Determination of an Unknown Amino Acid
Abstract
Experiment 11 used a titration curve to determine the identity of an unknown amino
acid. The initial
pH of the solution was 1.96, and the pKa's found experimentally were 2.0, 4.0, and 9.85.
The accepted
pKa values were found to be 2.10, 4.07, and 9.47. The molecular weight was calculated to
be 176.3 while
the accepted value was found to be 183.5. The identity of the unknown amino acid was
established to be
glutamic acid, hydrochloride.
Introduction
Amino acids are simple monomers which are strung together to form polymers (also
called proteins).
These monomers are characterized by the general structure shown in figure 1.
Fig. 1
Although the general structure of all amino acids follows figure 1, the presence of a
zwitterion is made
possible due to the basic properties of the NH2 group and the acidic properties of the
COOH group. The amine group (NH2) is Lewis base because it has a lone electron pair
which makes it susceptible to a coordinate covalent bond with a hydrogen ion. Also, the
carboxylic group is a Lewis acidic because it is able to donate a hydrogen ion (Kotz et
al., 1996). Other forms of amino acids also exist. Amino acids may exists as acidic or
basic salts. For example, if the glycine reacted with HCl, the resulting amino acid
would be glycine hydrochloride (see fig. 2). Glycine hydrochloride is an example of an
acidic salt form of the amino acid. Likewise, if NaOH were added, the resulting amino
acid would be sodium glycinate (see fig. 3), an example of a basic salt form.
Fig. 2
Fig. 3
Due to the nature of amino acids, a titration curve can be employed to identify an
unknown amino acid.
A titration curve is the plot of the pH versus the volume of titrant used. In the case
of amino acids, the
titrant will be both an acid and a base. The acid is a useful tool because it is able to
add a proton to the
amine group (see fig. 1). Likewise the base allows for removal of the proton from the
carboxyl group by
the addition of hydroxide. The addition of the strong acid or base does not necessarily
yield a drastic
jump in pH. The acid or base added is unable to contribute to the pH of the solution
because the protons
and hydroxide ions donated in solution are busy adding protons to the amine group and
removing protons
from the carboxyl group, respectively. However, near the equivalence point the pH of the
solution may increase or decrease drastically with the addition of only a fraction of a
mL of titrant. This is due to the fact that at the equivalence point the number of
moles of titrant equals the number of moles of acid or base originally present (dependent
on if the amino acid is in an acidic or basic salt form). Another point of interest on a
titration curve is the half-equivalence point. The half-equivalence point corresponds to
the point in which the concentration of weak acid is equal to the concentration of its
conjugate base. The region near the half-equivalence point also establishes a buffer
region (Jicha, et al., 1991). (see figure 4).
Fig. 4
The half-equivalence point easily allows for the finding of the pKa values of an
amino acid. A set
pKa values can be extremely helpful in identifying an amino acid. Through a manipulation
of the
Henderson-Hasselbalch equation, the pH at the half-equivalence point equals the pKa.
This is reasoned
because at the half-equivalence point the concentration of the conjugate base and the
acid are equal.
Therefore the pH equals the pKa at the half-equivalence point (see figure 5.)
Fig. 5 [base]
pKa= pH - log -------
[acid]
[base]
log -------- = log 1 = 0
[acid]
therefore, pH = pKa
However, many substances characteristically have more than one pKa value. For each
value, the
molecule is able to give up a proton or accept a proton. For example H3PO4 has three pKa
values. This is
due to the fact that it is able to donate three protons while in solution. However, it
is much more difficult
to remove the second proton than the first. This is due to the fact that it is more
difficult to remove a
proton from a anion. Furthermore, the more negative the anion, the more difficult to
remove the proton.
The trapezoidal method can be employed to find the equivalence points as shown if
figure 6. The
volume of titrant between two equivalence points is helpful in the determination of the
molecular weight
of the amino acid.
Fig. 6
The purpose of experiment 11 is to determine the identity of an unknown amino acid
by analyzing a
titration curve. The experiment should lend the idea that the following may be directly
or indirectly
deduced from the curve-- the equivalence and half equivalence points, pKa values, the
molecular weight
and the identity of the unknown amino acid.
Experimental
The pH meter was calibrated and 1.631 grams (.0089 moles) of the unknown amino acid
was weighed and placed in a 250-mL volumetric flask. About 100 mL of distilled water was
added to dissolve the solid. The flask was gently swirled and inverted to insure a
complete dissolution of the solid. The solution was diluted with distilled water to the
volume mark on the flask. Then, one buret was filled with 0.100 M HCl stock solution and
another buret was filled with 0.100 M NaOH. A pipet was used to add 25.00 mL of the
unknown amino acid solution to a 100-mL beaker. The solution's initial pH was
established to be 1.96 by the pH meter. The electrode was left in 100-mL beaker with the
unknown amino acid solution. In the accurate titration curve, the acid was added in 0.5
mL increments until the pH of the solution was 1.83. As the titrant was added the pH of
the solution was recorded on a data sheet. Also, a graph of pH versus the mL of titrant
added was plotted. After the addition of the acid, a new 25 mL aliquot of unknown
solution was added to a clean 100-mL beaker. The base was then used to titrate the
solution. It was added in 0.20 to 1.0 mL increments depending on the nature of the
curve. (The nature of the curve was somewhat expected because previously an experimental
titration curve was established. This curve used increments of up to 2.0 mL.) The base
was added until the pH reached 12.03.
Results
Table 1 shows the pH endpoints for both the titration with the acid as well as with
the base. It also shows the initial pH. Table 1 also shows the experimentally
determined and accepted molecular weight and pKa values for the glutamic acid,
hydrochloride. Tables 2 and 3 show the amounts of base and acid added to the unknown
solution (respectively) and the pH which corresponds to that amount. Figures 7 and 8
represent the exploratory titration and the accurate titration curves (respectively).
Figure 9 represents the structure of the unknown amino acid, glutamic acid,
hydrochloride.
Table 1
pH of endpoints pKa values (experimental) pKa values (accepted) initial pH Molecular
weight identity of unknown
1.83 2.0 2.10 1.96 176.3 (expt.) Glutamic acid, hydro-chloride
12.03 4.0 4.07 183.5 (accepted)
9.85 9.47
Table 2
Accurate Titration for NaOH Fig.9
total mL of 0.10 M NaOH pH of solution
0.00 1.96
1.0 2.05
3.0 2.26
5.0 2.5
7.0 2.84
9.0 3.28
10.0 3.53
11.0 3.77
13.0 4.14
14.0 4.39
15.0 4.56
15.5 4.66
16.0 4.78
16.5 4.93
17.0 5.13
17.5 5.63
17.7 5.99
17.8 6.52
18.0 7.93
18.2 8.18
18.4 8.50
18.5 8.56
19.0 8.83
21.0 9.44
22.0 9.62
23.0 9.82
23.5 9.93
24.0 9.98
24.5 10.12
25.0 10.21
25.5 10.37
26.0 10.52
26.5 10.69
27.0 10.86
27.5 11.06
28.0 11.22
28.5 11.37
29.0 11.41
29.5 11.53
30.0 11.58
31.0 11.71
33.0 11.85
36.0 12.03
Table 3
Accurate Titration for HCl
total mL of 0.10 M HCl pH of solution
0.00 1.96
0.5 1.93
1.0 1.91
1.5 1.87
2.0 1.85
2.5 1.83
Discussion
The initial pH of the unknown solution was 1.96. This information was helpful
in determining the identity of the unknown amino acid because only a three of the nine
unknowns were acidic salts. (Acidic salt forms of amino acids are capable of having pH
values of this degree.) However, more information was required before the determination
could be conclusive. The unknown produced three equivalence points and therefore, three
pKa values. Therefore, one of the three remaining amino acids one could be omitted from
the uncertainty, because it contained only two pKa values. However, after examining the
pKa values of the unknown, it was apparent that they were remarkably similar to those of
glutamic acid, hydrochloride. The unknown's pKa values were 2.0, 4.0, and 9.85, while
the glutamic acid's pKa values were 2.10, 4.07, 9.47. At this point, the identity of the
amino acid was conclusive. However, as a precautionary measure, the molecular weight of
the amino acid was calculated and found to be 176.3 amu. The calculated value
corresponds well with the known value of 183.5 amu.
There are a few errors that can be held accountable for the small deviation from the
accepted values.
First, the pH meters never reported a definite value; most times the meter would report a
floating number. Therefore, one have no way of knowing which reported pH was more
correct. Also, the method by which the equivalence points was extremely crude. It
called for a series of rough of estimations. These estimations then led to the
equivalence point. Then the use of the equivalence point was used to determine the
half-equivalence point. This point was then used to find the pKa. The deviance from
accepted values of the pKa values occur because of the compounded series of crude
estimates which were required. Likewise, the deviance of the calculated molecular weight
can be attributed to these crude vehicles, because the change in volume (between
equivalence points) were used in calculation.
Conclusion
The identity of an unknown amino acid was determined by establishing a titration curve.
The
equivalence and half-equivalence point, the pKa values, and the molecular weight were
directly or
indirectly found through the titration curve. The equivalence points were found through
a crude method
known as the trapezoidal method. The establishment of the equivalence points gave rise
to the half
equivalence points and the D volume (used in calculating the molecular weight). The
half-equivalence
points were directly used to find the pKa values of the unknown. The molecular weight
could also be
calculated. This data led to the determination of the identity of the unknown amino
acid--glutamic acid,
hydrochloride.
References
Jicha, D.; Hasset, K. Experiments in General Chemistry; Hunt: Dubuque, 1991:37-53.
Kotz, J.C.: Treichel , P. Jr. Chemistry and Chemical Reactivity; Harcourt-Brace: Fort
Worth, 1996; 816- 837.
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