Applied Science Department

Applied Biology Section

TITRATION CURVE OF AMINO

ACIDS
Safety Precautions

• Always wear a laboratory coat made of protective material in

the laboratory while carrying out an experiment.

• Do not start the work until a brief introduction is given by your

lecturer.

• Students need to handle different types of scientific

equipment’s; therefore, a careful handling of them is necessary.

• Operate the instruments only under strict supervision.

• In case of any electrical hazard please inform the lab instructor

and lecturer.
Objectives

• To understand the acid base behavior of an amino acid.

• To determine the titration curve for an amino acid.
• To use this curve to estimate the pKa values of the ionizable
groups of the amino acid.
Introduction

• Amino acids in solution are found to exist as zwitter ions at

neutral pH and are amphoteric molecules that can be titrated
with both acid and alkali.

• Titration curves are obtained when the pH of given volume of

a sample solution varies after successive addition of acid or
alkali.
Relationship between pH and Pi

• When the pH is less than Pi the amino acids will have a net
positive charge (H+ is added).

• When pH is greater than Pi, the amino acid has a net negative
charge (H+ is removed).

• The PKa values of the ionizable groups determine the Pi of a

protein or amino acid. In this experiment we are finding out
the titration curve of the amino acid Glycine
Materials required
Equipment/ Chemical/Glassware Concentration/Quantity

Hydrochloric acid 0.1 M

Sodium Hydroxide 0.1 M

pH meter

Glycine 0.1 M

Burettes 2

Stirrer

Standard Buffer pH=4, pH=7, pH =10

Method_ titration with acid

1. Pipette out 20ml of the amino acid glycine solution into a

100ml beaker.

2. Standardize the pH meter using the standard buffer

solutions.

3. Determine the pH of the amino acid solution.

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

5. Continue adding the acid until the pH falls to 1.6

Method_ titration with alkali

1. Wash thoroughly the pH electrode in distilled water.

2. Take 20 ml of amino acid solution in another beaker and

check its pH.

3. Now titrate the amino acid solution by adding 0.3ml of 0.1M

NaOH until the pH reaches 12.5.

4. Plot the titration curve using the values recorded and find
the pKa values.
Graph

• Plot a graph by taking OH- equivalents on X axis and PH on the

Y axis.

• There will be two points on the graph where the buffering

capacity will be the maximum.

• These points are considered as

• pka-1(COOH group)
• and pKa-2 (NH2)

• Then the Pi of the amino acid will be =

• PKa 1(COOH) + pka2 ( NH2) /2