CPB 30103 1

BIOCHEMICAL ENGINEERING

Experiment 1
Preparation of Buffer Solution
INTRODUCTION
Maintenance of pH is vital to all cells. Cellular processes such as metabolism are
dependent on the activities of enzymes. In turn, enzyme activity is markedly influenced
by pH. Consequently, changes in pH would be disruptive to metabolism. Hence the pH of
a living system is maintained with buffers. In humans, the pH of blood is maintained
between 7.35 and 7.45. Interestingly, 5 liters of blood contains enough buffering capacity
to absorb 150 ml of 1M acid. The principal buffers in blood are bicarbonate, proteins
(including hemoglobin and oxyhemoglobin) and phosphates.
THEORY
Buffers are solutions that tend to resist changes in their pH as acid or base is added.
Typically, a buffer system is composed of a weak acid and its conjugate base. A solution
of weak acid that has a pH nearly equal to its pK a, by definition, contains an amount of
the conjugate base nearly equivalent to the weak acid. Addition of H+ then has a little
effect because it is absorbed by the following reaction:
H+ + A- HA
Similarly, any increase in [OH-] is offset by the process
OH- + HA A- + H2O
The pH of a solution can be calculated, provided pKa and the concentrations of the weak
acid HA and conjugate base A- are known. The calculation can be done by HendersonHasselbalch Equation. This equation provides a general solution to the quantitative
treatment of acid-base equilibria in biological system.

LABORATORY PROCEDURE
Apparatus
1.
2.
3.
4.

pH meter
Micropipette and tips
Beakers
Measuring cylinder

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BIOCHEMICAL ENGINEERING

5. Spatula
6. Stirring hotplate and stir bars
Procedure
Waste and Spills:
All of the chemicals used in this experiment can be discarded via drain disposal with
running tap water. Wet towels can be used to clean the spill. Rinse your glassware with
distilled water before the experiments starts to avoid chemical contamination.
pH meter:
The pH electrode of the pH meter is very fragile. Always keep the pH electrode moist. It
is recommended to store the electrode in a solution of 4 M KCl. If 4 M KCl is not
available, use a pH 4 or 7 buffer solution. DO NOT store electrode in distilled or
deionized waterthis will cause ions to leach out of the glass bulb and render your
electrode useless. After storage, you may notice white KCl crystals forming outside your
electrode. This will not interfere with measurements. Simply rinse the electrode and blot
dry before use. Electrodes should be rinsed between samples with distilled or deionized
water. Never wipe an electrodewiping can cause erroneous readings due to static
charges. Blot the end of the electrode with lint-free paper to remove excess water.
A. Preparation of the Buffer Solution
a) Prepare 0.1 M of citric acid solution and 0.1 M of sodium citrate solution.
b) Work out the calculation for the preparation of 500 ml 0.1 M buffer with a
pH of 5.0 by using 0.1M citric acid and 0.1 M sodium citrate (pK a of citric
acid = 4.76). Refer to the example below.

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BIOCHEMICAL ENGINEERING

c) Combine the appropriate volumes of two solutions to get the correct ratio
of base/ acetic acid.
d) Place 25 ml of the buffer in a 100 ml beaker. Measure the pH of the
prepared buffer with pH meter.
B. Effectiveness of the Buffer Solution
a) Transfer 50 ml of distilled water to two 100 ml beakers respectively. To
one of the beaker, add 1.0 ml of 1M HCl and add 1.0 ml of 1M NaOH to
the other. Measure the pH.
b) Repeat procedure (a) by replacing distilled water with the prepared buffer
solution.
RESULTS AND DISCUSSION
o Show your calculation on the buffer preparation.
o Report the initial pH of the buffer. Explain if there is any difference with the
expected pH.
o Discuss the error of pH measurements.
o Construct a table and compare the pH changes resulting from the addition of 1 M
HCl and 1 M NaOH, respectively, to pure water and to the buffer solution. Is your
solution resistant to the pH change? Please explain.
QUESTIONS
1. Explain the theory of pH measurement.
2. Define buffer capacity and the factors affecting the buffer capacity.
3. Discuss the importance of pH buffer in industrial system.