CHROMATOGRAPHIC SEPARATION AND IDENTIFICATION OF AMINO ACIDS
Purpose:
The purpose of this lab is to use chromatographic techniques to separate and identify a mixture of
amino acids.
Discussion:
 Summarize general chromatography procedures from last year.
 How will paper chromatography be applied to separate and identify a mixture of amino acids?
 How will the ninhydrin test be incorporated?
Objectives:
In this lab, you will…
1. use paper chromatography to separate a mixture of amino acids.
2. Use experimental Rf values to identify the amino acids in the mixture.
Materials:
Equipment:
Chromatography paper, 20 x 10 cm
Graphite pencil
9 small test tubes
600 mL beaker
Watch glass
100 mL graduated cylinder
Toothpicks
Hot plate
Ruler
Plastic pipets
Chemicals:
Amino acid solutions, 1%:
Alanine
Arginine
Asparagine
Aspartate
Glycine
Lysine
Methionine
Tyrosine
Unknown (AAUK1-10)
Ninhydrin solution, 2%
Chromatography solvent
Safety:
 Wear safety goggles at all times.
 Only handle the very edges of the chromatography paper with your fingers. The ninhydrin
solution is used to detect fingerprints at crime scenes, so any fingerprints left on the paper will be
colored and may interfere with your amino acids spots.
 The organic solvent contains n-butyl alcohol (flammable) and glacial acetic acid (causes serious
burns). Handle with care, and use in a well ventilated area.
Procedure:
1. Remember, only handle the very edges of the chromatography paper with your fingers. On a 20 x
10 cm piece of chromatography paper, use a pencil to draw a straight line one centimeter from the
bottom of the paper.
2. Place nine pencil dots on the line spaces about 2 cm apart.
3. Write the abbreviation for each of the amino acid solutions under each of the nine dots.
4. Place a plastic pipet full of each amino acid solution into the nine test tubes. Place a toothpick
into each of the test tubes and allow the solution to soak into the test tube.
5. Gently touch the tip of the toothpick to the chromatography paper directly on top of the dot.
Repeat three times for each amino acid. Repeat with the remaining amino acid solutions.
6. With the pencil line facing outward, roll the chromatography paper into a loose cylinder.
Carefully staple the cylinder together. Reminder: only handle the very edges of the paper.
7. Pour 20 mL of the chromatography solvent in the 600 mL beaker.
8. Place the paper cylinder pencil line down into the beaker containing the chromatography solvent.
Cover the beaker with a watch glass.
9. Allow the chromatography paper to “develop” until the solvent level is about one centimeter from
the top of the paper.
10. Remove the paper and mark the solvent height with a pencil line before the solvent front
evaporates. Carefully remove the staples. Allow the paper to dry completely.
11. Spray the paper with ninhydrin solution. Allow it to dry completely.
12. If the sample spots do not show up, turn the hot plate on low. Hold the paper about 10 cm over
the surface of the hot plate for a few minutes until the color develops. Do not scorch the paper.
13. Place a pencil dot at the center of each amino acid spot. The unknown should have multiple
spots.
Cleanup:



Pour the remaining solvent into the organic waste container in the fume hood.
Throw the used micropipets in the trash.
Clean all glassware used in the lab.
Analysis/Calculations:
1. The distance the solvent travels may vary slightly for each spot, so measure the distance it
traveled above every spot. Record in Data Table 2.
2. Measure the distance from the pencil line to the pencil dot in the middle of each spot for each
amino acid sample. Record in Data Table 2.
a. Include the chromatogram in the lab notebook as Data Table 1.
3. Calculate the Rf value for each amino acid sample. Show work for one. Record in Data Table 2.
4. Identify the components of the unknown amino acid mixture.