Analysis of Aspartame and its Hydrolysis Products by
~ h i n k a ~ chromatography
er
Alfred R. Conklin
Wllmington College, Wllmington, OH 45177
Undergraduate lahoratory exercises involving the separation and identification of amino acids have been developed
(1-4). Also experiments that demonstrate the hydrolysis of
prvreinsund simple polypeptidesand thesubsequent identification of amino arids have been reported ( 4 . 5). These
exercises use a variety of thin-layer add colu~nchromatographic procedures and a variety of solvent systems.
For some years I have been developing a lahoratory experiment that would allow students to separate and identify
amino acids found in food. This exercise is for u s e in a
nonmajors or general chemistry laboratory. Because the
background of the students in such courses is limited, the
experiment must deal with a simple protein or polypeptide
that most students would know about. An additional ohjective would be for this experiment to serve as an introduction
to chromatographic techniques.
Accomplishing these objectives is complicated because of
the variety of amino acids found in common proteins. Of
course, small polypeptides, such as oxytocin, bradykinin, or
encephalins could he used. However, these are expensive,
potentially dangerous, and not commonly known by students. Thus, what I am looking for is a polypeptide that is
readily available, safe for students to use, and recognized by
them.
There is a common dipeptide, actually the methyl ester of
a dipeptide, that can be used for such an exercise. This is
asnartame. better known bv the student as Nutrasweet.
Aspartame and its hydrolysis products can easily be separated and identified usinrr s i m ~ l ethin-laver chromatomaphy (6). Other chromato&ap& separation methodsare
equally suitable and can be used to separate aspartame from
other constituents in food (7). However, thin-layer is preferred for freshman laboratories because i t is simple and
fast. Both aspartame and its hydrolysis products, except for
methanol, are easily detected by common amino acid visualizing reagents.
Aspartame, aspartylmethylphenylalanine, is readily hydrolvzed under acid conditions to its constituent Darts.
. . aspar& acid, phenylalanine, and methanol. This reaction occurs r a ~ i d l va t elevated temDeratures and slowlv hut measurabl; with long storage a t room tempera&e.
The
hydrolysis can be represented
Commercial preparations of solid Nutrasweet, that is, not
as part of drinks, contains components in addition to aspartame. The common powder contains aspartame, dextrose,
silicon dioxide, cellulose, etc. One preparation also contains
the amino acid leucine. Although most of the additional
components pose no problem in analysis, leucine is difficult
to separate from phenylalanine. Fortunately, its occurrence
is infrequent enough so as not to pose a serious problem in
most cases.
Solutions of aspartic acid, leucine, and phenylalanine containing 30 mgl25 mL Hz0 work well as standards. For aspartame a solution containing 30 mgI25 mL H20 of pure aspartame or 90-180 mgI25 mL H20 of commercially available
tabs or granular (powder) Nutrasweet is adequate. Using
these concentrations, one spot (0.5 r L approximately 1mm
in diameter) will contain enough amino acid or aspartame to
allow easy detection after chromatography and visualization.
Chromatography can be carried out using either cellulose
or silica gel thin-layer plates. Separation is faster on the
former. Plastic-hacked thin-layer sheets allow students t o
cut microscope slide size "plates" for the analysis (see figure). This size plate gives adequate separation while allowing maximum speed and economy. For more complex separations, that is, partially hydrolyzed beverage samples, a
longer plate cut to fit into a tall form heaker (58 X 140 mm)
works well.
Plates can be spotted using either Pasteur pipets pulled
out to make a capillary tip, capillary tubes pulled t o a finetip, or a microliter syringe. I t is a good idea to have students
practice spotting a previously used or a waste piece of plate
before starting to spot a plate for development.
Elution is carried out with one of two eluants depending
on the type of plate used. A mixture of n-hutanol-acetic
acid-water (4:1:5 vlv) is a good eluant system for cellulose.
For silica gel a solution of n-butanol-acetic acid-water ( 6 2 2
or 12:2:2 vlv) is a good eluant.
Plates are spotted and spots allowed to dry, then the
plates are placed in a developing chamber containing an
appropriate eluant. Development, which is typically carried
out in a slide-staining (Copin) jar, or a beaker as described
above, is allowed to continue until the solvent front is within
a millimeter of the top of the plate. Plastic wrap makes a
suitable cover when a heaker is used as a developing chamber.
The dried plates are sprayed with either a0.2% solution of
ninhydrin (1,3,3-triketohydrindenehydrate) in acetone or
0.10% isatin in acetone. Sprayed plates are allowed to dry
and are heated t o hasten the reaction. An infrared lamp
works as well or better than putting the plates in the oven t o
heat.
Both visualizing reagents work well. Ninhydrin gives a
typical purplish color upon reaction with amino acids and
with asDartame. Isatin Droduces different colors with differen1 amkoacids. Wr ha;e,on occasion, been unable toohtnin
\,isualization with old solutions of ninhydrin. Thus. if no
spots are observed after heating, check the activity of your
visualizing reagent.
Volume 64 Number 12 December 1987
1065
These s a m e nrocedures c a n
be used to look for aspartame
or its decomposition ~ r o d u c t s
in beverages o r other foods.
However, beverages are dilute
solutions, s o 12 to 15spots (2+
cL), w i t h d r y i n g b e t w e e n
spotting, m u s t b e m a d e in t h e
s a m e place. Although drying
between spotting i s t i m e consuming, it does n o t t a k e long if
t h e spots a r e dried using a
hair dryer.
Experimental
Obtain a cellulose thin-layer
sheet and cut out several microscope-sire picrrs ro be used in the
analysis. T h r fhin-layer <heel
s h d d not be touched with fingers or anything else, as foreign
material on the olate mav adv e r 4 y aflrcf the nnnlysis. Make a
small mark alcmg the side 1u.r
pencil or scrape off a little of the
layer) of the plate about 1 cm
from the bottom. Use the mark as
a guide far spotting. All spots
should be placed an equal distance from the bottom of the
plate.
A 15- X 6.5cm thin-layer plate
Obtain or make solutions, as
showing the separation of aspartic described above, of aspartic acid,
acid (A), phenylalanine (B), leucine phenylalanine, and aspartame
(C). aspartame (D), and a minure (El (commercial Nutrasweet can be
used as the aspartame standard).
of A, B. and D.
Also obtain a sample of food containing aspartame.
Spot plates with a capillary pipet; allow spots to dry before placing in the eluant. If several spottings are necessary, all spots should
be dried thoroughly hetween spotting. Place plates in an appropriate development chamber containing about 5 mm of eluant, and
allow elution to continue until the eluant is within 5 mm of the top of
the plate. Mark the top of the eluant (the line between the wetted
and unwetted thin layer) as soon as the plate is removed. Once the
eluant has evaporated, which can happen very rapidly, it may be
imoossihle to determine the too of the eluant.
Once
-~~~~
drv.
~ ~ -the
~-~~
* , nlates
*~~~~~~~
are soraved with the aoorooriate
~ . .. visualizine"
reagent. Plates should he just wetted when spraying. All solvent is
allowed to evaporate before heating the plates. Heating is done,
C for about 2-3 min or under an infrared
either in an oven at 105 '
~
~~~~
~~
in the Figure
Distance
moved lcml
Eluant front
Aspartic acid
Phenylalanine
Leucine
Aspamme
19
7.9
14.3
15
16
0.38
0.75
0.79
0.83
lamp until spots just appear. Spots will darken somewhat with time
but should be dark enough at this paint to allow collection of data.
Treatment of Results
Prepare a table of data including distances moved by spots and
Ris. The solvent front is measured from the spotting mark to the top
of the solvent front. Also measure the movement of the spots from
the starting mark to the middle of the respective spot. The Rt is then
calculated as follows:
~~~~~
~.~
~~.
~
1066
~
Data Obtalned from Thin-Layer Plate Shown
~
Journal of Chemical Education
where
S1 = distance spot 1moved,
Er= distance eluant front moved, and
Rn = Rt spot 1.
This same procedure would be continued until you obtain atable of
R{s for all spots. The table is an example of the type of data obtained
for the thin layer plate as shown in the figure.
Now obtain a fresh and an old partially hydrolyzed sample (a
fresh sample brought to a boil will suffice as an old sample) of an
aspartame containing beverage and carry out a thin Layer analysis.
Prepare a table of the spots and their Ris. From this table identify
the comoonents found. Usine this tvoe
.. of olate.
. . would vou exuect
the ~ ( s ' t obe the same as th&e obtained on the short& plates, or
different? What do you find?
Acknowledgment
I would like to t h a n k R o n F e r g u s o n f o r his h e l p i n developing this exercise.
Literature
Clted
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3. Hurlbut. J. A,: Raika,T.J. J.Chem. Educ. 1978.55, 794.
4. Fierer. L.: Fieser, M. Bosie Organic Chumistry: Heath: Bmton, 1959.
5. Deuer.D. F.J. ChemEduc. 1976.52. 338.
6. Sherma. J.;Cupin,S.: Fullweiler,d.Am.Lnb. 1985,March, 131.
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~&kowrki,L. J.: Warren, F. V.. Jr. J. Chem.Edue. 1985,62.6