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 Foil to cover beakers
ruler

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:
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
Salter’s A2 Engineering Proteins

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