BIOLS 315

Biochemistry Lab. No. 1

Amino Acids Titration

1) Titration of a weak acid with a strong base

Materials

0.1M acetic acid

0.1M KOH
Burette (25ml)
pH meter

Method

1) Standardize the pH meter using standard buffers at pH 4 and 7.

2) Pipette 50 ml of 0.1m acetic acid into a 100ml beaker.
3) Place a clean stirring bar into the beaker and place the beaker on a
magnetic stirrer.
4) Insert the pH meter electrode into the acetic acid containing beaker.
5) Fill the burette with 0.1 M KOH and adjust it over the acetic acid
beaker.
6) Start the titration by adding 1-2 ml KOH increments.
7) Record the pH.
8) Tabulate your data and continue the titration.
9) Stop the titration against KOH until the pH reaches 12.
10) Remove the pH electrode from the solution and wash it thoroughly
with distilled water.

2) Titration of amino acids with a strong base

Materials

0.1M alanine solution (pH ~ 1-1.5)

0.1M Glutamic acid solution (pH ~ 1-1.5)
Unknown amino acids (pH ~ 1-1.5)
0.1M KOH
Burette (25ml)
pH meter

1
Method

1) Standardize the pH meter using standard buffers at pH 4 and 7.

2) Pipette 50 ml of 0.1M amino acid into a 100ml beaker.
3) Place a clean stirring bar into the beaker and place the beaker on a
magnetic stirrer.
4) Insert the pH meter electrode into the acetic acid containing beaker.
5) Fill the burette with 0.1 M KOH and adjust it over the acetic acid
beaker.

6) Start the titration by adding 1-2 ml KOH increments.

7) Record the pH.
8) Tabulate your data and continue the titration.
9) Stop the titration at pH 10-11.
10) Remove the pH electrode from the solution and wash it thoroughly
with distilled water.

Lab Assignment Instructions

1) Calculate the number of moles of acetic acid before the addition of

KOH.

No. of moles = M x V/L = 0.1 x 50 x 10-3 = 5 x 10-3

2) Calculate the number of moles of base added.

No. of moles of base added = M x V/L = 0.1 x 0.5 x 10-3 = 0.05 x 10-3

3) Calculate the number of moles of unreacted –COOH.

No. moles unreacted –COOH (No. of moles of acetic acid before the
addition of KOH) – No. of moles of base added = 5.0 x 10-3 - 0.05x10-3 =
4.95x10-3.

4) Plot the titration curve of acetic acid with KOH. Convert the number of
mls of KOH added to number of moles and plot them on the (X-axis)
against the pH values on the (Y axis).

5) Find out the Inflection point in which the number of moles of base
added = 2.5 x 10-3 and the number of moles of unreacted –COOH = 2.5 x
10-3.

2
6) Find out the first inflection point in which the number of moles of base
added = 2.5 x 10-3 and the number of moles of unreacted –COOH = 2.5 x
10-3. Find out the second inflection point in which the number of moles of
base added = 2.5 x 10-3 and the number of moles of unreacted –NH3+ =
2.5 x 10-3. Find out the third inflection point in which the number of
moles of base added = 2.5 x 10-3 and the number of moles of unreacted –
R group = 2.5 x 10-3.

Example of titration of a weak acid with a strong base

KOH Volume of No. moles No. moles

added pH acid of base unreacted
(ml) (ml) added -COOH
0 1.5 50 0 5.0x10-3
0.5 50.5 0.05x10-3 4.95x10-3
1.5 51.5 0.15x10-3 4.85x10-3
3.5 53.5 0.35x10-3 4.65x10-3

25.0 2.5x10-3 2.5x10-3

~10-12 5.0x10-3 0

3
Example of titration of amino acid (2-3 titrable groups) with a strong
base

No. No. moles No. of

KOH Volume No. moles
moles of unreacted moles of
added pH of acid unreacted
base – R group unreacted
(ml) (ml) -COOH
added (COOH) –NH3+
0 1.5 50 0 5.0x10-3 - -
0.5 50.5 0.05x10-3 4.95x10 -3
- -
1.0 51.5 0.15x10-3 4.85x10 -3
- -
2.0 53.5 0.35x10-3 4.65x10 -3
- -
- -
- -
- -
25.0 2.5x10-3 2.5x10 -3
- -
- -
- -
- -
- -
50.0 100 5.0x10-3 0 - -
50.5 100.5 0.05x10-3 4.95x10 -3
-
51.5 101.5 0.15x10-3 4.85x10 -3
-
53.5 103.5 0.35x10-3 4.65x10 -3
-
-
-
2.5x10-3 2.5x10 -3
-
-
-
5.0x10-3 0 -
- -
- -
- -
- -