Experiment 1: Amino Acids as Ampholytes

Quennie Lynn Dueas & Glenn Vincent Tumimbang

Biochem 34.1 HEJ, Mr. Avvin Pelovello
Abstract
Keywords:

Biochem 34.1: Amino Acids as Ampholytes

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I. Introduction
Acids may be defined as proton donors
and bases as proton acceptors. Each acid
has a characteristic tendency to lose its
proton in aqueous solution. The stronger
the acid is, the greater its tendency to
dissociate its proton. Strong acids such as
hydrochloric, sulphuric, and nitric acids
almost completely dissociated to form ions
in solution, as do strong bases NaOH and
KOH. They are called strong electrolytes.
On the other hand, those not completely
ionized when dissolved in water are called
weak electrolytes. Acetic acid, a weak
acid, is a good example (Garrett &
Grisham, 2010). These are ubiquitous in
biological systems and play important
roles in metabolism and its regulation (Neil
& Cox, 2013).
Buffers are solutions that tend to resist
changes in their pH as acid or base is
added. Typically, a buffer system is
composed of a weak acid and its
conjugate base. It is at the pK a that the
buffer system shows its greatest buffering
capacity, thus, the components are
chosen such that pKa of the weak acid is
close to the pH of interest. Biologically,
maintenance of pH close to 7.0 is vital to
all cells otherwise, cellular processes such
as metabolism are disrupted (Garrett &
Grisham, 2010). Two especially important
biological buffers are the phosphate and
Carbonic Acid
the bicarbonate system.
Phosphoric
The cytoplasm of most cells contains
highAcid
Aspartic Acid
concentration of proteins. Amino acids are
considered as ampholytes. They can act
as both acids and bases. 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 positively. Amino
acids without charged groups on their side
chains exist in neutral solutions as
zwitterions
with
no
net
charge.

Biochem 34.1: Amino Acids as Ampholytes

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Zwitterions, which are compounds that

have a both positive charge and a
negative charge have come into wide use
as buffers more recently. They are usually
considered less likely to interfere with
biochemical reactions than some of the
earlier buffers (Campbell & Farrell, 2015).
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 (Neil & Cox,
2013).
II. Experimental
The group was required to prepare 50 mL
of 0.1 M phosphate buffer with a pH of 4.
Table 1. pKa values of weak acids
Pk1
pK2
4.74
6.1
10.4
3.1
4.1
1.97
7.0
2.1
3.9
2.3
9.6
In accordance to the table, the pK 2 of
phosphoric acid which is equivalent to 7.0,
closest to the desired pH 4.0, was used as
the pKa in the Henderson- Hassellbach
equation. This equation was used in order
to calculate the ratio of salt (Na 2HPO4) and
the acid (NaH2PO4). The calculated
amounts of the acid and salt were mixed

6
1
9
9

and dissolved in ample amount of distilled

water. A pH meter was used to monitor
the pH of the buffer solution. If the
obtained pH is higher, 0.1 M HCl was
added and if the pH is lower, 0.1 M NaOH
was added necessarily in order to achieve
the desired pH value. The solution was
then 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 bottle. The buffer solution was
properly labelled with the reagents name,
its pH, the groups number and 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 glycine (pH 1.0) were

Biochem 34.1: Amino Acids as Ampholytes

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supposed to be prepared but the only

available reagent was the phosphoric acid.
The solution was titrated with 0.5 mL
increments of 0.1 M NaOH introduced
through a burette attached to an iron
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
notebook until a pH of 13.0 is reached.
Finally, to observe the titration curve of
phosphoric acid, the pH values were
plotted against the volume of 0.1 M NaOH.
Theoretical titration curves of aspartic acid
and glycine were researched.