Thin Layer Chromatography of Amino Acids
Aim: to identify the amino acids in an unknown mixture.
Safety
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Perform the experiment in a well-ventilated room as the solvents used are corrosive and flammable.
Wear safety goggles and tie long hair back.
Wear gloves when spraying ninhydrin as it can stain the skin.
Spray ninhydrin in the fume cupboard as it is harmful.
Equipment/Materials
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1% solutions of amino acids
Unknown mixture of amino acids
TLC cellulose slide
Prepared solvent containing butanol, ethanoic acid and water (highly flammable, harmful and
corrosive)
Ninhydrin spray (harmful)
Beaker and clingfilm
Capillary tubes
Access to warm oven
Procedure
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Take the thin layer chromatography side and identify the front (matt, not shiny) side.
Make a small mark about 1.5cm from the bottom of the left hand side.
Mark with a pencil the spots (in line with the pencil mark) where you will apply each amino acid (your
teacher will tell you which amino acids, and how many spots to draw).
4. Use the capillary tube to put one spot of amino acid solution onto the slide at the appropriate point.
5. Let the spot dry then add more of the same amino acid to the same spot.
6. Repeat this with any remaining solutions you are asked to use.
7. Pour the solvent into the beaker until it just covers the surface of the beaker.
8. Put the slide into the beaker and cover the beaker with cling film.
9. Allow the solvent to travel at least 10cm up the slide.
10. Remove the slide before the solvent reaches the top. Use pencil to mark the position of the solvent
front. Add your initials in pencil at the top.
11. Allow the slide to dry.
12. Your teacher will spray your slide with ninhydrin, then place it in a warm oven for the spots to
develop.
Analysis
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Measure the distance travelled by the solvent (from the initial mark at 1.5cm to the solvent front).
Measure the distance travelled by each amino acid (from the initial mark to the centre of the spot).
Find the Rf (retardation factor) value of each amino acid using the formula:
Rf = distance travelled by amino acid
distance travelled by solvent
Record the Rf value of each amino acid in a suitable table.
Compare the Rf values of the spots in the mixture with those for each amino acid to identify the
components of the mixture.
Questions
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What would happen if the solvent was higher than the applied spots of solute when the
chromatogram was put into the beaker?
What are the stationary and mobile phases in thin layer chromatography?
Why does the beaker have to be covered?
What advantages does TLC have in comparison with paper chromatography?