OBJECTIVE

1. To separate Amino Acid Mixture by Paper Chromatography

INTRODUCTION
Chromatography is a simple and quick procedure that gives a quick answer as to how many
components in a mixture. It is also used to support the identity of a compound in a mixture
when the Rf of the mixture is compared with the Rf of the known compound.
Amino acids are the building block of protein. There are 20 amino acids used by cells in our
body for protein biosynthesis. The word chromatography means color-writing. The name of
was chosen at the beginning of last century when the technique was first used to separate
colored components from the plant leaves.
In this experiment, paper chromatography technique will be used to separate amino acid
mixture. Before spotting the standards as well as mixture, paper chromatography needs to be
prepared. All the solute including unknown mixture, alanine, leucine and methionine will
move through the paper and form the spots. The identify of the mixture can be determined by
comparing the retention factor (Rf) of the standards and the individual spots in the mixture.
Retention factor can be calculated by using this formula,
distance travelled by the solute
Rf=
distance travelled by the solvent

PROCEDURE
1. A line was drew 3.5 cm from the end of a strip of chromatography paper. Filter paper
Whatman No. 1 was used.
2. Four points were marked with a pencil on the line starting 3 cm from the edge and
leaving a space of about 3 cm in between each point.
3. The amino acid mixture was dropped using a capillary tube to obtain the diameter of
less than 5 mm. When the first drop has dried, another drop of mixture is added on the
spot and let dried again.
4. The chromatography paper was placed into the chromatographic tank or beaker,
which contains solvent mixture. (butanol : glacial ethanoic acid : water , 12:3:5)
5. The solvent was allowed to move upward through the chromatography paper until it
reaches about 5 cm from the top end. The paper was taken out the paper and dried the
solvent in the fume cupboard using the dryer provided.
6. The chromatography paper was sprayed with ninhydrin and then the paper was placed
in the oven at 100℃ for about 5 minute to dry off the solvent.
7. The chromatography paper was examined for the various spots on it. The spots were
compared for amino acid mixture with the standard amino acid for identification
8. The distance from the original point to where it stops was measured. The R f was
calculated for each of the amino acid present.
RESULT
Distance travelled by amino acid mixture (cm)
1. 0.9 cm
2. 2.9 cm
3. 5.9 cm
Distance travelled by pure (standard) amino acids (cm)
1. Alanine = 0.6 cm
2. Methionine = 2 cm
3. Leucine = 4.3 cm
Distance travelled by the solvent = 13.1 cm

Retention Factor, R f
distance travelled by the solute
Rf=
distance travelled by the solvent

0.9
R f for spot 1=
13.1
¿ 0.0 7 cm
2.9
R f for spot 2=
13.1
¿ 0.22 cm
5.9
R f for spot 3=
13.1
¿ 0.45 cm
0.6
R f for Alanine=
13.1
¿ 0.05 cm
2.0
R f forMethionine=
13.1
¿ 0.15 cm
4.3
R f for Leucine=
13.1
¿ 0.33 cm
DISCUSSION
The purpose of this experiment was to spot standard amino acids and mixture to separate the
unknown mixture into individual component which is alanine, methionine and leucine. To
conduct this experiment, paper chromatography was used. The spots will appear when the
paper was sprayed with ninhydrin and dried for 5 minutes. The distance travelled by the
solvent and solute was measured.
For pure (standard) amino acids which is alanine, methionine and leucine, leucine travelled
the longest which is 4.3 cm followed by methionine with 2.0 cm and the last one is alanine
with 0.6 cm. for unknown mixture, spot 3 travelled the longest with distance 5.9 cm and
followed by spot 2 and spot 1 with distance 2.9 cm and 0.9 cm respectively. Then, by using
the obtained distance of the solvent and solute, the detention factor, Rf was calculated with
the formula
distance travelled by solute
Rf=
distance travelled by solvent
Using the formula, the R f value for alanine calculated to be 0.05 cm followed by methionine
with R f value 0.15 cm and leucine with 0.33 cm. The R f value for each spots of unknow
mixture was calculated to be 0.07 cm, 0.22 cm and 0.45 cm respectively. By comparing the
R f value of known mixture with the unknown mixture of amino acids, we can say that the
unknown amino acid mixture contains alanine, methionine and leucine because of the
retention factor for each spots were closed with the retention factor of the standards.
The result might be slightly different from the actual value due to several error that happened
during the experiment such as the paper chromatography was over immersed in the
chromatography tank and the spots were immersed in the solvent that caused the spots
smudged with the solvent.

CONCLUSION
In conclusion, the amino acid mixture can be separated using the paper chromatography and
known to contain alanine, methionine and leucine.

REFERENCES
1. Coşkun, Ö. (2016). Separation Tecniques: CHROMATOGRAPHY. İstanbul Kuzey

Klinikleri. https://doi.org/10.14744/nci.2016.32757

2. Libretexts. (2021a). 25.4: Analysis of Amino Acids. Chemistry LibreTexts.

https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Basic_Principles_of_Org
 anic_Chemistry_(Roberts_and_Caserio)/

25%3A_Amino_Acids_Peptides_and_Proteins/

25.04%3A_Analysis_of_Amino_Acids#:~:text=Thus%20a%20mixture%20of

%20amino,(Figure%2025%2D4).

3. Admin. (2022f). RF Value - Definition, Calculation, Explanation & Factors

affecting RF Values. BYJUS. https://byjus.com/chemistry/rf-value/#:~:text=The

%20Rf%20(retardation%20factor,solvent%20under%20the%20same%20conditions.