CHM160L Biochemistry 1 Laboratory

4th Quarter SY 2018-2019

Separation of Amino Acids in Thin Layer Chromatography

Nanette D. Santos1, Amarrador, Jelsea, Cortez Mariene-syne Edisa P., Sardome, Rachelle, 2Victoria, Jane Xuan B.
1Professor, 2Student, CHM160L/E01, School of Chemical, Biological, and Materials Engineering and Sciences, Mapua University

ABSTRACT

Thin layer chromatography (TLC) is an easy, convenient and inexpensive way to determine how many components are in a
mixture and, in many instances, can be used to identify the components as well. The objective of this experiment is to separate
amino acids using thin layer chromatography. A spot of the sample is placed on a sheet of glass treated with an absorbent
substance. The glass is then placed in a solvent that will travel up the absorbent surface and cause the solid to move out of the
liquid with it. Different solids will move different distances on the sheet, but the distance will remain constant no matter how
many times chromatography is done. This distance is calculated into an amount called the Rf value, which can be used to
determine the identity of the substance.

Keywords: Thin layer chromatography, amino acids, Rf value, polarity, solublity, distance, molecular weight

INTRODUCTION 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,
Chromatography is by far the most useful general group of taking care that the sample spot/zone is not immersed in
techniques available for the separation of closely related the solvent. As the solvent moves towards the other end of
compounds in a mixture. Here the separation is effected by the strip, the test mixture separates into various
differences in the equilibrium distribution of the components components. This is called as the development of TLC
between two immiscible phases, viz., the stationary and the plates. The separation depends on several factors; (a)
mobile phases. These differences in the equilibrium solubility: the more soluble a compound is in a solvent, the
distribution are a result of nature and degree of interaction faster it will move up the plate. (b) attractions between the
of the components with these two phases. The stationary compound and the silica, the more the compound interacts
phase is a porous medium like silica or alumina, through with silica, the lesser it moves, (c) size of the compound,
which the sample mixture percolates under the influence of the larger the compound the slower it moves up the plate.
a moving solvent (the mobile phase). There are a number
of interactions between the sample and the stationary The plate is removed after an optimal development time
phase and these have been well exploited to effect the and dried and the spots/zones are detected using a suitable
separation of compounds. location reagent. An important characteristic used in thin
layer chromatography is Rf value.
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 Figure 2.1

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 CHM160L Biochemistry 1 Laboratory
4th Quarter SY 2018-2019

MATERIALS AND METHODOLOGY

The materials used in the experiment are TLC silica plate,

mobile phase 1-butanol, glacial acetic acid, water, known
solutions of amino acids, unknown solutions of amino acids,
glass capillaries, and beaker.

For the experiment proper, because fingerprints can leave

significant quantities of protein and therefore ruin the TLC
plate (and your results), the researchers wore gloves for
this experiment. TLC sheet was obtained and handled by
the edges to avoid touching the coated surface. A light
pencil line was drawn about 2.5 cm from the bottom of the
plate across the narrow side. These will be the places
where the performers will spot their amino acid samples on
the plate. Each mark was labeled so that they can be Figure 2.2
identified later.
Sample No. Distan Name Rf Original
A small sample of amino acid solution were spotted onto
ce Rf
each mark with a capillary tube. Repeat for the following
Values
known amino acids and the unknown. The plate is then
1 4.8 cm Phenylalanine 0.685 0.68
allowed to dry. A solvent is then added to the bottom of a
2 4.2 cm Tyrosine 0.60 0.45
large beaker so that the entire bottom edge of the TLC plate
will be sitting in solvent (about 1 cm in the bottom). The 3 4.7 cm Leucine 0.67 0.73
beaker was then covered. 4 2.5 cm Alanine 0.35 0.38
Unknown 1 2.5 cm Alanine 0.35 0.38
Afterwards, the TLC plate was placed in the beaker, sample Unknown 2 4.5 cm Leucine 0.642 0.73
side down and then covered. The researchers waited at Table 2.1
least for an hour until it travelled to the marked spot in the
silica plate. After the solvent travelled up to the marked In this experiment, the RF value was determined using the
point in the plate the researchers remove the TLC plate equation below:
carefully and immediately draw a thin pencil line at the
solvent front: the line which defines how far the solvent has
traveled. They then allowed the chromatogram to dry
completely.
Rf value is the distance a substance travels related to the
They then take the plate to the hood, spray lightly, but distance the solvent travels. By separation of amino acids in
evenly, with a ninhydrin solution. They allowed to dry, and thin layer chromatography, it was found out that different
heat with a hair dryer. Amino acids present in the mixture amino acids travel in different distances.
should appear as purple or orange/brown spots. A circle
was drawn around the outside edge of each spot and the The obtained value in sample 1 coincides with the original
center of each spot with a pencil. value of phenylalanine with just a slight variation as shown
Finally, the distance (cm) travelled from starting line to the in Table 2.1. The obtained Rf value in sample 4 also was in
top of the solvent line was measured to obtain the distance agreement with the original value of alanine with just a 0.03
traveled by the solvent. difference. The Rf value obtained in sample 2 was
completely different with that of the original Rf value of
RESULTS AND DISCUSSIONS tyrosine and a slight variation of Rf value in sample 3 was
also observed.
The results in the Thin Layer Chromatography Silica Plate
is in Figure 2.2 The separation depends on several factors; (a) solubility:
the more soluble a compound is in a solvent, the faster it

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 CHM160L Biochemistry 1 Laboratory
4th Quarter SY 2018-2019

will move up the plate. (b) attractions between the that of the obtained known sample and was not compared
compound and the silica, the more the compound interacts with the theoretical value.
with silica, the lesser it moves, (c) size of the compound,
the larger the compound the slower it moves up the plate. By doing so, it was observed that the unknown sample
comprises of alanine and leucine.

CONCLUSIONS AND RECOMMENDATIONS

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.
Figure 2.3
The relative extent to which solute molecules move in a
Phenylalanine is known to be a hydrophobic nonpolar chromatography experiment is indicated by Rf values. The
amino acid due to the enormous size of its R group as Rf value for a component is defined as the ratio of the
shown in figure 2.3. This means that it is not likely to be distance moved by that particular component divided by the
soluble in water. Its molecular weight is 165 Da. But having distance moved by the solvent.
said this, the data showed that phenylalanine was able to
travel 4.8 cm despite of its size and polarity. This REFERENCES
occurrence may be because of the weak attraction between
phenylalanine and the silica. Leucine is also a hydrophobic 1. Reachdevices.com. (2019). TLC of aminoacids
nonpolar amino acid due to its R group that consists of and short peptides. [online] Available at:
hydrocarbons. This amino acid was able to travel 4.7 cm, http://www.reachdevices.com/TLC_aminoacids.ht
almost the same as phenylalanine. Leucine‘s molecular ml [Accessed 24 Jun. 2019].
weight is about 131 Da, and is way lesser than
phenylalanine’s distance travelled. Leucine may also 2. Steane, R. (2019). Chromatography of amino
posses a weak interaction with silica given that it is a acids. [online] Biotopics.co.uk. Available at:
https://www.biotopics.co.uk/as/amino_acid_chrom
nonpolar amino acid.
atography.html [Accessed 24 Jun. 2019].
Tyrosine is known to be a polar amino acid, with 181 Da as
its molecular weight. It has a greater molecular weight than 3. https://www.promega.com/~/media/files/resource
phenylalanine making it travel less than that of the s/technical%20references/amino%20acid%20abb
phenylalanine has travelled. Also, one reason that it reviations%20and%20molecular%20weights.pdf
travelled way lesser than that of the phenylalanine is [Accessed 24 Jun. 2019]
because of its polarity, it is a polar amino acid which means
that it is highly soluble in the solvent used.
4. Voet, Donald., Fundamentals of Biochemistry., “Amino
Alanine is known to be a nonpolar amino acid that is Acids”., John Wiley & Sons, Inc. 2nd Ed, 2006
hydrophobic due to its R group; a hydrocarbon. It is highly
acceptable that this amino acid would only be able to travel
at 2.5 cm. It may have a small molecular weight; 89 Da, but
the factor that affected the distance it travelled is clearly
because of its polarity.

The unknown samples were determined by comparing the

obtained values of Rf using the unknown sample between

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