Experiment 1: Amino Acids as Ampholytes

2011-35493, 2011-85007
Biochem 34.1 HEJ, Sir Marvin Pelovello

I. Abstract
Buffers are able to resist changes in pH due to the presence of a weak acid or weak base and its conjugate. They are
important in living systems in maintaining the biological pH of 7.2 to 7.4 for various enzymatic activities and metabolic
processes. In the experiment, phosphate buffers with different pH values of 2.0, 4.0, 6.7, 7.4, and 9.0 were prepared.
In the preparation of pH 4.0 phosphate buffer, 0.7819 grams of the Na2HPO4H2O was used. The titration curves of
three amphoteric substances; phosphoric acid, aspartic acid (acidic amino acid), and glycine (non-polar amino acid);
were obtained. Only the experimental titration curve of phosphoric acid was determined, which showed three buffer
regions as governed by its pKa values. The results showed no significant differences to the theoretical titration curve of
phosphoric acid. Similarly, the theoretical titration curve of glycine and aspartic acid were compared based on their
respective pKa values. Glycine showed two buffer regions while aspartic acid showed three. The titration curves also
follow their pKa values, demonstrating their acidic and basic properties.

II. Keywords: buffers, Henderson-Hasselbach equation, ampholytes, glycine, aspartic acid

III. Introduction positively. Amino acids without charged groups on their

Acids may be defined as proton donors and side chains exist in neutral solutions as zwitterions with
bases as proton acceptors. Each acid has a no net charge. Zwitterions, which are compounds that
characteristic tendency to lose its proton in aqueous have a both positive charge and a negative charge
solution. The stronger the acid is, the greater its have come into wide use as buffers more recently. They
tendency to dissociate its proton. Strong acids such as are usually considered less likely to interfere with
hydrochloric, sulphuric, and nitric acids almost biochemical reactions than some of the earlier buffers
completely dissociated to form ions in solution, as do (Campbell & Farrell, 2015).
strong bases NaOH and KOH. They are called strong The general objectives of the study is to
electrolytes. On the other hand, those not completely understand the acid/base properties of amino acids
ionized when dissolved in water are called weak and buffers. The experiment specifically aims to
electrolytes. Acetic acid, a weak acid, is a good prepare buffer solutions of certain pH and
example (Garrett & Grisham, 2010). These are concentration, construct and interpret the titration
ubiquitous in biological systems and play important curves of phosphoric acid, aspartic acid, and glycine,
roles in metabolism and its regulation (Nelson & Cox, and describe the buffering action of the three
2013). aforementioned solutions.
Buffers are solutions that tend to resist
changes in their pH as acid or base is added. Typically, IV. Experimental
a buffer system is composed of a weak acid and its The group was required to prepare 50 mL of
conjugate base. It is at the pKa that the buffer system 0.1 M phosphate buffer with a pH of 4.
shows its greatest buffering capacity, thus, the
components are chosen such that pKa of the weak acid Table 1. pKa values of weak acids
is close to the pH of interest. Biologically, maintenance Acid pK1 pK2 pK3
of pH close to 7.0 is vital to all cells otherwise, cellular
processes such as metabolism are disrupted (Garrett & acetic acid 4.74
Grisham, 2010). Two especially important biological carbonic acid 6.1 10.4
buffers are the phosphate and the bicarbonate system. citric acid 3.1 4.1 6.4
The cytoplasm of most cells contains high phosphoric acid 1.97 7.0 12.5
concentration of proteins. Amino acids are considered aspartic acid 2.1 3.9 9.8
as ampholytes. They can act as both acids and bases. glycine 2.3 9.6 9.8
In a free amino acid, the carboxyl group and amino
group of the general structure are charged at neutral
pHcarboxylate portion negatively and the amino group

BIOCHEM 34.1: AMINO ACIDS AS AMPHOLYTES 1

 In accordance to the table, the pK2 of [42 ]
phosphoric acid which is equivalent to 7.0, closest to 4.0 = 7.0 +
[2 4 ]
the desired pH 4.0, was used as the pKa in the 0.1 = [42 ] + [2 4 ]
Henderson-Hasselbach equation. This equation was [42 ]
used in order to calculate the ratio of salt (Na2HPO4) 1103 =
[2 4 ]
and the acid (NaH2PO4). The calculated amounts of the
110 [2 4 ] = [42 ]
3
acid and salt were mixed and dissolved in ample
1103 (0.1 [42 ]) = [42 ]
amount of distilled water. A pH meter was used to
1104 = [42 ] + 1 103 [42 ]
monitor the pH of the buffer solution. If the obtained
1 104
pH is higher, 0.1 M HCl was added and if the pH is [42 ] =
lower, 0.1 M NaOH was added necessarily in order to 1 + 1 103
156.01
achieve the desired pH value. The solution was then [42 ] = 9.99 105 0.05
1
transferred to a 50 mL volumetric flask and was filled [ ] = .
to the mark with distilled water. For storage in the
freezer at 4C, it was transferred to a recycled plastic
[2 4 ] = 0.1 9. 99 105
bottle. The buffer solution was properly labelled with
156.01
the reagents name, its pH, the groups number and [2 4 ] = 0.0999 0.05
1
section, and the date it was prepared. [ ] = .
In separate Erlenmeyer flasks, 10 mL of 0.1 M
of H3PO4, 0.1 M aspartic acid (pH 1.0), and 0.1 M The assigned buffer solution to the group has
glycine (pH 1.0) were supposed to be prepared but the a pH of 4.0. Due to the very small amount of the weak
only available reagent was the phosphoric acid. The acid needed, only the salt was used in the preparation.
solution was titrated with 0.5 mL increments of 0.1 M
NaOH introduced through a burette attached to an iron
[2 2 4 2 ] = 0.7793 + 7.79 104
stand with a clamp. After each base increment was
[ ] = .
added, the corresponding pH value was determined by
a pH meter and was listed down religiously on a
Part B. Titration Curve of an Acid
notebook until a pH of 13.0 is reached. Finally, to
Maintaining the pH at a constant value is done
observe the titration curve of phosphoric acid, the pH
with buffers. This is possible due to the presence of
values were plotted against the volume of 0.1 M NaOH.
appreciable amounts of both the acid and its conjugate
Theoretical titration curves of aspartic acid and glycine
base. This is acquired at pH values near the pKa of the
were researched.
acid of interest. Through its titration curve, its buffer
capacity can be observed. The net electric charge and
V. Results and Discussion
pH of the solution of the solution can also be
determined through the titration curve of amino acids
Part A. Buffer Preparation
(Nelson & Cox, 2013).
The amount of salt and weak acid needed to
During preparation, the observed pH may be
prepare a pH 4 phosphate buffer was computed. The
different from the calculated pH due to the
Henderson-Hasselbach equation was used.
temperature of the environment. In some cases, it may
be due to the nature of the solution itself. Certain weak
3 4 + + 2 4 acids and their conjugate bases are already acting as
2 4 + + 42 buffers.
42 + + 43 Amino acids have a very characteristic
Eq. 1 property to act as both an acid and a base due to the
[ ] presence of both positive and negative charges. They
= +
[] are called amphoteric substances (acid-base property)
Eq. 2 and zwitterionic (positive, negative charges). Aspartic
This equation is used to determine the acid is an example of acidic amino acid while glycine is
properties of buffer solutions that are used to control a non-polar amino acid (Nelson & Cox, 2012). These
the pH of reaction mixtures (Campbell, 2011). properties can be observed from their theoretical
titration curves. These amino acids can act as buffers

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in a pH range of 1 of their pKa values; that is, their
presence in protein cells and tissues help stabilize the
pH in the bloodstream and in intercellular spaces for
proper enzymatic activity and other chemical
processes. On the other hand, the titration curve of
phosphoric acid shows its triprotic nature. A conjugate
base is produced for every proton loss.
12
8 are observed. Aspartic acid, on the other hand, which is
pH
4 buffer regions. Both of their zwitterionic forms can be
0
0 4 8 12 16 20 24
titrant volume (0.1M NaOH)
Figure 1. Experimental titration curve of phosphoric
acid.
This can be observed from the three buffering
regions observed as more titrant is added.
Only the theoretical titration curves of aspartic
acid and glycine were observed. Glycine is also a weak
diprotic acid. At low pH values, the predominant
species of glycine is the fully protonated form, +H3N-
CH2-COOH. As more titrant is added, the first pKa value
shows that both the fully protonated form and the
deprotonated carboxyl group are present in equal
amounts. This is at the pKa value of 2.34. Before
reaching the second pKa value, an important point
called the isoelectric point or pI is reached. This is
obtained by determining the midpoint of the two pKa
values. At the pKa of 5.97, the deprotonation of the
carboxyl group is completed. As the titration proceeds,
the second pKa value of 9.60 is reached. This is the
point where +H3N-CH2-COO- and H2N-CH2-COO- are
present in equal amounts. As soon as the titration
proceeds into completion, the fully deprotonated form
of glycine is the predominant species in the solution
(Garret & Grisham, 2010).
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Figure 2. Theoretical titration curve of glycine.
Another amino acid in the experiment is
aspartic acid. It is a triprotic amino acid. Based on its
theoretical titration curve, three buffer regions are
readily observable.
Comparing the titration curves of glycine and
aspartic acid can be done based on their pKa values. It
can be observed that their pKa values demonstrate the
buffering capacity of these amino acids. In the case of
glycine, which is has two pKa values, two buffer regions
a triprotic acid with three pKa values, shows three
pinpointed at the isoelectric point obtained at a certain
pH range as their acidic and basic properties are both
demonstrated.
Figure 3. Theoretical titration curve of aspartic acid.
One of the most important biochemical
equilibria is the pH balance in the body. It affects both
the substrates and the enzymes, determining the rate
at which these chemical activities, or even flow of
electricity, proceed in biological systems. The pH, by
itself, can have an effect on the ionization of acidic and
basic amino acids, which results to changes in protein
structures, as well as enzymatic activity and protein
recognition (Nelson & Cox, 2012).
In the laboratory, buffers are also essential in
regulating pH. The process of gradually adding known
amounts of reagent to a solution with which the
reagent reacts while monitoring the results is called a
titration (Berg, Tymoczko, & Stryer, 2012). The titration
curve illustrates the progressive dissociation of weak
acid. In addition, the shape of the titration curve of any
weak acid is described by the Henderson-Hasselbach
equation, which is important for understanding buffer
action and acid-base balance in the blood and tissues
of vertebrates. This equation enables us to deduce
some important quantitative relationships between pH,
pKa, and buffer concentration (Nelson & Cox, 2013).

VI. Conclusion and Recommendations

Buffers are very important in biological
systems. It prevents interferences in biological
processes even under the presence of pH-affecting by-
products. It is also used to control certain reactions
that occur at specific pH ranges, which is needed in
some laboratory experiments.
Amino acids are amphoteric substances that
have acidic and basic properties. This characteristic
property of amino acids makes them one of the most
important substances needed in the body. The
presence of different functional groups, namely an
amino group head and a carboxyl group tail, determine
its tendency to donate a hydronium ion in certain pH
changes. This resistance to drastic changes in pH was
successfully observed with the use of the experimental
(phosphoric acid) and theoretical (glycine, aspartic
acid) titration curves of these polyprotic acids. This, in
turn, showed the importance of buffers in biological
systems.
It is recommended that other titration
techniques should be employed to determine the
titration curves of the amino acids in the experiment.
Other common amino acids in the body, such as basic
amino acids and polar amino acids, can also be used
to determine their buffer capacity.

VII. References

Biochemistry laboratory manual. Retrieved from:

http://www.chem.fsu.edu/chemlab/bch4053/
character/titration/

Berg, J., Tymoczko, J., and Stryer, L., (2012).

Biochemistry (5th ed.). WH Freeman, New York.

Campbell, M., and Farrel, S., (2015). Biochemistry (8th

ed). Brooks/Cole.

Garret, R., and Grisham, C., (2010). Biochemistry (5th

ed.)

Nelson, D., and Cox, M., (2013). Lehninger principles of

biochemistry (6th ed.).

Proteins, peptides, and amino acids. Retrieved from:

http://www2.chemistry.msu.edu/faculty/reusc
h/virttxtjml/proteins.htm

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