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Experiment 1: Preparation of Buffers

I. Water
Water is the medium in which life on earth is based. In our cells, about 70% is water.
Thus, water is the medium in which life on earth is based. Biochemical reactions occur in
water. Thus, biological activity is affected by the acidity or alkalinity of the medium in
which they occur.
However, water itself can function as an acid or a base (amphoteric). It can accept or
donate a hydrogen ion (H+) during chemical reactions (amphiprotic). Thus, the H+
concentration may affect the conformation or structure of substances. Remember that
the conformation of biomolecules affects their activity. Correlate this information with
what you learned in the lecture.
II. Importance of Buffers in Biological Systems
Biological systems have evolved such that cells are able to regulate their pH levels to
maintain long term biological activity. These efficient control processes prevent drastic
changes in H+ concentration even after metabolic processes that introduce acidic or
basic substances are effectively neutralized. Thus, mechanisms to regulate the pH of the
fluids and tissues of living organisms within the limits consistent with life and normal
function are of primary importance.
The mechanisms involved include (but not necessarily limited to)
i. the buffer systems in the body;
ii. the action of the kidneys that excrete acids and bases in the urine; and
iii. the respiratory mechanism in which H+ ion concentration in the body fluids is
regulated by the rate of CO2 elimination in the lungs.
III. Applications of Buffers
They are widely employed in the laboratory to control the pH of culture media for
microorganisms and tissues. Buffers are also used for many chemical reactions requiring
controlled pH including those catalyzed by enzymes.
The use of buffers is an important part of many industrial processes like fermentation,
electroplating, manufacture of leather, photographic materials, and dyes. They are also
extensively used in analytical chemistry and especially in the calibration of pH meters.
IV. The Nature of Buffers
Buffered solutions, or simply buffers, are solutions whose pH do not change drastically
when small amounts of strong acid (or strong base) are added. A buffer solution is one
that resists drastic changes in pH when small amounts of acid or base are added. Recall
the common-ion effect in equilibrium reactions. When strong acid is added to the
solution, the conjugate base in the buffered solution reacts with it. When a strong base
is added, then the acid component of the buffer reacts. The net effect is that the [H +]
concentration does not change much.
V. Composition of Buffers
Commonly met buffer solutions are those containing weak acids and their salts. An
example of this buffer solution is an aqueous solution of HOAc in the presence of
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NaOAc. A combination of a primary and a secondary salt capable of furnishing ions that
can act as weak acid and base can act as a buffer solution. An example is NaH2PO4 -
Na2HPO4 buffer. Other solutions which can show buffer action are those containing a
weak base plus one of its salts, an example of which is an aqueous solution of NH3 and
NH4Cl, solutions of ampholytes like amino acids and proteins, and solutions of strong
acids and bases.
Buffer solutions made up of weak bases and their salts are not commonly employed
because of the volatility and instability of the bases and the dependence of their pH on
their p K w which is markedly affected by temperature changes.
It is also important to ensure that the chosen buffer components do not interfere with
the main reactions one wishes to study.
VI. The Henderson-Hasselbalch Equation
The pH of the buffer solution made up of a weak acid and its salt can be calculated with
the use of the Henderson-Hasselbalch equation also known as the buffer equation:
[salt ]
pH= p K a + log
[acid ]
This equation states that the pH of a solution containing a weak acid and its salt is
determined by the value of the p K aof the acid and by the ratio of the salt/acid
concentrations in the solutions.
VII. Factors to Consider in Buffer Selection
The buffer components should be chosen such that they do not interfere with the
biochemical reactions one wishes to study. Some enzymes are sensitive to the
counterions of the acids or bases used. This may be a deciding factor on whether the
Na+ or K+ salt would be used, for example. If the reaction will be studied
spectrophotometrically, the potential for interference should also be considered.
The addition of neutral salts to buffers changes the pH of the solution by altering the
ionic strength. Thus, the ionic strength of a buffer solution may be raised to a certain
level by the addition of 0.02 M NaCl or KCl. Changes in the ionic strength () and hence
in the pH of a buffer solution may be also brought about by dilution.
The addition of water in moderate amounts does not alter the ratio of the buffer
constituents upon which the pH of the solution depends. If the dilution is too great,
however, it will alter the activity of the buffer constituents especially that of anions
involved to such degree as to alter their effective ratio. Another reason is that dilution
decreases the reserve acidity and alkalinity of the buffer. Also, water itself can act as a
weak acid or base.
Temperature also affects pH of buffers. While the pH of acetate buffers increases with
increase in temperature, that of boric acid-sodium borate decreases. Here, we also
observe the effect of temperature on the pH of most basic buffers. The pH of these
buffers changes more markedly with temperature change than those of acidic buffers
owing to p K a , which appears in the buffer equation. Both K w and K a change
significantly with temperature change.
VIII. Examples of Buffers in Biological Systems
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Buffer systems of the body are made up of weak acids and their salts as well as proteins.
Below are some samples of human body fluids and their buffer systems:

*Hb = hemoglobin
IX. Preparation of Buffers
1. Choose the weak acid whose p K a is closest to the desired pH. This insures maximum
buffer capacity.
2. Also consider the compatibility of the weak acid with cellular systems.
3. Volatile components are to be avoided.
4. Consider the individual concentration of the buffer salt and acid needed to obtain a
suitable buffer capacity. Concentration ranges from 0.05 M to 0.50 M. Buffer capacity
ranges from 0.01 to 0.10. Use as high a concentration as is compatible with other
features of the system. Too high concentration of salt frequently inhibits activity of
enzymes and other physiological systems. The solubility of the buffer component may
also limit the concentration, which can be employed.
5. From the Henderson-Hasselbalch equation calculate the ratio of salt and weak acid
required to obtain the desired pH. This buffer equation is satisfactory within a pH range
of 4 to 10.
6. Mix the calculated amount of acid and salt solutions.
7. Check the pH using pH meter and, if necessary, adjust with a small amount stock salt or
acid solution if the pH obtained is lower or higher than the desired.
X. The p K a of some commonly used buffer acids:

XI. Sample Problem: Prepare 200.0 ml of a 0.10 M buffer at pH 4.50 from 0.20 M stock solutions
of the acid and its salt. What volumes of the stock acid and the stock salt solutions should
you choose?
1. If we limit our choices to the acids and their salt solutions to the ones listed above, then
the appropriate acid is HOAc ( p K a = 4.74 which is closest to the desired pH of 4.50) and
the appropriate base is NaOAc.
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2. Compute for the volumes of the acid and the salt using the Henderson-Hasselbalch
equation
[salt ]
pH= p K a + log
[acid ]

Something to think about: Consider the situation wherein the target concentration of the
buffer is less than the concentration of the available stock acid and base solutions. What
extra calculations do you need to do? What extra procedural steps do you need to
perform?
XII. Experimental Concerns
Since we are in the distance learning situation right now, no extra procedures shall be
provided. No new laboratory skills are being introduced in this experiment. It is assumed
that you learned proper solution preparation skills and pH measurement skills from your
analytical chemistry laboratory classes.
XIII. Assignment
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Using the Henderson-Hasselbalch equation, calculate the volumes of the acids, bases,
and water for dilution in the preparation of the following buffered solutions. Assume
that 0.20 M stock solutions of HOAc, H3PO4, and citric acid and their conjugate bases are
all available. Do not forget SigFig rules in your calculations

Target Target Target Identity Volume Identity Volume Volume

pH concentration volume of acid of acid of base of base of
of buffer (M) (ml) (ml) (ml) water
needed
for
dilution
5.00 0.200 100.0
5.00 0.100 100.0
5.00 0.010 100.0
7.21 0.200 100.0
7.21 0.100 100.0
7.21 0.010 100.0
8.21 0.200 100.0
8.21 0.100 100.0
8.21 0.010 100.0

XIV. Questions
1. In the laboratory, it will be expected that the actual pH of the prepared solutions would
not equal the target pH using the solutions as provided to the students. Cite as many
factors as you can that would contribute to such situation. Explain how each of the
errors you cited would contribute to the error. Exclude ‘human error’ from your list.
2. List specific ‘human errors’ that could contribute to the failure in achieving the desired
pH for the target buffered solutions. Justify whether you think the ‘human error’ is
acceptable or not.