Core Practical 17 Lab 

Aims

To carry out a practical to explore the techniques associated with paper chromatography.

To separate and identify the amino acids present in an unknown sample. 

Equipment

● Gas jar ● Chromatography paper ● Pencil ● Ruler ● Gloves ● Chromatography solvent ● Capillary tubing ● Ninhydrin spray ● Hair dryer ● oven set at 110°C ● samples

1.      You are provided with a solution of albumen (egg white) that has been incubating with the enzyme trypsin for three days. The result is a solution of amino acids.

2.      Wearing eye protection, place your gas jar in the fume unit and pour solvent in to a depth of about 3 cm. Place the lid on the gas jar and leave it in the fume unit so that the atmosphere inside the gas jar becomes saturated with solvent fumes.

3.      Wash and dry your hands.

4.      Take a piece of chromatography paper - it should be long enough for about 5 mm to dip into the solvent and the other end should extend out of the top of the gas jar by about 20 mm. Handle the paper as little as possible throughout the experiment.

5.      Draw a faint pencil line about 40 mm from one end of the chromatography paper.

6.      Using the capillary tubing, draw up amino solution and place a tiny dot in the centre of the line.

7.      Wait for it to dry.

8.      Place another dot of amino acid solution on top of the first one using the capillary tubing and wait for it to dry.

9.      Repeat this at least six times letting it dry between applications until you have a very concentrated dot of amino acid solution. Try to keep the dot as small as possible by being patient and letting it dry fully between applications.

10.   Carefully lower the paper into the gas jar, making sure it does not touch the sides and allow it to just dip into the solvent. Fold the top of the paper over and replace the lid. Leave for several hours until the solvent has moved to within 2 cm of the top of the paper.

11. After several hours and once the solvent has moved to within 2 cm of the top of the paper, carefully remove the chromatography paper from the gas jar.

12.      Without damaging the paper, draw another pencil line marking the point where the solvent moved to and then hang the paper within the fume unit to dry.

      13.      Once the chromatography paper is dry, wearing gloves and eye protection, spray the paper carefully with ninhydrin         reagent.

      14.      Use heat as directed by your teacher to develop the chromatogram. If you continue to apply heat after the paper is dry, the amino acids will start to show up as purple spots which will become denser with more heat.

15.      Each purple spot represents one or more amino acid. Outline each spot with pencil and mark the centre of each one

16.      In order to identify them, a measurement called the Rf value is used. 

17.      Measure the distance that the solvent moved.

18.      Measure the distances that all of the amino acids moved and record in a suitably designed table.

19.      Calculate the Rf value for each amino acid and use the table below to try to identify them and record in the table.