Learning objectives

 Define pH, pK and pH scale and mention their importance.

 Define acid, base, strong acid and weak acid.
 Define buffer. State the body fluid buffers with their basic mechanism of action.
 State Handerson Hasselbach equation and its importance.

pH and its conception

Ionization of water:
Water molecules can function as both acids and bases. One water molecule (acting as a
base) can accept a hydrogen ion form second one (acting as acid). A hydronium ion (H3O+) and
Hydroxide ion (OH-) are formed.

The process is often simplified to show the ionization of just one water molecule into a
hydrogen ion and a hydroxide ion.
H2O ⇌ H+ + OH−
In pure water, the concentration of hydrogen and hydroxide ions are equal to one another.
Any aqueous solution in which [H+] = [OH-] is said to be neutral. For any neutral solution at 25°C,
each of these ions has a concentration of 1.0 × 10−7 M
pH: The letters pH stands for the power of the hydrogen ion.
Definition: The negative logarithm of the hydrogen ion concentration of a solution,
concentration is expressed in term of molarity.
pH = − log [H+]
pH Scale
pH Scale:
In pure water or a neutral solution [H+] = 1× 𝟏𝟎−𝟕 M
We know,
pH = − log [H+]
pH = − log (1× 𝟏𝟎−𝟕 )
pH = − log (1) - log (𝟏𝟎−𝟕 )
pH = 0 – (-7)
pH = 7
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 So,
pH = 7 means, [H+] = 1×10-7 Mol/L
pH = 14 means, [H+] = ?
pH = 1 means? [H+] = ?
Few information:
pH = 7 --------------- Neutral
pH = <7 -------------- Acidic
pH = >7 -------------- Alkaline

Typically, the concentrations of H+ in water in most solutions is between a range of 1 M

(pH = 0) and 10-14M (pH=14). Hence pH scale ranges from 0 to 14.
Importance and significance of pH
Importance of pH:
Normal pH is required for-
1. To maintain enzyme activity;
2. To maintain Native structure;
3. To maintain Conformation of biomolecules;
4. To maintain Neuro-muscular activity;
5. To maintain cellular viability. E.g. Na+K+ATPase pump is maintained by pH.
Significance of pH:
pH signifies whether the solution is acidic or basic in nature.
pH of different body fluids
 Blood = 7.35 – 7.45 (average 7.4)
 CSF = 7.2 – 7.4
 Pancreatic Juice = 7.5 – 7.54
 Bile = 7.4 – 8.5
 Saliva = 6.4 – 7
 Urine = 6 – 6.8
 Gastric Juice = 1.5 – 3

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 Acidemia and Alkalemia
Acidemia:
A blood pH below the lower reference interval.
Alkalemia:
A blood pH above the higher reference interval.
Methods of determination of pH
Non-specific methods:
 Litmus paper
 Indicators
 Buffers
Specific methods:
 pH meter
 Gas electrode
 Calomel electrode
 Hydrogen electrode
 Biochemistry analyzer
Law of mass action
Definition:
When the reaction is reversible and is in equilibrium, the rate at which a reaction take place
at constant temperature and pressure is directly proportional to the product of the concentration of
the reacting substances.
Example-
V1
A+ B C + D
V2
According to Law of mass action,
V1 α [A] × [B]
V1 = K1 [A] × [B]
Similarly,
V2 ∞ [C] × [D]
V2 = K2 [C] × [D]
K1 and K2 are the proportionality constant of respective reaction.
At equilibrium,
V1 = V2
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Or, K1 [A] x [B] = K2 [C] x [D]
K𝟏 [C] ×[D]
Or, = [A] ×[B]
K𝟐

[C] ×[D]
Or, K = [A] ×[B]

[K is an equilibrium constant or dissociation constant]

Significance of Law of mass action:
1. It explains the basis of biochemical reaction.
2. It expresses the concept of pH.
pK:
pK is the negative logarithm of dissociation constant.
So, pK = - log K
Henderson- Hasselbalch Equation (HHE)
Definition:
The relationship between pH, pK and the concentration of an acid and it’s conjugated base can
be described an convenient equation, called Henderson- Hasselbach equation.
The equation is –
[𝑪𝒐𝒏𝒋𝒖𝒈𝒂𝒕𝒆 𝒃𝒂𝒔𝒆]
pH = pK + log
[𝑨𝒄𝒊𝒅]

A weak acid, HA, ionizes as follows:

HA H+ + A-
[𝐻+][𝐴⁻]
Or, K = [Applying the law of mass action]
[𝐻𝐴]

Or, [H+] [A⁻] = K [HA]

[HA]
Or, [H+] = K [Dividing both side by A-]
[A⁻]
[𝐻𝐴]
Or, Log [H+] = LogK (Taking log on both side)
[A⁻]
[𝐻𝐴]
Or, Log [H+] = LogK + Log [A⁻]
[𝐻𝐴]
Or, -Log [H+] = - logK - Log {multiplying by -1}
[A⁻]
[𝐻𝐴]
Or, pH = pK - log [A⁻]
[A⁻]
Or, pH = pK + log [𝐻𝐴] [removing minus sign]

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 [𝐶𝑜𝑛𝑗𝑢𝑔𝑎𝑡𝑒 𝐵𝑎𝑠𝑒]
So, pH = pK + log
[𝐴𝑐𝑖𝑑]

Importance of HHE:
1. pH of a buffer solution can be calculated.
2. The equation is useful to determine the acid base status of the body.
When pH = pK:
[𝐶𝑜𝑛𝑗𝑢𝑔𝑎𝑡𝑒 𝐵𝑎𝑠𝑒]
pH = pK + log [𝐴𝑐𝑖𝑑]

When conjugate base and acid components are present at equal concentration, their ratio
become one.
pH = pK + log 1
We know that, log 1 = 0. In that case, pH = pK.
Acid
Definition:
An acid is a substance which donate proton (H+) in solution.
HCl H+ + Cl-
Types:
According to the dissociation, acids are of two types-
1. Strong acid
2. Weak acid
Strong acid:
Strong acids are those acids which dissociates quickly and completely. e.g. HCl, H2SO4,
H2NO3
Weak acid:
Weak acids are those acids which dissociates slowly and partially. e.g. H2CO3, Acetic acid.

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Note:
➢ Strong acids that produce in our body is HCl.
➢ Weak acid that produces in our body is H2CO3.
➢ H2CO3 is formed in- Lung, kidney, stomach, RBC.
➢ In our body where carbonic anhydrase enzyme is present, H2CO3 is formed there.
𝑪𝒂𝒓𝒃𝒐𝒏𝒊𝒄 𝑨𝒏𝒉𝒚𝒅𝒓𝒂𝒔𝒆
CO2 + H2O → H2CO3
Base
Base:
A base is a substance which accept proton in solution.
Bases may be charged particle (e.g. Cl-, HCO3-) or may be without charge (NH3).
Cl- + H+ HCl
Conjugate base and acid
Conjugated base of an acid:
It is the remaining part of an acid after donating proton. e.g. Cl- (conjugated base of HCl)
Conjugated acid of a base:
It is the acid formed by a base after accepting proton. e.g. HCl acid is the conjugated acid
of chloride.

Alkali
Alkali:
The metallic hydroxides such as NaOH and KOH are commonly referred as alkali.
Buffer
Definition:
Buffer is a mixture of weak acid and it’s salt, which tends to maintain the normal hydrogen
ion concentration when limited amount of acid or alkali is added to it.
Buffer pair:

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 Buffers always remain in pair. Conventionally buffer pair is symbolized as-
𝐁¯(𝐜𝐨𝐧𝐣𝐮𝐠𝐚𝐭𝐞𝐝 𝐛𝐚𝐬𝐞)
𝐇𝐁( 𝐰𝐞𝐚𝐤 𝐚𝐜𝐢𝐝)
Examples of some buffer pair:
Buffer system Weak acid Conjugate base pK
Bicarbonate buffer H2CO3 HCO3- 6.1
Phosphate buffer H2PO4- HPO42- 6.8
Protein buffer HPr Pr- 6.4-7.8
Hemoglobin buffer HHb Hb- 6.6-7.8
Ammonia buffer NH4 NH3+ 9.0

Distribution of body buffers

Blood buffers: Plasma/ECF buffer:
 Bicarbonate buffer  Bicarbonate buffer
 Hemoglobin buffer  Phosphate buffer
 Phosphate buffer  Plasma protein buffers
 Plasma protein buffer
ICF buffer:
RBC buffers:
 Protein buffer
 Hemoglobin buffer  Bicarbonate buffer
 Bicarbonate buffer  Phosphate buffer
 Phosphate buffer
Kidney/Urinary buffer:
 Ammonia buffer
 Bicarbonate buffer
 Phosphate buffer

How buffer acts

Buffer acts by converting the strong acid into corresponding weak acid and strong base
into corresponding weak base and thereby minimizes the effect of strong acid or strong base on
pH of a solution.
Example:
 Conversion of strong acid (e.g. HCl, H2SO4) into a weak acid: When a strong acid is
added to a buffer solution (e.g. bicarbonate buffer), then the buffer base reacts with strong
acid converting it into weak acid.
HCl + NaHCO3 H2CO3 + NaCl
(Strong acid) (Weak acid)

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  Conversion of strong base (NaOH) into weak base: When a strong base is added to a
buffer solution, then the buffer acid reacts with strong base converting it into weak base.
NaOH + H2CO3 NaHCO3 + H2 O
(Strong base) (Weak base)
“Bicarbonate buffer is the most potent buffer in plasma”- justify
In spite of low pK (6.1) & low concentration, Bicarbonate buffer is the most potent buffer
in plasma because-
1) The two elements of bicarbonate buffer system, HCO3- & CO2 are regulated respectively
by the kidneys and lungs. As a result of this regulation, pH of the ECF can be precisely
controlled by the relative rate of removal and addition of HCO3- by the kidney and also the
rate of removal of CO2 by the lungs.
2) It has high buffering capacity (60% of total body buffering activity).
3) Its conjugate base (HCO3-) concentration is 20 times more than it’s acid (H2CO3).
4) It is an open-end system and has a very high and broad adaptation to acid base disturbance.
5) It has wide field of buffering activity (both in ECF and ICF).
6) It works in cooperation with hemoglobin buffer.
Ratio of HCO3- and H2CO3 to maintain pH at 7.4:
20:1
Maintenance of constant blood pH
There are three mechanisms by which the relative consistency of blood pH is maintained.
They are-
1. Blood buffer system
2. Respiratory system
3. Renal system
Blood buffer system
It is the 1st line defense & acts within seconds. The blood contains three buffer systems-
Bicarbonate, phosphate and protein buffer. The base component of buffer combines with free H+
of metabolic acids & is converted into weak acids which dissociates very little. Thus, blood
buffers change pH in the body.

B-(buffer base) + H+ (from metabolic acid)

HB (weak acid)

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For example-
HCO3- (buffer base) + H+ (from metabolic acid)

H2CO3 (weak acid)

But blood buffers cannot remove H+ from body. They only keep the H+ “tied up” until lungs &
kidney remove the H+ from the body.
Respiratory buffer system
It is the 2nd line defense & acts within minutes. The respiratory system regulates the
concentration of CO2. Lungs excrete H2CO3 in the form of CO2 through respiration. CO2 is
produced by metabolism which binds with H2O to form H2CO3 (volatile acid) in blood. H2CO3
comes to lungs and dissociates into H2O & CO2. Then this CO2 is excreted through lungs by
expiration.
𝑪𝒂𝒓𝒃𝒐𝒏𝒊𝒄 𝑨𝒏𝒉𝒚𝒅𝒓𝒂𝒔𝒆
H2CO3 → H2O + CO2
Renal buffer system
It is the 3rd line defense and & acts within hours or days and regulates plasma bicarbonate
concentration by the kidneys. Kidney maintains normal pH by excreting either acidic or alkaline
urine. But as the body is usually a net acid producer, kidney usually excretes acidic urine.
This occurs by-
1) Excretion of H+ ions
2) Reabsorption of HCO3-
3) Generation of new HCO3-

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