are generally far sweeter than ordinary sugar. Thus the sweetener itself makes up only a
small portion of the contents of a commercial packet containing the sweetener. The remainder consists of a variety
of substances, depending on the particular sweetener and on the manufacturer. One popular sweetener is aspartame,
whose structure is shown in Figure 28-1. It is the methyl ester of a dipeptide, formed from the amino acids aspartic
O
HO
H 2N
phenyl alanine
methyl ester
O
H
N
OCH3
O
aspartic acid
Figure 28-1. Aspartame
acid and phenylalanine. Aspartame has the ability to form a transition metal complex with copper(II) ions, as shown
in Figure 28-2. L is an abbreviation for ligand, water in this case. This complex strongly absorbs some wavelengths
O
O
H 3CO
O
N
H
H H
N
O
L
O
Cu2+
N
H
L
H
N
O
H O
OCH3
O
Figure 28-2. Aspartame-Cu(II) Complex
of visible light, resulting in a deep blue color for its solutions. In this experiment (based on a recent publication:
Journal of Chemical Education, 84, 1676, 2007) we will use this fact to distinguish sweeteners containing
aspartame from those containing two other artificial sweeteners, saccharin and sucralose, as well as from ordinary
table sugar, sucrose. Saccharin, sucralose, and sucrose do not form this type of complex with Cu(II). The deep blue
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color of the complex allows us to first determine which sweetener contains aspartame. Subsequently, we will make
a quantitative determination of the amount of aspartame present in that sweetener product for comparison with the
manufacturer’s claim. The copper(II) ions will be provided in a solution known as a modified biuret reagent. This
solution is also blue in color, but the shade is substantially different from that of the aspartame complex, and the
intensity of the color is much less. The procedure described below allows us to determine both the presence of the
aspartame and its quantitative content despite this minor interference.
TECHNIQUE
A simple visual test is adequate to determine the presence of aspartame. You will place solutions of each of the
sweeteners, sucrose, and an aspartame standard solution in the depressions of a spot plate and then add the biruet
reagent to each. You can easily determine which commercial product contains aspartame by comparing the
resulting solutions with each other and with the aspartame standard solution.
You will then use the colorimeter to carry out a quantitative determination of the amount of aspartame in the
commercial sweetener. The analysis uses the Lambert-Beer Law (sometimes called just Beer’s Law),
A = ·b·C
(28-1)
in which A is the absorbance at wavelength ,  is the molar absorptivity, b is the path length of the light through
the sample in cm, and C is the molar concentration of the absorbing species. (Review experiment 6 for more
information.) You will determine which LED in the colorimeter to use by first measuring the absorption spectrum
of the deep blue complex with the spectrometer, then comparing that with the emission wavelengths of the red,
green, and blue LEDs (see below).
You will measure the absorbance with the colorimeter for a series of standard solutions of aspartame you
prepare from a standard stock solution and then prepare a Beer’s Law plot of the results. You will then prepare and
measure solutions of the commercial sweetener containing aspartame. Comparison of the absorbance of these
solutions with the Beer’s Law plot will enable you to determine the amount of aspartame present.
EQUIPMENT NEEDED
colorimeter
cuvets for colorimeter (2) (all four sides are
clear)
spectrometer
cuvets for spectrometer (2) (two sides are
"frosted")
beakers
spot plate
disposable pipets
2 mL volumetric pipet
5 mL graduated pipet
10 mL volumetric flask
pipet pump
medium test tubes
balance
CHEMICALS NEEDED
distilled water
modified biuret solution
~2.0  10–2 M aspartame stock solution
packets of various sweeteners
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PROCEDURE
Spot Tests of Sweeteners
1. Obtain packets of each of the four different types of sweeteners and a spot plate. Obtain ~20mL of the modified
biuret solution in a clean dry beaker. In a separate beaker, obtain ~20mL of the aspartame stock solution (note
and record the concentration on the bottle).
2. Pour the contents of each sweetener packet into a separate medium test tube. Fill each test tube ~half full with
distilled water, and agitate the test tube until the sweetener is dissolved.
3. Into separate wells of the spot plate place a portion of each of the sweetener solutions you prepared. Into
another well place distilled water, and into a final well place the aspartame stock solution.
4. To each well containing solution add 3-4 drops of the modified biuret solution. Record your observations.
Preparing Standard Solutions
5. Using a graduated pipet, deliver 1.00mL of aspartame stock solution into a 10mL volumetric flask. Using a
volumetric pipet, deliver 2.00 mL of the modified biuret solution into the flask. Fill to the mark with distilled
water, and transfer the solution into a clean, dry, labeled, medium test tube.
6. Repeat the previous step to prepare four additional standard solutions containing 2.00, 3.00, 4.00 and 5.00 mL
of the aspartame stock solution. Each of these solutions should contain 2.00mL of biuret solution. Calculate
and tabulate the aspartame concentration in each of these standard solutions.
7. Prepare a blank solution by adding 2.00mL of the biuret solution to the volumetric flask and diluting to the
mark with distilled water (do not add any of the aspartame solution).
8. The solutions should stand for at least 15 minutes so that the color develops fully; while you are waiting, you
can obtain the absorption spectra of the blank and one of the aspartame solutions.
Setting Up the Workstation
9. Press the MAIN MENU button on the workstation, select SPECTROSCOPY, then ABSORPTION(1), and
press DISPLAY.
10. Rinse and then fill one of the spectrometer cuvets with distilled water. Rinse the other spectrometer cuvet
with a small amount of the blank solution, then fill it to within ~1 cm of the top.
Obtaining the Absorption Spectra (blank and standard)
11. At the spectrometer, press STATION NUMBER, your station number, then ENTER.
12. Place the black cuvet in the sample holder, and press ZERO on the spectrometer.
13. Place the reference cuvet (containing distilled water) in the sample holder, then press REFERENCE on the
spectrometer.
14. Remove the reference cuvet from the sample holder and replace it with your cuvet containing the blank
solution, making sure that the arrow on the cuvet is aligned properly. Press SAMPLE on the spectrometer.
15. Remove the sample cuvet, return to your workstation, and print the spectrum using PRINT standard. Label
the printed spectrum as the blank solution.
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16. Pour the blank solution from the sample cuvet into the Inorganic Waste container, then fill the cuvet with the
standard solution containing the highest concentration of aspartame. Repeat steps 11-15 to measure, print, and
label the absorption spectrum for this standard solution.
Setting Up the Colorimeter
17. Connect the colorimeter to one of the 9-pin inputs on the station. (MAKE SURE NO OTHER PROBES ARE
CONNECTED TO THE STATION.) Fill two cuvets with distilled water and cap them.
18. Press MAIN MENU on the workstation. Press the function key listed for COLORIMETRY/
FLUOR./TURB./LUM., then the function key for COLORIMETRY.
19. In order to determine which LED in the colorimeter to use for this experiment, compare the absorption
spectrum you measured for the standard solution above with the emission spectra for the three LED choices in
the colorimeter, shown in Figure 28-3 below. Choose the color which is most strongly absorbed by the
standard solution.
Colorimeter LED Emission Spectra
4000
3500
Blue LED
Red LED
Green LED
3000
Intensity
2500
2000
1500
1000
500
0
-500
400
450
500
550
600
650
700
Wavelength/nm
Figure 28-3. Colorimeter Emission Spectra
20. Press the function key for the chosen LED, and then the function key for KINETICS (we will not be
performing a kinetics experiment; we will simply be using the colorimeter to measure the solution
absorbance.).
21. Fill both of the colorimeter cuvets with distilled water, place them in the reference and sample holders in the
colorimeter, slide the colorimeter lid closed and press ENTER. An adjustment of 0%T and 100%T will now be
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made, requiring about 10 seconds. When this process is finished, press DISPLAY. The displayed absorbance
value should be zero.
Measuring Absorbance of Standard Solutions
22. Remove the cuvets from the colorimeter and discard the water from each. Rinse one cuvet with a small amount
of the blank solution, then fill it with this solution and place the cuvet in the reference cell holder in the
colorimeter (labeled with an R).
23. Rinse the other cuvet with a small amount of the standard solution containing 1 mL of aspartame stock
solution, then fill it with this solution and place it in the sample cell holder in the colorimeter (labeled with an
S).
24. Close the colorimeter lid and record the absorbance reading in your notebook, along with the calculated
aspartame concentration in the standard solution (See step 6.).
25. Repeat steps 23-24 with the remaining aspartame standard solutions.
26. Go to the computer and enter your absorbance and aspartame concentration values using the ‘Beer’s Law’
program. Print the calibration curve.
Measuring Absorbance of Commercial Sweetener Solutions
27. You should have found that one of the commercial sweeteners tested contains aspartame. Weigh out between
0.3 and 0.4g of this sweetener. Record the exact mass used.
28. Transfer the sweetener sample to a 10mL volumetric flask. Add ~5mL of distilled water and shake until the
sweetener is dissolved.
29. Using a volumetric pipet, deliver 2.00 mL of the modified biuret solution into the 10mL volumetric flask. Fill
to the mark with distilled water, and transfer the solution into a clean dry medium test tube.
30. Repeat steps 27-29 twice to prepare two additional sample solutions.
31. After waiting 15 minutes to allow the color to develop, measure and record the absorbance of each of the
three sample solutions using the colorimeter.
Waste Disposal
32. All colored (copper-containing) solutions should be placed in the Inorganic Waste container. All leftover
sweetener solutions may be poured down the drain.
RESULTS
1. Record your observations for the spot plate tests on the Report Sheet.
2. Record your colorimetric absorbance data for the standard solutions on the Report Sheet, and present the Beer’s
Law plot (calibration curve) for this data.
3. Record the mass and absorbance data for each of the sample solutions prepared from the sweetener packet
containing aspartame.
4. Using the calibration curve, calculate the concentration (in M) of aspartame in each of the sample solutions.
Recall from the Introduction to Spectroscopy experiment that the slope of the calibration curve = εb and that
absorbance and concentration can be related by A = εbC. Show a sample calculation.
5. Determine the amount of aspartame per packet of sweetener. Since each packet contains ~1 g of sweetener,
express this value in mg aspartame/g sweetener.
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EXPERIMENT 28
REPORT SHEET
Name: ____________________________________
Date:__________
Partner: __________________________________
Spot Plate Observations
Sugar:
Equal:
Sweet N Low:
Splenda:
Water:
Aspartame stock solution:
Standard Solutions
Concentration of aspartame stock solution
Volume Stock Solution used,
mL
Color LED light used
Concentration of standard
solution
Absorbance
Show sample calculation for concentration of standard solution:
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Sample Solutions
Mass of sweetener
used, g
Absorbance
Concentration of
sample solution
mg aspartame per
gram sweetener
Average:
Show sample calculation for concentration of sample solution:
Show sample calculation for mg aspartame per gram sweetener:
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