EXP 6: Dentification of Amino Acid by Paper Chromatography

Amino Acid

• building blocks of proteins.

• molecular formula has two functional groups

  • carboxylic group that imparts acidic properties to the substance

  • amino group that imparts its basic property

Carboxylic Group (-COOH)

imparts acidic properties

Amino Group (NH3+)

imparts basic property

Zwitterion Compounds

contain both a positive and a negative charge as illustrated below

250

no. of identified amino acids

20; 10

__ aa commonly found in plants and animals, __in humans because they cannot be synthesized by the body

Chromatography

• a technique that depends on the rate of ligation of the different amino acids

• also used in the separation and quantitative analysis of peptides, nucleotides, lipids, and carbohydrates

Stationary Phase

the filter paper’s role in chromatography

Mobile Phase

the organic solvents’ role in chromatography

Paper Chromatography

In this method, a sample of an amino acid is applied as a small spot near one edge of a piece of chromatography paper.

The edge of the paper is then placed in a shallow layer of solvent mixture in a chromatographic tank.

Filter Paper

a good material for the stationary phase because many substances that are soluble in water or other mixed solvents are more or less tightly adsorbed onto the fibers of the paper

1. Substances not adsorbed on the paper → move at the same rate as the solvent

2. Substances bound very tightly to the paper → not move very far

3. Substances that interact weakly with paper → move, but slower than the solvent

As the solvent is drawn up into the paper by capillary action, three possible actions will take place:

water + non-polar solvent

solvent mixture’s usual components (several)

Capillary Action

the process where the mobile phase (solvent) moves up the stationary phase (like paper or a porous material) against gravity due to the combined cohesive forces within the liquid and adhesive forces between the liquid and the stationary phase

• the water in the mixture binds to the hydrophilic paper (cellulose) and creates a liquid stationary phase of many small water droplets

• The non-polar solvent continues to move up the paper forming a liquid mobile phase.

As the solvent mixture moves up the paper by capillary action,

An amino acid with a polar R-group

• will be more soluble in water than in the non-polar solvent

• so it will dissolve more in the stationary water phase

• and will move up the paper only slightly

An amino acid with a hydrophobic (non-polar) R-group

• will be more soluble in the mobile non-polar solvent than in water

• so it will move up the paper with the non-polar solvent.

Ninhydrin Solution

used and sprayed on the paper to make the position of the amino acids clearly visible

Rf (Retention Factor) Value

• quantity that defines the movement of amino acids (constant)

• measures the movement of an amino acid compared to the movement of the solvent

Origin

At the start of chromatography, the amino acid is spotted and this is called ______

Factors that Affect the Location of the Amino Acid

• Temperature

• pH and solvent concentration

• Time of chromatography

1. 0.5% Aspartic acid

2. 0.5% Lysine

3. 0.5% Phenylalanine

4. 0.5% proline

5. 0.5% unknown solution

6. distilled water

7. n-butanol

8. lacial acetic acid

9. 0.2% Ninhydrin solution

Chemicals

0.5% Aspartic Acid

1st spot

0.5% Phenylalanine

2nd spot

0.5% Proline

3rd spot

0.5% Lysine

4th spot

0.5% Unknown Substance

5th spot