Experiment Number 4

Colorimetric Assay of Aspartame in Commercial Sweeteners

Marissa Steck

SCH 143-03 General Chemistry Ⅱ Laboratory

October 15, 2020

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Ⅰ. TITLE:

Experiment #4, Colorimetric Assay of Aspartame in Commercial Sweeteners (Quantitative

Spectroscopy)

Ⅱ. OBJECTIVES:

The purpose of this experiment is to determine the concentration of Aspartame in commercial

sweeteners by using colorimetric assay and Beer’s Law with Biuret-Aspartame solutions.

Ⅲ. THEORY:

This experiment requires laboratory equipment. The necessary equipment includes a Vernier
Colorimeter, a volumetric flask, eight cuvettes, test tubes, a pipet, a biuret reagent (Copper Ⅱ), a
packet of Essential Aspartame Sweetener, and a packet of Equal Original Sweetener.

The main goal of this experiment is to find the mass of aspartame per gram of sweetener.
Aspartame is a dipeptide derivative that is commonly found in commercial sweeteners.
Aspartame has a molecular formula of ​C1​ 4​H1​ 8​N2​ ​O5​ . ​In this particular experiment, the calculated
mass of aspartame in the sweeteners will be compared with the true value of aspartame in
sweeteners, 37 mg.
(​https://www.uab.edu/shp/nutritiontrends/recipes-food-facts/food-facts/artificial-sweeteners​) The
chemical structure of aspartame is as followed:

https://www.researchgate.net/figure/Structure-of-aspartame-N-L-a-aspartyl-L-phenylalanine-1-methyl-ester_fig1_6011307

A biuret reagent is used to determine the concentration of Aspartame. Aspartame binds with
copper (Cu ​Ⅱ) ions and acts as ligands due to the N in the amino group and O in the carboxylate
group, resulting in a dark blue color.

The experiment requires an analysis known as colorimetric assay. Colorimetric assay is an

analysis where the concentration of a complex is determined based on its absorbance of light. To
achieve this, a Vernier Colorimeter is used. A Colorimeter, is a spectrometer that shines light of
four different wavelengths of a sample, and then measures the absorbance of light by the
chemical sample. In this particular experiment, the colorimeter will use the 635 nm (red)
wavelength. The absorbance of visible light is typically opposite of the visible color. The amount
of light is directly proportional to the concentration of the solution that the light is passing
through, which is supported by the Beer’s Law. The Beer’s law Plot shows the correlation
between absorbance and concentration. In this particular experiment, the plot compares the
standard solutions of the aspartame-Cu(Ⅱ) with the aspartame-Cu(Ⅱ) solution with sweeteners.
A volumetric flask is used to mix the solutions of aspartame standard solutions with the
aspartame-Cu(Ⅱ) complex. The cuvettes hold the six different aspartame standard solutions and
are filled with stock aspartame. A pipet transfers all the reagents. The test tubes allow the
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solutions to be inverted to mix, as well as to golf excess solution. The packet of Essential
Aspartame Sweetener and the packet of Equal Original Sweetener are the two sweeteners used to
measure the amount of aspartame in them.

Ⅳ. PROCEDURE:

For the experiment, when conducting the analysis of standard solutions a colorimeter was
required. A Vernier Colorimeter was set to a wavelength (635 nm, red). The first solution
prepared was the “blank” sample. This was done by adding the biuret solution (2.00 mL) to a
volumetric flask (10.00 mL) and diluted to volume with water. A pipet transferred the reagent
into a cuvette, where the cuvette was then filled with blank solution. Next, the five aspartame
standard solutions were prepared. For the aspartame standard (0.50 mg/mL), stock aspartame
solution (1.00 mL) was added to a volumetric flask (10.00 mL) and into a cuvette. From there,
the absorbance for each sample (blank, 0.50 mg/mL, 1.00 mg/mL, 1.50 mg/mL, 2.00 mg/mL,
2.50 mg/mL) was collected after waiting at least fifteen minutes for the samples to react and
recorded. This process was repeated three times, giving a total of three readings for each of the
six samples. Lastly, it was diluted to volume 10.00 mL with water. The solutions remained blue.

For the analysis of the unknown solutions, two solutions of sweetener were used. The brand of
sweetener 1 was a packet of Essential Everyday Aspartame Sweetener, no calories. The
ingredients of Essential Everyday Aspartame Sweetener include dextrose with maltodextrin,
aspartame, and acesulfame potassium. The mass of sweetener 1 was collected. The Essential
Everyday Sweetener (0.4219 g) was put into a volumetric flask (10.00 mL). To dissolve the
sweetener powder, water (5 mL) was added, and then the biuret reagent was added (2.00 mL).
Lastly, it was diluted to volume with water, and sealed with parafilm. The parafilm allowed the
solution to be inverted to mix. After fifteen minutes, a cuvette was filled with the solution and
placed into the Colorimeter (635 nm) . From there, the absorbance of the solution for sweetener 1
was measured and recorded. This process was repeated three times, giving a total of three
readings for sweetener 1. The entire process was also repeated for sweetener 2. Sweetener 2 was
a packet of Equal Original, 0 calorie Sweetener. The ingredients of Equal Original, 0 calorie
Sweetener include dextrose with maltodextrin, aspartame, and acesulfame potassium. The mass
of sweetener 2 was collected. The Equal Original (0.3969 g) was put into a volumetric flask
(10.00 mL). To dissolve the sweetener powder, water (5 mL) was added, and then the biuret
reagent was added (2.00 mL). Lastly, it was diluted to volume with water, and sealed with
parafilm. The parafilm allowed the solution to be inverted to mix. After fifteen minutes, a cuvette
was filled with the solution and placed into the Colorimeter (635 nm). This process was repeated
three times, giving a total of three readings for sweetener 2.

Ⅴ. DATA:

Table 1. Absorbance Readings for Standard Aspartame Solutions at 635 nm

Standard Soln Absorbance Absorbance Absorbance Absorbance

(mg / mL) A1 A2 A3 Average

Blank 0.001 0.001 0.001 0.001

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0.50 0.101 0.099 0.100 0.100

1.00 0.209 0.208 0.218 0.212

1.50 0.296 0.315 0.293 0.301

2.00 0.394 0.395 0.392 0.394

2.50 0.490 0.492 0.509 0.497

Table 2. Mass of Sweetener Samples

Brand of Sweetener Mass (g)

Essential Everyday Aspartame Sweetener 0.4219

no calories

Equal Original 0 calorie Sweetener 0.3969

Table 1. Absorbance Readings for Commercial Sweetener Solutions at 635 nm

Brand of Absorbance Absorbance Absorbance Absorbance

Sweetener Rdg Rdg Rdg Average
A1 A2 A3

Essential 0.378 0.374 0.384 0.379

Everyday
Aspartame
Sweetener

Equal Original 0.219 0.217 0.217 0.218

Sweetener

Ⅵ. CALCULATIONS:

The Beer's Law Plot shows the relationship between absorbance and concentration. To determine
the amount of aspartame (concentration) the equation in the Beer’s Law Plot can be used.
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To determine the concentration of aspartame in the Essential Sweetener, the trendline equation
from the Beer’s Law Plot is used. The mass of the Essential Sweetener and the average
absorbance of the Essential Sweetener is also needed.

Mass: 0.4219g Avg. Absorbance: 0.379

Equation: y = 0.1972x + 0.0043
0.379 = 0.1972x + 0.0043
0.3747 0.1972x
0.1972 = 0.1972
X = ​1.900 mg / mL (concentration of solution)

To determine the concentration of aspartame in the Equal Sweetener, the trendline equation from
the Beer’s Law Plot is used. The mass of the Equal Sweetener and the average absorbance of the
Essential Sweetener is also needed.
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Mass: 0.3969g Avg. Absorbance: 0.218

Equation: y = 0.1972x + 0.0043
0.218 = 0.1972x + 0.0043
0.2137 0.1972x
0.1972 = 0.1972
X = ​1.0837 mg / mL (concentration of solution)

To determine the mass of aspartame in the Essential sweetener solution (mg), the solution
concentration and volume will be used with stoichiometry.

Solution Concentration: 1.900 mg / mL

1.900 mg
1 mL × ( 101mL ) = ​19.00 mg Aspartame

To determine the mass of aspartame in the Equal sweetener solution (mg), the solution
concentration and volume will be used with stoichiometry.

Solution Concentration: 1.0837 mg / mL

1.0837 mg
1 mL × ( 101mL ) = ​10.837 mg Aspartame

To determine the concentration of aspartame in the Essential sweetener packet (mg/g), the mass
of aspartame in the sweetener solution will be divided by the mass of the sweetener.

19.00 mg Asp
0.4219 g = ​45 mg/g Aspartame

To determine the concentration of aspartame in the Equal sweetener packet (mg/g), the mass of
aspartame in the sweetener solution will be divided by the mass of the sweetener.

10.84 mg Asp
0.3969 g = ​27.31 mg/g Aspartame

To calculate the average of the class data for Group A, I took the students with Equal and
Essential Sweeteners values, added them and divided them by the total number of values.
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Name Aspartame in
Sweetener (mg/g)

Kristi Kada Equal: 21.11 mg/g

Essential: 16.61 mg/g

Taylor Holmes Equal: 15.84mg/g

Essential: 18.43mg/g

Marissa Steck Equal: 27.3 mg/g

Essential: 45.0 mg/g

Alyssa Powell Equal: 21.57 mg/g

Essential: 31.71mg/g

Jameson Kutz Equal: 33.44 mg/g

Essential: 25.15 mg/g

Elizabeth Varner Equal: 22.72 mg/g

Essential: 17.45 mg/g

Mackenzie English Equal: 29.34 mg/g

Essential: 26.39 mg/g

Vaneris Fuentes Equal: 31.50 mg/g

Essential: 33.97 mg/g

LiLing Caldwell Equal: 17.40 mg/g

Essential:19.60 mg/g

Elizabeth Varner Equal: 22.72 mg/g

Essential: 17.45 mg/g

Essential Sweetener Class Average:

16.61 + 18.43 + 45.0 + 31.71 + 25.15 + 17.45 + 26.39 + 33.97 +19.60 +17.45
10 = ​25.18

Equal Sweetener Class Average:

21.11 + 15.84 + 27.3 + 21.57 + 33.44 + 22.72 + 29.34 + 31.50 + 17.40 + 22.72
10 = ​24.29

To determine the percent error of the Equal and Essential Sweeteners and the class average, The
absolute theoretical (true) value was subtracted from the absolute experimental value. Then that
number was divided by the theoretical value, and multiplied by 100.
True value: 37 mg ​Artificial Sweeteners - SHP - Nutrition Trends

Essential Sweetener % Error:

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|45.0− 37|
× 100 = ​21.7%
37
Equal Sweetener % Error:
|27.31 − 37|
× 100 = ​26.2%
37
Essential Sweetener class average % Error:
|25.18−37|
× 100 = ​31.9%
37
Equal Sweetener class average % Error:
|24.29−37|
× 100 = ​34.4%
37
VII. DISCUSSION:

The experiment was completed successfully. All of the solutions reacted as they were expected
to. The results of the experiment for the Essential Sweetener Solution were 1.900 mg / mL for
the concentration, and had a mass of 19.00 mg of Aspartame. The concentration of aspartame in
the Essential sweetener packet (mg/g) was 45.0 mg/g.​ ​The results of the experiment for the Equal
Sweetener Solution were 1.0837 mg / mL for the concentration, and had a mass of 10.837 mg of
Aspartame. The concentration of aspartame in the Equal sweetener packet (mg/g) was 27.31
mg/g. The result for the concentration of aspartame in the Equal sweetener packet (27.31 mg/g)
was good. However, the concentration of aspartame in the Essential sweetener packet (45.0
mg/g) was not good. The number seems extremely high compared to the true value (37 mg/g)
and the class average (25.18 mg). That being said, the percent error was extremely high vs the
true value at 21.7% error​ ​and the class average at 31.9% error. The percent error for the Equal
sweetener packet (27.31 mg/g) was also high compared to the true value at 26.2%, and the class
average at 34.4% Possible sources of errors could have been using too much sweetener, or not
leaving the solutions in the Calorimeter long enough. To improve the results of this experiment,
next time I would make sure to get a very accurate mass close to the expected value (.4000g),
and I would make sure to leave all of the solutions in the Calorimeter for the same amount of
time. For this experiment, additional experimental work could be calculating the concentration of
the other ingredients in the sugar packet. Such as, dextrose with maltodextrin and acesulfame
potassium. Over the course of this experiment, I now have a better understanding of how to
calculate concentration and using the percent error formula. I also learned that there can be a
various amount of aspartame among each packet of sweetener. In addition, I learned that it is
extremely important to label equipment, and what solutions are in what cuvette because all of the
solutions look very similar, it is very easy to become confused and record incorrect data.