Essay/Term paper: Determination of an unknown amino acid from titration

Essay, term paper, research paper:  Science Research Papers

Determination of An Unknown Amino Acid From Titration


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.