Buffer solutions - Brief introduction

Definition “Solutions which resist changes in pH when

small quantities of acid or alkali are added.”

Acidic Buffer (pH < 7) made from a weak acid + its sodium or potassium salt
ethanoic acid sodium ethanoate

Alkaline Buffer (pH > 7) made from a weak base + its chloride
ammonia ammonium chloride

Uses Standardising pH meters

Buffering biological systems (eg in blood)
Maintaining the pH of shampoos
 Buffer solutions - action

It is essential to have a weak acid for an equilibrium to be present so that ions can be
removed and produced. The dissociation is small and there are few ions.

CH3COOH(aq) CH3COO¯(aq) + H+(aq)

relative concs. HIGH LOW LOW

NB A strong acid can’t be used as it is fully dissociated and cannot remove H +(aq)

HCl(aq) ——> Cl¯(aq) + H+(aq)

Adding acid
Any H+ is removed by reacting with CH3COO¯ ions to form CH3COOH via the
equilibrium. Unfortunately, the concentration of CH 3COO¯ is small and only a few H+
can be “mopped up”. A much larger concentration of CH 3COO¯ is required.

To build up the concentration of CH3COO¯ ions, sodium ethanoate is added, which

dissociates completely.

CH3COO¯Na+ (aq) —> CH3COO¯(aq) + Na+(aq)

relative concs. LOW HIGH HIGH
.
 Buffer solutions - action
We have now got an equilibrium mixture which contains a high concentration of both the
undissociated weak acid, CH3COOH(aq), and its conjugate base, CH3COO¯(aq).

CH3COOH(aq) CH3COO¯(aq) + H+(aq)

relative concs. HIGH LOW LOW

CH3COO¯Na+ (aq) —> CH3COO¯(aq) + Na+(aq)

relative concs. LOW HIGH HIGH

Adding alkali
This adds OH¯ ions
These react with the small concentration of H + ions: H+(aq) + OH¯(aq) H2O(l)
Removal of H+ from the weak acid equilibrium means that more CH 3COOH will dissociate
to form ions to replace those being removed.
CH3COOH(aq) CH3COO¯(aq) + H+(aq)
As the added OH¯ ions remove the H+ from the weak acid system, the equilibrium moves
to the right to produce more H+ ions.
(There needs to be a large concentration of undissociated acid molecules to be available)
 Buffer solutions - ideal concentration

The concentration of a buffer solution is important

If the concentration is too low, there won’t be enough CH 3COOH and CH3COO¯
to cope with the ions added.

Summary
For a buffer solution one needs ...

large [CH3COOH(aq)] - for dissociating into H+(aq) when alkali is added

weak acid (equilibrium shifts to the right)

large [CH3COO¯(aq)] - for removing H+(aq) as it is added

conjugate base (equilibrium shifts to the left)

This situation can’t exist if only acid is present; a mixture of the acid and salt is used.

The weak acid provides the equilibrium and the large CH 3COOH(aq) concentration.
The sodium salt provides the large CH3COO¯(aq) concentration.

One uses a WEAK ACID + its SODIUM OR POTASSIUM SALT

 Alkaline buffer solutions - action

Alkaline buffer
Very similar but is based on the equilibrium surrounding a weak base; AMMONIA

NH3(aq) + H2O(l) OH¯(aq) + NH4+(aq)

relative concs. HIGH LOW LOW

but one needs ; a large conc. of OH¯(aq) to react with any H+(aq) added
a large conc of NH4+(aq) to react with any OH¯(aq) added

There is enough NH3 to act as a source of OH¯ but one needs to increase the
concentration of ammonium ions by adding an ammonium salt.

Use AMMONIA (a weak base) + AMMONIUM CHLORIDE (one of its salts)

 Factors Affecting pH of а Buffer
The pH of а buffer solution is determined by two
factors:
• 1. The value of pK: The lower the value of pK, the
lower is the pH of the solution.
• 2. The ratio of salt to acid concentrations: Actual
concentrations of salt and acid in а buffer solution
may be varied widely, with по change in рН, so long
as the ratio of the concentrations remains the same.
 BUFFER EQUATION
(Henderson – Hasselbalch equation)
 For Acid Buffers:
The pH of acid buffer can be calculated from the
dissociation constant, Ka of the weak acid and the
concentrations of the acid and salt used.
• The dissociation expression of the weak acid can be
represented as:
• HA ↔ H+ + A-
• Ka = [H+] [A-] / [HA]
Or
• [H+] = Ka [HA] / [A-] ------------- (1)
• A weak acid is only slightly dissociated, and its
dissociation is further depressed by the addition of
the salt (XA) which provides A- ion (common ion
effect) as a result the equilibrium concentration of
the unionized acid is nearly equal to the initial
concentration of the acid. The equilibrium
concentration of A- is assumed to be equal to the
initial concentration of the salt added since it is
completely dissociated. Therefore, in above
equation (1), we represent concentration of A- by
salt concentration.
 • [H+] = Ka. [Acid] / [Salt] --------- (2)
• Taking log on both sides, we get:
• log[H+] = logKa + log [Acid] / [Salt] 
• multiplying both sides by –ve sign,
• -log[H+] = -logKa - log [Acid] / [Salt]
• As -log[H+] = pH & -logKa = pka
• pH = pka - log[Acid] / [Salt] OR
pH = pka + log[Salt] / [Acid] ---------- (3)

Eq. (3) is called as Henderson – Hasselbalch equation.

It helps in calculating the pH value of buffer solution,
if the concentrations of acid as well as that of the salt are known.
 For Basic Buffers:
Buffer equation for basic buffer can be calculated in
same way as that
for acidic buffers.
Consider a basic buffer composed of a mixture of weak
base (BOH)
and its salt (BA). The dissociation constant for base can
be written as,
BOH ↔ B+ + OH-
Kb = [B+] [OH-] / [BOH]
OR
[OH-] = Kb [BOH] / [B+] ------------- (1)
• A weak base is only slightly dissociated, and its dissociation
is further depressed by the addition of the salt (BA) which
provides B+ ion (common ion effect) as a result the
equilibrium concentration of the unionized base is nearly
equal to the initial concentration of the base. The
equilibrium concentration of B+ is assumed to be equal to
the initial concentration of the salt added since it is
completely dissociated. Therefore, in above equation (1), we
represent concentration of B+ by salt concentration.
 [OH-] = Kb. [Base] / [Salt] --------- (2)
Taking log on both sides, we get:
log[OH-] = logKb + log [Base] / [Salt]
multiplying both sides by –ve sign,
-log[OH-] = -logKb - log [Base] / [Salt]

As -log[OH-] = pOH & -logKb =

pkb
pOH = pkb – log [Base] / [Salt]
Or
pOH = pkb + log[Salt] / [Base] ---------- (3)
 
 Significance of Henderson – Hasselbalch equation:
By this equation, the pH of a buffer solution can be
calculated from the initial concentrations of the weak
acid and the salt provided when ka is given. However, the
Henderson equation for a basic buffer will give pOH, and
so pH can be calculated as;
pkw = pH + pOH

or pH = pkw – pOH
pH = 14 – PoH

Also, the dissociation constant of a weak acid (pka) or a

weak base (pkb) can be calculated by measuring the pH
of a buffer solution containing equimolar concentrations
of the acid (or base) and the salt.
 BUFFER CAPACITY
• The buffer capacity of a buffer solution is “a measure of its magnitude of
its resistance to change in the pH on an addition of an acid or a base.”
• Buffer capacity is also referred as buffer index, buffer value, buffer
efficiency or buffer coefficient.
• The buffer capacity represented by ‘β’ may also be defined as:
• “The ratio of the increment (amount added) of strong acid or base to the
small change in pH (ΔpH) brought about by this addition”.
 
• β = ΔA or ΔB / ΔpH
•  
• Where, ΔA or ΔB represents the small increment (in gram equivalents /
litre of strong acid or base added) to the buffer to bring about a pH
change of ΔpH.
• According to the above equation, a solution has a buffer capacity of 1
when one litre of it requires one gram equivalent of a strong acid or
base to change the pH by one unit. So, smaller the pH change in a
solution upon the addition of an acid or base, greater is the buffer
capacity and vice versa.
Buffer Systems in Body Fluids

Figure 27.7
 ACIDS PRODUCED IN THE BODY
• Carbonic acid (H2CO3):
It is the chief acid produced in the body in the course of oxidation in the cells.
Oxidation of C-compounds resulting in CO2 production.
• Sulphuric acid (H2SO4 ):
Produced during oxidation of S-containing amino acids, e.g. cysteine/cystine and
methionine.
• Phosphoric acid:
Products of metabolism of dietary phosphoproteins, nucleoproteins,
phosphatides and hydrolysis of phosphoesters.
• Organic acids:
Abnormal production and accumulation of certain intermediary organic acids
from oxidation of carbohydrates, fats and proteins, under certain circumstances,
e.g. pyruvic acid, lactic acid, acetoacetic acid, β -OH-butyric acid, etc.
 MECHANISMS OF REGULATION OF pH

The mechanisms of regulation of blood pH involves the following

factors:
(a) “Front-line” defense: They are mainly:
•Buffer systems in the blood: Which restricts pH change in body
fluids.
• Respiratory mechanisms: Regulation of excretion of CO 2 and
hence, regulation of H2CO3 concentration in EC fluid.
(b) “Second-line” defense: This is achieved by kidneys (Renal
mechanisms). Ultimate excretion of excess of acid or base and
thus ultimate regulation of concentration of H+ and HCO –3 ions in
EC fluid.
 Bicarbonate Buffer System
• Contains two ingredients: (1) a weak acid, H2CO3, and (2) a
bicarbonate salt, such as NaHCO3.
• H2CO3 is formed in the body by the reaction of CO2 with H2O
catalyzed by the enzyme carbonic anhydrase is present. This enzyme
is especially abundant in the walls of the lung alveoli, where CO2 is
released; carbonic anhydrase is also present in the epithelial cells of
the renal tubules, where CO2 reacts with H2O to form H2CO3.
• H2CO3 ionizes weakly to form small amounts of H+and HCO3–.
• The second component of the system, bicarbonate salt, occurs
predominantly as sodium bicarbonate (NaHCO3) in the extracellular
fluid. NaHCO3 ionizes almost completely to form HCO3– and Na+
• When a strong acid such as HCl is added to the bicarbonate buffer
solution, the increased H+ released from the acid (HCl --> H + + Cl–) is
buffered by HCO3– to form the very weak acid H2CO3, which in turn
forms CO2 and H2O.The excess CO2 greatly stimulates respiration,
which eliminates the CO2 from the extracellular fluid.
• The opposite reactions take place when a strong base, such as sodium
hydroxide (NaOH), is added to the bicarbonate buffer solution.
NaOH + H2CO3  NaHCO3 + H2O
• In this case, the OH– from the NaOH combines with H2CO3 to form
additional HCO3–.Thus, the weak base NaHCO3 replaces the strong
base NaOH. At the same time, the concentration of H2CO3 decreases
(because it reacts with NaOH), causing more CO2 to combine with H2O
to replace the H2CO3.
• The net result, therefore, is a tendency for the CO2 levels in the blood
to decrease, but the decreased CO2 in the blood inhibits respiration and
decreases the rate of CO2 expiration.The rise in blood HCO3– that
occurs is compensated for by increased renal excretion of HCO3–.
Normal ratio in blood NaHCO3/ H2CO3 = 20/1
Advantages of bicarbonate buffer system:
Bicarbonate buffer system is efficient as compared to other
buffer systems as:
• It is present in very high concentration than other buffer
systems. (26 to 28 millimole per litre)
• Produces H2CO3, which is a weak acid and volatile and CO2
is exhaled out.
• Hence, it is a very good physiological buffer and acts as a
front line defence.
Disadvantage:
• As a chemical buffer, it is rather weak,
• pKa is further away from the physiological pH.
 PHOSPHATE BUFFER SYSTEM
• Plays a major role in buffering renal tubular fluid
and intracellular fluids.
• The main elements of the phosphate buffer system
are H2PO4– and HPO42-.
• When a strong acid such as HCl is added to a
mixture of these two substances, the hydrogen is
accepted by the base HPO42- and converted to
H2PO4–.
HCl + Na2H2PO4 NaH2PO4 + NaCl
• The result of this reaction is that the strong acid,
HCl,is replaced by an additional amount of a weak
acid, NaH2PO4, and the decrease in pH is
minimized.
• When a strong base, such as NaOH, is added to the
buffer system, the OH– is buffered by the H2PO4–
to form additional amounts of HPO42- + H2O.
NaOH + NaH2PO4 Na2HPO4 + H2O
 Protein buffer system
• The plasma proteins and hemoglobin together constitute
theprotein buffer system of the blood.
• The buffering capacity of proteins is dependent on the pK of
ionizable groups of amino acids.
• The imidazole group of histidine (pK = 6.7) is the most
effective contributor of protein buffers.
• The plasma proteins account for about 2% of the total
buffering capacity of the plasma.
• Hemoglobin of RBC is also an important buffer. lt mainly
buffers the fixed acids, besides being involved in the transport
of gases (O2 andCO2).
 Buffering action of proteins

In acidic medium:
• Protein acts as a base, NH2 group takes up H+ ions from the medium
forming NH+3,Proteins become +vely charged.

In alkaline medium:
• Proteins act as an acid. Acidic COOH gr dissociates and gives H+,
forming COO–.
• H+ combines with OH–to produce a molecule ofwater, proteins
become –vely charged.
 Hemoglobin as a Buffering Agent

• The buffering capacity of Hb, as of any protein, depends on the number of

dissociable buffering groups, viz. acidic COOH gr, basic-NH2 gr, Guanidino
group and most important is imidazole group, which varies with the pH of
the medium. With the pH range of 7.0 to 7.8, most of the physiological
buffering action of Hb is due to the “imidazole” group of amino acid
“histidine”. Each molecule of Hb contains 38 mols of Histidine. In α -chain,
histidine at 87 position, and in β-chain, histidine at 92 position is
directly linked with Fe ++ of “haem” .
• Imidazole contains two groups 1.Fe ++ containing group which is
concerned with carriage of O2 , and 2.Imidazole N2 group, which can give
up H+ (proton) and accept H+ depending on the pH of the medium.
• Thus, buffering capacity of Hb is due to the presence of “Imidazole” nitrogen
group which remains dissociated in acidic medium and conjugate base forms.
• Oxygenated Hb is a stronger acid than deoxygenated Hb. On oxygenation,
the imidazole N2 group acts as acid and donates protons in the medium.
• Deoxygenated Hb is less acidic, less dissociable and imidazole N 2 group
acts as ‘base’ and takes up protons from the medium.
• Acidity of the medium favours delivery of O2
• Alkalinity of the medium favours oxygenation of Hb
• In the case of “Imidazole” group of histidine, which is intimately
associated with the Fe ++ of Hb, its strength as a buffer is affected by
changes in degree of oxygenation of Hb. When O2 is removed (in
deoxygenated Hb), the imidazole group is rendered “less acidic”,
consequently less dissociated, removing a H + from solution
andbecoming electrically +ve. This effect is reversed with increased
oxygenation of Hb.
 Sequence of events that occur in lungs and
tissues

• 1. In the Lungs
The formation of oxy-Hb (HbO2 ) from deoxygenated Hb (HHb), must
release H+ ions, which will react with HCO–3 to form H2CO3. Because of
Low CO2 tension in the lungs, the equilibrium then shifts towards the
production of CO2, which is continually eliminated in the expired air.
• 2. In tissues
Due to reduced O2 tension, local acidity, and aided by CO2 Böhr effect,
Oxy-Hb (HbO2) dissociates delivering O2 to the cells and deoxygenated Hb
(HHb) is formed. At the same time, CO2 produced as a result of metabolism in
the cells, is hydrated to form H2CO3, which ionizes toform H+ and HCO–3 .
Deoxygenated Hb (HHb) acting as an anion, accepts the H+ ions, forming
so-called acid-reduced Hb (HHb).Very little change in pH occurs because the
newly arrived H+ ions are buffered by formation of a very weak acid.
 Isohydric Transport of CO2

• At a pH of 7.25, one mol. of oxy-Hb donates 1.88 mEq

of H+; on the other hand, one mol. of reduced Hb,
because it is less ionized, donates only 1.28 mEq H+. It
may, therefore, be calculated that at the tissues a
change of one mol.of oxy-Hb to reduced Hb allows 0.6
mEq H+ to be bound(buffered), so that these newly
formed H+ ions do not bring about a change in pH.
This circumstance as it relates to the role of Hb buffers
is sometimes referred to as isohydric transport of CO2
 ROLE OF RESPIRATION IN ACID-BASE
REGULATION

Participation of the respiratory mechanism, in the

regulation of acid-base balance is depend upon:
• The sensitivity of the respiratory centre (RC) in
medulla oblongata to very slight changes in pH and
pCO2, and
• The ready diffusibility of CO2 from the blood, across
the pulmonary alveolar membrane, into the alveolar
air. Hence, the lungs should be healthy so that
diffusion of CO2 take place properly.
• An increase in blood pCO2 will results in increase in pulmonary
ventilation (stimulation of respiratory centre), which
increases also with slight increases in H+ ion concentration
of the blood (acidosis). The excess CO2 is thereby promptly
removed from the ECF in the expired air.
• A decrease in blood pCO2↓ or H+ ion concentration (alkalosis),
causes depression of respiratory centre,with consequent slow
and shallow respiration(hypoventilation) resulting to retention
of CO2 in the blood until the normal pCO2 and pH are
restored.
• This respiratory mechanism, therefore, tends to maintain the
normal B-H CO3/H2CO3 ratio in the EC fluids.
 RENAL MECHANISMS FOR REGULATION
OF ACID-BASE BALANCE
Kidneys also affect acid-base equilibrium:
• By providing for elimination of non-volatile acids viz.Lactic acid, H2SO4,
ketonebodies, etc. after being buffered with cations (principally Na+) are
first removed by glomerular filtration.
•Body cannot afford to lose Na+, being extremely important. It is recovered
in the renal tubules by reabsorption in exchange of H+ ions which are
secreted. It is recovered as NaHCO3(“alkali reserve”).
• There are three mechanisms by which the above is achieved.
A. Bicarbonate mechanism
B. Phosphate mechanism
C. Ammonia mechanism
 A. Bicarbonate Mechanism
• Mobilisation of H+ ions for tubular secretion is accomplished by ionisation of
carbonic acid (H2CO3) which itself is formed from metabolic CO2 and H2O.
• This reaction CO2 + H2O ⇔ H2CO3
• is catalysed by the enzyme (Zn-containing enzyme) carbonic anhydrase, present in
renal tubular epithelial cells. Site: In proximal tubular epithelial cells, the exchange of
H+ ions proceed first against sodium bicarbonate. 
• Under normal conditions, the rate of H+ secretion is about 3.50 millimoles per
minute, and the rate of filtration of HCO3– ions is about 3.49 millimoles per minute.
Hence, all the HCO3– ions are normally reabsorbed, while a slight excess of H+ ions
remains in the tubules to react with other substances and to be excreted in urine.
Above mechanism provides:
• For complete reabsorption of all of NaHCO3,
• Reduction of H+ ion load of plasma with little change in pH of urine.
 Carbonic Anhydrase (CA)

• It is a Zn-containing metalloenzyme. It specifically catalyses the removal of

CO2 from H2CO3, however, the reaction is reversible. At the tissues, the
formation of H2CO3 from CO2 and H2O is also accelerated by CA.

Source:
1. RB cells: Where associated with Hb; never found in plasma.
2. In most of the tissues, where catalyses formation of H2CO3 from H2O and
metabolic CO2
3. In parietal cells of stomach: Where the enzyme is involved in secretion of HCl.
4. In renal tubular epithelial cells
5. Recently, it has been demonstrated in small quantities Muscle tissue,Pancreas
and
6. Spermatozoa.
 Factors Affecting Bicarbonate Reabsorption

1.Influence of CO2 tension (pCO2):

• Increase in pCO2↑, accelerates formation of H2CO3 and thus increases the H+ secretion
by renal tubular epithelial cells, which facilitate reabsorption of HCO3–.
• Decrease in pCO2↓ will do the reverse.
2. Variations in the body store of K+:
• The intracellular concentration of K+ and NOT of plasma is the factor that controls the
HCO3– absorption. When K+ is administered (in excess), K+ rapidly enters into the cell in
exchange of H+ ions. H+ ions leaving the cells are buffered by bicarbonate of ECF as a
result plasma bicarbonate content is reduced. Hence, renal tubular epithelium, secretes
less H + ions and thus less HCO–3 is absorbed. Bicarbonate excretion in urine becomes
more and urine becomes alkaline, though ECF is acidic.
3. Variations in plasma level of Cl–:
• Increase or decrease of Cl– concentration in plasma causes decrease or increase of HCO–
3 concentration respectively. Increase in (Cl–) ↑ causes decrease in (HCO–3) ↓and vice
versa.
4. Variations in secretion of adrenocortical hormones:
• In Cushing’s syndrome, plasma [HCO–3] increases with augmented HCO–3 reabsorption
 B.PHOSPHATE MECHANISM
• Both disodium hydrogen phosphate (Na2HPO4, alkaline PO4) and
monosodium dihydrogen PO4(NaH2PO4, acid phosphate) are present
in the plasma. The pH of the urine is determined by the ratio of these
two phosphates. In plasma, concentration of Na2HPO4 exceeds that of
NaH2PO4 and the ratio is maintained to 4:1. But in urine,the
concentration of NaH2PO4 exceeds that of Na2HPO4 and the ratio
becomes 9:1.Glomerular filtrate of pH 7.4 is converted to a urine
having pH –6.0 or even as low as 4.8. After all the HCO–3 has been
reabsorbed by the mechanism stated above, H+ secretion proceeds
against Na2HPO4. The exchange of Na+ion for secreted H+ion changes
Na2HPO4 to NaH2PO4 with consequent increase in acidity of urine,
resulting in decrease in pH . Site: Operates in “distal tubule” of kidney
 C. Ammonia Mechanism
• A third mechanism operates in the distal renal
tubule cells, for the elimination of H+ions and
the conservation of Na+, by production of NH3
by the renal tubular epithelial cells.
• Source of NH3 in Distal Tubular Epithelial Cells
• 1.NH3 is produced by the hydrolysis of
Glutamine by the enzyme Glutaminasewhich
is present in these cells.
• 2.In addition to above, if the cells require NH3
more
• •NH3 can also be formed from other amino acids
by oxidative deamination by L-amino acid oxidase
• .•NH3 can also be formed from glycine by
glycine-oxidase.
• The NH3 thus formed forms NH+4 ions by
combining with H+ ions and NH+4 ions can
exchange Na+ ion from NaCl.
• NH+4 ions formation
• •NH3 can diffuse into the tubular filtrate and there forms NH+4 ions in
combination with H+ions.
• •NH3 can combine with H+ions inside the cells and then NH+4 ions
come into tubular filtrate. This probably is not the principal mechanism
as NH+4 ions are less readily permeable to tubular epithelial cells
• The NH3 production is greatly increased in metabolic acidosis and
negligible in alkalosis. It is also observed that activity of renal
glutaminase is enhanced in acidosis.The NH3 mechanism is a valuable
device for the conservation of fixed base. Under normal conditions, 30 to
50 mEq of H+ions are eliminated per day, by combination with NH3 and
about 10 to 30 mEq, as titratable acid, i.e.buffered with PO4.
 Four Basic Types of Imbalance
• Metabolic Acidosis
• Metabolic Alkalosis
• Respiratory Acidosis
• Respiratory Alkalosis
 Previous year questions
• Maintenance of pH in blood
• buffer solution
• buffer system in blood
• pH and its biological importance