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Experiment no. 2
Analysis of Amino Acids by Paper Chromatography

I. Objectives

1. To separate amino acids using the technique of paper chromatography.

2. To identify the color reactions of different amino acids with the detection reagent.

II. Introduction

Chromatography is a technique for analyzing or separating mixtures of substances into

their components. There are various forms of chromatography techniques. Most of these
techniques involve two distinct phases; the stationary phase and the mobile phase. In paper
chromatography, the stationary phase is a liquid which is adsorbed on the cellulose paper
whereas the mobile phase is a mixture of solvents suitable for separating the components in a
sample. The relative affinity of a substance for each phase depends on properties such as
molecular weight, structure and shape of the molecule, and the polarity of the molecule.

In this experiment, very small volumes of solutions containing amino acids will be
applied using capillary tube. After the solutions have been applied, the paper will be placed
inside the chromatographic chamber for development. As the mobile phase rises on the paper
it will eventually encounter the “spots” of amino acids. The fate of each amino acid in the
mixture now depends on the affinity of each substance for the mobile and stationary phases.
It is these differences in the amino acid affinities that lead to the separation. Identification of
the amino acid is done by spraying with Ninhydrin solution. The Ninhydrin forms a blue
violet complex with an amino acid except for proline/hydroxy proline which gives a yellow
color complex.

Amino acids play central roles both as building blocks of proteins and as intermediates in
metabolism. The 20 amino acids that are found within proteins convey a vast array of
chemical versatility.
In these experiments students will identify amino acids by qualitative analysis and paper
chromatography.

III. Materials

A. Equipment
(1) 250-mL Beaker Whatman # 1 paper
(1) Capillary tube Gloves
Aluminum foil (5) 10-mL Test tubes
 B. Reagents
(1mg/mL) Alanine Ninhydrin solution (4 mg/mL)
(1mg/mL) Leucine Unknown amino acid mixture
(1mg/mL)Lysine Solvent system(1-butanol, acetic acid, water (12:3:5)
(1mg/mL)Cystein (1mg/mL) Arginine
(1mg/mL)Tryptophan (1mg/mL) Glycine
(1mg/mL) Tyrosine

IV. Procedure

A. Preparation of Detection Reagent

Prepare 100 mL Ninhydrin solution with a concentration of 2 mg/mL.

B. Preparation of Amino Acid Solution

Prepare 20 mL (1mg/mL) solution each of the amino acid.

C. Qualitative Analysis of Amino Acids

1. Test for Amines

Prepare the following test tubes.
Test tube 1: contains 10 drops of Ninhydrin solution and 5 drops of Alanine.
Test tube 2: contains 10 drops of Ninhydrin solution and 5 drops of Lysine.
Test tube 3: contains 10 drops of Ninhydrin solution and 5 drops of Tyrosine
Heat the three test tubes in a water bath and observe the change in color.

2. Test for Amides

Prepare the following test tubes:

1. Test tube 1: contains 10 drops of 20% NaOH, and 5 drops of glutamine
2. Test tube 2: contains 10 drops of 20% NaOH and 5 drops of Alanine.
Heat the test tubes in a water bath. While heating, place a strip of red litmus paper at the
mouth of the tube. Record your observations.

3. Test for substituted benzene rings.

Prepare the following test tubes:

Test tube 1: contains 5 drops of Tyrosine, and 5 drops of concentrated nitric acid.
Test tube 2: contains 5 drops of Tryptophan, and 5 drops of concentrated nitric acid.
Test tube 3: contains 5 drops of Alanine, and 5 drops of concentrated nitric acid.
Heat the test tubes in a water bath for 3 minutes. Add 10 drops of 20% NaOH and
record your observations.
 4. Test for Thiol groups.

Prepare the following test tubes:

Test tube 1: contains 5 drops of Cystein and 10 drops of DTNB(5,5’-dithiobis(2-
nitrobenzoic acid).
Test tube 2: contains 5 drops of Leucine and 10 drops of DTNB (1mg/mL).
Allow to stand for 5 minutes. Record your observationS.

5. Paper Chromatography
1. Prepare a 8 x 6 cm paper chromatogram using Whatman # 1 paper.
2. Draw a pencil line 1.5 cm from the edge of the paper.
3. Make 6 marks (0.5 cm apart).
4. Using a capillary tube draw your sample from the 5 samples of amino acid (Ala,
Leu, Lys, Cys, Trp) and the unknown mixture of amino acid.
5. Apply it to the mark you made. Samples applied should be small drops, giving a
wet spot ¼ to 3/8 inch in diameter. Apply the sample 3x drying the spot between
applications.
6. Staple the ends of the paper and be careful not to touch the main area of the
chromatogram (wear gloves). Transfer the paper into a wide-mouth quart jar
containing 15mL of solvent mixture (1-butanol, acetic acid, and water in 12:3:5
ratio). Develop the chromatogram for 1 h.
7. After 1 h, remove the paper from the chamber and let it dry inside the hood.
8. Spray or wet the chromatogram with ninhydrin solution (2mg/mL in ethanol) and
place it in the oven for 30 minutes to develop the color.

Experiment No. 2
Analysis of Amino Acids by Paper Chromatography

Name: Nicole Dicen, Shery Empinado, Jester Garcia Date Performed:___________________

Year and Section: BSN1A Date Submitted: September 14, 2022
A. Qualitative Tests for Amino Acids
1. Test for Amines Observations
Test Tube 1: Alanine After 1min and 40secs of heating, it turned
violet in color but not fully.

Test Tube 2: Proline (Lys)
Test tube 3: Tyrosine
2. Test for Amides
Test Tube 1: Arginine (Gln)
Test Tube 2: Alanine
3. Test for Aromatic Amino Acids
Test Tube 1: Tyrosine
Test Tube 2: Tryptophane
Test tube 3: Alanine
4. Test for Thiols
Test Tube 1: Cysteine
Test Tube 2: Leucine
Paste the paper chromatogram
Finally, after 3 min and 48 secs of heating,
the violet color became more visible,
continuously did so until it became deep
violet.
After 3 min and 48 secs of heating, it
started to turn violet.
Turned purplish after few minutes.
No observable changes after few minutes.
After adding more tyrosine, it finally
turned violet in color.
Observations
Red litmus paper turned blue.
No observable changes happened. The
litmus paper remained red.
Observations
After heating for 3mins, it turned yellow in
color.
When NaOH was added, the yellow color
turned orange.
Originally yellow, after heating for 3mins,
it turned more yellow in color.
After adding NaOH, the solution became
orange in color.
No observable changes even after heated
for 3mins.
Even after adding NaOH, there’s still no
visible changes.
Observations
No observable changes.
No observable changes.

Questions
1. Give the color reactions of the following amino acids,
Arginine, Tyrosine, Tryptophan, Alanine, and Cystein
Arginine-
Tyrosine- Orange
Tryptophane- Orange
Alanine- Violet
Cystein-
2. Calculate the Rf values of the known and unknown amino acids.

3. Explain why there are two spots for Cystein.

4. Identify the composition of your unknown amino acid mixture.

5. Give the structure of Ninhydrin.