Unit 2 assignment 2 template – Chromatography
Introduction of chromatography
You are demonstrating different chromatographic techniques to separate and identify components
in mixtures. One of these techniques is paper chromatography and you will be using this to separate
mixture of plant pigments and identify them.
Equipment
Gas jar
Chromatography paper
Pencil
Ruler
Gloves
Amino acid mix
Individual amino acids
Solvent
Capillary tubing
Ninhydrin spray
Oven set at 110°C
Method
1. Wearing eye protection, pour solvent into your gas jar to a depth of about 1cm. Place
the lid on the gas jar and put in the fume cabinet so that the atmosphere inside the gas
jar becomes saturated with solvent fumes. Wash and dry your hands.
2. Take 1 pieces of chromatography paper - they should be long enough for about
0.5cm to dip into the solvent and the other end should extend out of the top of the gas jar by
about 2c. Use gloves to handle the paper, and try to only touch the sides.
3. Draw a faint pencil line about 3cm from one end of the chromatography paper.
4. Using the capillary tubing, draw up amino solution and place 4 tiny dots on the line.
Wait for it to dry. Use the piece of paper for the amino acid mixture, one dot for pure
alanine, one dot for pure glycine, and one dot for pure lysine,
5. Place another dot of amino acid solution on top of the first one using the capillary
tubing and wait for it to dry.
, 6. 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.
7.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 until the solvent has moved to within 2cm of the top of the paper.
8. Teacher/Technician: After several hours and once the solvent has moved to within
2cm of the top of the paper, carefully remove the chromatography paper from the gas jar.
9. Teacher/Technician: Without damaging the paper, draw another pencil line
marking the point where the solvent moved to and then hang the paper somewhere warm to
dry.
10.Teacher/Technician: Once the chromatography paper is dry, take it back to the
fume cupboard wearing gloves and eye protection.
11.Teacher/Technician: Spray the paper carefully with ninhydrin reagent.
12. Teacher/Technician: 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.
13. Teacher/Technician: Each purple spot represents one or more amino acids.
Outline each spot with a pencil and mark the centre of each one.
18. Measure the distance that the solvent moved.
19. Measure the distances that all of the amino acids moved and record in a suitably
designed table.
20. Calculate the Rf value for each amino acid and use the table
Introduction of chromatography
You are demonstrating different chromatographic techniques to separate and identify components
in mixtures. One of these techniques is paper chromatography and you will be using this to separate
mixture of plant pigments and identify them.
Equipment
Gas jar
Chromatography paper
Pencil
Ruler
Gloves
Amino acid mix
Individual amino acids
Solvent
Capillary tubing
Ninhydrin spray
Oven set at 110°C
Method
1. Wearing eye protection, pour solvent into your gas jar to a depth of about 1cm. Place
the lid on the gas jar and put in the fume cabinet so that the atmosphere inside the gas
jar becomes saturated with solvent fumes. Wash and dry your hands.
2. Take 1 pieces of chromatography paper - they should be long enough for about
0.5cm to dip into the solvent and the other end should extend out of the top of the gas jar by
about 2c. Use gloves to handle the paper, and try to only touch the sides.
3. Draw a faint pencil line about 3cm from one end of the chromatography paper.
4. Using the capillary tubing, draw up amino solution and place 4 tiny dots on the line.
Wait for it to dry. Use the piece of paper for the amino acid mixture, one dot for pure
alanine, one dot for pure glycine, and one dot for pure lysine,
5. Place another dot of amino acid solution on top of the first one using the capillary
tubing and wait for it to dry.
, 6. 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.
7.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 until the solvent has moved to within 2cm of the top of the paper.
8. Teacher/Technician: After several hours and once the solvent has moved to within
2cm of the top of the paper, carefully remove the chromatography paper from the gas jar.
9. Teacher/Technician: Without damaging the paper, draw another pencil line
marking the point where the solvent moved to and then hang the paper somewhere warm to
dry.
10.Teacher/Technician: Once the chromatography paper is dry, take it back to the
fume cupboard wearing gloves and eye protection.
11.Teacher/Technician: Spray the paper carefully with ninhydrin reagent.
12. Teacher/Technician: 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.
13. Teacher/Technician: Each purple spot represents one or more amino acids.
Outline each spot with a pencil and mark the centre of each one.
18. Measure the distance that the solvent moved.
19. Measure the distances that all of the amino acids moved and record in a suitably
designed table.
20. Calculate the Rf value for each amino acid and use the table
