LABORATORY REPORT

CHM421
(ANALYTICAL CHEMISTRY)

NAME:
STUDENT NO.:
GROUP: AS2464A1
EXPERIMENT NO: 9
EXPERIMENT TITLE: SEPARATION OF AMINO ACID MIXTURE BY PAPER
CHROMATOGRAPHY
ABSTRACT
In this experiment, paper chromatography separation technique was used to spot standard amino
acids and mixtures, to separate the unknown mixture to the individual component and to identify
the component in the unknown mixture. Amino acids used in this experiment were alanine, leucine
and methionine. All standard amino acids were calculated the Rf value of each. The unknown
mixture first spot, second spot and third spot was found to be alanine, methionine and leucine
respectively.

OBJECTIVES
To spot standard amino acids and mixtures, to separate the unknown mixture into individual
components and to identify the components in the unknown mixture.

INTRODUCTION
Chromatography is a popular technique for separating, identifying, and determining chemical
components in complex mixtures. In this experiment, paper chromatography separation technique
was used. All the solutes which are an unknown mixture, Alanine, Leucine and Methionine will
move through the paper, drawn by capillary action. The pace at which solutes move as spots is
determined by how much time they spend in the stationary or mobile phases, as defined by their
partition coefficient (Rf value). The solutes will follow the solvent at different rates depending on
the solubility in solvent (MP) and the degree of retention by paper (SP). The distance traveled by
the solute will be used to calculate the Rf value.

METHOD
A line was drawn with pencil 5 cm from the end of a strip of filter paper. Filter paper Whatman
No.1 was used (20 cm ✖ 20 cm). Four points were marked with pencil on the line starting 4 cm
from the edge and leaving a space of 4-5 cm in between each point. The amino acids mixture was
dropped using a capillary tube to obtain the diameter of less than 5 mm. Put another drop on the
spot when the first drop dried and let it dry again. The same standard was done on the other three
points. The filter paper was placed in the chromatographic tank or beaker, which contains a solvent
mixture (Butanol : Glacial ethanoic acid : Water, 12 : 3 : 5). The solvent was allowed to move
upward through the filter paper until it reached about 5 cm from the top end. The filter paper was
taken out and dried in the fume cupboard. The filter paper was sprayed with ninhydrin. The paper
was placed in the oven at 100 C for 5 minutes. The filter paper was examined for various spots on
it. The spots for the amino acids mixture were compared with standard amino acids for
identification. The distance from the original point was measured to where it stops for each spot.

The Rf was calculated for each of the amino acids present.

DISCUSSION
In order to separate the unknown mixture, paper chromatography separation technique was
conducted. There are an unknown mixture and three standard amino acids used which are alanine,
methionine and leucine. The distance traveled for each spot was measured. For the standard amino
acid, leucine traveled the longest which is 4.1cm, followed by methionine and alanine which is
3.3cm and 2.2cm. For the unknown mixture, the longest distance was found to be the third spot
followed by the second and first spot. The third spot was 4.3cm, the second spot was 3.4cm and
the first spot was 2.4cm. For the retention factor, Rf, leucine travels near the top of the paper which
is 0.43. Alanine travels near the bottom of the paper which is 0.23 while methionine travels
between the middle of the paper which is 0.34. The unknown mixture contains alanine, methionine
and leucine.

Rf

The precaution in this experiment was to make sure before the paper is placed in the solvent to be
run, the drop applied to it must be allowed to dry. Other than that, the drop must not be so close to
the paper's bottom that it is submerged in the solvent. Next, make sure the filter paper does not
touch the spotting area of paper, only the edge can be touched. Last but not least, the development
tank should be close enough to the development chamber that the development solvent is saturated.

CONCLUSION
In conclusion, all the objectives were achieved. The unknown are separated into individual
components and are identified as alanine, leucine and methionine.
RESULT
Standard Amino acid; Alanine, Leucine, Methionine

Paper chromatography

a. Distance travelled by amino acids mixture (cm)

1. 2.4 cm
2. 3.4 cm
3. 4.3cm
b. Distance travelled by pure (standard) amino acid (cm)
1. Alanine – 2.2 cm
2. Leucine – 4.1 cm
3. Methionine – 3.3 cm

c. Rf
Amino Acids Mixture
1. 2.4cm

2. 3.4cm
 3. 4.3cm

Pure (Standard) Amino Acids

1. Alanine – 2.2 cm

2. Leucine – 4.1 cm

3. Methionine – 3.3 cm

QUESTIONS
1. Define stationary phase and mobile phase.
Stationary phase is a layer or a coating on the supporting medium that interacts with the
analytes while mobile phase is a solvent that flows through the supporting medium.

2. Explain why the chromatography tank has to be fully covered during the process. During
the procedure, the chromatography tank must be completely covered to ensure that the
atmosphere in the beaker is saturated with solvent vapour.
 3. Briefly explain why the purple colour of the spots appear after spraying with ninhydrin.
Since amino acids are colorless, ninhydrin was used to dye the spot. Ninhydrin will give a
purple colour to the amino acid spot.

4. Why did some amino acid travel to the top of the paper and others remained near the bottom
of the paper? Explain in terms of the attractions of the particles for either the mobile liquid
phase or the solid paper stationary phase.

Solutes that are more soluble in mobile phase and weakly retained by stationary phase will
travel to the top of paper while solute that is less soluble in mobile phase and strongly
retained by stationary phase will remain near the bottom of the paper.

REFERENCES

Pinto, C., Anand, R., & Cejka, P. (2018). Methods to Study DNA End Resection II: Biochemical
Reconstitution Assays. Methods in Enzymology, 67–106.

https://doi.org/10.1016/bs.mie.2017.11.009

Mohamed Ahmed Sayed. (2021, October 24). A review of Chromatography: principles,

Classification, Applications. ResearchGate; unknown.
https://www.researchgate.net/publication/355493136_A_review_of_Chromatography_prin
ciples_Classification_Applications

Toennies, G., & Kolb, J. J. (1951). Techniques and reagents for paper
chromatography. Analytical Chemistry, 23(6), 823-826.