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