Buffered Solutions
Objective:
Buffering of weak acid/weak base solutions is very important, especially in biological chemistry. In
this experiment you will demonstrate the buffer effect to yourself, and will investigate a situation in
which a buffered solution may arise.
Introduction:
A buffered solution is one that does not change its pH significantly when a strong acid or base is
added to it. Buffered solutions typically consist of an approximately equimolar mixture of a
conjugate acid-base pair. For example, the following mixtures would be expected to act as
buffered solutions:
0.10 M HC2H3O2 (acid) and 0.10M NaC2H3O2 (conjugate base)
0.25 M NH3 (base) and 0.20 M NH4Cl (conjugate acid)
The two components of the buffered solution do not have to be present in exactly equal amounts,
but there must be comparable amounts of the components for the buffer to have a significant
capacity to resist changes in its pH.
A buffered solution is able to resist changes in its pH when strong acids or strong bases are
added because the components of the buffered solution are able to react chemically with such
added substances. If the added strong acid or strong base is chemically consumed by one of the
components of the buffered solution, then the acid or base will not have an effect on the total
hydrogen ion concentration (pH) of the solution.
For example, for the HC2H3O2/ NaC2H3O2 buffer above, the weak acid portion of the buffer will
react with any added strong base, and the conjugate base portion of the buffer will react with any
added strong acid.
HC2H3O2 + NaOH  NaC2H3O2 + H2O
C2H3O2- + HCl  HC2H3O2 + ClBuffered solutions are vitally important in the physiology of living cells. Many biochemical
reactions are extremely sensitive to pH, and will not take place if the acidity of the physiological
system is outside a very narrow range. For example, if a few drops of acid are added to whole
milk, the milk will almost instantly “curdle” as the protein in the milk precipitates. The change in
the pH of the milk when the acid is added is enough to cause the disruption of the structure of the
milk protein so that it is no longer soluble in water.
In this experiment you will demonstrate the ability of buffered solutions to resist changes in pH,
and will investigate a common situation in which buffered solutions arise.
Safety Precautions:
 Safety eyewear approved by your institution must be worn at all times while you are in
the laboratory, whether or not you are working on an experiment.
 Assume that all the acid and base solutions used in this experiment are corrosive to the
eyes, skin, and clothing. Wash immediately if spilled and inform the instructor. Clean up
all spills on the bench top.
Apparatus/Reagents Required
pH 7 buffer concentrate, universal indicator and color chart, 3 M HCl, 3 M NaOH, and dropper
bottles of 1.0M HCl, 1.0M acetic acid, and 1.0M NaOH, albumin (egg white)
Buffered Solutions Lab
Procedure:
1. The buffer effect
 Place approximately 50mL of distilled water into each of two small beakers.

To one beaker of distilled water, add 1mL of concentrated pH 7.00 standard
reference buffer.

Add 5 drops of universal indicator to each of the beakers. Record the color of the
liquid in each beaker after the indicator is added. Use the color chart provided for
the indicator to record the pH of the water and of the buffered solution.

To each beaker, add 1 drop of 3 M HCl solution and stir to mix. Record the color
of the solution in each beaker. Use the indicator color chart to record the pH of
the two solutions. Which solution underwent the larger change in its pH when the
acid was added?

To each beaker, add 2 drops of 3 M NaOH solution (one drop of the NaOH is to
neutralize the acid which was added previously) and stir to mix. Record the color
of the solution in each beaker. Use the color chart provided for the indicator to
record the pH of each solution. Which solution underwent the larger change in its
pH when the base was added?
2. Buffering During Titrations
 In Part 1 above, you demonstrated the buffer effect on a buffered solution that
was purposely prepared. Buffered solutions may also arise in situ during titrations
of weak acids or bases, which may make the endpoint of the titration less sharp.
If we were to titrate a solution of the strong acid HCl with a sodium hydroxide
solution, the pH would change drastically and suddenly when we had reached
the point where sufficient NaOH had been added to react with the HCl present.
On the other hand, if we were to titrate a solution of the weak acid acetic acid
with sodium hydroxide, the change in pH at the endpoint would be much less
sudden or drastic. As sodium hydroxide is added to acetic acid, the acetic acid is
converted to sodium acetate, and a buffered solution arises.

Set up 2 clean, dry test tubes in a rack. Add 20 drops of 1.0M HCl to one of the
test tubes and 20 drops of 1.0M acetic acid to the other.

Add 1 drop of universal indicator to each of the test tubes and mix. Use the color
chart provided with the indicator to record the pH of each of the solutions before
any change is made to them.

Obtain a dropper bottle of 1.0M NaOH solution, and prepare to titrate each of the
acid samples. Use the indicator color chart to record the pH of the solutions after
each drop of NaOH has been added (give your best estimate of the pH if the
color seems intermediate to two of the colors on the chart).

Titrate the acid samples in parallel so that you can compare them: that is, add a
drop of NaOH to the HCl solution and record the pH, then add a drop of NaOH to
the acetic acid solution and record its pH.

Continue adding NaOH dropwise until the pH has reached a value of 10.

Using Graph paper from the back of this manual construct two graphs (one for
each titration): plot the pH of the solution at each point in the titration versus the
Buffered Solutions Lab
number of drops of NaOH that had been added to reach that point. Draw a
smooth curve through your data points (do not “connect the dots”).

Notice the difference in the shape between the two curves: the strong acid/strong
base titration shows a sharp, sudden increase in pH after approximately 20 drops
of the NaOH has been added. The weak acid/strong base titration shows a much
more gradual change in pH as the NaOH was added, reflecting the fact that the
system was buffering.
3. Proteins as Buffers
 Proteins are constructed of long chains of linked amino acid molecules. An amino
acid molecule contains both a weak acid group (the carboxyl group) and a weak
base group (the amino group). Since amino acids have both acidic and basic
properties, solutions of proteins behave as buffered solutions when strong acids
or strong bases are added.
H

H2N—C—C=O
 
R OH

Place approximately 10 mL of water in a small beaker. Add a tiny amount of egg albumin
approximately the size of the head of a match. Stir the mixture vigorously to dissolve as
much albumin as possible. Decant approximately 5 mL of the albumin solution into a
clean test tube (avoid transferring any undissolved albumin from the beaker to the test
tube.)

Set up the test tube containing the albumin and a second test tube containing an equal
amount of water in a test tube rack. Add 1 drop of universal indicator to each of the test
tubes and mix. Use the color chart provided with the indicator to record the pH or each of
the solutions before any change is made to them.

Add 1 drop of 1M HCl to each test tube. Record the color of the indicator. Then add 2
drops of 1M NaOH to each test tube. Record the color of the indicator. Did the albumin
solution appear to behave as a buffered solution?
Buffered Solutions Lab
Pre-laboratory Questions
1. Use your textbook to write a specific definition for a buffered solution.
2. Give two examples of mixtures that would behave as buffered solutions, and show how (by
writing equations) the components of each of your solutions would consume added strong acid
(HCl) and added strong base (NaOH). Do not use examples discussed in either this lab manual
or in your textbook.
3. Buffered solutions are especially important in biological systems. Why? Give two examples of
buffered solutions in biological systems.
Buffered Solutions Lab
Results/Observations
1. The Buffer Effect
Color of distilled water + indicator ___________________________________________
Color of buffered solution + indicator _________________________________________
pH of distilled water ________________ pH of buffered solution _________________
pH’s after adding HCl: distilled water _______________ buffer __________________
pH’s after adding NaOH: distilled water ______________ buffer _________________
Which solution underwent the larger changes in pH when either HCl or NaOH was added? Why?
2. Buffering During Titrations
Initial pH of HCl solution __________________________________________________
Drops
1
2
3
4
5
6
7
pH
Drops
8
9
10
11
12
13
14
pH
Buffered Solutions Lab
Drops
15
16
17
18
19
20
21
pH
Initial pH of acetic acid solution _____________________________________________
Drops
1
2
3
4
5
6
7
pH
Drops
8
9
10
11
12
13
14
pH
Drops
15
16
17
18
19
20
21
pH
3. Proteins as Buffers
Did the albumin solution appear to exhibit the properties of a buffered solution (compared to
water) when strong acid and strong base were added? Explain your observations.
Question:
Describe in your own words how the shapes of your two “titration curves” differ as determined in
Part 2 of the experiment. Why does one curve show a sudden, dramatic increase in pH, whereas
the other curve shows a much more gradual increase in pH?
Buffered Solutions Lab