Laboratory Report No.

2
Analysis of Amino Acids by Paper Chromatography

Esconde, Yvonne Keithlene J. BS Nursing 1A

Grp. 3 / Thurs (7:00–7:30 PM) Aug. 23, 2018
Rating: ________

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 absorbed 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 tubes. 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 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/hydroxyl 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 this experiment, 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
• Solvent System [1-butanol, acetic acid, water (12:3:5)]
• (1 mg/mL) Alanine • (1 mg/mL) Tyrosine
 • (1 mg/mL) Leucine • (1 mg/mL) Arginine
• (1 mg/mL) Lysine • (1 mg/mL) Glycine
• (1 mg/mL) Cystein • (4 mg/mL) Ninhydrin Solution
• (1 mg/mL) Tryptophan • Unknown amino acid mixture
IIII. Methodology (Schematic Diagram)
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 (1 mg/mL) solution for each of the amino acid.

C. Qualitative Analysis of Amino Acids

1. Test for Amines

2. Test for Amides

3. Test for substituted Benzene Rings

4. Test for Thiol groups

5. Paper Chromatography

V. Data and Results

1. Test for Amines Observations

Test Tube 1: Alanine Dark blue; Quickest to react

Test Tube 2: Proline Red; slowest to react
Test Tube 3: Tyrosine Blue

2. Test for Amides

Test Tube 1: Arginine (Glytomine) Blue

Test Tube 2: Alanine Red

3. Test for Aromatic Amino Acids

Test Tube 1: Tyrosine Dark yellow

Test Tube 2: Tryptophane Light orange
Test Tube 3: Alanine No reaction

4. Test for Thiols

Test Tube 1: Cystein No reaction

Test Tube 2: Leucine No reaction

Paste the paper chromatogram

VI. Discussion

For this experiment, the group observed the color reactions of the amino acids when mixed
with reagents. On the test for Amines, Alanine was the quickest to react, turning a dark
shade of blue immediately when allowed to stand for around 3 minutes, followed by
Tyrosine, which had a lighter shade of blue. Proline was the slowest to react, to the point
where the researchers almost thought nothing would happen, but it eventually turned a
reddish-brown sort of color. The Test for Amides yielded a blue color reaction from
Glytomine, and red instead of the previous dark blue for Alanine. Tyrosine and
Tryptophane had similar color schemes for the test for Aromatic Amino Acids, the former
having a dark yellow shade while the latter leaned more towards orange. Alanine, this time,
had no reaction. There was also no changes in the substances for the Test for Thiols. Both
Cystein and Leucine remained the same color.

VII. Conclusion

The chemical versatility of the various amino acids were proven through the plethora of
reactions observed during the initial tests and on the paper chromatogram. Their distinct
properties were implied and emphasized upon by the color reactions that occurred during
boiling, and the different spots that appeared after the chromatogram had finally dried at the
end of the experiment. It made identifying the compounds of the mixtures way easier, and
helped in the computing for the Rf values of the amino acids utilized in the experiment.

VIII. Assessment (Q&A)

1. Color reactions of the ff:

• Arginine – Blue
• Tyrosine – Dark yellow
• Tryptophane – Light orange
• Alanine – Dark Blue
• Cystein – No change in color

2. Compute the Rf values of the known and unknown amino acids.

3. Explain why there are two spots for Cystein.

It has a very reactive sulfhydryl group at its side chain. This puts cystein in a
special position, and the disulfide property allows for the two spots to appear.
4. Identify the composition of your unknown amino acid mixture.

5. Give the structure of Ninhydrin.

IX. References
Leluk, J. (n.d.). Why Cysteine is special? Retrieved
from http://www.cryst.bbk.ac.uk/pps97/assignments/projects/leluk/project.htm
Santa Cruz Biotechnology. (2018). Ninhydrin reagent, 2% solution [photograph]. Retrieved
fromhttps://www.scbt.com/scbt/product/ninhydrin-reagent-2-solution

X. Certification / Conforme

Esconde, Yvonne Keithlene J.

Signature over printed name

August 30, 2018

Date Submitted: