UST- FMS Department of Biochemistry, Molecular Biology and Nutrition

Academic Year 2021-2022

EXPERIMENT NO. 2

TITRATION OF AMINO ACIDS

All amino acids are amphoteric electrolytes (ampholytes), that is, they contain at least one
acidic (carboxyl) and one basic (amino) group. These ionizable groups act as weak acids or bases,
giving off or taking protons as the pH of the solution changes during titration. Some amino acids also
contain ionizable R groups, e.g., amino, carboxyl, guanidinium, p-hydroxyphenyl, imidazole, etc.
These affect the behavior of the amino acids during titration.

This experiment is a study of the reaction of typical amino acids with hydrogen ions. As an
acid or base is added to the amino acid solution, a change in pH is observed. Soren Peter Sorensen,
a Danish biochemist who established the concept of pH, however observed that the end point of
titrating a solution of an amino acid with standard alkali is not reached without the addition of
neutralized formaldehyde.

It is the purpose of this experiment to determine the acid base behavior of the amino acid
during titration with an alkali and acid. The effect of formaldehyde on the titration curve of the amino
acid will also be determined. The experiment consists of the addition of increments of acid or base to
a given amino acid solution and measuring the pH of the solution after each addition. The titration
curve is then plotted using the pH values on the ordinate and equivalents of HCl and NaOH on the
abscissa.

The experiment is divided into four 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) (3)
titration of the formaldehyde-treated amino acid with NaOH

Materials/Reagents:
0.1 N NaOH Pipettes, beakers, burettes
0.1 N HCl Neutralized formaldehyde
0.1 M glycine solution pH meter
0.1 M lysine solution
0.1 M aspartic acid solution

Procedure:
1. Take two burettes or 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 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 mL at a time and determining the pH after each
addition, until a total of 20 mL is reached. In addition, measure the pH at 5 mL and 15 mL
volumes.

4. Titrate the second solution in the same manner as the 1st (procedure #3), using instead,
0.1N NaOH, until 20 mL is reached and measure the pH at 5 mL & 15 mL volumes.

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 (#3 & #4) but add 5 mL of neutralized formaldehyde into each of the
amino acid solutions of glycine and aspartic acid and 10 mL of formaldehyde into the lysine
solution before determining the pH of the solution. Titrate the solutions, as in procedure (#3 & #4).

7. Using a graphing paper, plot pH vs. the equivalent acid/base with the pH values on the ordinate
and milliequivalents (mEq) of HCl and NaOH on the abscissa. (one amino acid per plot with values
with and without formaldehyde). Solve for the pI and pK values of your amino acid. (0.1N of
standard acid/base: 1.0 ml = 0.1 mEq; hence 10 ml = 1mEq)

8. Record your results as follows:

Page 1 of 5
 UST- FMS Department of Biochemistry, Molecular Biology and Nutrition
Academic Year 2021-2022
EXPERIMENT NO. 2

TITRATION OF AMINO ACIDS

Name: _____________________________________ Facilitator: ___________________

RESULTS FOR NEUTRAL AMINO ACID

Titration with HCl (Acid)

0.1N HCl mEq pH 0.1N HCl mEq pH

(ml) (ml)
w/o HCHO w/ HCHO w/o HCHO w/ HCHO

0 12

2 14

4 15

5 16

6 18

8 20

10

Titration with NaOH (Base)

0.1N mEq pH 0.1N mEq pH

NaOH NaOH
(ml) w/o HCHO w/ HCHO (ml) w/o HCHO w/ HCHO

0 12

2 14

4 15

5 16

6 18

8 20

10

Page 2 of 5
 UST- FMS Department of Biochemistry, Molecular Biology and Nutrition
Academic Year 2021-2022
EXPERIMENT NO. 2

TITRATION OF AMINO ACIDS

Name: _____________________________________ Facilitator: ___________________

RESULTS FOR ACIDIC AMINO ACID

Titration with HCl (Acid)

0.1N HCl mEq pH 0.1N HCl mEq pH

(ml) (ml)
w/o HCHO w/ HCHO w/o HCHO w/ HCHO

0 12

2 14

4 15

5 16

6 18

8 20

10

Titration with NaOH (Base)

0.1N mEq pH 0.1N mEq pH

NaOH NaOH
(ml) w/o HCHO w/ HCHO (ml) w/o HCHO w/ HCHO

0 12

2 14

4 15

5 16

6 18

8 20

10

Page 3 of 5
 UST- FMS Department of Biochemistry, Molecular Biology and Nutrition
Academic Year 2021-2022
EXPERIMENT NO. 2

TITRATION OF AMINO ACIDS

Name: _____________________________________ Facilitator: ___________________

RESULTS FOR BASIC AMINO ACID

Titration with HCl (Acid)

0.1N HCl mEq pH 0.1N HCl mEq pH

(ml) (ml)
w/o HCHO w/ HCHO w/o HCHO w/ HCHO

0 12

2 14

4 15

5 16

6 18

8 20

10

Titration with NaOH (Base)

0.1N mEq pH 0.1N mEq pH

NaOH NaOH
(ml) w/o HCHO w/ HCHO (ml) w/o HCHO w/ HCHO

0 12

2 14

4 15

5 16

6 18

8 20

10

Page 4 of 5
 UST- FMS Department of Biochemistry, Molecular Biology and Nutrition
Academic Year 2021-2022
EXPERIMENT NO. 2

Discussion 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?

2. Compare the values obtained when the amino acid was titrated with HCl and NaOH both in the
absence and presence of formaldehyde. How do you account for these differences among the
amino acids?

3. At which pH will an amino acid exert its maximum buffering capacity? Why? Where in your graph
is the buffering region for your amino acid?

4. From the titration curve of an amino acid, can you determine the nature of its R group, i.e., basic,
acidic or neutral? Give examples of amino acids that behave in the similar manner as glycine,
aspartate and lysine.

5. Using the chemical structure of the amino acids, illustrate the stepwise titration of the 3 amino
acids used in the experiment, starting from the most acidic species to the most basic species.
Indicate the different amino acid species in the titration curve that you have constructed. Label all
parts of your plots.

Page 5 of 5