Chem. Educator 2009, 14, 1–4
(web)1
Acid Hydrolysis of Aspartame and Identification of its Products by Thin
Layer Chromatography
Amanda Melberg, Paige Adejare, and William H. Flurkey*
Department of Chemistry, Indiana State University, Terre Haute, IN 47809, [email protected]
Received January 26, 2009. Accepted July 1, 2009.
Abstract: Aspartame, a sugar substitute, is a modified peptide composed of aspartic acid and phenylalanine
methyl ester. Hydrolysis of aspartame can produce a variety of products, including α-L-aspartyl-L-phenylalanine,
L-phenylalanyl-α-L-aspartic acid, L-aspartic acid, L-phenylalanine, and L-phenylalanine methyl ester. Many of
these products can be identified using thin layer chromatography. The products from acid hydrolysis of
aspartame were followed over a six-hour period using thin layer chromatography. Conversion of aspartame into
the peptide aspartyl phenylalanine was observed, followed by conversion of the peptide into aspartic acid and
phenylalanine. No conversion of aspartame into phenylalanine methyl ester or a β isomer of aspartyl
phenylalanine was observed. This experiment uses common techniques to demonstrate hydrolysis of a peptide
and to identify the products obtained from acid hydrolysis of a peptide.
Introduction
Aspartame, commonly known as Nutrasweet, is a lowcalorie sweetner used as a sugar substitute. Its serendipitous
discovery by a chemist at the G.D. Searle and Company in
1965 and introduction to consumers in 1981, led to a large
increase in products containing sugar substitutes [1]. In some
ways this led to the development of new sugar substitutes such
as sucralose, acesulfame K, alitame, neotame, and stevioside to
name a few. Not long after the introduction of aspartame,
reviews on the properties/stability and safety issues of
aspartame appeared in the literature [2–3].
Aspartame is a modified peptide (α-L-aspartyl-Lphenylalanine methyl ester, αAPM) containing two common
amino acids, aspartic acid and phenylalanine. The peptide can
be completely hydrolyzed under highly acidic or basic
conditions, generating free amino acids and methanol. Several
studies have examined the hydrolysis of aspartame under
neutral pH conditions [4], in aqueous solutions [5–6], and in
acidic conditions [7–9]. Historically, determination of
aspartame in food products has been carried using HPLC and
thin layer chromatography (TLC) [10–12].
Aspartame
has
been
the
subject
of
several
chemistry/biochemistry lab experiments. For example,
aspartame has been used in a sweetness test for a biochemistry
lab [13]. Analysis of aspartame in soft drinks has been carried
out using UV spectroscopy, HPLC, capillary electrophoresis
and mass spectrometry [14–16]. The kinetics of aspartame
hydrolysis and degradation has also been followed using
HPLC [8]. This latter experiment is very informative, but may
not be practical for many undergraduate labs in terms of time
and equipment. Lastly, analysis of aspartame and/or its
hydrolysis products by TLC have been used as a lab exercise
to demonstrate the composition of aspartame [17–18]. In one
of these aspartame lab experiments, aspartic acid,
phenylalanine, leucine, and aspartame were subjected to TLC
to illustrate the composition and polarity of aspartame and its
products, but hydrolysis of aspartame was not performed [17].
In another aspartame lab experiment, hydrolysis of aspartame
was carried out under basic conditions, but no actual TLC
chromatograms were given to identify the products or to show
migration patterns [18].
Conceptually, our primary goal was to carry out acid
hydrolysis of aspartame in order to demonstrate the
composition of a small peptide. A secondary goal was to
monitor the progress of the hydrolysis reaction within a threehour time frame using a relatively inexpensive visual method
of analysis. A third goal was to demonstrate that separation of
the products (amino acids) by TLC is based on their polarity.
Specifically, the products from acid hydrolysis of aspartame
were monitored using TLC over an extended time period to
show formation of the peptide aspartyl phenylalanine (DF)
followed by hydrolysis of this peptide into aspartic acid (D)
and phenylalanine (F). This experiment clearly demonstrates
conversion of a reactant into a product(s).
This experiment was developed by students participating in
undergraduate research. It has been carried out by dietetic
students in a sophomore/junior level one semester survey of
biochemistry course and lab. Because these students have only
had one year of general, organic, and biochemistry less
emphasis was spent of the chemistry of the reaction and more
time was spent on demonstrating the composition of
Nutrasweet. This experiment can be easily adapted for a senior
level biochemistry lab in a variety of formats and will be tested
out using a variety of hydrolysis conditions. We anticipate this
experiment could also be used in a general education chemistry
course to demonstrate the biological composition of a familiar
consumer product.
We believe this experiment could be a valuable, interesting,
and practical addition to a biochemistry lab course because the
reactants and products can be easily visualized, it is simple and
can be completed within two 3 hour lab periods, and it could
potentially be used in a wide variety of undergraduate
chemistry or biochemistry lab courses ranging from
introductory to advanced levels.
© 2009 The Chemical Educator, S1430-4171(09)0xxxx-x, Published on Web 12/31/2009, 10.1333/s00897092231a, xxxxxxaa.pdf
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Chem. Educator, Vol. 14, No. X, 2009
Flurkey et al.
Table 1. Rm values of NutraSweet and its products
Sample
D
F
DF
αAPM
FD
FMe
βAPM
1.5 hr spots
Brown
Red
Blue
Experimental
Rm
0.11
0.36
0.25
0.36
0.31
0.48
0.3
0.36
0.25
0.111
α APM
Fme, D
↑↓
α DF
←
DKP
→
ά FD
↓
↑↓
↓
D, F
β APM
D, F
Figure 1. Diagram of potential products from hydrolysis of
α aspartame.
O
NH 2 H
N
HO
O
O
OCH 3
O
O
O
OCH3
NH 2 O
aspartame,  APM
-L-aspartyl-L-phenylalanine methyl ester
NH 2 H
N
HO
H
N
HO
-aspartame,  APM
-L-aspartyl-L-phenylalanine methyl ester
O
O
OH
H
N
HO
O
O
OH
NH 2 O
-L-aspartyl-L-phenylalanine,  DF
-L-aspartyl-L-phenylalanine,  DF
O
O
H2 N
NH 2
O
H2 N
OH
OCH 3
Chemicals. Aspartame, β-aspartame, α-aspartyl-phenylalanine, βaspartyl-phenylalanine, phenylalanyl-aspartic acid, aspartic acid,
phenylalanine, and phenylalanine methyl ester were obtained from
Sigma Chemical Company (St. Louis, MO) or Bachem Americas, Inc.
(Torrance, CA). Silica gel thin layer chromatography plates (silica gel
G 60 F, 250 μM, 10  20 cm) on a glass backing were from EMD
chemicals. All other chemicals were of reagent grade.
Procedure. Aspartame (4 mg/ 0.4 mL) was dissolved in 2 M HCl
in a 1 mL glass vial (Kontes) and placed in an aluminum heat block
set at 95–100o C. Alternatively, the sample can be placed in a plastic
microfuge tube used for thermocyclers. Aliquots of 10 μL were
removed at various time intervals (0, 0.5, 1, 2, 4, 6, 18 hours), placed
into microfuge tubes and immediately transferred to a freezer. Care
should be used in removing samples from the heat block since the
glass vials are very hot. These aliquots can be removed by the
instructor or by students after the 3 hour lab is over, especially if the
lab begins in the morning. Samples were thawed at room temperature
and then mixed well. 0.5 μL from each hydrolysis sample was spotted
on a silica gel TLC plate using a 1 μL syringe. The syringe was rinsed
four times with water before spotting another sample. One μL
capillary tubes from Drummond can also be used to spot samples.
Standards were prepared fresh on the day of the lab at a concentration
of 1 mg per 0.1 mL of 2 M HCl. Standards included α-aspartame,
aspartic acid, phenylalanine, α-aspartyl-phenylalanine, phenylalanylaspartic acid, phenylalanine methyl ester, β- aspartyl phenylalanine,
and, in some cases, β-aspartame. One packet of Equal, containing
approximately 38 mg of aspartame, was dissolved in 5 mL of 2 M
HCl and also used as a standard in some experiments. The samples
and standards spotted on the TLC plate (10  10 cm or 10  20 cm)
were allowed to dry, and the plates were then placed in a mini - TLC
tank containing butanol: acetic acid: water (6:1:1) as a solvent [17].
The solvent was allowed to move up the plate until it reached near the
top of the plate, requiring approximately 1.5-2 hours. The plates were
removed and dried with a hair dryer. The plates were then dipped in a
0.5 % ninhydrin/ethanol solution and dried again with a hair dryer.
The plates were then placed in an oven at 110o C for about 5 min for
color development. The color and position of any spots were noted.
Patterns of the stained samples were clearly visible on the day of
staining. However, the color intensity of the individual spots varied
with the time of heating and gradually faded away with time. Some
TLC plates were scanned using a UMAX Astra 4950 scanner and
stored as digital images. We have increased the intensity of the spots
in the original chromatograms using the photo-imaging software in
order to make stained samples in the figures more visible.
Alternatively, the TLC plates can be viewed under UV light at either
254 nm or 365 nm and images captured with suitable photodocumentation systems.
OH
HO
Results and Discussion
O
L-phenylalanine, F
L-aspartic acid, D
H
N
O
L-phenylalanine methyl ester, Fme
O
O
OH
OH
O
HO
O
N
H
H 2N
N
H
O
diketopiperazine, DKP
methylenecarboxyl-6-benzyl-2,5-diketopiperazine
L-phenylalanyl-L-aspartic acid, FD
Figure 2. Structural drawings of α aspartame and its potential
products after hydrolysis.
A variety of products can be formed during hydrolysis of
aspartame depending on the conditions (flow diagram in
Figure 1). The structural representations of these products are
shown in Figure 2. Samples taken at various time intervals
during hydrolysis may yield the peptide aspartyl-phenylalanine
(DF), phenylalanine methyl ester (Fme), aspartic acid (D), and
or phenylalanine (F). Complete hydrolysis of aspartame should
yield aspartic acid, phenylalanine, and methanol as products.
The standards used in this experiment were subjected to
TLC and stained with ninhydrin to localize any compounds
with free amino groups (Figure 3). We tested a variety of TLC
solvents, TLC media, hydrolysis conditions, and staining
methods using these standards to identify conditions suitable
for the lab experiment (data not shown). Each of the standards
showed a slightly different mobility except for phenylalanine
© 2009 The Chemical Educator, S1430-4171(09)0xxxx-x, Published on Web 12/31/2009, 10.1333/s00897092231a, xxxxxxaa.pdf
Acid Hydrolysis of Aspartame and Identification of its Products…
Figure 3. Thin layer chromatography of amino acid and peptide
standards TLC was carried out as described in the experimental
section. (D) L-aspartic acid; (F) L-phenylalanine; (α DF) α-L-aspartylL-phenylalanine; (α FD) L-phenylalanyl-α-L- aspartic acid; (Fme) Lphenylalanine methyl ester; (α APM) α aspartame; (β APM) β
aspartame; (β DF) β L-aspartyl-L-phenylalanine. Samples were
subjected to TLC for one hour and then stained with ninhydrin.
Figure 4. Hydrolysis of aspartame and thin layer chromatography of
the products Hydrolysis of aspartame and TLC were carried out as
described in the experimental section. Samples were withdrawn at 0
min, 30 min, 60 min, 90 min, 2 hours, 4 hours, and 6 hours and then
subjected to TLC for one hour followed by staining with ninhydrin.
Standards: L-aspartic acid (D), L-phenylalanine (F), α-L-aspartyl-Lphenylalanine (ά-DF), aspartame (α-APM), L-phenylalanyl-α-Laspartic acid (ά-FD), L-phenylalanine methyl ester (Fme), and β
aspartame (β-APM) were also subjected to TLC on the same plate at
the same time.
(F) and aspartame (APM). These two compounds have similar
mobility but do have slightly different stain colorations. The
standards in Figure 3 indicate that one can differentiate
between DF, FD, D, F, and Fme using TLC. Therefore, one
should be able to follow the products formed during hydrolysis
using TLC. β APM and β DF have different migration patterns
and staining patterns compared to their corresponding α
isomers (Figure 3). Thus, one should also be able to detect any
potential isomerization of these two compounds during
hydrolysis. If APM (either α or β isomer) is dissolved in 2 M
HCl shortly before the experiment, it does not show partial
conversion into DF, D and F. However, we have noticed
partial hydrolysis of APM if the samples are stored in 2 M
HCL for several hours at room temperature before the
experiment or TLC is started.
An example of acid hydrolysis of APM and the products
formed during hydrolysis is shown in Figure 4. Rm values
were determined for the standards and the products detected at
the 1.5 hr time point. Although one can use Rm values to
Chem. Educator, Vol. 14, No. X, 2009
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identify the products, it is much easier to observe the spot
patterns and coloration for identification of compounds since
some of the products and starting material have similar Rm
values. At zero time, APM is the primary component observed.
At 30 min, the peptide DF is clearly visible as well as the
amino acids, D and F, compared to the migration pattern and
Rm values of the standards. This pattern would be expected if
partial hydrolysis had occurred. At 30 min, there does not seem
to be any Fme present, suggesting that hydrolysis of the ester
group has taken place before hydrolysis of the peptide bond.
As the time of hydrolysis increases, smaller amounts of the
peptide DF are present and increasing amounts of D and F are
apparent. In other words, the intensity of the spot
corresponding to DF decreases and the intensities of the spots
corresponding to the products D and F increase. Because
αAPM and F have similar mobilities and Rm values under
these TLC conditions we can only assume the amount of APM
is decreasing with time. Complete hydrolysis of APM usually
required longer than two hours under our experimental
conditions. In addition, as hydrolysis occurred there did not
seem to be a rearrangement or conversion of DF into FD or
into β APM or β DF (compare Figure 1 and Figure 4). These
compounds have different colorations and Rm values.
Although some of the hydrolysis products may be derived
from DKP, this compound is not detected by ninhydrin
staining at the μg level [12]. Therefore, we do not observe
DKP as an intermediate product during the hydrolysis.
Under basic conditions APM forms DKP and/or DF and
methanol [4, 7, 9]. In preliminary trials for this experiment we
also tried to carry out hydrolysis of APM using 2 M KOH and
observed that complete hydrolysis took much longer than when
using acidic conditions. We did, however, observe the
formation of D and F, but APM and DF showed similar
mobilities during TLC, making it difficult to monitor the
disappearance of APM and DF with time (data not shown).
Not only did hydrolysis take longer, but the spots were
somewhat distorted on the TLC plates. Therefore, we did not
pursue base hydrolysis of Nutrasweet further for use as an
experiment in the biochemistry lab. However, during the time
it takes for chromatography to finish, it might be advantageous
to discuss possible mechanisms of acid and base catalysis of
Nutrasweet.
Under mildly acidic and neutral pH conditions, an
intramolecular cyclization of APM with loss of methanol
occurs to form DKP as the primary product [4–6, 9, 19].
Hydrolysis of the methyl ester without cyclization yields the
peptide DF as well as the free amino acids D and F due some
hydrolysis of the peptide bond. Mildly acidic and neutral pH
conditions can also lead to the formation of the β isomers of
APM and DF [4, 6, 7]. In preliminary trials we have also tried
hydrolysis using mildly acidic conditions (citrate buffer pH
4.5) and neutral conditions (phosphate buffer pH 7.0), but
observed little hydrolysis of APM and little conversion to D
and F (data not shown). In addition, we did not observe any
isomerization of APM to β APM which has been reported
under similar conditions [4, 6, 7].
In more acidic hydrolysis conditions (pH < 2) DF should
form from loss of methanol [7, 9]. In addition, less DKP
should be formed by intramolecular cyclization. The peptide
bond of DF is then hydrolyzed to yield D and F. This is very
similar to what we observed in our experiments using our
acidic hydrolysis conditions and analysis by TLC. In summary,
under acidic conditions, we observed formation of DF, D and
© 2009 The Chemical Educator, S1430-4171(09)0xxxx-x, Published on Web 12/31/2009, 10.1333/s00897092231a, xxxxxxaa.pdf
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Chem. Educator, Vol. 14, No. X, 2009
F within 30 min of hydrolysis (Figure 4). We did not observe
formation of any β isomers of APM or DF, nor did we observe
isomerization of DF into FD. This is also consistent with the
hydrolysis products expected under more acidic conditions.
Our lab procedure for acid hydrolysis of aspartame is more
instructive than simply performing TLC using D, F, and APM
to illustrate conversion of aspartame into its component amino
acids [17]. We believe it may also be more useful than
hydrolysis using KOH [18] since we have noticed that this
hydrolysis reaction takes longer and the products do not
separate as well by TLC (data not shown). In addition, the
results from this experiment are not as instrumentation
intensive as those reported by Williams et. al. for examining
the kinetics of APM hydrolysis (8). Our protocol is well suited
for monitoring several products from hydrolysis of aspartame
and can be carried out in the three-hour time blocks commonly
used in many educational laboratories. This experiment also
fulfills our intent to demonstrate peptide composition and
hydrolysis of a peptide using a familiar commercial
sweetner/modified peptide. It also simulates the process that is
probably occurring during digestion using peptidases and
indicates how amino acids are derived from APM.
Depending on the nature of the course in which it is used,
this laboratory exercise could be modified or extended in a
variety of ways to illustrate various concepts in chemistry and
biochemistry. It could be modified to compare acid and base
hydrolysis of APM, to quantify the reactants and products of
hydrolysis by densitometry, or to look for the formation of
different products (such as the β isomers) using neutral and
mildly acidic conditions. This same procedure could be carried
out using β APM and either acid or base hydrolysis to
determine if the reactions result in conversion to any α
isomers. One could also use a peptidase(s) to carry out this
reaction and compare the products to acid hydrolysis. Lastly,
this experiment could be coupled with model building, either
using a computer or with ball and stick/space filling molecular
models. While the students are waiting during the hydrolysis
and/or TLC part of the experiment, this model building
exercise could be used to illustrate which bonds are broken and
what products are formed.
Flurkey et al.
specialized equipment and can be completed within time
constraints common to many laboratory settings. The
experiment can be used in beginning and advanced
chemistry/biochemistry classes in a variety of formats and
provides an introduction to separation and characterization of
amino acids and peptides.
Acknowledgments. We would like to thank Dr. Jennifer
Inlow for a critical evaluation of this manuscript and Dr.
Richard Kjonaas for providing the figures using Chem Draw.
References and Notes
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We have described a biochemistry lab experiment that
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amino acids was followed over an extended time period using
thin layer chromatography. This experiment does not use
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© 2009 The Chemical Educator, S1430-4171(09)0xxxx-x, Published on Web 12/31/2009, 10.1333/s00897092231a, xxxxxxaa.pdf