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Thin layer chromatography
CHE 303 L
Joao Paulo Toledo
Abstract
The purpose of this activity was to apply thin layer chromatography (TLC) to identify
amino acids present in an unknown sample. This technique of separation needs a stationary
phase immobilized, and an organic solvent. The sample, either liquid or dissolved in a volatile
solvent, is deposited as a spot on the stationary phase. The constituents of a sample can be
identified by simultaneously running standards with the unknown. The amino acids presented
difference in the affinity with the solvent and the amino acids present in the unknown sample
were found.
Introduction
Amino acids are crystalline solids with surprisingly high melting points. Decomposition
and melting tend to be in the 200 - 300°C range. For the size of the molecules, this is very high.
A general structure of an amino acid has both a basic amine group and an acidic carboxylic acid
group [1].
There is an internal transfer of a hydrogen ion from the -COOH group to the -NH2 group
to leave an ion with both a negative charge and a positive charge. This is called a zwitterion.
A zwitterion is a compound with no overall electrical charge, but which contains separate
parts which are positively and negatively charged. This is the form that amino acids exist even in
the solid state. Instead of the weaker hydrogen bonds and other intermolecular forces that you
might have expected, you actually have much stronger ionic attractions between one ion and its
neighbors. These ionic attractions take more energy to break and so the amino acids have high
melting points for the size of the molecules [1].
Amino acids are generally soluble in water and insoluble in non-polar organic solvents
such as hydrocarbons. This reflects the presence of the zwitterions. In water, the ionic attractions
between the ions in the solid amino acid are replaced by strong attractions between polar water
molecules and the zwitterions. This is much the same as any other ionic substance dissolving in
water. The extent of the solubility in water varies depending on the size and nature of the "R"
group [1].
Thin layer chromatography (TLC) is a chromatography technique used to separate non-
volatile mixtures.[2] Thin layer chromatography is performed on a sheet of glass, plastic, or
aluminum foil, which is coated with a thin layer of adsorbent material, usually silica gel,
aluminum oxide, or cellulose. This layer of adsorbent is known as the stationary phase.
First a sample must be applied on the plate, then a solvent or solvent mixture, the mobile
phase, is drawn up the plate via capillary action. The separation is achieved as different analytes
ascend the TLC plate at different rates [3].
Differences in their attraction to the stationary phase and differences in solubility in the
solvent make different compounds in the sample mixture travel at different rates. By changing
the solvent, or perhaps using a mixture, the separation of components, measured by the distance
moved by compound/distance moved by solvent, can be adjusted [4].
Thin layer chromatography can be used to monitor the progress of a reaction, identify
compounds present in a given mixture, and determine the purity of a substance. Some examples
of these applications include: analyzing fatty acids, detection of pesticides or insecticides in food
and water, analyzing the dye composition of fibers in forensics, assaying the radiochemical
purity of radiopharmaceuticals, or identification of medicinal plants and their constituents [5]
Methodology
The plates must be handled only by their edges because fingerprints can be detected by
ninhydrin giving a false result of masking a true one.
1. By very gently using a pencil, (the resin rubs off the plate rather easily) mark a
baseline on the TLC plate 1.0cm from the bottom of the plate. Mark spots on this line 1.0cm
apart (and 1.0cm from the plate edges).
2. Transfer 1.0L of amino acid solution onto the plate at a pencil mark and allow it
to dry. Repeat this once more for that same amino acid on the same spot (you're loading the spot
with a reasonable amount of the amino acid). Repeat this for each of the remaining amino acids
(including the unknown). Make note of which amino acid is at which spot.
3. Carefully place the spotted plate into the solvent chamber so that the solvent level
is just below your baseline. Close the lid on the chamber.
4. Allow the chromatogram to develop until the solvent front is 0.5cm from the top
edge of the plate (about 90 min).
5. Remove the chromatogram from the chamber and immediately mark the solvent
front all the way across the plate with a pencil. Allow the chromatogram to dry completely.
6. Spray the plate lightly but thoroughly with ninhydrin solution under a fume hood
and allow drying. If the spots don't develop, incubate at about 60 ° C for 5 min.
7. Note the color of each spot. Draw a small line across each spot in the middle of
the spot.
8. Measure and record the distance from that line to the baseline. Measure and
record the distance of the solvent front line directly above the spot to the baseline. Determine the
Rf values for each amino acid from the distance data.
9. Compare the spots and Rf values for the unknown amino acids with those for the
known amino acids to determine the identity of the unknown amino acid.
Results
As shown in figure 1, leucine is the amino acid with greater affinity for three different
solvents, while arginine is the amino acid with lower affinity for the three different solvents. In
the three solvents, the unknown sample presented to be composed by arginine and cysteine. The
composition of the unknown could be observed by the Rf and the color of the dot in the three
plates, yellow for cysteine and purple for arginine.
Figure 1. TLC plate for 4 amino acids and one unknown sample in three different solvents. A= arginine, B=
cysteine, U= unknown, C= glutamic acid and D= leucine. Dark dots represent purple and light dots represente
yellow.
Discussion
The experiment proved a substitution in the polar solvent often results in a change in
resolution, while a change in the less-polar solvent results primarily in a change in Rf of the
sample components, which is used to distinguish if the analyte is more attracted to the solvent
than the stationary phase. Controlled particle size can bring faster separations and improve
resolution.
A good buffer has a pH between its pKa -1 and pKa +1. So the rage for each amino acid
to be a good buffer would be 1.48-3.48 and 10.51-12.51 for arginine, 0.96-2.96 and 7.18-9.18
for cysteine, 1.19-3.19, 3.25-5.25 and 8.67-10.67 for glutamic acid, and 1.36-3.36 and 8.60-
10.60 for leucine.
Ninhydrin degrades amino acids into aldehydes, ammonia, and CO2 (carbon dioxide)
through a series of reactions; the net result is ninhydrin in a partially reduced form hydrindantin.
Ninhydrin then condenses with ammonia and hydrindantin to produce a blueish-purple pigment.
This result is usually associated with primary amino acids. In these amino acids, the N is free to
react with ninhydrin. However, in proline, the N is not available for reaction as it is locked in the
ring structure. Therefore no ammonia is produced, so no blue color is presented (figure 2).
Figure 2. Reaction between ninhydrin and proline
This is a good method to determine constituents of a mixture. The method was validated
since the unknown could have its composition determined, when compared with the amino acids
known.
References
1. "An Introduction to Amino Acids." An Introduction to Amino Acids. Jim Clark, Aug. 2007. Web. 09 Dec. 2013 < http://www.chemguide.co.uk/organicprops/aminoacids/background.html >.
2. Harry W. Lewis and Christopher J. Moody (13 Jun 1989). Experimental Organic Chemistry: Principles and Practice (Illustrated ed.). WileyBlackwell. pp. 159–173.
3. A.I. Vogel, A.R. Tatchell, B.S. Furnis, A.J. Hannaford, and P.W.G. Smith. Vogel's Textbook of Practical Organic Chemistry (5th ed.).
4. Fair, J. D.; Kormos, C. M. J. Chromatogr. A 2008, 1211(1-2), 49-54.5. Reich, E.; Schibli A. High-performance thin-layer chromatography for the analysis of
medicinal plants (Illustrated ed.). New York: Thieme, 2007.