FACULTY OF APPLIED SCIENCES

LABORATORY REPORT

ANALYTICAL CHEMISTRY
(CHM 421)

Name of student Amirah Ishami Binti Ishak

Matrix Number 2022835624

Course code AS245

Title of the experiment Separation of Amino Acid Mixture by Paper

Chromatography

Experiment number Experiment 9

Instructor’s (lecturer’s) name Dr. Mohd Azizi Nawawi

Date of the experiment 15/12/2022

Abstract
The goal of this experiment is to use paper chromatography to separate amino acid mixtures and to
identify components in the unknown amino acid combination. The separation approach is based on
the compound's ability to travel through a supporting medium. Analysing, identifying, filtering, and
measuring separable mixtures are all part of this technique.

The stationary phase is a liquid or solid that is fixed in one location for the procedure, whereas the
mobile phase is the solvent moving through the stationary phase. Because of surface tension
interaction from the pressures, adhesive and cohesive allow the mobile phase to move up until it
reaches the stationary phase in paper chromatography.

To achieve a diameter of less than 5 mm, the amino acids combination was dropped using a capillary
tube. The solvent was allowed to rise through the chromatography paper until it reached a point
around 5cm from the top. The paper was removed, and the solvent was dried using the dryer
included in the fume cupboard. Ninhydrin was sprayed on the chromatography paper. The
numerous locations on the chromatography paper were investigated.

This experiment also employs chromatography paper in order to perform a separation process.
Amino acid, alanine, leucine, and methionine are the four points on the chromatography paper. The
chromatography paper is then placed in a chromatographic tank or beaker with a solvent mixture of
(Butanol: Glacial ethanoic acid:Water, in the ratio of 12:3:5).The 𝑅𝑓 for the first spot of pure amino
acid mixture from the bottom (edge) of the filter paper is 0.00857, 0.2286 for the second spot, and
0.3429 for the third spot. As a result, Alanine has an 𝑅𝑓 of 0.0986, Leucine has an 𝑅𝑓 of 0.3521, and
Methionine has an 𝑅𝑓 of 0.2535.

𝑅𝑓 can be calculated using this formula:

𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒

𝑅𝑓 = 𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡

Objectives
1. To separate amino acid mixture by paper chromatography.
2. To identify components in the unknown amino acid mixture.

Introduction
This experiment shown the technique of doing separation by paper chromatography. Before
spot the standards as well as the mixture, the paper was prepared. The identity of the mixture
be determined by comparing the retention factor, 𝑅𝑓 of the standards and the individual spots in
the mixture. Retention factor can be calculated by this formula:

𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒

𝑅𝑓 = 𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡
Method
A line was drawn with pencil 5 cm from the end of a strip of filter paper. The filter paper Whatman
No.1 (20 cm × 20 cm) was used. Four points was marked with pencil on the line starting 3 cm from
the edge and a space of 3 cm was leave in between each point.

The amino acids mixture was dropped using a capillary tube to obtain the diameter of less than 5
mm. Another drop was put on the spot after the first drop has dried and let the another drop dry
again. 3 drops were place on each spot. This step was repeated for the standard (known sample), 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 chromatography paper until it reaches about 5 cm from the top end. The chromatography paper
was taken out and was dried in fume cupboard using dryer provided.

The chromatography paper was sprayed with ninhydrin. Then, the paper was placed in the oven at
100° for about 5 minutes to dry off the solvent.

The chromatography paper was examined for the various spots in it. The spots for amino acids
mixture was compared with the standard amino acids for identification. Lastly, the distance from the
original point to where it stops for each spot was measured. The 𝑅𝑓 for each of the amino acid
present.

Data
Distance traveled by amino acids mixture (cm).

1. 0.6 cm
2. 1.6 cm
3. 2.4 cm

Distance traveled by pure (standard) amino acids (cm).

1. Alanine = 0.7 cm
2. Leucine = 1.9 cm
3. Methionine = 1.8 cm

Results and Discussion

𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒
𝑅𝑓 = 𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡

0.6 𝑐𝑚 0.7 𝑐𝑚
𝑅𝑓 𝑜𝑓 𝑎𝑚𝑖𝑛𝑜 𝑎𝑐𝑖𝑑 𝑚𝑖𝑥𝑡𝑢𝑟𝑒 1 = 𝑅𝑓 𝑜𝑓 𝐴𝑙𝑎𝑛𝑖𝑛𝑒 =
7.0 𝑐𝑚 7.1 𝑐𝑚

= 0.0857 = 0.0986

1.6 𝑐𝑚 1.8 𝑐𝑚
𝑅𝑓 𝑜𝑓 𝑎𝑚𝑖𝑛𝑜 𝑎𝑐𝑖𝑑 𝑚𝑖𝑥𝑡𝑢𝑟𝑒 2 = 𝑅𝑓 𝑜𝑓 𝑀𝑒𝑡ℎ𝑖𝑜𝑛𝑖𝑛𝑒 =
7.0 𝑐𝑚 7.1 𝑐𝑚

= 0.2286 = 0.2535

2.4 𝑐𝑚 2.5 𝑐𝑚
𝑅𝑓 𝑜𝑓 𝑎𝑚𝑖𝑛𝑜 𝑎𝑐𝑖𝑑 𝑚𝑖𝑥𝑡𝑢𝑟𝑒 3 = 𝑅𝑓 𝑜𝑓 𝐿𝑒𝑢𝑐𝑖𝑛𝑒 =
7.0 𝑐𝑚 7.1 𝑐𝑚

= 0.3429 = 0.3521

Based on this experiment, the mixture of amino acid was spotted on the chromatography paper.
Then, the mixture of amino acid was compared with the standard solution by finding their 𝑅𝑓 .
The retention factor (𝑅𝑓 ) for the first spot of amino acid mixture from the below (edge) of the
chromatography paper is 0.0857 , second spotted is 0.2286 and the third one is 0.3429. Hence ,
the retention factor (𝑅𝑓 ) for the Alanine is 0.0986 which is higher than 0.0986 of amino acid
mixture 1, Leucine is 0.3521 which is close to 0.3429 of amino acid mixture 3 and Methionine is
0.2535 which is also close to 0.2286 of amino acid mixture 2.

Some errors might occur in this experiment. One of it is when the solvent does not moveup in a
perfect line. Next, the chromatography paper is not handled from the edges to there was
contaminating the results. Furthermore, the chromatography paper is kept in horizontal in the
chromatography tank and ninhydrin is sprayed outside the fume hood, so the smell
mightspread in the lab.
The precautions that should be taken in this experiment is to make sure that the line on the
chromatography paper is drawn using a pencil to avoid the ink to be in the solvent. Next, ensure
the spots is not allowed to touch the solvent in the chromatography tank and make sure the
chromatography paper is handled from the edges so as to prevent contaminating the results.

Conclusion
As the conclusion, the purpose of this experiment which is to spot standard amino acid and the
mixture, to separate the unknown mixture into individual components and to identify the
component in the unknown mixture was achieved. As the conclusion, the purpose of this experiment
which is to separate the amino acid mixture by chromatography paper and to identify components
in the unknown amino acid mixture was achieved. As a result, amino acid mixture 1 is a Alanine,
amino acid mixture 2 is a Leucine and amino acid mixture 3 is a Methionine.

References
Harper College. (2020). Retention Factors. Retrieved on January 7, 2023, from
http://dept.harpercollege.edu/chemistry/chm/100/dgodambe/thedisk/chrom/wback3.htm

Palmer, C. (2020). What is Chromatography and How it Does it Work?. Retrieved from
https://www.coleparmer.com/tech-article/what-is-chromatography

Chromatography. (2023). Bitesize. Retrived on January 7, 2023, from

https://www.bbc.co.uk/bitesize/guides/zp2wrwx/revision/3