ANALYTICAL TECHNIQUES
TOPIC : BUFFERS
INTRODUCTION
• A buffer is an aqueous solution consisting of a mixture of a weak
acid and its salt (acidic buffer) or a weak base and its salt (basic
buffer) that resists a change in pH on the addition of either acid or
base.
• Its pH changes very little when a small amount of strong acid or
base is added to it and thus it is used to prevent changes in the pH
of a solution.
• In practice, a buffer solution consists of an aqueous mixture of a
weak acid and its conjugate base.
• The conjugate base component would neutralise any hydrogen
ions generated during an experiment whilst the unionised acid
would neutralise any base generated.
• Types of Buffer Solutions
• Buffers are broadly divided into two types –
1. Acidic buffer solutions
2. Alkaline buffer solutions
Acidic buffer
• these solutions are used to maintain acidic environments.
• Acid buffer has acidic pH and is prepared by mixing a weak
acid and its salt with a strong base.
• pH of these solutions is below seven
• These solutions consist of a weak acid and a salt of a weak
acid.
• An example of an acidic buffer solution is a mixture of
sodium acetate and acetic acid (pH = 4.75).
• CH3COOH(Weak Acid ) +CH3COONa (Salt)
ALKALINE BUFFER
• These buffer solutions are used to maintain basic conditions. Basic buffer has a basic pH and
is prepared by mixing a weak base and its salt with strong acid. The aqueous solution of an
equal concentration of ammonium hydroxide and ammonium chloride has a pH of 9.25.
• The pH of these solutions is above seven
• They contain a weak base and a salt of the weak base.
• An example of an alkaline buffer solution is a mixture of ammonium hydroxide and
ammonium chloride (pH = 9.25).
PHYSIOLOGICAL BUFFER
• Physiological Buffers are chemicals used by the body to
prevent sudden, rapid changes in the pH of a fluid. As
buffers are most able to resist changes in pH when the pH
of the solution is close to the unique pH of the buffer.
Consequently, physiological buffers must be chemicals
whose pH are near the normal blood pH which ranges from
7.37 – 7.42. The primary buffers of the ECF appear to be
inorganic phosphate (pH 6.8) and bicarbonate (pH 6.1).
• The most important examples of physiological buffers
system are as follows:-
i) Bicarbonate buffer
ii) Phosphate buffer
iii) Oxyhaemoglobin buffer
• Buffering capacity:
• The efficiency of a buffer in maintaining a
constant pH on addition of acid or base is
referred to as buffering capacity. It mostly
depends on the concentration of buffering
component.
• The maximum buffering capacity is achieved
by keeping same concentration of acid and its
salt or base and its salt.
• Properties of good buffer:
• The buffer should be non-toxic
• The buffer should be able to penetrate cell
membrane and should not absorbs light at UV
or visible region
• Buffer should have adequate buffering
capacity
• Buffer should not form insoluble complex with
any anions or cations in the reaction.
• How does a buffer work?
• 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)(1)(1)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)(2)(2)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 NaFNaF to the solution will, however, increase the
concentration of F- in the buffer solution, and, consequently, by Le Chatelier’s 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, HFHF, 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.
• 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 NaFNaF to the solution will,
however, increase the concentration of F- in the buffer solution, and,
consequently, by Le Chatelier’s 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, HFHF, 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)(3)(3)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)(4)(4)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.
Handerson-Hasselbalch Equation
• Preparation of Acid Buffer
• Consider an acid buffer solution, containing a weak acid (HA) and its salt (KA) with
a strong base(KOH). Weak acid HA ionizes, and the equilibrium can be written as-
• HA + H2O ⇋ H+ + A−
• Acid dissociation constant = Ka = [H+] [A–]/HA
• Taking, negative log of RHS and LHS
• pH of acid buffer = pKa + ([salt]/[acid])
• The equation is the Henderson-Hasselbalch equation, popularly known as the
Henderson equation.
• Preparation of Base Buffer
• Consider base buffer solution, containing a
weak base (B) and its salt (BA) with strong
acid.
• pOH, can be derived as above,
• pOH of a basic buffer = pKb +
log ([salt]/[acid])
• pH of a basic buffer = pKa – log ([salt]/[acid])
• Significance of Handerson Equation
Role of buffer in vitro
• Acid-base buffer helps in tissue or organ preservation ( eg
phosphate buffer)
• In tissue culture, the optimum pH is maintained by buffers.
For example HEPES buffer is widely used in cell culture
because it is better at maintaining physiological pH despite
change in CO2 concentration in media.
• HEPES also maintains enzyme structure and function at
low temperature
• Buffer are used in fermentation process
• It is also used for setting correct condition for dyes used in
coloring fabrics
• Buffer is used to calibrate pH meter
Role of buffer in vivo
There are three buffer system in human body to maintain acid-balance.
• Bicarbonate buffer
• Phosphate buffer
• Protein buffer
1 .Bicarbonate buffer:
• Sodium bicarbonate and carbonic acid constitute bicarbonate buffer. It is
the most predominant buffer system in plasma.
• It is extracellular buffer
• It helps in maintaining blood pH 7.4
2. Phosphate buffer:
• Sodium dihydrogen phosphate and disodium hydrogen phosphate
constitute phosphate buffer.
• It is intracellular buffer
Protein buffer:
• The plasma protein and haemoglobin together
constitute protein buffer system of blood.
• The buffering capacity of protein buffer depends upon
pKa of amino acids.
• The imidazole group of histidine (pKa= 6.7) is the most
effective contributor of protein buffer.
• Haemoglobin of RBCs is also an important buffer. Hb
binds to H+ ions and helps to transport CO2 as HCo3-
ion with minimum change in pH. In lungs Hb combines
with O2 then H+ ion is released and combine with
HCO3- to form carbonic acid (HCO3-). Later carbonic
acid dissociates to release CO2.