Identification of Amino Acids

(Titration, Ninhydrin Test, &

TLC)

By Grant Akalonu
Instructor: Binh Ngyuen
CHE 447-01
Grant Akalonu Identification of Amino Acids

Abstract:

Amino acids can be characterized using various methods that distinguish them based on
intrinsic properties of that amino acid. In this experiment the titration curves for various amino
acids were determined, and used to estimate their pKa. In addition, TLC and ninhydrin staining
was used to distinguish between the amino acids and their Rf values were calculated.

Introduction:

Amino acids are monomers of proteins, which is an important macromolecule in

biochemistry. The structure of an amino acid, is composed of a carbon (known as the -carbon)
covalently bonded to a hydrogen, an amino group, (-NH2) a carboxyl group (-COOH) and a R
group which is unique to each of the 20 amino acids. The general structure of an amino acid pH
7 is shown below:

Fig.1: General Structure of an Amino Acid at pH 7

The amino group and carboxyl group of all amino acids, as well as the R group of some amino
acids possess acidic hydrogens. The structure that a specific amino acid takes in an aqueous
solution depends on the pH of the solution. Each amino acid has at least 2 pKas which can be
used to determine the concentration of a specific structure of the amino acid. There is a specific
pH for each amino acid at which the net charge on it will equal 0. This pH is known as its
isoelectronic pH, and all of the amino acid will exist in its zwitterionic form. If the pH of the
solution is higher than the isoelctronic pH, then the amino acid will bear a net negative charge. If
the pH is lower, it will bear a net positive charge.

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Grant Akalonu Identification of Amino Acids

Fig 2: General structures of an amino acid in neutral, acidic and basic solution.

Fig 3:Titration Curve for a Diprotic Amino Acid

The titration curve above corresponds to the titration curve of a diprotic amino acid. At point A,
the amino acid exists in its completely protonated form and has a net positive charge. At point B,
the pH of the solution is equal to the pKa of the carboxyl group, and the solution contains half of
the fully protonated amino acid and half of the neutral amino acid. At point C, the amino acid has
a charge of zero and is in its zwitterionic form. At point D, the pH is equal to the pKa of the
amino group and the solution consists of half neutral amino acid and half deprotonated amino
acid. At point E, the amino acid has been fully deprotonated and bears a negative charge.
In this experiment, the titiration curves for Lysine, Aspartate and Theronine were
determined. Using the results of the titration, and the Henderson-Hasselbach equation, the pKa
values for all ionizable groups were determined. Their structures are shown below.

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Grant Akalonu Identification of Amino Acids

Lysine Threonine Aspartic Acid

Another method of identifying amino acids is by the use of thin layer chromatography,
(TLC). It provides qualitative information about a mixture of amino acids. This experiment is
performed on a sheet of aluminum, which is coated with a layer of silica gel. The layer of silica
gel acts as the stationary phase. The solvent mixture acts as the mobile phase and is applied to
the plate with the sample. Due to the differences in the polarities of the amino acids in the
mixture, they will travel at different rates along the TLC plate. Due to the fact that amino acid
solutions are colorless, a ninhydrin staining agent is used to identify different amino acids. The
ninhydrin solution is applied to the TLC plates, and its reaction with the amino acid is
summarized in the figure below.

Fig.4: Reaction of Ninhydrin with an -amino acid.

After staining the TLC plate with ninhydrin, the amino acid can be identified qualitatively by the
intensity of the color produced. The calculated Rf values were also used to identify the amino
acid in the mixture.

Materials & Methods:

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Grant Akalonu Identification of Amino Acids

1) Amino Acid Titration:

First, the amino stock solutions were prepared.

Table 1: Amino acid stock solutions.

Amino Acid Mass Molecular Moles Used to Volume Concentration
(g) Weight Make the Solution
(g/mol)
Lysine 0.477 146.19 0.01 100 0.1
Threonine 0.122 119.12 0.01 100 0.1
Aspartic Acid 0.135 133.11 0.01 100 0.1
Table 1: Amino acid stock solutions.

10 mL of each stock solution was poured in to 100 mL beakers, with 25 mL of distilled

water. The solutions were stirred with magnetic stirring rods.
The pH electrodes were washed and calibrated using the amino acid solutions
The beaker, burette, and electrode was set up as shown in the figure below.

Fig.5 Titration Experimental Setup

The titrant was 0.3 M NaOH, and the pH of the solution was recorded after every 0.5 mL
of base was added to the beaker.

2) Thin Layer Chromatography/Ninhydrin Test

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Grant Akalonu Identification of Amino Acids

A standard solution (1mg/mL) for each of the 20 amino acids was prepared in 0.01 M
phosphate buffer (pH=8.0) with DMSO as additional organic solvent.
0.5 ml of each amino acid was used for TLC analysis.
0.5 mg of each amino acid was weighed, then 5 drops of organic solvent (DMSO) and 0.5
mL of PBS were added. 5 ml vials were used to preserve the solution of each amino acid.
The positions of the amino acid spot on the TLC plate were marked.
The solvent (mobile phase) was prepared by mixing propanol with distilled water, at a
1:1 ratio.
The chromatography chamber was prepared by adding 10-15 mL of the solvent and
allowing it to sit for 10-15 minutes.
A graduated 5L pipette was used to spot samples On the TLC plates. The samples were
allowed to dry and then covered with Parafilm.

Fig.6: Chromatography chamber with TLC plates.

After 45 minutes, the plates were removed and marked at the points where the
solvent front had traveled. After the solvent evaporated, the plate was sprayed
with ninhydrin stain.
The plates were then heated at 110C until spots were revealed.
The Rf values were then calculated for each amino acid using the following
equation:

Rf = (1)

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 Grant Akalonu Identification of Amino Acids

Results

Part 1: Amino Acid Titrations:

1) Aspartate Volume(mL) pH
0 1.16
0.5 1.3
1 1.5
14
1.5 1.75
2 2.09
pka3
12 2.5 2.5
3 2.93
3.5 3.32
10 4 3.73
4.5 4.67
5 8.66
8
5.5 9.72
pH

6 10.04
6 6.5 10.4
7 10.84
pka2
7.5 11.3
4 pka1 8 11.68
8.5 11.93
2
9 12.07
9.5 12.23
10 12.3
0 10.5 12.36
0 2 4 6 8 10 12 14 16 18 11 12.41
Volume (mL) 11.5 12.47
12 12.51
Fig.7: Aspartic Acid Titration Curve
12.5 12.55
13 12.59
13.5 12.61
14 12.65
14.5 12.71
15 12.73
15.5 12.75
16 12.77
16.5 12.79
17 12.81

Table 4: Aspartate Titration

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 Grant Akalonu Identification of Amino Acids

2) Lysine
Volume
(ml) pH
0 0.76
14
0.5 0.88
pka3 1 0.98
12 1.5 1.1
pka2 2 1.26
10 2.5 1.44
3 1.62
3.5 1.95
8
4 2.56
pH

4.5 7.55
6 5 8.45
5.5 8.92
pka1 6 9.19
4
6.5 9.44
7 9.7
2
7.5 9.95
8 10.25
0 8.5 10.45
0 2 4 6 8 10 12 14 16 18
9 10.66
Volume (mL)
9.5 10.85
Fig 8: Lysine Titration Curve 10 11.11
10.5 11.3
11 11.5
11.5 11.67
12 11.81
12.5 11.94
13 12.03
13.5 12.14
14 12.22
14.5 12.29
15 12.34
15.5 12.44
16 12.46
16.5 12.52
Table 3: Lysine Titration.

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 Grant Akalonu Identification of Amino Acids

3) Threonine

Volume
14 (mL) pH
0 0.96
12 0.5 0.99
pka2 1 1.05
1.5 1.2
10
2 1.32
2.5 1.5
8 3 1.72
3.5 2.02
pH

4 2.61
6
4.5 7.73
5 8.5
4 pka1 5.5 8.8
6 9.06
6.5 9.31
2
7 9.57
7.5 9.9
0 8 10.58
0 2 4 6 8 10 12 14
8.5 11.53
Volume of OH- (mL)
9 11.86
Figure 9: Threonine Titration Curve 9.5 12.07
10 12.18
10.5 12.27
11 12.35
11.5 12.46
12 12.53
12.5 12.56
13 12.6
Table 4. Theronine Titration

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Grant Akalonu Identification of Amino Acids

Discussion & Conclusion

Aspartate Lysine Threonine

Volume of NaOH at 2.18 3.5 2.0
First Endpoint (mL)
pH at first endpoint 2.3 1.3 1.4
First pKa 2.3 1.3 1.4
Volume of NaOH at 3.4 6.6 6.6
second endpoint
pH at Second 3.2 9.5 9.5
Endpoint
Second pKa 3.2 9.5 9.5
Volume of NaOH at 7 9.2 n/a
3rd endpoint
Third pKa 10.8 10.4 n/a
pI 2.69 9.95 5.6
Table 5. Estimated Isoelectronic pH and pkas for each amino acid .

pKa1 pKa2 pKa3 pI

Aspartate Experimental 2.18 3.2 10.8 2.69
value
Literature value 1.88 3.65 9.60 2.77
% error 13.7% 12.3% 12.5% 2.97%
Lysine Experimental 1.3 9.5 10.4 9.95
value
Literature value 2.18 8.95 10.53 9.74
% error 40.3% 6.14% 1.234% 2.11
Threonine Experimental 1.4 9.5 n/a 5.6
value
Literature value 2.11 9.62 n/a 5.87
% error 33.65% 1.24% n/a 0.046%
Table 6: Percent error of pka and pI values.

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Grant Akalonu Identification of Amino Acids

Part 2: Thin Layer Chromatography

Fig.6: TLC plates after staining with ninhydrin. The identities of each amino acid are indicated by their one letter
codes at the bottom of the plates.

Amino Acid Distance Moved from Distance Moved from Rf Value

the Origin by Amino the Origin by the
Acid (cm) Solvent
(cm)
Tryptophan 3.5 4.0 0.88
Tyrosine 2.4 4.3 0.56
Phenylalanine 3.4 4.3 0.79
Isoleucine 3.4 4.2 0.81
Histidine 0.6 4.3 0.14
Lysine 0.4 4.3 0.93
Asparagine 2.5 4.3 0.58
Aspartate 2.7 4.3 0.63
Glutamate 2.8 4.3 0.65
Glutamine 3.0 4.2 0.71
Valine 3.0 4.2 0.71
Alanine 3.4 4.0 0.85
Glycine 2.5 4.2 0.60
Leucine 0.6 4.0 0.15
Methionine 2.7 4.3 0.63
Threonine 2.2 4.3 0.51
Cysteine 0 4.2 0
Arginine 0.4 4.3 0.093
Proline 2.2 4.0 0.55
Serine 2.6 4.0 0.65
Table 5: TLC Results.

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Grant Akalonu Identification of Amino Acids

Discussion & Conclusion

The accepted experimentally determined pkas and pIs were estimated by observing the titration curves and
estimating the pH that corresponds to the middle of each buffer zone. There was a considerable amont of error in some
of the determined pka values, particularly the first pkas of lysine and threonine. Due to the fact that they were
determined simply by looking at the graph, this introduces a considerable amount of human error and can affect the
results. Also the pH in each titration curve does not start at the lowest possible pH which introduces error, particularly
in the measurement of the initial pkas. For the lysine titration curve, the separation between pkas 2 and 3 is not clear,
this could be due to base being added at increments that are too large to observe them. If this experiment were to be
repeated, more acid would be added initially to lower the stating pH as much as possible, and smaller increments of
base would be added to the amino acid solutions.

The results of thin layer chromatography show that the aromatic amino acids, Tryptophan, tyrosine, and
phenylalanine had the highest Rf values. Amino acids with branched and non-polar side chains such as Leucine,
Iosleucine, and Valine also had relatively high Rf values. This is an expected result, due to the fact that these amino
acids are non-polar and will not adhere to the very polar solvent and therefore move up the TLC plate. On the other
hand amino acids that are polar, such as Arginine, Lysine, and Cystiene stayed close to the solvent and had low Rf
values. There appears to be an error regarding Leucine however, it is an amino acid with a branched non-polar side
chain, and is expected to move up the TLC plate and only has a Rf value of 0.15. This indicates some form of error
introduced to the experiment in which the Leucine sample was introduced to a polar contaminant. There is also an
error with Lysine, as it has two spots on the TLC plate. This could be due to a mistake when spotting the amino acids
onto the TLC plate before submerging it in the mobile phase.

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