Experiment 2

Titration of
Amino Acids

Sundusit Yangvisit
Edwin Hojillla
Jayson Doria
Group 4 Renan Difuntorum
Dandelo De Guzman
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 Sorensen’s Formol Titration

 In view of the presence of the amino group, it is

not possible to titrate and estimate the total
acidity of an amino acid solution, since the H+ ion
formed by the ionisation of –COOH will be taken
up by –NH2 and be present as –NH3+. However,
an amino acid solution could be titrated to the
complete neutralisation point after the addition of
excess of formalin (solution of formaldehyde in
water).
 Formaldehyde converts the basic –NH2 group of
the amino acid forming a neutral dimethylol
derivative thus overcoming the interference by
–NH2 in the titration. After the addition of
formaldehyde, an amino acid behaves as any
ordinary organic acid. Thus, formalin makes
available the entire H+ ions during titration
against alkali.
 Purpose

to determine the acid base behavior of

select amino acids during titration with an
alkali and acid. The effect of formaldehyde
on the titration curve of amino acids will
also be determined.
 The experiment is divided into 4 parts:

(1) titration of the amino acid with HCl,

(2) titration of the amino acid with NaOH,
(3) titration of the formaldehyde-treated
amino acid with HCl and
(4) titration of the formaldehyde-treated
amino acid with NaOH.
 Materials/Reagents

 0.1N NaOH
 0.1N HCl
 0.1M glycine solution
 0.1M lysine solution
 0.1M aspartic acid solution
 Neutralized formaldehyde
 Procedure

1. Take two pipettes and fill the first with 0.1N HCl and the second
with 0.1N NaOH.

2. Into each of the two beakers, introduce 10.0 mL of the amino acid
solution and measure the resulting pH of the solution.

3. Titrate the first solution with 0.1N HCl adding 2.0 mL at a time and
determining the pH after each addition, until a total of 10.0 mL is
reached (for glycine and aspartic acid) or 20.0 mL (for lysine). In
addition, measure the pH at 5 mL and 15 mL volumes.
 Procedure

4. Titrate the second solution in the same manner as the 1st using
instead 0.1N NaOH, until 10.0 mL is reached (for glycine and lysine)
or 20.0 mL is reached (for aspartic acid).

5. Plot the pH (ordinate) vs. the equivalent acid/base (abscissa). One

mL of acid/base=0.1 mEq of acid/base. (Show how this value was
obtained).

6. Repeat the above procedure, but add 5.0 mL of neutralized

formaldehyde solution into each of the amino acid solutions before
determining the pH of the solution. Titrate the solutions.
 Procedure

7. Plot pH vs. the equivalent acid/base on the same graph

as above. Construct your titration curves on graphing
paper. Solve for the pI and pKa values of your amino
acid.

www.themegallery.com
 1 mL of acid/base = 0.1 mEq
of acid/base
mEq = atomic weight (g)
valence x 1000

0.1 N HCl = 0.1 M HCl = (0.1 mol/L)(36.5

g/mol) = 3.65 g/L

0.1 mEq = 0.1 x (36.5 g / (1 x 1000) = 0.1

x 0.0365 g = 0.00365 g
Glycine
Aspartic Acid
Lysine
QUESTIONS
 1. What can account for Sorensen’s
discovery that the endpoint of titration
between an amino acid and a standard
alkali (without formaldehyde) is not
reached?
 ANSWER

The free amino group at the alpha-carbon acts

as a base and interferes with the end point of
the titration using a standard alkali.
Formaldehyde in excess is needed to modify
the basic free amino group to modify it to a
neutral group, a dimethylol derivative, which
allows for the endpoint to be reached.
2. When an amino acid is titrated with an
acid, example HCl, both with and without
formaldehyde. How do you account for
this?
 ANSWER

In the presence of formaldehyde, tritration

curve was slight lower. This is due to the
fact that formaldehyde ties down the
amino groups, making the carboxyl
hydrogen ion more available
3. Draw the titration curve of glycine and
point the areas of maximum buffering
capacity, the point of least buffering
capacity.
ANSWER
Maximum buffering capacity (pink)
when pH = pKa +/- 1. In this
region there is nearly equal
amounts of proton donors and
acceptors, i.e. weak acid/base and
conjugate base/acid.

Least buffering capacity at the pI

because both the amino and
carboxyl group are protonated and
deprotonated, respectively.
Therefore any minute addition of
H+ or OH- will result in a large
change in pH.
 4. From the titration curve of an amino
acid, can you determine the nature of its
R group, explain why?
 ANSWER

Yes,
Yes a reactive side group can be determined from the titration
curve of an amino acid. When the amino acid is titrated and
graphed, three buffering regions will be developed. The extra
buffering region aside from the alpha-amino (pKa = approx. 2)
and alpha-carboxyl (pKa = approx. 9) can be used to determine
the identity of the unknown side chain. If the R group has:
pKa < pH, it is basic
pKa > pH, it is acidic
pKa = pH, neutral because zwitterion forms
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