Nursing Biochemistry (NurBio) 1

Laboratory Manual

Laboratory Procedure Activity

pH and Buffers 1

INTRODUCTION

pH

The pH of a solution is the common logarithm of the reciprocal of the hydrogen ion
concentration expressed as:
1
pH = log
H+

Pure water is slightly ionized and at 25C contains 0.0000001 or 10-7 moles of H+ per
liter. The pH of water is therefore:

1
pH = log
10-7

pH = log 107

pH = 7

If the hydrogen ion concentration, H+ of a solution is greater than the pure water, the
pH is a smaller number than 7. Such a solution is acidic. Conversely, if the H+ is less
than that in pure water, the solution is basic. The range of pH values falls between 0
and 14.

pH is detected and measured by using a pH meter, but the most common detectors are
the color changes of acid-base indicator. An acid-base indicator is a weak organic acid.
The ionized and unionized forms of the indicator have different colors:

HIn + H2O H+ + In-

Acidic Color Basic Color

When the indicator is placed in acid solution the equilibrium is shifted to the left and HIn
form predominate. In alkaline solution the In- form predominate. The color of
the solution depends upon the ration of HIn to In and is related to pH in the following
equation:

Natural Sciences Department, College of Science and Information Technology

Ateneo de Zamboanga University, Zamboanga City, Philippines
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Laboratory Manual

 In- 
Log10 = pH – pKa
 HIn

The color of the indicator solution is therefore a measure

Buffers

A buffer solution is one that resists a change in pH when a small amount of acid or base
is added. A buffer solution contains a weak Bronsted acid, HA and its conjugate base, A -.
An example is the mixture of acetic acid and sodium acetate.

If a small amount of strong acid is added to the buffer solution, some of the anions from
the buffer salt will combine with the added protons to form more undissociated acid:

H+ (from outside source) + A- (buffer salt) HA

Similarly, if a small amount of base is added to the buffer solution, some of the buffer
acid reacts with the OH- to form more buffer salt:

OH- (from outside source) + HA (buffers acid) H2O + A-

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 concentration of the conjugate base to the concentration of the weak
acid. The most effective buffer is one with equal concentrations of basic and acidic
components. This mixture could furnish equal amounts of basic or acidic components to
react with added acid and alkali respectively.

Buffers are significant in biochemistry. An excellent example is its effect in blood. Blood
contains phosphates, carbonates, and proteins as buffering components, and has a
constant pH between 7.35 and 7.45 even though there are large amounts of acids, and
bases constantly introduced by digestion of foods, absorption, muscular activity and
respiration.

APPARATUS/MATERIALS CHEMICALS/REAGENTS

Borrow: Request:
1 vial pH paper 20 mL 0.1M sodium acetate
1 unit pH meter 20 mL phosphate buffer pH 7
1 Bunsen burner 20 mL albumin
3 mL 0.1N sodium hydroxide
3 mL 0.1N hydrochloric acid
Natural Sciences Department, College of Science and Information Technology
Ateneo de Zamboanga University, Zamboanga City, Philippines
Nursing Biochemistry (NurBio) 3
Laboratory Manual

Bring:
30 mL defribinated blood
30 mL flesh milk
30 mL freshly voided urine
Distilled water

PROCEDURE

A. pH Determination

1. Prepare the following samples:

a. Defribinated blood
b. Fresh milk
c. Freshly voided urine

2. Determine the approximate pH of the samples using the pH paper by following

these steps:
a. Dip a piece of pH paper in the sample for about 10 seconds. Remove the pH
paper and place on a watch glass.
b. Match the color produced in the pH paper with the color chart to determine
the pH of the sample.
c. Record the results.
3. Determine the accurate pH of the samples using pH meter by following these
steps:
(Note: If the portable pH meter is to be used, check whether the batteries are in
place before using it.)

a. Dip the electrode(s) of the pH meter into the standard buffer solution.
b. Turn the knob to position “pH” and check if the pointer gives the correct
reading of the pH of the buffer solution. If the reading is not correct, turn the
standardization knob until the accurate reading is obtained.
c. Remove the buffer solution and rinse the electrode/s with distilled water.
Wipe the electrode/s with tissue paper.
d. Dip the electrode/s into the first solution and turn the knob to position “pH”.
Get the pH reading on the scale of the instrument and record it.
e. Turn back the knob to “off” position and remove the electrode(s) from the
solution.
f. Rinse the electrode(s) with distilled water and wipe it dry.
g. Repeat the test with other samples.

(Note: Keep the electrode/s immersed in distilled water when not in use.)

Natural Sciences Department, College of Science and Information Technology

Ateneo de Zamboanga University, Zamboanga City, Philippines
Nursing Biochemistry (NurBio) 4
Laboratory Manual

B. Buffers

1. Prepare duplicate samples of the following:

a. 10 mL of freshly boiled and cooled distilled water
b. 10 mL 0.1M sodium acetate solution
c. 10 mL phosphate buffer pH 7.0
d. 10 mL albumin solution

2. Determine the original pH of each sample.

3. To the first set add 0.10 mL of 0.1N NaOH to each sample. Determine the pH
using the pH meter. Record your results.

4. To the second set add 0.10 mL of 0.1N HCl to each sample. Determine the pH
using the pH meter. Record your results.

QUESTIONS

1. What is the normal pH of blood? Milk? Urine?

2. What is the biochemical relevance of pH?
3. Calculate the pH of the following solutions?
a. 0.01M HCl
b. 2.2 x 10-3 M NaOH
4. Which sample exhibited buffer action? Explain.
5. What are the buffer systems in whole blood and plasma?

PROPER WASTE DISPOSAL

1. Dispose all used reagents into the sink under running water.
2. Place/pour ALL UNUSED EXCESS CHEMICALS in the containers on the instructor’s
table.

Natural Sciences Department, College of Science and Information Technology

Ateneo de Zamboanga University, Zamboanga City, Philippines
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Laboratory Manual

Name : Subject/Section :
Course/Year: Date Performed :
ID Number:

Data Sheet Activity

pH and 1
Buffers

A. pH Determination

Test Samples pH Paper Reading pH Meter Reading

There is no pH determination due 7.93
1. Defibrinated Blood to the pigment in blood affecting
the result.
5 or 6 6.64-6.67
2. Fresh Milk
Between 6 and 7 6.76
3. Fresh Urine

B. Buffers

Test Solutions Original pH pH with 0.1 mL pH with 0.1 mL

0.1N NaOH 0.1N HCl
7.03 10.02 3.25
1. Distilled Water

7.53 10.18 6.42

2. 0.1M Sodium
Acetate

7.13 7.19 7.04

3. Phosphate Buffer
pH 7.0

7.13 7.94 6.97

4. Albumin Solution

Natural Sciences Department, College of Science and Information Technology

Ateneo de Zamboanga University, Zamboanga City, Philippines