ASSIGNMENT CA-2

NUMERICAL METHODS USED IN

BIOLOGY (BTY 187)

Submitted to: Submitted by:

Dr. Madhuri Girdhar 1. Mansi Sharma
11915656
A-12
2. Fathima Sasna
11913834
A-13
3. Prateek Bhardwaj
11914020
A-14
4. Girish Sawhney
11912634
A-15
5. Ankita Mishra
11915656
A-16
 INTRODUCTION
Acid and base play a major role in the fields of chemistry as every chemical reaction is
categorized under acid-base reaction, but redox reactions. These are not just the important
laboratory chemical reactions but also have a huge practical importance such as our bodily
functions like ionic transport for neural impulse, digestion in stomach at acidic pH and basic
digestion in intestine. Other major physiological function being regulated by acid-base
reaction is homeostasis.

ACIDS AND BASES AND THEIR TYPES

For a long time, the term ‘acid’ and ‘base’ were being used to describe inorganic and organic
compounds.

All acids compulsorily have hydrogen as their constituent element in the form of proton (H+)
Examples – Hydrochloric acid (HCl), Sulphuric acid (H2SO4) and Nitric acid (HNO₃).

All bases are either metallic oxide (O2–) or metallic hydroxide (OH-) or metallic ammonia
solution. Examples – Copper oxide (CuO), Sodium hydroxide (NaOH) and Ammonium
hydroxide (NH4OH).

ACIDS

Acids are defined as a compound which contains one or more hydrogen atoms and when
dissolved in water, produce hydronium ions (H 3O +) the only positively charged ions. At first,
the acid molecule furnishes hydrogen ion (or proton), i.e., H + ion in aqueous solution. But this
H+ ion cannot exist independently. Therefore, it combines with water molecules to form
hydronium ion (H3O+). This hydrated ion can now exist independently in solution.

Hydrogen is common in all acids.

 Some naturally occurring acids
Acetic acid Vinegar
Citric acid Citrus fruits
Butyric acid Rancid butter
Hydrochloric acid Gastric juice
Formic acid Sting of bees and ants
Lactic acid Sour milk
Maleic acid Apples
Oleic acid Olive oil
Oxalic acid Tomatoes
Stearic acid Fats
Uric acid Urine

CLASSIFICATION OF ACIDS

Acid can be classified in different ways.

1. Depending on their sources

1. Organic acids: Acids which are obtained usually from plants are called organic
acids.
They contain carbon atom also along with hydrogen atom.
Examples: Oxalic acid (COOH2), Acetic acid (CH3COOH).
They are weak acids and they do not ionize completely in solution. So they contain
ions as well molecules.
2. Inorganic acids (mineral acids): Acids which are obtained usually from minerals
known as inorganic acids. They do not contain carbon (except carbonic acid H 2CO3).
Examples: Hydrochloric acid (HCl), Sulphuric acid (H 2SO4), Nitric acid (HNO3).
They are strong acids. They ionize completely in solution producing a high
concentration of hydronium ions [H 3O] +.

Carbonic acid (H2CO3) is a weak mineral acid. It turns blue litmus pink. It is non-corrosive,
and so used in soft drink. Acids which contain oxygen along with hydrogen and some other
element are oxy-acids. Examples: Nitric acid (HNO 3 ), Sulphuric acid (H 2SO4). Hydracids
contain hydrogen and a non-metallic element, and no oxygen. Examples: Hydrochloric acid
(HCl), Hydrobromic acid (HBr).
 2. Depending on their concentration

Concentration of an acid means the amount of acid present in a definite amount of its aqueous
solution.

Concentrated acid: An acid which contains a very small amount of water or no water is called
concentrated acid.

Dilute acid: An acid which contains far more amount of water than its own mass is known as
dilute acid.

In order to dilute an acid, pour acid into water in amounts and stir constantly. Water is not
added to acid as it is an exothermic process and in this process so much heat is produced that
splashing of acidic solution may occur; also the container may break which can be fatal to the
person. Concentration of an acid simply tells us the amount of water present in the acid and
not at all the strength of the acid. Strength of an acid is the measure of concentration of
hydronium ions it produces in its aqueous solution. Thus dil. HCl is stronger acid than highly
concentrated acetic acid.

The Strength of an acid depends on the degree of ionization [α] and concentration of
hydronium ions [H3O] + produced by that acid in aqueous solution.

No.of acid molecules ionised

Degree of ionization (α) = *100
Total no.of acid molecules present in aqueos solution

If the degree of ionization (α) for an acid and base in aqueous solution is greater than 30%, it
is strong and if it is less than 30%, it is weak.

3. Depending on their Basicity

The basicity of an acid is defined as the number of hydronium ion (H 3O) + that can be
produced by the ionization of one molecule of that acid in aqueous solution.
Therefore we have,
i) Monobasic acids: Acids which on ionization in water produce one hydronium ion
per molecule of acid are known as monobasic acids.
For examples: Hydrochloric acid
HCl + H2O → H3 O+ + Cl-
Other similar examples are: HBr, HNO 3, HI, CH3COOH, etc.
Monobasic acids ionize in one step and so form only one normal salt.
NaOH + HCl → NaCl + H2O
ii) Dibasic acids: Acids which, on ionization in water, produce two hydronium ions
(H3O)+ per molecule of the acid, are known as dibasic acids.
Examples: Sulphuric acid (H2SO4), Oxalic acid (H2C2O 4), Sulphurous acid
(H2SO3), Carbonic acid (H2 CO3)
Dibasic acids ionize in two steps, as shown below:
 H2SO4 + H 2O ⇌ H3O + + HSO4 -
HSO4 - + H2 O ⇌ H3O + + SO42-
H2CO3 + H2O ⇌ H3O + + HCO3-
HCO3- + H 2O ⇌ H3O + + CO32-

Dibasic acids have two replaceable hydrogen ions; therefore they form one acid salt or one
normal salt.

NaOH + H2SO4 → NaHSO4 (acid salt) + H2 O

2NaOH + H2SO4 → Na2 SO4 (normal salt) + 2H2O

iii) Tribasic acids: Acids which, on ionization in water, produce three hydronium
Ions per molecule of acid are called tribasic acids.
Examples: Phosphoric acid (H3 PO4), Citric acid (CH2 COOH-COOH-CH2COOH)
They ionize in three steps, as shown below:

(a) H3PO4 + H2O ⇌ H3O+ + H2PO4-

(b) H2 PO4- + H2O ⇌ H3O + + HPO42-
(c) HPO42- + H2O ⇌ H3O + + PO43-

Tribasic acids can form two different acid salts and one normal salts and one
normal salt, because they have three replaceable hydrogen ions.

NaOH + H3PO4 → NaH2 PO4 + H2O

(Monosodium dihydrogen phosphate)
2NaOH + H3PO4 → Na2 PO4 + 2H2O
(Disodium
Hydrogen phosphate)
3NaOH + H3PO4 → Na3 PO4 + 3H2O
(Trisodium phosphate)

BASES

A base is either a metallic oxide or a metallic hydroxide or ammonium hydroxide which

reacts with hydronium ions of an acid to form salt and water only. For example, CuO
(metallic oxide), Mg (OH) 2 (metallic hydroxide), etc.

Base + Acid → Salt + Water

CuO + 2HCl → CuCl2 + H 2O
Mg(OH)2 + H2SO4 → MgSO4 + 2H2O
Al(OH)3 + 3HNO3 → Al(NO3)3 + 3H2O
NH4OH + HCl → NH4 Cl + H2O
Basic Oxide: A basic oxide is a metallic oxide which contains the ion O2- and reacts with an
acid to form salt and water only. For example: Lead (Ⅳ) oxide is a metallic oxide which
reacts with hydrochloric acid to produce Lead (Ⅱ) chloride (a salt) and water, but the word
only excludes it from the class of bases, because chlorine is also produced.

PbO2(s) +4HCl (aq) → PbCl2 (aq) +Cl2 (g) + 2H2O

Thus Lead (II) oxide PbO2 is not a base.

Basic Hydroxide: It is a metallic hydroxide which contains OH - and will react with an acid to
give salt and water only. For example: Sodium hydroxide (NaOH) and Aluminium hydroxide
(Al (OH)3).

NaOH + HCl → NaCl + H2O

CLASSIFICATION OF BASES

1) On the basis of their strength

(a.) A strong base: It undergoes almost complete ionization in aqueous solution to
produce a high concentration of OH - ion.
Examples: Sodium hydroxide (NaOH) and Potassium hydroxide (KOH)
NaOH (aq) ⇌ Na+ + OH- (aq)
KOH (aq) ⇌ K+ + OH - (aq)
(b.) A weak base: It undergoes only partial ionization in aqueous solution to produce a
low concentration of OH - in solution.
Examples: Ammonium hydroxide (NH 4OH) and Calcium hydroxide (Ca (OH)2).
NH4OH (aq) ⇌ NH4 + + OH- (aq)
Ca (OH)2 (aq) ⇌ Ca2+ (aq) + 2OH- (aq)
2) On the basis of their acidity
The acidity of a base: The number of hydroxyl ions [OH] - which can be produced per
molecule of the base in aqueous solution or the number of hydrogen ions (of an acid) with
which molecules of that base will react to produce salt and water only is known as acidity
of the base.
(a.) Monoacidic base: It is a base that dissociates in aqueous solutions to produce one
OH- ion per molecule of that base. For example: NaOH, KOH and NH 4OH.
NaOH → Na+ + OH -
(b.) Diacidic base: It is a base that dissociates in aqueous solutions to produce two OH -
ions per molecule of that base. For example: Ca(OH) 2 and Cu(OH)2.
Ca(OH)2 (aq) ⇌ Ca2+ + 2OH -
Cu(OH)2 (aq) ⇌ Cu2++ 2OH-
(c.) Triacidic base: It is a base that dissociates in aqueous solutions to produce three OH-
ions per molecule of that base (or contains three replaceable hydroxyl ions per molecular
of the base). For example: Al(OH)3 and Fe(OH)3.
 For water insoluble hydroxides, acidity is equal to the number of replaceable OH - ions in
one molecule of that base.

DIFFERENT CONCEPTS FOR ACID-BASE DETERMINATION

There have been a number of concepts given for the identification and categorization of
different chemicals under acid or base.

Arrhenius was the first to give the concept of acid and base. He described acid as a chemical
substance which gives a proton or H + ion in an aqueous solution while the bases were
described as the producers of OH - ions in the aqueous solution. While this is the basic and the
correct description of acid and base, it could only recognize the limited number of chemical
substances which contain hydrogen or hydroxide ions in them, eg. HCl, H 2 SO4, NaOH,
Ca(OH)2 etc.

Then the Bronsted-Lowry model was introduced which described acid-base on the account
of proton donor or acceptor. The proton donors are the acid whereas the proton acceptors are
the base. Although it expanded the acid-base determination area in chemistry but it failed to
explain why the metallic ions made water acidic.

This was explained by the Lewis method. According to him, acids are the chemicals that can
accept electron pairs while the bases are the chemical substances that can donate the electron
pairs.

IONIZATION OF STRONG ACIDS AND BASES

Strong acids are the chemicals which ionize completely (approx 100%) in an aqueous
solution, e.g. HCl completely ionizes to H 3O + and Cl- when present in an aqueous solution.

 HCl ⇌ H3O + + Cl−

H3O+ is simply a proton, hence similar as H +. So the dissociation of HCl is simply into H +
and Cl- when present in water. For a strong acid, the anion formed after dissociation is the
conjugate base for that acid. So for HCl, Cl- is the conjugate base.

 HCl ⇌ H+ + Cl−
 Substances are known to be the strong bases if they ionize to almost 100% in an
aqueous solution i.e. which yields OH -. E.g. KOH dissociates to K+ and OH -
 KOH ⇌ K+ + OH −

.The cation formed by the dissociation of a base is known as the conjugate acid for the
respective base. So, for KOH, K+ is the conjugate acid which is weak as the respective base
is strong.

The H + and OH - ion concentration are the major parameters for determining the strength of
an acid or base. These parameters give rise to the concept of pH and pOH.
The meaning of p is that whenever you see a “p” in front of a value, like pH, pKa, pKb it
means you're dealing with a -log of the value following the "p". For example, pKa is the -log
function works, a smaller pKa means larger Ka. This general rule applies for all of the
parameters, like pH, pOH, pKa and pKb.

Ka and pKa
Ka is the acid dissociation constant; pKa is simply the -log of this constant. The acid
dissociation constants are usually expressed in terms of moles per liter (mol/L). A general
acid, say HA dissociates as:

HA ⇋ H+ + A-

[H+][A−]
Ka =
[HA]

pKa = -log [Ka]

A larger Ka value indicates a strong acid because it means the acid is largely dissociated into
its ions. A large Ka value also means the formation of products in the reaction is favored. A
small value of Ka means little of the acid dissociate, so weak acids have a Ka range from 10 -2
to 10-14 . The pKa gives the same information, just in a different way. A strong acid will have
a low pKa value. Weak acids have a pKa ranging from 2-14.

Kb and pKb
Kb is the base dissociation constant; pKb is simple the – log of the Kb value. The Kb is also
expressed in terms of moles per litre (mol/litre). A general base, say BOH dissociates as:

BOH ⇋ B + + OH-

[OH−][B+]
Kb =
[BOH]

pKb = -log [Kb]

A large Kb value indicates the high level of dissociation of a strong base. A lower pKb value
indicates a stronger base.

pKa and pKb are related by the simple relation:

 pKa + pKb = 14

pH
The measure of presence of H + ions in a substance is known as the pH. It is one of the most
important parameters to measure the strength of an acid or base. More the pH less will be the
strength of an acid and more will be the basic character.

The pH is calculated by the negative logarithm of hydrogen ion concentration to the base of
10.

pH = −log[H+]

pOH
The measure of presence of OH - ions in a substance is known as the pOH. It is one of the
most important parameters to measure the strength of a base. More the pOH more will be the
acidic character and lesser will be the basic character.

pOH is calculated by the negative logarithm of the number of hydroxide ions to the base of
10.

pOH = −log[OH-]
 RELATION BETWEEN pH and pOH
Since pH is the measure of hydrogen ion concentration and the pOH is the measure of
hydroxide ion concentrations.

Derivation
Self ionization of water can be given as:

2H2O ⇌ H3O+ + OH−

Since;
[H3O+] [OH−] = Kw

So the relation between pH & pOH is:

pH + pOH = pKw

As:

pH = −log[H +],
pOH = −log[OH−]
pKw = −logKw

pKw depends on temperature and pressure. At STP it is equals to 14. So;

pH + pOH = 14

Ion Concentration Solution Type pH pOH

[H+] > [OH-] Acidic pH < 7 pOH > 7

[H+] < [OH-] Basic pH > 7 pOH < 7

[H+] = [OH-] Neutral pH = 7 pOH = 7

 SIGNIFICANCE OF pH & pOH

 pH plays a major role when it comes to the agricultural fields. pH of a soil is of

utmost importance as for growing crops because misbalance in the soil’s pH can harm
and retard the plant growth.
 Aquatic ecosystems too need to have a proper pH. Every organism has a particular
survival chance under certain environmental conditions. So, change in pH can be
fatal, especially in the aquatic ecosystem.
 Regulation of all the enzymatic activities in our body is also dependent on pH.
Enzymes are highly active at a certain pH known as optimum pH. Below or above this
pH the enzymatic activity slows down eventually leading to the denaturation of the
enzyme.
 Other significant areas for pH can be the food industry, water treatment, aqua
cultures, brewing and wine making, etc.
 Kw
Water molecules can function as both acids and bases. One water molecule acting as a base accept a
hydrogen ion from the second water molecule which is acting as an acid. This process is called self
ionization of water.

H2O ⇋ H+ + OH -

However, the hydroxonium ion is a very strong acid and hydroxide ion is a very strong
base. As fast as they are formed they react to form water again. The net effect is that
equilibrium is setup

H2O (l) + H 2O (l) ⥃ H3O+ + OH -

This equilibrium is written in simplified form as follows,

H2O (l) ⇋ H+ + OH -

Kw is known as the ionic product of water. It is the equilibrium constant for the ionization
of water. Kw varies with temperature .It's value is usually taken as 1x10 -14 mol2 dm-6 at
room temperature.

DERIVATION OF Kw
Water dissociates as follows,

H2O (l) ⇋ H+ + OH-

Equilibrium constant for this reaction,

[H+][OH−]
Keq =
[H2O]

As liquid or gas phases are not included in equilibrium equation, Kw does not include
concentration of water which is a pure liquid.
At room temperature, pH of pure water is 7. Therefore hydroxonium ion concentration is
10-7mol dm- 3. For any sample of pure water, molar concentrations of hydroxonium ion and
hydroxide ion are equal. Therefore hydroxide ion concentration is also 10 -7mol dm-3 .

KW = [H+][OH-]

Kw = [10-7]*[10-7]
 Kw = 10-14

Value of Kw varies with temperature. It's value is usually taken as 1x10 -14 mol2 dm-6 at
room temperature.

RELATIONSHIP BETWEEN IONIZATION CONSTANT (Kw), pH AND pOH

We know that

KW = [H+][OH-]

Taking – log on both sides

-log KW = -log [H+] -log [OH-]

It gives

pKw = pH + pOH

Here, pKw is defined as the negative logarithmic function of the water ionisation constant.

The value of pKw can be calculated as,

pKw = - log [10-14]

pKw = 14

at room temperature the value of pKw is 14.

FACTORS AFFECTING THE VALUE OF pKw

Temperature
The value of pKw decreases as the temperature increases from the melting point of ice to a
minimum temperature of 25°C, after which it increases up to the critical point of water at
374°C.
 Curve showing relation between
temperature and pKw

Pressure
The value of pKw increases with the decrease of pressure.

Curve showing relation between

pKw and Pressure at a constant
temperature

Ionic strength of the electrolyte

In case of 1:1 electrolytes like NaCl the value of pKw first decreases and then increases when
ionisation is almost complete but in case of 1:2 electrolytes like CaCl2 the value of pKw
decreases with increase in ionic strength of the electrolyte.

Curve showing relation between

pKw and ionic strength for a 1:1
electrolyte like NaCl.
BUFFERS AND THEIR SIGNIFICANCE IN MOLECULAR BIOLOGY
Buffers are solutions which resist the change in pH on addition of a strong acid or base.
Sometimes also referred to as pH buffers or hydrogen ion buffer it mostly consists of an
aqueous solution of a weak acid or a weak base and its conjugate base of conjugate acid. The
buffer solutions are a very important part of any molecular biology laboratory as these serve
as media for enzyme and protein extraction. Enzymes and proteins are especially very
sensitive to the changes in pH and a varying pH may result in temporary loss of activity or
complete denaturation. Artificial buffers make sure none of it happens and the enzyme or the
protein can be worked upon easily. Some natural buffers exist in the human body as well, like
the most commonly cited buffer system present in the blood. It is a bicarbonate buffer system
which prevents the blood pH from changing significantly on removal or addition of O 2 and
CO2 from the blood cells.

TYPES OF BUFFERS

Buffers are mainly divided into two types- Acidic and Alkaline buffer solutions.

Acidic buffers are solutions which have a pH less than 7 and are mainly made up of a weak
acid and its salt. The most common example is the acetate buffer. It contains acetic acid
(CH3COOH) as the weak acid and mostly has Sodium acetate (CH3COO -Na+) as the salt. It
has a working pH of about 4.75.

CH3COOH H+ + CH3COO–

CH3COONa Na+ + CH3COO-

Alkaline buffer solutions of basic buffers are those which have a pH greater than 7 and are
mainly made up of a weak base and its salt. The most commonly cited example is the
ammoniate buffer. It contains ammonium chloride (NH 3Cl) as the weak base and ammonium
hydroxide (NH3OH) as the salt. It has a working pH of about 9.25.

NH3OH ⇋ OH - + NH3+

NH3Cl ⇋ NH 3+ + Cl-

PRINCIPLES OF BUFFER ACTION

A buffer by definition should resist any and all change in pH of the solution i.e. should
maintain a near constant pH. The main reason for this “resistance” of a buffer solution is due
to the equilibrium which is maintained between the weak acid or base and its salt.

For an acidic buffer:

Assume a weak acid HA and its conjugate base A- as

HA ⇋ H+ + A- (eqn 1)
When a strong acid (eg. HCl) is added to the solution, it dissociates as –

HCl ⇋ H + + Cl-

The newly released H+ ions are neutralised by the A- ions present in the solution, thus the pH
change is resisted as the concentration of H + ions remain fairly constant. The equilibrium in
eqn1 shifts towards the left according to Le Châtelier's principle.

When a strong base (eg. NaOH) is added to the solution it dissociates as –

NaOH ⇋ Na+ + OH-

The released OH - ions are neutralised by the H + ions present in the solution, thus the pH
remains constant as the concentration of H + ions is maintained by the shifting of the eqn1
equilibrium towards the right, maintaining the H + concentration fairly constant and resisting
the pH change.

For basic buffers:

Assume a weak base BOH and its conjugate acid B + as

BOH ⇋ B+ + OH - (eqn 2)

When a strong acid (eg. HCl) is added to the solution, it dissociates as –

HCl ⇋ H + + Cl-

The released H+ ions are neutralised by the OH - ions present in the solution. This ensures that
the pH remains constant and the buffer functions properly. The equilibrium in eqn 2 shifts
right leading to the release of more OH - ions and thus maintenance of the pH.

When a strong base (eg. NaOH) is added to the solution it dissociates as –

NaOH ⇋ Na+ + OH-

The newly released OH- ions are neutralised by the B+ ions present in the solution and the pH
remains constant, almost. The equilibrium of eqn 2 shifts left leading to a decrease in the OH -
concentration and thus the pH is maintained.

APPLICATIONS OF BUFFER

Due to their ability to resist the change in pH the buffers play a crucial role in those systems
where the pH plays an important role in maintain the very integrity and functioning of the
system.

The most common example is the buffer system in blood. Blood is the liquid transport system
in the body, the proper functioning of which ensures a constant supply of nutrients and O2 to
the tissues and proper removal of CO 2 and wastes from there. The pH of the blood should
theoretically fluctuate as O2 is exchanged to and from the tissues but it doesn’t as a
bicarbonate buffer system is present in the blood. The blood buffer is constituted of a mixture
of carbonic acid (H2 CO3) and bicarbonate ion (HCO3-) which is present in the plasma. This
buffer ensures a constant pH between 7.35 and 7.45. The increase in pH above this range is
called alkalosis and decrease below this range is known as acidosis. Both conditions on the
long term are lethal and require immediate medical attention.

In molecular biology labs artificial buffers are used to preserve the functionality of the
extracted enzymes as these are very sensitive to pH and may even denature the enzyme.
Usually the Phosphate Buffer Saline (PBS) is used for this purpose which maintains the pH at
7.4.

In certain industries like dying and colouring industry the buffers are used to maintain a
constant pH for the dyes to function properly.

Some commonly used buffers in molecular biology are:

1. TRIS (tris(hydroxymethyl)aminomethane) or (2-amino-2-(hydroxymethyl)propane-

1, 3-diol)

2. HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)

3. TES (2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid)

 HENDERSON–HASSELBALCH EQUATION

The Henderson – Hasselbalch equation is used to calculate the pH of a buffer solution. It was
introduced by the American biological chemist Lawrence Joseph Henderson in 1908 but the
equation was mathematically incomplete until in 1917 the Swedish physiologist Karl Albert
Hasselbalch re-introduced this formula in logarithmic terms giving it more mathematical
solidity and correctness.

Generally the equation is expressed as-

[conjugate acid]
pH = pKa + log
[acid]

Here, pH is the negative logarithmic function of H + ion concentration.

pH = -log [H +]

pKa is the negative logarithmic function of the dissociation constant of the weak acid.

pKa = -log [Ka]

[Dissociation product]
Ka =
[weak acid]

The square brackets ([]) means the concentrations of the entities are to be considered.

The equation is based upon four basic assumptions which are to be taken into account each
time the equation is used.

1. The acid or base should dissociate as follows:

HA ⇋ H+ + A-
BOH ⇋ B+ + OH-
Here the species dissociates into only two species one is the conjugate acid or base
and second is the either of H+ or OH- ions. In case the acid is polybasic or the base is
polyacidic, the subsequent Ka values must be differing by 3 for the equation to be
valid on that species.

2. The self ionisation of water is to be not considered while applying the equation. Water
being a polar molecule ionises as –

This assumption not valid after the pH value of 10 or when concentration of solution
becomes less than 10-5 mol/litre as beyond these values there is significant fluctuation
in the pH values due to the ionisation of water.
 3. It is assumed that the salt present in the buffer dissociates completely to form the
respective ions.
MA ⇋ M+ + A-
The Ka of this reaction is 1.

4. The activity coefficient of the entire equilibrium should be constant under

experimental conditions. The activity coefficient is defined as the measure for
deviations from ideal behaviour of a mixture of components. The constant value of
this ensures that there are no unwanted interactions in the reaction mixture apart from
those intended.

DERIVATIONS

For acidic solutions:

Assume a weak acid HA

The acid dissociates as:

HA ⇋ H+ + A-

Here HA is the weak acid with concentration [HA] and A - is its conjugate base with
concentration [A-]

Ka of the reaction would be:

[H+][A−]
Ka =
[HA]

Multiplying log on both sides:

log Ka = log {[H+][A−]

[HA]
}

Multiplying -1 on both sides

-log Ka = -log {[H+][A−]

[HA]
}

Here -log Ka = pKa and -log [H+] is pH.

Rearranging the equation gives:

[HA]
pH = pKa – log
[A−]
Flipping the RHS gives the exact equation for acids.
[A−]
pH = pKa + log
[HA]

OR
[CONJUGATE BASE]
pH = pKa + log
[ACID]

For basic solutions

Assume a weak base BOH

The base dissociates as:

BOH ⇋ B + + OH-

Here BOH is the weak base with concentration [BOH] and B + is the conjugated acid with
concentration [B +].

Kb of the reaction will be:

[OH−][B+]
Kb =
[BOH]

Multiplying log on both sides

log Kb = log {[OH−][B+]

[BOH]
}

Multiplying -1 on both sides

-log Kb = -log {[OH−][B+]

[BOH]
}

Here –log Kb is pKb and –log [OH -] is pOH.

Rearranging equation
[BOH]
pOH = pKb – log
[B+]
Flipping RHS gives the exact equation
[B+]
pOH = pKb + log
[BOH]

OR
[CONJUGATE ACID]
pOH = pKb + log
[BASE]

The main application of the Henderson – Hasselbalch equation is for the calculation of pH of
solutions containing a weak acid or a weak base along with its salt i.e. a buffer solution.
 CONCLUSION
Acids and bases are very significant in chemistry as well as molecular biology. Hence
understanding their chemistry becomes a task of primary importance. The very definition of
acids and bases include two moieties namely H + and OH -. The molecule which can furnish
H+ is an acid and the ones which can furnish OH - are bases, this definition although still
acceptable has its own flaws. There are many molecules which do not have either of these but
function perfectly as acids and bases. This ambiguity was resolved with newer concepts of
acids and bases and this process of refining the definition is still an ongoing process. The
strength of an acid or a base i.e. how acidic an acid is or how basic an base is determined by
the concentrations of H + and OH- ions in the solution media respectively. This gives rise to
the concepts of pH and pOH which are measures of the above stated strength of acids and
bases. Important parameters of acids and bases are their dissociation constants Ka and Kb
respectively which again define the strength of an acid and a base. These although are simply
the equilibrium constants of the dissociation reactions but these are very important as these
are of more practical application in molecular biology while preparing buffer solutions. The
significance of pH and pOH is in the biological domains where the delicate balance of the H +
and OH- concentrations regulates many cellular pathways. Buffers are of great significance
to molecular biologists as these provide a suitable media for extraction of proteins and
enzymes without damaging their biological functionality. The Henderson – Hasselbalch
equation gives us the ability to calculate the pH of a buffer solution thus simplifying the
tedious calculations which are actually required to create a buffer of a give pH. Thus acid and
base chemistry influence every sphere of our lives and it is very important to understand
every aspect of it to successfully harness the potentials of acids and bases.
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