PHYSICAL BIOCHEMISTRY

BUFFER

A buffer is a special solution that stops massive changes in pH levels. Every buffer that is made
has a certain buffer capacity, and buffer range. The buffer capacity is the amount of acid or base
that can be added before the pH begins to change significantly. It can be also defined as the
quantity of strong acid or base that must be added to change the pH of one liter of solution by
one pH unit. The buffer range is the pH range where a buffer effectively neutralizes added acids
and bases, while maintaining a relatively constant pH.

A solution whose pH is not altered to any great extent by the addition of small quantities of
either an acid or base is called buffer solution.

Buffer is also defined as the solution of reserve acidity or alkalinity which resists change of pH
upon the addition of a small amount of acid or alkali.

Many chemical reactions are carried out at a constant pH. In nature, there are many systems that
use buffering for pH regulation. For example, the bicarbonate buffering system is used to
regulate the pH of blood, and bicarbonate also acts as a buffer in the ocean.

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.

COMPOSITION OF BUFFER

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)

MECHANISM

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 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, 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.

SELECTING PROPER COMPONENTS FOR DESIRED pH

Buffers function best when the pKa of the conjugate weak acid used is close to the desired
working range of the buffer. This turns out to be the case when the concentrations of the
conjugate acid and conjugate base are approximately equal (within about a factor of 10). For
example, we know the Ka for hydroflouric acid is 6.6 x 10-4 so its pKa= -log(6.6 x 10-4) = 3.18.
So, a hydrofluoric acid buffer would work best in a buffer range of around pH = 3.18.

For the weak base ammonia (NH3), the value of Kb is 1.8x10-5, implying that the Ka for the
dissociation of its conjugate acid, NH4+, is Kw/Kb=10-14/1.8x10-5 = 5.6x10-10. Thus, the pKa for
NH4+ = 9.25, so buffers using NH4+/NH3 will work best around a pH of 9.25. (It's always the
pKa of the conjugate acid that determines the approximate pH for a buffer system, though this is
dependent on the pKb of the conjugate base, obviously.)

When the desired pH of a buffer solution is near the pKa of the conjugate acid being used (i.e.,
when the amounts of conjugate acid and conjugate base in solution are within about a factor of
10 of each other), the Henderson-Hasselbalch equation can be applied as a simple approximation
of the solution pH, as we will see in the next section.

CHARACTERISTICS OF BUFFER SOLUTION

(i) It has a definite pH.

(ii) Its pH does not change on standing for long periods of time.

(iii) Its pH does not change on dilution.

(iv) Its pH is slightly changed by the addition of small quantity of an acid or base.
Types of buffer solutions

(a) Acidic Buffer:

It is formed by the mixture of weak acid and its salt with a strong base.

Examples: (i) CH3COOH + CH3COONa, (ii) HCN + NaCN, (iii) Boric acid + Borax etc.

(b) Basic Buffer:

It is formed by the mixture of a weak base and its salt with strong acid.

Examples: (i) NH4OH + NH4Cl, (ii) NH4OH + NH4NO3, (iii) Glycine + Glycine hydrochloride

(c) Simple Buffer:

It is formed by a mixture of acid salt and normal salt of a polybasic acid,

example Na2HPO4 + Na3PO4

Or a salt of weak acid and a weak base. Example: CH3COONH4

Buffer Actions

(a) Acidic Buffer:

It is the mixture of CH3COOH and CH3COONa in aqueous solution.

CH3COOH ⇋ CH3COO– + H+ (incomplete dissociation)

CH3COONa → CH3COO– + Na+ (complete dissociation)

H2O ⇋ H+ + OH– (incomplete dissociation)

Action of acid: when a drop of stong acid (HCl) is added in the above buffer solution H+ ions
combine with CH3COO- ions to form feebly ionised CH3COOH. Whose ionisation is further
suppressed due to common ion effect. So pH of the solution unaltered.
Action of base: when a drop of strong base (NaOH) is added to the above buffer solution it react
with free acid to form undissociated water molecules. So pH of the solution unaltered.

CH3COOH + OH– ⇋ CH3COO– + H2O

(b) Basic Buffer:

It is the mixture of NH4OH and NH4Cl in aqueous solution.

NH4OH ⇋ NH4+ + OH– (incomplete dissociation)

NH4Cl → NH4+ + Cl– (complete dissociation)

H2O ⇋ H+ + OH– (incomplete dissociation)

Action of acid: when a drop of HCl is added, the added H+ ions combine with NH4OH to form
undissociated water molecules. So the pH of buffer is unaffected.

NH4OH + OH– ⇋ NH4+ + H2O

Action of base: when a drop of NaOH is added, the added OH – ions combine with NH4+ ions to
form feebly ionised NH4OH. It is further suppressed due to common ion effect. So the pH of
buffer is unaffected.

Hendersion’s Equation (pH of buffer)

(a) Acidic Buffer:

It is a mixture of CH3COOH and CH3COONa

CH3COOH ⇋ CH3COO– + H+

CH3COONa → CH3COO– + Na+

By the law of chemical equilibrium, Ka = {[CH3COO–] [H+]} / [CH3COOH]

∴ [H+] = {Ka [CH3COOH]} / [CH3COO–]

Taking negative log both sides, we obtain that

– log[H+] = – log Ka – log {[CH3COOH]/[CH3COO–]}

pH = pKa + log {[CH3COO–]/[CH3COOH]}

pH = pKa + log {[salt] / [acid]}

This equation is known as Hendersion’s Equation

Where, Ka = dissociation constant

[CH3COO–] = initial concentration of salt

[CH3COOH] = initial concentration of acid

(b) Basic Buffer:

It is a mixture of NH4OH and NH4Cl

NH4OH ⇋ NH4+ + OH–

NH4Cl → NH4+ + Cl–

By the law of chemical equilibrium, Kb = {[NH4+] [OH–]} / [NH4OH]

∴ [OH–] = {Kb [NH4OH]} / [NH4+]

Taking negative log both sides, we obtain that

– log [OH–] = – log Kb – log {[NH4OH] / [NH4+]}

pOH = pKb + log { [NH4+] / [NH4OH]}

pOH = pKb + log {[salt] / [base]}

This equation is known as Hendersion’s Equation

Where, Kb = dissociation constant

[NH4+] = initial concentration of salt

[NH4OH] = initial concentration of base

pH + pOH = 14

BUFFER CAPACITY

Buffer capacity is defined as the number of moles of acid or base added in one litre of solution as
to change the pH by unity.

Buffer capacity (Φ) = No. of moles of acid or base added to 1 litre solution/change in pH

Φ = ∂b /∂(pH)

Where ∂b – No. of moles of acid or base added to 1 litre

∂(pH) – change in pH

APPLICATIONS OF BUFFER IN CHEMISTRY

(i) Buffers are used in industrial processes such as manufacture of paper, dyes, inks, paints,
drugs, etc.

(ii) Buffers are also employed in agriculture, dairy products and preservation of various types of
foods and fruits.

(iii) It is used to determine the pH with the help of indicators.

(iv) Blood is the natural buffer, it maintenance of pH is essential to sustain life because enzyme
catalysis is pH sensitive process. The normal pH of blood plasma is 7.4.

(v) For the removal of phosphate ion in the qualitative inorganic analysis after the second group
using CH3COOH + CH3COONa buffer.

APPLICATION OF BUFFER IN BIOCHEMISTRY

BUFFERS IN THE HUMAN BODY

Blood contains large amounts of carbonic acid, a weak acid, and bicarbonate, a base. Together
they help maintain the bloods pH at 7.4. If blood pH falls below 6.8 or rises above 7.8, one can
become sick or die. The bicarbonate neutralizes excess acids in the blood while the carbonic acid
neutralizes excess bases.

Another example is when we consume antacids or milk of magnesia. After eating a meal with
rich foods such as sea food, the stomach has to produce gastric acid to digest the food. Some of
the acid can splash up the lower end of the esophagus causing a burning sensation. To relieve
this burning, one would take an antacid, which when dissolved the bases buffer the excess acid
by binding to them.

Blood

Human blood contains a buffer of carbonic acid (H2CO3H2CO3) and bicarbonate anion
(HCO−3HCO3−) in order to maintain blood pH between 7.35 and 7.45, as a value higher than
7.8 or lower than 6.8 can lead to death. In this buffer, hydronium and bicarbonate anion are in
equilibrium with carbonic acid. Furthermore, the carbonic acid in the first equilibrium can
decompose into CO2CO2 gas and water, resulting in a second equilibrium system between
carbonic acid and water. Because O2O2 is an important component of the blood buffer, its
regulation in the body, as well as that of O2O2, is extremely important. The effect of this can be
important when the human body is subjected to strenuous conditions.

In the body, there exists another equilibrium between hydronium and oxygen which involves the
binding ability of hemoglobin. An increase in hydronium causes this equilibrium to shift towards
the oxygen side, thus releasing oxygen from hemoglobin molecules into the surrounding
tissues/cells. This system continues during exercise, providing continuous oxygen to working
tissues.

In summation, the blood buffer is:

H3O++HCO−3↽−−⇀H2CO3+H2OH3O++HCO3−↽−−⇀H2CO3+H2O

With the following simultaneous equilibrium:

H2CO3↽−−⇀H2O+CO2H2CO3↽−−⇀H2O+CO2

Buffers are used often in biological research to maintain pH of specific processes. This can be
especially useful when culturing bacteria, as their metabolic waste can affect the pH of their
medium, consequently killing the sample. For example, a buffer of cacodylic acid
(C2H7AsO2C2H7AsO2) and its conjugate base is used to make samples which will undergo
electron microscopy. Another buffer, tricine (C6H13NO5C6H13NO5), is used to buffer
chloroplast reactions.