TITRATION CURVES

OF AMINO ACIDS
FOR THIS EXPERIMENT WE HAVE
USED AMINO ACID - GLYCINE

CREATED BY SANSKAR VIRMANI

SUBJECT – BIOTECHNOLOGY
What are
titration curves?
Titration curves are obtained
when the pH of given volume of a
sample solution varies after
successive addition of acid or
alkali.
The curves are usually plots of pH
against the volume of titrant
added or more correctly against
the number of equivalents added
per mole of the sample.
This curve empirically defines
several characteristics.
How to plot a titration
curve ?
Pipette out 20ml of the amino acid solution into a 100ml beaker.
Standardize the pH meter using the standard buffer solutions.

Determine the pH of the amino acid solution.

Add 0.3ml of 0.1M HCl from the burette and record the pH after each addition.

Continue adding the acid until the pH falls to 1.6

Wash thoroughly the pH electrode in distilled water.

Take 20 ml of amino acid solution in another beaker and check its pH.

Now titrate the amino acid solution by adding 0.3ml of 0.1M NaOH until the pH

reaches 12.5.
Plot the titration curve using the values recorded and find the pKa values.
Because pKa is a log10 scale, a 1.0 unit change in pH on either side of the
pKa will be associated with a tenfold change in the ratio of the associated
and dissociated species, and a 2.0 unit change in pH on either side of the
pK  will be associated with a 100-fold change in the ratio.
Titration curves of different amino
acids!
 TYPICAL APPARATUS FOR AMINO ACID
TITRATION!
(1) A variety of apparatus you might come across, particularly the pipette (3)
for measuring accurately volumes of solutions to be analyzed by titration with
a standard solution in a burette (4) with the chemicals mixing in a conical flask
used for titrating amino acids.
 HOW DO AMINO ACIDS
GET THEIR CHARGES

 A zwitterion is a molecule with functional groups, of which at least one has a

positive and one has a negative electrical charge. The net charge of the entire
molecule is zero.
Amino acids are the best-known examples of zwitterions. They contain an amine
group (basic) and a carboxylic group (acidic). The -NH2 group is the stronger base,
and so it picks up H+ from the -COOH group to leave a zwitterion.
Chirality and Optical Rotation

General structure for the α-amino acids. Stereoisomers are shown in

their L and D forms. Note the position of the α-carbon.
Because carbon can form four valence bonds, the α-carbon of an amino acid can be
viewed as being at the center with its four substituents located at the corners of a
tetrahedron. When a carbon atom has four different substituents, two distinct spatial
arrangements are possible. Fisher projections are used to depict
the L and D isomers.
In a Fisher projection, bonds pointing horizontally are viewed as coming out of the
plane on which they are depicted, whereas those pointing vertically go below the
plane. A zwitterion is also depicted, wherein the arrows designate the potential
balance and interaction between the positive (+) charge of the amino group and the
negative (−) charge of the carboxylate group.
 These ionizations follow
the Henderson-
Hasselbalch equation
 

 When the concentration of the unprotonated form equals that of the

unprotonated form, the ratio of their concentrations equals 1, and log
1=0. Hence, can be defined as the pH at which the concentrations of the
protonated and unprotonated forms of a particular ionizable species are
equal. The pKa also equals the pH at which the ionizable group is at its
best buffering capacity; that is the pH at which the solution resists
changes in pH most effectively.
 Precautions
 Always clamp the electrode in order
to avoid the bumping of stir bar with
it during stirring.
 Make sure that the electrode is
properly immersed in the beaker
containing amino acid solution,
otherwise the pH reading will be
erratic.
Results of the experiment !
20 ml of 0.1M Glycine is pipetted out for titrations with NaOH and HCl separately.
Initial pH of 0.1M Glycine = 6.22

Volume of 0.1M Volume of 0.1M

HCl added NaOH added
pH pH
[ml] [ml]

0.3 3.76 0.3 8.05

0.6 3.47 0.6 8.21
0.9 3.09 0.9 8.42
1.2 2.96 1.2 8.45
22.2 1.63 3.9 9.22
22.5 1.62 10.2 10.22
22.8 1.61 18.0 11.22
23.1 1.60 22.2 12.22
Titration Curve Plotted for Volume Of
HCl
Titration Curve Plotted for Volume Of
NaOH