LAB REPORT

BCB 202L

Experiment number: 05
Title of the experiment: Thin Layer Chromatography (TLC) of Amino Acids

Submitted by, Submitted to,

Name & ID: Munasib Ahmed | 1910269 Dr. Md Omar Faruk
Lecturer
Department of Life Sciences
Independent University, Bangladesh
Aim of the Experiment: Understanding the Principles of Thin Layer Chromatography

Principle:
This layer chromatography is a separating technique that separates polar molecules.
For this it requires TLC plates which contain silica gel. We know Amino acids are
negatively charged particles. The silica gel itself is polar and contains hydroxyl groups
at the end. The silica gel is crucial during the mobile phase when the samples move
from the line of origin to the solvent line. The hydroxyl groups will interact with the
samples and will slow down the movement of the sample amino acid. More polar
molecules will interact more with the silica gel and will move more slowly.
How far the component travels tells us about the chemical properties of the substance.
The distance the solvent line travels are the distance the sample travels are measured
and the Rf value is obtained. The Rf value is used to determine the type of amino acid.

Materials:

● Amino acids

● Butanol

● Acetic acid

● Ninhydrin

● Na2CO3 for Cystine, Phenylalanine and Tyrosine

● TLC plate

● TLC chamber

● Fume hood

Method:
● An amino acid solution will be prepared with the concentration of 5 mg/mL
● In that solution, Na2CO3 for Cystine, Phenylalanine and Tyrosine will be added
● The line of origin must be drawn on the TLC plate with a pencil. It should be 1cm
above the bottom of the plate.
● Just above the line, a blob of 1µl will be smeared from the solution with a
capillary tube. A distance of 0.5 cm must be maintained between the adjacent
blobs to avoid overlapping
 ● The solvent must be transferred to a separating chamber. In that chamber, the
TLC plate will be submerged up to the line of origin. The chamber must have a
lid to minimize evaporation of volatile substances.
● The plate must be eluted with a mixture of n-butanol:acetic acid:water 3:1:1(by

volume). Once the TLC plate has been spotted and ready to go, it will be placed

inside the developing chamber. The plate should be placed upright, slightly

inclined against the wall of the container. The solvent should not touch the spots

when the separation first begins. When the solvent front is about 0.5 to 1 cm

from reaching the top, the elution must be stopped.

● The spots will not be visible as the amino acids are colorless. They must be
sprayed with Ninhydrin. This will give purple spots.
● The distance traveled by each spot will be divided by the distance traveled by the
solvent. This will give the Rf value. The value in comparison with the chart will
help us identify the amino acid.

Results:

RfLys = 1 ➗4
= 0.25

RfLeu = 2.7 ➗4
= 0.675

RfGly = 1.7 ➗4
= 0.425

RfVal = 2.4 ➗4
= 0.6

RfAla = 1.9 ➗4
= 0.475

RfAsp = 1.5 ➗4
= 0.375
Discussions:

The line of origin and the other labellings are always done with pencil. This is because
the ink does not dissolve and affect the results.

Apart from identifying the type of amino acid, chromatography is used to determine
the purity of the sample and to conclude if the reaction has been completed or not.
Fig: Finding the Rf value

Conclusions:

The amino acids present in the sample are Lysine, Leucine, glycine, valine, alanine and
aspartic acid.