Aim:
To
separate and identify the amino acids in mixture by thin layer chromatography
Introduction:
Thin layer chromatography
is a simple, quick and inexpensive technique to identify the compound of
interest in a mixture. The first
reported use of a thin layer chromatography was in 1938 by two Russian
scientists, N.A. Izmailov and M.S. Schreiber. This technique is highly useful
in research laboratories to separate, identify and characterize the unknown
compound. 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.
Materials Required:
Reagents:
Individual amino acid solution (2% each)
Solvent mixture of normal butanol, acetic acid
and water in the ratio 4:1:5 by volume.
Ninhydrin reagent.
Requirements:
TLC plate.
TLC chamber.
Capillary tubes.
Reagent spray bottle.
Conical flasks.
Beakers.
Procedure :
1. A uniform layer of silica gel was applied on
clean glass plate/slide with the help of an
applicator
(spreader). Thickness of layer 0.25mm
was made.
2. TLC plates were activated by heating in an
oven at 100-150 C for about half an hour.
3. Poured the solvent mixture
in to the TLC chamber and closed the chamber.
4. The chamber was not
disturbed for about 30 minutes so that the atmosphere in the jar became
saturated with the solvent.
5. By pencil gently drawn
a straight line across the plate approximately 2 cm from the bottom.
6. Using a capillary tube,
a minute drop of amino acid was spotted on the line. Allowed the spot
to dry.
7. Spotted the second
amino acid on the plate [enough space provided between the spots].
8. Repeated the above
step for spotting the unknown acid.
9. The plate was placed
in the TLC chamber (immersed the plate such that the line is above the
Solvent).
10. Allowed capillary
action to draw the solvent up the plate until it is approximately 1 cm from
the end.
11. Removed the plate
and immediately drawn a pencil line across the solvent top and allowed
the plate to dry
12. Sprayed the dry
plate with ninhydrin reagent.
14. Dried the plates in
hot air oven at 105°C for 5 min. [Ninhydrin react with the faded spots
of amino acids and made them visible as
purple coloured spots.]
15. Then marked the
center of the spots on the plates, measured the distance of the center of the
spots from the origin and calculate the
Rf values.
Result:
The given amino acids
were identified as ______ based on Rf
value and results were tabulated.
The Rf values with butanol-acetic acid- water solvent: alanine 0.24, glutamic acid 0.25, glycine 0.2, leucine 0.58, valine 0.4, lysine 0.58, tyrosine 0.42.
Ref:
vlab.amrita.edu,. (2011). Separation of Amino Acids by Thin Layer
Chromatography.
