Experiment 2

Preparations

Aspartic Acid

1) Measure accurately 1g of Aspartic acid.

2) Put it into a test tube and pour in 100ml of distilled water

EQUAL

1) EQUAL (Aspartame) can be purchased from shops. EQUAL is a type of food sweetener.

2) Open the packet of EQUAL and pour it into a container.

3) Measure accurately 0.1g of EQUAL.
4) Put it into a test tube and pour in 1ml of distilled water.

EQUAL in Acid

1) Measure accurately 0.1g of EQUAL.

2) Put it into a test tube and pour in 1ml of 6M HCl (Hydrochloric Acid)

3) After that, heat the solution in a water bath for 30 minutes.

4) Note that while heating, the solution may turn brown due to sugar content.

Standards

1) Place the Aspartic acid, EQUAL (Aspartame), EQUAL in Acid (Hydrolysed Aspartame) on a test tube rack.

2) Put a few glass pipettes too.

3) It should look like the picture on the right.

Thin Layer Chromatography Slides

1) A microscope slide is needed to make a TLC slide.

2) Scoop 2 spatulaful of Silica gel into 2ml of distilled water.

3) Stir well.

4) Use a pipette to put some of the solution onto the slide. Ensure that the solution covers the surface of the slide throughly and has to be as thin as possible.

5)Bake the slide at 130^oC for at least 10 minutes.

Ninhydrin in Spray Gun

1) Obtain a spray bottle from anywhere like shops or supermarkets.

2) To prepare the solution, measure exactly 0.2g of ninhydrin and place it into 100ml of Acetone.

3) Pour the solution into the spray bottle

Amino Acid Chromatography

Aim: To investigate if EQUAL (Aspartame) can be separated using the method known as hydrolysis

Problem Statement: Will Phenylalanine appear above the Aspartic acid in the hydrolysed Aspartame?

Hypothesis: Phenylalanine should appear above the Aspartic acid in the hydrolysed Aspartame

Procedure:

- Paper Chromatography

1) Cut out a piece of chromatography paper measuring 5.5cm by 10 cm.
2) Draw a line across the entire paper using pencil at about 1.5cm from the bottom of the paper.
3) Use a glass pipette and dip it into the amino acids. As it is removed from the liquid, note that there will be some liquid in the tip of the pipette.

4) Put the tip on another piece of chromatography paper to remove excess liquid.

5) When the amount of liquid left in the pipette is very little, spot it onto the line on the chromatography paper prepared earlier. Try to keep the liquid in its minimum amount.

6) Repeat this process for every amino acid.

7) After that is done, place the chromatography paper into the solvent that is prepared.

8) Let the solvent run until it reaches about 3 quarters of the paper. When it reaches that point, remove it from the solvent.

9) Dry the paper with a hairdryer

10) When it is completely dry, spray it with the ninhydrin solution prepared earlier.

11) After it is sprayed, dry the chromatography paper using the hairdryer. As it dries, purple colours form on the paper

-Thin Layer Chromatography (TLC)

1) Place the slide on a piece of clean paper.

2) Estimate a distance of 1cm away from the bottom of the slide. Make markings on the paper.

3) Use a glass pipette and dip it into the amino acids. As it is removed from the liquid, note that there will be some liquid in the tip of the pipette.

4) Put the tip on a piece of chromatography paper to remove excess liquid.

5) When the amount of liquid left in the pipette is very little, spot it onto the line on the chromatography paper prepared earlier. Try to keep the liquid in its minimum amount.

6) Repeat this process for every amino acid.

7) After that is done, gently place the slide into the solvent that is prepared.

8) Let the solvent run until it reaches about 3 quarters of the paper. When it reaches that point, carefully remove it from the solvent.

9) Bake it in the oven at 130^oC until it dries but not burnt.

10) After that, carefully remove it from the oven and spray it with ninhydrin. Ensure that the spray is fine and does not create a hole on the slide.

11) Bake it again in the oven at the same temperature. Make sure that it does not burn the sugar. When all the colours appear clearly, remove it from the oven.

Observation:

The separation of the proteins is much clearer in the TLC method because it is much more sensitive. Phenylalanine appears much clearer in TLC than chromatography paper

The hydrolysed EQUAL (Aspartame) has two spots, Phenylalanine, the higher spot, and Aspartic acid, the lower spot.

Inference: When hydrolysed, Aspartame is separated into Phenylalanine and Aspartic acid

Variables:
1) Constant: Aspartame, Aspartic Acid
2) Manipulated: Hydrolysed Aspartame (Time taken)
3) Responding: Detection of Phenylalanine and Aspartic acid

Conclusion: Hydrolysis method can be used to separate the bond between two amino acids