Titrimetric Analysis of Amino Acids and Peptides

Paulo T. Carpio
HUB32

De La Salle University-Dasmariñas
Dasmariñas, Cavite, Philippines

ABSTRACT

The acid-base behavior of amino acids and peptides were analyzed with the use of
titration. A 0.25g sample of unknown amino acid and aspartame in the beaker were
added each of HCl to produce an acidic solution and then titrated with the addition of
every 0.200ml of 0.200M NaOH. The pH of the acidic solutions was measured from 1.5
until it reaches pH 12 and it was recorded every addition of the base. The total volume of
the base added was 22.6ml on the unknown and 19.8ml on the Aspartame. A titration
curves were constructed based on the pH values and volume added. The identity of the
sample revealed that the unknown amino acid was Aspartic acid. A titrimetric profile of
the amino acid and aspartame were drawn. The pI value for Aspartic was 2.64 with
11.40% error and Aspartame has pI of 2.71 with 4.91% error. The experimental pKa
values and pI showed no significant difference from the theoretical values.

INTRODUCTION

Proteins are the indispensible agents of biological function and amino acids are the building
block of proteins. Amino acid is an organic compound containing an amino group (-NH2) and a
carboxyl group (-COOH). Since amino acids contain both an acidic and a basic group, they
undergo an intramolecular acid-base reaction and exist primarily in the form of a dipolar ion, or a
zwitterion.(2) In addition, amino acids are amphoteric or they can react either as acid or base
depending on the circumstances.
Peptide is a molecule that consists of two or more amino acid linked by bonds between the
amino group and the carboxyl group which is known as a peptide bond. Although their structures
are less complex than the larger protein molecules, peptides have significant biological activities.
Titration is a useful tool in determining the reactivity of amino acid side chains. Because
amino acids contain an ionizable group, the predominant ionic form of these molecules in solution
depends on pH. Titration of amino acid illustrates the effect of pH on amino acid structure.(3) It is
also useful in determining the isoelectric pH of the sample. While the titration curve reveals the
pKa of the various prototropic groups in amino acids and peptides and their respective pI values.
(1)

MATERIALS AND METHODS

A 0.250g sample of the unknown amino acid powder that was placed on a 250ml beaker was
approximately weighed using an analytical balance. Then a volumetric pipette was use to add a
25.0ml of distilled water on the beaker to dissolve the powder sample. Small increments of
0.200M HCl were added with the aid of a syringe to totally dissolved the sample while stirring.
The pH was measured and the addition of HCl was repeated until the pH reached 1.50 and
become acid solution. Then the solution was titrated with 0.20ml increments of 0.200M NaOH.
Stirred, then the pH was measured and recorded at each amount of base that was added. The
procedure was repeated until the pH of the solution reached 12.0.
All the procedures were repeated using commercial aspartame as the new sample. A titration
curves of the unknown sample and that of aspartame were constructed using a MS Excel
program. Then the unknown sample of amino acid was identified based on the pKa values and pI
that were seen in the constructed titration curves. When the unknown sample was identify, a
titrimetirc profile of it and that of aspartame were drawn. Then the percent error of the pKa and pI
were calculated.

RESULTS AND DISCUSSIONS

The Identity of the unknown amino acid sample was identified as Aspartic acid based on its
pKa and pI value. It was based on the closest pKa of amino acid in the table of pKa values for
protropic groups in amino acid. The added base on the unknown sample until it reached pH 11.34
was 22.6ml. The titrimetric analysis for the unknown amino acid and titrimetric profile of Aspartic
acid were shown below:

Titrimetric Analysis of Unknown Amino Acid

A. Titration Curve of the Unknown Amino Acid

B. Identity of Unknown Amino Acid

Experimental Theoretical %Error

pKa1 2.06 2.09 1.44
pKa2 3.21 3.86 16.84
pKa3 9.55 9.82 2.75
pI 2.64 2.98 11.40
Identity of Unknown Amino Acid: Aspartic acid
 Figure 1.1 Titrimetric Profile of Aspartic Acid

From adding NaOH, the pH of the acidic solution rises slowly and it determines that the
acidic sample resist the change in pH. Based also on the pI of the amino acid, it can be a buffer
on physiologic or basic pH.
During titration the carboxylate, -CO 2-, is the one that acts as the basic site and accepts a
proton in acid solution, and it’s the ammonium cation, -NH3+, that acts as the acidic site and
donates a proton in a base solution. In a strongly acidic solution it is present mainly in the form in
which the carboxyl group is uncharged. Under this circumstance the molecule’s net charge is +1,
because the ammonium group is protonated. Lowering the H+ concentration results in the
carboxyl group losing its proton to become a negatively charged carboxylate group. At this point,
there is no net charge and is electrically neutral. The pH at which this occurs is called the
isoelectric point (pI). Because there is no net charge at the isoelectric point, amino acids are least
soluble at this pH.(3) As the titration continues, the ammonium group loses its proton and the
molecule then has a net negative charge. The isoelectric point is when the amino acid is
zwitterionic.
Aspartame or Aspartylphenylalanine dipeptide, is a low-calorie sweetener which is which is
200 times sweeter than sucrose. Aspartic acid and phenylalanine are the amino acids that
bonded to produce a aspartame. The process is illustrated in Figure 2.1

Figure 2.1 Aspartame Formation

 The total volume of NaoH added until pH of the Aspartame reached 11.24 was 19.8ml. the
titrimetric analysis of the Aspartame were shown below:

Titrimetric Analysis of Aspartame

A. Titration Curve of Aspartame

B. pKa Values and pI of Aspartame

Experimental Theoretical %Error

pKa1 1.83 1.83 0
pKa2 3.58 3.86 7.25
pKa3 10.04 9.82 2.24
pI 2.71 2.85 4.91
 Figure 1.2 Titrimetric Profile of Aspartame

Based on the percent error on the pKa and pI values of the unknown sample and the
aspartame, there is almost no significant differences in the theoretical and experimental values
except on the pKa2 of the unknown sample that exceeded on 10% error. Some possible source of
errors on the experiment was the inaccurate plotting of pKa values on the titration curve. If the
pKa was wrong, the identity of the unknown sample will be incorrect.
The rapid rise in aspartame’s popularity can be attributed to the many benefits aspartame
provides to the many calorie-conscious consumers. It has a taste very similar to sugar, enhances
the flavors, it does not promote tooth decay, there is a scientific studies show that aspartame is
beneficial in weight control and it is helpful for individuals with diabetes that allow them to satisfy
their taste for sweets without affecting blood sugar which helps them to comply with a healthful
meal plan. In addition, consuming products with aspartame can result in fewer calories, which
helps people with diabetes, manage their weight. Under increased temperature, such as baking
technique, aspartame hydrolyzes into its respective amino acids. Food products with a higher pH,
result in the degeneration of this sweetener. In beverages that are marketed in a powdered form,
the aspartame undergoes non-enzymatic browning reaction, referred to as Maillard reaction,
thereby resulting in disappearance of flavor and aroma. When consumed within the
recommended intake, it is also advised for pregnant mothers. It substitutes their craving for
sweets, thereby keeps a check on the calories.(4)
But adverse side effects of aspartame consumption includes loss of memory, seizures,
headache, blindness, protruding eyes, ringing sound in the ear, palpitation, depression, lack of
sleep, breathlessness, diarrhea, and skin rashes. Aspartame changes the ratio of amino acids in
the blood, blocking or lowering the levels of serotonin, tyrosine, dopamine, norepinephrine, and
adrenaline. Therefore, it is typical that aspartame symptoms cannot be detected in lab tests and
on x-rays. Textbook disorders and diseases may actually be a toxic load as a result of aspartame
poisoning.

REFERENCES

(1) Legaspi, G.A. 2009. Essentials of Biochemistry Laboratory

(2) McMurry, J. Simanek E. 2008. Fundamentals of Organic Chemistry. 6th edition. The
Thompson Corporation
(3) McKee, T. McKee, J.R. 2003. Biochemistry-The Molecular Basis of Life. 3rd Edition.
McGraw-Hill Companies, Inc
(4) Retrieved 7 July 2010 from http://www.diethealthclub.com/articles/24/diet-and-
wellness/the-truth-about-aspartame.html