Identifying amino acids by chromatography
Apparatus
- Empty glass coffee jar
- Lid for glass jar
- Strip of chromatography paper
- Capillary tubes
- Staples
- Oven
- Fume cupboard
- Ninhydrin solution
- Lysine
- Leucine
- Valine
- Glycine
- Threomine
- X acid
- Y acid
- Solvent- 4 parts butan-1-01, 1 part ethanoic acid and 1 part distilled water.
Method
- Fetch and set up equipment.
- Pour a small quantity of the solvent into the glass jar.
- Cover tank with lid.
- On the chromatography paper, make a light pencil mark across the paper 2 cm’s from one end.
- Using pencil, place the names of the acids on the paper so you can identify them afterwards.
- Practice using the capillary tubes on normal filter paper
- Using the capillary tubes, place a small amount of each acid on its position along the line on the filter paper.
- Make the spots no bigger than 2 mm’s if possible.
- Let the paper dry for a few minutes in air.
- Roll the chromatography paper into a cylindrical form.
- Staple the ends together about a third of the way in from each edge.
- Make sure the edges do not touch, however, otherwise the solvent will flow more rapidly at that point and form an uneven front.
- Quickly place the chromatography paper into the glass jar so it stays saturated in the solvent.
- Cover the glass jar back up.
- Leave to stand for about half an hour, or until the ‘solvent front’ has rised sufficiently.
- Remove the chromatography paper and mark the solvent front with a pencil.
- Place it on bench to dry.
- When most of the solvent has evaporated rip off the staples and place it in a fume cupboard upside-down to dry.
- When all the solvent has evaporated completely spray the chromatography paper lightly but completely with the ninhydrin solution.
- Place the chromatography paper in the oven at 100- 110 degrees for about 10 minutes- or until all the spots have developed.
- Take the chromatography paper out the oven.
- Circle the spots with pencil.
