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THE PHILIPPINE WOMEN’S UNIVERSITY

Biochemistry Preparation and Performance Rating
Laboratory Experiment/s A. Before the Activity
(5 pts) Attendance ____
(10 pts) Lab. Gown/Apron ____
(5 pts) Lab Manual ____
B. During the Activity
(10 pts) Organization/Orderliness____
(10 pts) Cooperation/Behavior ____
(10 pts) Knowledge (Quiz) ____
(50pts) Total  _______

Written Report
A. Group
(30 pts) Data/Results ____
B. Individual
(5 pts) Readability ____
(10 pts) Answer to question/s ____
(5 pts) Neatness ____
(50 pts) Total 
Experiment No. ___ Overall All Total  ______

Buffer Solution and pH

Water is very important to living organism. It makes about 70% of the human body and
other animals. It serves as the medium in which biochemical reactions take place. All biological
processes are affected in marked degrees by the acidity or alkalinity of the medium in which
they occur. Hydrogen ion concentration may affect the conformation or structure of substances
and the concentration of molecular and charged species in solution. It is therefore necessary to
regulate the hydrogen ion concentration.

The body is equipped which efficient control processes which prevent the variation in
hydrogen ion concentration upon the introduction of acidic or basic substances during the
metabolic processes.

A buffer solution is one that resists change in pH when a small amount of acid or base in
added. A buffer solution contains a weak acid and its conjugate base or a weak base and its
salt.

The pH of buffer solution can be calculated with the sue of Henderson – Hasselbalch
equation, also known as the buffer equation.

There are two factors that determine the effectiveness or capacity of a buffer solution.
The first is the molar concentration of the buffer components. The buffer capacity is directly
proportional to the concentration of the buffer components. The second factor is the ratio of the
conjugate base to the concentration of basic and acidic components.

Objectives:

At the end of the activity, the students should be able to:

1. Determine the pH of given sample using pH paper and pH meter.

2. Apply the Henderson-Hasselbalch equation in preparing buffer solutions.

Materials and Reagents:

 Graduated cylinder  0.20 M HAc

 2 of 10 mL pipette  0.20 M NaAc
 Aspirator  0.10 M NaAc
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 5 of 250 mL beaker  0.10 M HAc
 pH meter  0.01 M NaOH
 Spatula  0.01 M HCl
 Stirring rod
 10 test tubes
 Test tube rack
 Watch glass
 2 of 1mL pipette

Procedure:

A. Determination of pH
1. Prepare the following
a. .fresh milk
b. Freshly voided urine
c. Pineapple juice
d. Softdrink (colorless)
e. Vinegar
f. Toothpaste
g. Water
2. Determine the approximate pH of the different sample using the pH paper by
following these steps.
a. Dip a piece of pH paper in the sample for about 10 sec. Remove the
pH paper and place it in a watch glass.
b. Match the color produced in the pH paper with the color chard to
determine the pH of the sample.
c. Record the results in your report sheet.
3. Determine the pH of the sample using the pH meter by following the steps.
a. Dip the electrodes of the pH meter into standard and buffer solution (pH
= 4 and pH = 7.0)
b. Press the pH scale and check if the instrument gives the correct reading
of the pH of standard buffer solution.
c. Remove the buffer solution and rinse the electrode with distilled water.
Wipe the electrodes with tissue paper.
d. Dip the electrodes into the first sample and get the pH reading on the
instrument and record it in the report sheet.
e. Turn the instrument off and remove the electrode from the test solution.
f. Rinse the electrodes with distilled water and wipe it dry using a tissue
paper.
g. Repeat the test with the remaining samples.
Note: Keep the electrodes immersed in distilled water when not in use. Be
extra careful in using the electrode because it’s fragile

B. Preparation of Buffer Solution

Prepare 50 mL of 0.1 M acetate buffer, pH = 4.5 using 0.20 M stock solution of
acetic acid and sodium acetate.

This buffer solution is prepared by mixing 16 mL of 0.20 M acetic acid and 9.0
mL of 0.20 M sodium acetate. Dilute with 25mL pf distilled water. Set this solution
aside for Part C

Note: The volume of the acid and the salt is calculated using the Henderson-
Hasselbalch equation.

C. .Buffering Capacity
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1. Prepare the following samples:
a. Distilled water (freshly boiled and cooled)
b. 0.10 M NaAc
c. 0.10 M HAc
d. 0.10 M acetate buffer, pH = 4.5 (prepared in part B)
Pipette 20.0 mL of sample a, b, c, and d into separate beakers. Determine
initial pH or actual pH. Add 0.1 mL of 0.01 M NaOH to each sample and
determine the pH again (final pH). Record the initial and the final pH in your
report sheet.
2. Determine the Buffer Capacity of Acetate Buffer
Pipette 20.0 mL of the prepared acetate buffer in a beaker. Record its initial
pH as determined in part C #2. Add 2.5 mL of 0.1 N HCl. Mix wee and determine
their final pH. Calculate the buffer capacity using the following equation.

No. of equivalent of acid or base added

Buffer Capacity =
▲pH x volume of the buffer in liter

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Data Sheet

Set- Up

Data and Results:

A. Determination of pH
Samples pH using pH meter pH using pH meter
1. Fresh milk
2. Urine
3. Pineapple juice
4. softdrink
5. Vinegar
6. Toothpaste
7. Distilled water

B. Buffering Capacity
1. Buffer Capacity of sample a, b, c, and d
Solution Actual pH pH with NaOH ▲ in pH
a. distilled water
b. 0.10 M NaAc
c. 0.10 M HAc
d. acetate buffer

2. Buffer Capacity of Acetate Buffer

Solution Actual pH pH with HCl ▲ in pH Buffer
Capacity
Acetate
Buffer

Questions:

1. Why should distilled water be boiled and cooled before determining its pH?

2. What are the factors that affect buffer capacity?

3. At what pH does a buffer solution exhibit maximum buffer capacity?

Conclusion: