Title: To separate and identify a mixture of amino acids by paper chromatography

Objectives:

Precautions:

Concentrated ammonia solution is corrosive and gives off harmful vapour. Therefore you

Must:

 Wear safety glasses and gloves

 Keep bottles closed as much as possible

Ninhydrin sprays give off toxic fumes, Therefore you MUST:

 Work at fume hood

APPARATUS AND MATERIALS

10 ml measuring cylinder

400 ml beaker

Aluminium foil (to cover beaker)

Ethanol

Wash -bottle of distilled water

Ammonia solution, 0.880 NH3

Square chromatography paper, 12.5cm x 12.5cm

Pencil, ruler and paper clips/stapler

Capillary tubes

Aspartic acid solution, 0.01M

Leucine solution, 0,01M

Lysine solution, 0.01M

Mixture of two amino acids from above (labelled unknown)

Retort stands with clamps

Ninhydrin aerosol spray

Oven (105 C)
 PROCEDURE

In a fume hood, prepare the solvent mixture by pouring the following into 400ml beaker:

 24ml of ethanol
 3ml distilled water
 3ml 0.880 ammonia

Cover the beaker with aluminium foil. Swirl to mix the liquids and leave to stand.

Wearing disposable gloves, and handling it only by the top edge, place a square of
chromatography paper on a clean sheet of paper. With a pencil (not a pen) draw lines and labels
as shown in the diagram below.

Mixture Asp leusine lysine

5.4 Using capillary tubes place a small amount of each solution on the appropriate

labelled cross so that a spot, no more than 5mm across, appears on the paper.

5.5Using a fresh tube each time, repeat step 5.4 for each of the three solutions of

Single amino acids.

5.6Let the paper dry for a few minutes in air.

5.7Without touching it with your fingers, fold the chromatography paper into a

Cylindrical form and staple the two edges together in such a way that they do not touch.

5.8Place the cylindrical paper in in the 400ml beaker with the eluant. Cover the beaker with the
aluminium foil and leave until the eluant the top of the paper.

5.9Remove the paper and mark the level of the solvent. Place upside down to dry.

5.10 Remove staples and spray with ninhydrin solution. Allow to dry and then place

In the oven at 100 C for 10 mins.

5.11 Remove the paper from the oven and mark with a pencil the positions of the coloured

spots

5.12 Measure the distances from the origin line to the centres of the spots and record them in
the results table given.
6.0LABORATORY REPORT

6.1 As per report format

6.2Calculate an Rf value for each spot as follows

Rf = distance travelled by the compound from the origin

Distance travelled by the solvent from the origin

6.3For each amino acid, compare your two Rf values with each other. Determine

What the mixture is comprised of giving reasons.

6.4Why do Rf values change of giving reasons.

6.5Why is it so important to avoid touching the chromatography paper with your fingers?

6.6List precautions and also observations

7.0

Amino acid Distance travelled (cm)

Rf Value

By solvent by amino acid

Aspartic acid 8cm 2cm 0.25

leucine 8cm 5cm 0.625

lysine 8cm 1.8cm 0.225

mixture 8cm 2.2cm 0.275

Mixture made of asp and lysine

DISCUSSION

The thousands of different cellular proteins carry out distinct biological processes. The specific
process mediated by a protein is dependent on the protein’s three dimensional shape. Ultimately,
this three dimensional shape is dependent on the chemical structure of the protein. Proteins consist
of long polymers called polypeptides, strings amino acids linked together by peptide bonds.

All polypeptides are composed of the same set of twenty amino acids. Different proteins vary in the
order and number of amino acids in their polypeptide chains. All twenty amino acids share a
common structure called the “conserved region” of the amino acid. This conserved region consists of
a central carbon called the α-carbon. This α-carbon is linked to a carboxyl group, an amino group and
a hydrogen atom. These groups along with the α- carbon make up the “conserved region”. All
twenty amino acids have this structure. The α- carbon is also attached to a variable structure called
the R group. The R group is what differs among the twenty amino acids.

Chromatography is an analytical tool for distinguishing different biomolecule based on their

chemical properties. One of the oldest and most reliable forms of chromatography is paper
chromatography. In lab, a mixture of molecules is spotted on a piece of filter paper. The filter paper
is composed mostly of amino acid and is very hydrophilic. Next a hydrophobic organic solvent is
drawn up the paper by capillary action. As the solvent moves over the location of the biomolecule,
the biomolecule begins to move up the paper. The rate at which the biomolecule moves up the
paper is related to its relative affinity for the paper (which is hydrophilic) and the solvent (which is
hydrophobic). Hydrophobic molecules will move faster because they are more attracted to the
hydrophobic solvent than the hydrophilic paper. On the 5 other hand, hydrophilic molecules will
move slower because they are attracted more to the paper than the hydrophobic solvent.