BUFFERS
Rveyda AKN, Gebze Technical University, Turkey
Experiment 1
AIM
Buffer solutions play a large role in biochemical functions. Because buffers resist changes in pH levels,
they are used to regulate biological functions that only occur at certain pH levels. Buffers can also be
used to store compounds that would denature in more acidic or basic solutions.
INTRODUCTION
A buffer is a solution that can resist pH change
upon the addition of an acidic or basic
components. It is able to neutralize small
amounts of added acid or base, thus
maintaining the pH of the solution relatively
stable. This is important for processes and/or
reactions which require specific and stable pH
ranges. Buffer solutions have a working pH
range and capacity which dictate how much
acid/base can be neutralized before pH
changes, and the amount by which it will
change.
To effectively maintain a pH range, a buffer
must consist of a weak conjugate acid-base
pair, meaning either a. a weak acid and its
conjugate base, or b. a weak base and its
conjugate acid. The use of one or the other will
simply depend upon the desired pH when
preparing the buffer. For example, the
following could function as buffers when
together in solution:
Acetic acid (weak organic acid w/ formula
CH3COOH) and a salt containing its conjugate
base, the acetate anion (CH3COO-), such as
sodium acetate (CH3COONa)
Pyridine (weak base w/ formula C5H5N) and a
salt containing its conjugate acid, the
pyridinium cation (C5H5NH+), such as
Pyridinium Chloride.
Ammonia (weak base w/ formula NH3) and a
salt containing its conjugate acid, the
ammonium cation, such as Ammonium
Hydroxide (NH4OH).
A buffer is able to resist pH change because the
two components (conjugate acid and
conjugate base) are both present in
appreciable amounts at equilibrium and are
able to neutralize small amounts of other acids
and bases (in the form of H3O+ and OH-) when
the are added to the solution. To clarify this
effect, we can consider the simple example of
a Hydrofluoric Acid (HF) and Sodium Fluoride
(NaF) buffer. Hydrofluoric acid is a weak acid
due to the strong attraction between the
relatively small F- ion and solvated protons
(H3O+), which does not allow it to dissociate
completely in water. Therefore, if we obtain HF
in an aqueous solution, we establish the
following equilibrium with only slight
dissociation (Ka(HF) = 6.6x10-4, strongly favors
reactants):
HF(aq)+H2O(l)F(aq)+H3O+(aq)
We can then add and dissolve sodium fluoride
into the solution and mix the two until we
reach the desired volume and pH at which we
want to buffer. When Sodium Fluoride
dissolves in water, the reaction goes to
completion, thus we obtain:
NaF(aq)+H2O(l)Na+(aq)+F-(aq)
Since Na+ is the conjugate of a strong base, it
will have no effect on the pH or reactivity of the
buffer. The addition of NaF to the solution will,
however, increase the concentration of F- in
the buffer solution, and, consequently, by Le
Chteliers Principle, lead to slightly less
dissociation of the HF in the previous
equilibrium, as well. The presence of
significant amounts of both the conjugate acid,
HF and the conjugate base, F-, allows the
solution to function as a buffer. This buffering
action can be seen in the titration curve of a
buffer solution.
As we can see, over the working range of the
buffer. pH changes very little with the addition
of acid or base. Once the buffering capacity is
exceeded the rate of pH change quickly jumps.
This occurs because the conjugate acid or base
has been depleted through neutralization. This
principle implies that a larger amount of
conjugate acid or base will have a greater
buffering capacity.
If acid were added:
F(aq)+H3O+(aq)HF(aq)+H2O(l)
In this reaction, the conjugate base, F-, will
neutralize the added acid, H3O+, and this
reaction goes to completion, because the
reaction of F- with H3O+ has an equilibrium
constant much greater than one. (In fact, the
equilibrium constant the reaction as written is
just the inverse of the Ka for HF: 1/Ka(HF) =
1/(6.6x10-4) = 1.5x10+3.) So long as there is
more F- than H3O+, almost all of the H3O+ will
be consumed and the equilibrium will shift to
the right, slightly increasing the concentration
of HF and slightly decreasing the concentration
of F-, but resulting in hardly any change in the
amount of H3O+ present once equilibrium is re-
established.
If base were added:
HF(aq)+OH(aq)F(aq)+H2O(l)
In this reaction, the conjugate acid, HF, will
neutralize added amounts of base, OH-, and the
equilibrium will again shift to the right, slightly
increasing the concentration of F- in the
solution and decreasing the amount of HF
slightly. Again, since most of the OH- is
neutralized, little pH change will occur.These
two reactions can continue to alternate back
and forth with little pH change.
Henderson-Hasselbalch equation:
Classical buffer contains both a weak acid and
its conjugate base. Small amounts of acids or
bases added are absorbed by the buffer and
the pH changes only slightly. In the case of high
or low pH just solutions of strong acids or bases
are used - for example in the case of pH=1 acid
concentration is relatively high (0.1 M) and
small addition of acid or base doesn't change
pH of such solution significantly. How to
calculate the pH of a buffer solution containing
both acid and conjugate base?
or
This is so called Henderson-Hasselbalch
equation (or a buffer equation). It can be used
for pH calculation of a solution containing pair
of acid and conjugate base like HA/A-, HA-/A2-
or B+/BOH. For solutions of a weak bases
sometimes it is more convenient to use
equation in the form.

Both equations are perfectly equivalent and
interchangeable.The Henderson-Hasselbalch
equation is used mostly to calculate pH of
solutions created mixing known amounts of
acids and conjugate bases (or neutralizing part
of acid with a strong base). For example, what
is the pH of a solution prepared mixing
reagents so that it contains 0.1 M of acetic acid
and 0.05 M NaOH? Half of the acid is
neutralized, so concentrations of acid and
conjugate base are identical, thus the quotient
under logarithm is 1, the logarithm is 0 and
pH=pKa. This approach - while perfectly
justifiable in many cases - is dangerous, as it
creates false conviction that the equation can
be used this way always. That's not true. The
Henderson-Hasselbalch equation is valid when
it contains equilibrium concentrations of an
acid and a conjugate base. In the case of
solutions containing not-so-weak acids (or not-
so-weak bases) equilibrium concentrations can
be far from those predicted by the
neutralization stoichiometry.
An example of how to use the Henderson-
Hasselbalch equation to solve for the pH of a
buffer solution is as follows:
What is the pH of a buffer solution consisting
of 0.0350 M NH3 and 0.0500 M NH4+
(Ka for NH4+ is 5.6 x 10-10)? The equation for
the reaction is:
NH+4H++NH3
Assuming that the change in concentrations is
negligible in order for the system to reach
equilibrium, the Henderson-Hasselbalch
equation will be:
pH=pKa+log([NH3]/[NH+4])
pH=9.25+log(0.0350/0.0500)
pH = 9.095
MATERALS AND METHODS
10X Stock Buffer (25ml), Diluted buffer (25
ml), ddH2O (25ml), HCI(1-2), NaOH(13.5), 6
falcon tubes, pH meters, micropipet
1. After measuring the pH of your two buffer
samples, combine them to give
approximately 200 ml of your acetic acid-
sodium acetate buffer.
2. Transfer 20 ml of this combined buffer to a
clean 100 ml volumetric flask and make up
to volume by adding water. Mix well. You
now have two buffer solutions that buffer
solutions that differ five fold in their
concentrations.
3. Place a 25 ml sample of the diluted buffer
in a clean beaker. Measure the pH. Add 1
ml of 0,1 N NaOH. Mix well and measure
the pH. Continue adding the NaOH in 1 ml
increments and measuring the pH until the
pH is approximately 1 pH units above the
pKa for acetic acid(pKa for acetic acid=4,75)
4. Place a 25 ml sample of the diluted buffer
in a clean beaker. Measure the pH. Is the
same as the undiluted buffer? Add 0,5 ml
of 0,1 N NaOH. Mix well and measure the
pH. Continue adding the NaOH in 0,5 ml
increments and measuring the pH until the
pH is approximately 2 pH units above the
pKa for acetic acid.
5. Place a 25 ml sample of the diluted buffer
in a clean beaker. Measure the pH. Add 1
ml of 0,1 N HCI. Mix well and measure the
pH. Contiune adding the HCI in 1 ml
increments and measuring the pH until the
pH is approximately 1 pH unit below the
pKa for acetiic acid. At this point, star
adding the HCI in 0,5 ml increments and
measuring the pH until the pH is
approximately 2 pH units the below the pKa
for acetic acid.
6. Finally, place a sample of the diluted buffer
in a clean beaker. Measure the pH. Add 0,5
ml increments and measuring the pH until
the pH is approximately 2 pH units below
the pKa for acetic acid.
7. For further comparison place 25 ml of
distilled water n a clean beaker. Measure
 the pH. Add 1 ml of 0,1 N NaOH. Mix well
and measure the PH. Compare the change 6

8. in pH associated with addition of 1ml of 0,1 5

N NaOH to an unbuffered system to the 4

HCI (ML)
changes associated with similar additions 3
to your buffered systems. 2
9. Place another 25 ml sample of distilled
1
water in a clean beaker. Measure the ph.
0
Add 11 ml of 0,1 N HCI. Mix well and
0 5 10 15
measure the ph. Again compare the HCI (ML)
change in ph associated with addition of 1 Stock Buffer Diluted Buffer dH2O
ml of 0.1 N HCI to an unbuffered system to
the changes associated with similar
additions to your buffered systems. For NaOH (1000l);
* NaOH should be added until pH 7 is reached.
RESULT Stock Diluted dH2O
For HCI (1000l); Buffer Buffer
0. 5,02 5,08 8,75
Stock Diluted dH2O 1. 5,09 5,57 11,58
Buffer Buffer 2. 5,16 11,09
0. 5,02 5,05 5,37 3. 5,25
1. 4,92 4,69 2,82 4. 5,35
2. 4,84 3,52 5. 5,49
3. 4,78 2,79 6. 5,65
4. 4,7 7. 5,88
5. 4,62 8. 6,36
6. 4,54 9. 11,14
7. 4,46
8. 4,36
The pH vs. NaOH graphic according to above
9. 4,26
datas;
10. 4,16
11. 4,02
14
12. 3,28
12
13. 2,86
10
8
PH

The pH vs. HCI graphic according to above

6
datas;
4
2
0
0 5 10 15
NAOH (ML)
Stock Buffer Diluted Buffer dH2O
DISCUSSION
One of the most used things in experiments is
buffer at the appropriate pH. Therefore we
adjust pH of buffer according to buffer. pH pf
the water (7.5-8.5) closer slightly to base so, pH
of diluted buffer is bigger than pH of stock
buffer at first measurement. On the other hand
stock buffer is pure namely it doesnt have
another materials (example water). However
diluted buffer is not pure so, stock buffer is
more resistant to NaOH and HCI. NaOH is a
strong base and HCI is a strong acid thats way
the pH of the water drops quickly.
In the above experiment, the pH of the water
was very low at the first measurement. (for HCI
1000 l) The cause can be a measurement
error. Another reason for this may be
calibrating the pH meter.
REFERENCES
www.chembuddy.com
chem.libretexts.org
www.chemguide.co.uk
Albert and E.P Serjeant The determination of
Ionization Constants, Chapman and Hall, Ltd. ,
London(1971). This reference gives details of
research methods for determining pKas.
Greenstein, J.P. and M. Winitz, Chemistry of the
Amino Acids , vol I, Wiley and Sons, New York
(1961) pp482-491. The method for determining
pI is explained here.
West, E.S and W.R. Todd. Textbook of
Biochemistry, Macmillan, NY, 1955, p. 311.
Datain the table are from this reference.