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

holders0.2 % aqueous solutions of glycine, aspartic acid and phenylalanine

500 ml beakers

6 M HCl (aq) hairdryerspipettes and teats ninhydrin in butan-1-ol spraysmall test tube glovescapillary tubes for spotting Eluentchromatography 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: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 aspartame2 0.5 % aspartame solution3 0.2 % glycine solution4 0.2 % aspartic acid solution5 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:

ChromatographyChromatogram

Stationary phaseMobile phase

EluentSolvent front

Ninhydrin