Salter’s A2
Engineering Proteins
Hydrolysis of Aspartame
Aspartame contains two amino acids joined together by an amide (peptide) bond. In
this experiment you are going to hydrolyse aspartame to break the linkage and then
separate and identify the amino acids by chromatography.
Requirements:
0.5 % aqueous aspartame solution
heating apparatus and test tube holders
0.2 % aqueous solutions of glycine,
500 ml beakers
aspartic acid and phenylalanine
6 M HCl (aq)
hairdryers
pipettes and teats
ninhydrin in butan-1-ol spray
small test tube
gloves
capillary tubes for spotting
Eluent
chromatography paper, pencil and ruler
Foil to cover beakers
To Prepare Eluent of butan-1-ol (CARE harmful and flammable): glacial ethanoic acid
(CARE Corrosive; avoid inhaling the vapour): distilled water in a 5:1:4 ratio. Prepare
by shaking together in a separating funnel and keeping the top layer for use as the
eluent.
Method:
Salter’s A2
Engineering Proteins
Place 4 drops of 0.5 % aspartame solution and 4 drops of 6M HCl (aq) in a small test
tube. Cautiously heat the tube, boiling it gently for about 90 seconds. Allow the
contents to cool and label it hydrolysed aspartame.
For the chromatography:
With a pencil lightly draw a line along one end of the chromatography paper. Number
each column, and write your name on the top.
Using gloves carefully spot the following solutions on the relevant column:
1
hydrolysed aspartame
2
0.5 % aspartame solution
3
0.2 % glycine solution
4
0.2 % aspartic acid solution
5
0.2 % phenylalanine solution
Set the chromatogram to run in a large beaker in a mixture of 5:1:4
butan-1ol:ethanoic acid:distilled water (prepared as instructions). This solvent is
known as the eluent. Make sure the bottom of the paper is in the eluent but the spots
are not. Cover the beaker with foil to prevent evaporation. Remove from the beaker
when the solvent front is just at the top of the columns. Mark this point with a pencil
line.
Dry the chromatograms with a hair dryer and spray them with ninhydrin until they are
wet but not dripping. Dry with a hairdryer again. Circle the spots that appear.
Analyse your results to decide what amino acids are present in aspartame.
Key Words:
Chromatography
Chromatogram
Stationary phase
Mobile phase
Eluent
Solvent front
Ninhydrin
Salter’s A2
Engineering Proteins