Titration of Amino Acids and Peptides

Marc Renzo Mandocdoc

Katrina Miral
Joanna Jane Raboy
De La Salle University Dasmarias
Dasmarias, Cavite
ABSTRACT
Titration method and its effectivity to amino acid components were
demonstrated in this experiment. This process specifically analyzes the
acid - base behavior of amino acids and peptides as what the experiment
aimed all about. Aside from this, the experiment also intended to identify
the unknown amino acid considering its experimental pKa values by
means of a titration curve. A mixture of 0.25 g sample of unknown amino
acid and 15.0 ml of distilled water underwent an incremental addition of
0.200 M HCl and series of swirling to acidify the solution until 1.50.
Afterwards, it was titrated with 0.20 ml increments of 0.200 M NaOH until
a basic pH of 11.31 was achieved. A titration curve was plotted base on
recorded volume of every added NaOH and the corresponding pH of the
solution. The unknown amino acid yielded experimental pKa values of
2.03, 3.44, and 9.41 and a pI of 5.72. Results show that the unknown
amino acid was an Aspartic acid and can be used as a buffer on acidic
pH. The percentage error obtained ranges from 2.87% - 4.03%.
Differences between the experimental and theoretical pkA and pI values
are possibly due to inaccurate addition of NaOH, incorrect pH reading
and plotting of curve.
INTRODUCTION
Proteins are the most significant class of biochemical molecules. It is complex in
nature due to several amino acids components linked together by peptide bonds which
makes up a protein. Peptide is a molecule that consists of two or more amino acid linked
together by bonds between the amino group and the carboxyl group. Amino acid
contains an amino group (-NH2), a carboxyl group (-COOH) and a functional group (R). It
is considered amphoteric because of the existence of both positive (+) and negative (-)
charges in its side chain. They can react either as acid or base depending on the
circumstances. Twenty primary amino acids that serve as the backbone for most
proteins are summarized in Table1.

Amino Acid
Glycine
Alanine
Valine
Leucine
Isoleucine
Proline

Aliphatic
3 letter / 1 letter code
Gly / G
Ala / A
Val / V
Leu / L
Ile / l
Pro / P
Aromatic

Structure

Amino Acid
Phenylalanine
Tyrosine
Tryptophan
Serine
Threonine
Cysteine
Methionine
Aspartic
Glutamic
Lysine
Arginine
Histidine
Asparagine
Glutamine

3 letter / 1 letter code

Phe / F
Tyr / Y
Trp / W
Alcoholic
Ser / S
Thr / T
Sulfur containing
Cys / C
Met / M
Acidic
Asp / D
Glu / E
Basic
Lys / K
Arg / R
His / H
Amide
Asn / N
Gln / Q

Structure

Table1. The Standard Amino Acids.

Titration is a useful tool in determining the reactivity of amino acid side chains
and in portraying the acid base behavior of amino acids and peptides. Amino acids
contain ionizable groups and the predominant ionic form of these molecules in a solution
depends on the pH.

MATERIALS AND METHODS

The experiment was initiated 0.250 g of the unknown amino acid powder
weighed exactly by analytical balance and transferred the sample to 250-ml Erlenmeyer
flask. Next, 15.0 ml of distilled water was poured in each flask using the volumetric
pipette and added the small increments 0.200 M HCl to the dissolved sample and
swirled. Repetition of the preceded procedure until a pH of 1.50 achieved was observed.
Afterwards, titration follows in the acidified solution with 0.200 M NaOH by adding 0.20
ml incrementally until pH of at least 13.0 is reached. All the necessary data which
includes the vol. of NaOH added with its corresponding pH contributed to the solution
were collected for plotting of graph.

RESULTS AND DISCUSSION

Different amino acids constituting an amino group and carboxyl group vary in
their side chain group or the R group. Every amino acid can exist in an acidic or basic
form, depending on the pH of the solution in which the amino acid is dissolved.
Fig. 1 Titration curve of unknown amino acid (Aspartic Acid)

pKa = 9.41
pKa = 3.44
pKa = 2.03

Titration of an amino acid illustrates the effect of pH on amino acid structure. The
titration curve (Fig. 1) indicates the reaction of the unknown amino acid with hydrogen
ion. From the plotted curve, three inflection points of the curve was determined. These
three points represents the three structural features in amino acid: amino group NH 2;
carboxylic acid, COOH; and functional group, R. Table2 shows the pK a values
determined from the curve in contrast with the theoretical values of the identified amino
acid, specifically Aspartic acid.

Experimental

Theoretical

% Error

pKa1

2.03

2.09

2.87%

pKa2

3.44

3.86

10.88%

pKa3

9.41

9.82

4.18%

pI

5.72

5.96

4.0

Identity of Unknown Amino Acid: Aspartic Acid

Table2. Theoretical and Experimental pKa values of unknown amino acid.

At very low pH resulting from the addition of 0.200 M HCl, the unknown amino
acid gained a protonated carboxyl (COOH) and amino group (NH 2). Under these
conditions, the amino acid has a net positive charge of 1.50. Upon undergoing titration
with a strong base solution of 0.200 M NaOH, an increase in pH was observed. This
occurrence is due to the reaction of carboxyl group. It loses its proton and yields a
negatively charged carboxylate group (COO-). At this point, the unknown amino acid has
no net charge and is electrically neutral which actually distinguish its isoelectric point (pI)
positioned at the region between the pKa values of 2.03 and 3.44.The locating of pI is
based on the slope of the curve where pH change is slowest. Degree of curve is
proportional to the rate of change in pH. Therefore, lower degree of curve represents the
pI. As the titration continues, the next group to lose its proton is the functional group and
then ammonium group (NH3) followed. The summary of the proceeded statements were
illustrated below.
Fig. 2 Titrimetric profile of Aspartic acid base on the experimental pK a and pH values.

The classification of an amino acid as acidic or basic depends on the pK a of the side
chain. Aspartic acid is considered acidic because it has an extra carboxylic acid aside
from the carboxyl group that every amino acid has. This extra carboxylic acid has a very
low pKa value. The fact that amino acids, peptides, and proteins have different pK a
values give rise to the possibility that they can have different charges at a given pH. Like
for Aspartic acid, different charges and an order of diminishing charges can be observed
as shown in Fig. 2. Results on the experiment reveals the true nature and characteristics
of amino acids which can be concluded to be an amphoteric organic compounds due to
their capability of resisting drastic changes in pH,
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Campbell, M. and Farrel, S. (2012). Biochemistry. 7th Ed. Canada: Cengage

Learning.
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McKee, T. and McKee J. (2003). Biochemistry, the molecular basis of life. 3rd Ed.
New York: McGraw Hill.