TLC

Student Name: Jalil Haddadin
Student ID 1710132
Experiment # 7: Separation of Amino Acids by Thin Layer Chromatography.
Introduction
Chromatography is by far the most useful general group of techniques available for the separation of
closely related compounds in a mixture. Here the separation is effected by differences in the
equilibrium distribution of the components between two immiscible phases, viz., the stationary and the
mobile phases. These differences in the equilibrium distribution are a result of nature and degree of
interaction of the components with these two phases. The stationary phase is a porous medium like
silica or alumina, through which the sample mixture percolates under the influence of a moving
solvent (the mobile phase). There are a number of interactions between the sample and the stationary
phase and these have been well exploited to effect the separation of compounds.
Thin layer chromatographic (TLC) technique readily provides qualitative information and with careful
attention to details, it is possible to obtain quantitative data. Thin layer chromatography is a technique
used to separate and identify compounds of interest. A TLC plate is made up of a thin layer of silica
adhered to glass or aluminum for support. The silica gel acts as the stationary phase and the solvent
mixture acts as the mobile phase. In the ideal solvent system the compounds of interest are soluble to
different degrees. Separation results from the partition equilibrium of the components in the mixture.
In the simplest form of the technique, a narrow zone or spot of the sample mixture to be separated is
applied near one end of the TLC plate and allowed to dry. The strip or plate is then placed with this
end dipping in to the solvent mixture, taking care that the sample spot/zone is not immersed in the
solvent. As the solvent moves towards the other end of the strip, the test mixture separates into various
components. This is called as the development of TLC plates. The separation depends on several
factors;
a) solubility: the more soluble a compound is in a solvent, the faster it will move up the plate.
(b) attractions between the compound and the silica, the more the compound interacts with silica, the
lesser it moves,
(c) size of the compound, the larger the compound the slower it moves up the plate.
The plate is removed after an optimal development time and dried and the spots/zones are detected
using a suitable location reagent. An important characteristic used in thin layer chromatography is Rf
value.
The plate is removed after an optimal development time and dried and the spots/zones are detected
using a suitable location reagent. An important characteristic used in thin layer chromatography is Rf
value.
Chromatographic Separation of Amino acids:
The present experiment employs the technique of thin layer chromatography to separate the amino
acids in a given mixture.
All 20 of the common amino acids [standard amino acids] are a-amino acids. They have a carboxyl
group and an amino group bonded to the same carbon atom (the α- carbon). They differ from each
other in their side chains, or R groups, which vary in structure, size, and electric charge. The
interaction of the amino acids with the stationary phase like silica varies depending on their 'R' groups.
The amino acid that interacts strongly with silica will be carried by the solvent to a small distance,
whereas the one with less interaction will be moved further. By running controls [known compounds ]
alongside, it is possible to identify the components of the mixture.
Since amino acids are colourless compounds, ninhydrin is used for detecting them. To identify this,
after development, the TLC plate is sprayed with ninhydrin reagent and dried in an oven, at 105°C for
about 5 minutes. Ninhydrin reacts with α- amino acids that results in purple coloured spots [ due to the
formation of the complex - Rheuman's purple] [http://vlab.amrita.edu/?
sub=3&brch=63&sim=156&cnt=1]. Rf values can be calculated and compared with the reference
values to identify the amino acids. [The Rf value for each known compound should remain the same
provided the development of plate is done with the same solvent, type of TLC plates, method of
spotting and in exactly the same conditions].
Materials Required:
Reagents:
1. 2% solution of individual amino acids.

2. Solvent mixture of normal butanol, acetic acid and water in the ratio 12:3:5 by volume.
3. Ninhydrin reagent.
Requirements:
1. TLC plate.
2. TLC chamber.
3. Capillary tubes.
4. Reagent spray bottle.
5. Conical flasks.
6. Beakers.
Procedure:
1. Pour the solvent mixture in to the TLC chamber and close the chamber.
2. The chamber should not be disturbed for about 30 minutes so that the atmosphere in the
jar becomes saturated with the solvent.
3. Cut the plate to the correct size and using a pencil (never ever use a pen) gently draw a
straight line across the plate approximately 2 cm from the bottom.
4. Using a capillary tube, a minute drop of amino acid is spotted on the line.
5. Allow the spot to dry.
6. Spot the second amino acid on the plate [enough space should be provided between the
spots].
7. Repeat the above step for spotting the unknown acid.
8. Place the plate in the TLC chamber as evenly as possible and lean it against the
side(immerse the plate such that the line is above the solvent). Allow capillary action to
draw the solvent up the plate until it is approximately 1 cm from the end.
9. Remove the plate and immediately draw a pencil line across the solvent top.
10. Under a hood dry the plate with the aid of a blow dryer.
11. Spray the dry plate with ninhydrin reagent.
12. Dry the plates in hot air oven at 105°C for 5 min. [Ninhydrin will react with the faded
spots of amino acids and make them visible as purple coloured spots.]
13. After some time, mark the center of the spots, then measure the distance of the center of
the spots from the origin and calculate the Rf values.
Results
From the graph we can detect amino acid (o) and amino acid (p) and a micture(mix), we found that it
contains both amino acid (o) and (p),we can calculate the Rf value for each component and for the
mixture as follow:
Amino Acid Rf
Amino acid (o) R = 3/0.5= 0.6
f
Amino acid (p) R = 0.8/5 = 0.16

f
mixture R = 3/5 =0.6 , R =3/0.5= 0.6

f (O) f (p)
From Rf values we can clearly declare that the mixture is composed of amino acid (o) and amino acid
(p). And from that result we can see how this method is very useful especially in pharmaceutical
industrys.

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Student Name: Jalil Haddadin

Experiment # 7: Separation of Amino Acids by Thin Layer Chromatography.

1. 2% solution of individual amino acids.

Amino acid (p) R = 0.8/5 = 0.16

mixture R = 3/5 =0.6 , R =3/0.5= 0.6

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