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1 INTRODUCTION

1.1 INTRODUCTION TOPESTICIDES

Pesticides are chemical compounds that are used to kill pests, including insects, rodents,
fungi and unwanted plants. While the term pesticides is now somewhat considered as
synthetic chemical compounds. But some naturally occurring compounds or natural extracts
have been used as pesticides since ancient times Pesticides play a vital role in modern
agriculture by increasing food production, preventing crop loss, and enhancing the quality of
crops. In the face of challenges like pest resistance, climate change, and increasing global
populations, pesticides are an essential tool for farmers. They help ensure that crops grow in
healthy, pest-free environments, which leads to greater yields and fewer crop failures.

The use of pesticides also supports the economy by reducing the damage caused by pests,
increasing food availability, and allowing for the cultivation of diverse crops. Without
effective pesticide management, pest outbreaks could lead to a significant reduction in crop
production, which could, in turn, result in food shortages and economic loss.

Pesticide is a chemical or biological compound or agent (such as bacterium, or fungus)

that deters, incapacitates, kills, or otherwise discourages pests. Target pests
includes insects, plant pathogens, weeds, molluscs, mammals, fish, nematodes
(roundworms) and microbes that destroy property, cause nuisance, or spread disease, or
are disease vectors. Apart from that some pesticides have benefits; some also have
drawbacks, such as potential toxicity to humans and other species and living things.

Pesticides play a vital role in modern agriculture by increasing food production, preventing
crop loss, and enhancing the quality of crops. In the face of challenges like pest resistance,
climate change, and increasing global populations, pesticides are an essential tool for
farmers. They help ensure that crops grow in healthy, pest-free environments, which leads to
greater yields and fewer crop failures.

The use of pesticides also supports the economy by reducing the damage caused by pests,
increasing food availability, and allowing for the cultivation of diverse crops. Without
effective pesticide management, pest outbreaks could lead to a significant reduction in crop
production, which could, in turn, result in food shortages and economic loss.

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1.1.1 CLASSIFICATION OF PESTICIDES

The Pesticides can be classified in many ways on the basis of use, toxicity, mode of
entry, mode of action, chemistry and formulation. Some of them are described as
follows.
[Link]. Classification on the basis of use/ application: Refer Table1.1
Table 1.1: Classification of pesticides on the basis of use/ application
Class Use Example
To kill mites and ticks, or to
Acaricides disrupt their growth or Abamectin, Chlorpyrifos.
development
Algicide To kill or inhibit algae Diuron, Isoproturon
Prevent an insect or other pest
Anifeedants Azadirachtin
from feeding
Avicides To kill birds Fenthion, strychnine
To kill or inhibit bacteria in plants
Bactericides Streptomycin, Tetracycline.
or soil (derived from Bactria)
Bird
Act as the bird repellents Diazinon, Thiram,
repellents
Used to prevent, cure eradicate the Prothioconazole, Tebuconazole,
Fungicides
fungi Metalaxyl, Chlorothalonil
To kill plants, or to inhibit their
Herbicides Clodinafop
growth or development propargyl ,Phenmedipham,
Pendimethalin,
Lures pests to a trap, thereby
Insect
removing them from crops, Gossyplure, Gyplure, Muscalure
attractants
animals or stored products.
Insect Deters an insect from landing on a
Citronella oil, Permethrin
repellents human or an animal.
Insect
Disrupting the growth or
Growth Diflubenzuron, Buprofezin
development of an insect
regulator
Used to kill insects, or to disrupt
Insecticides Lambdacyhalothrin ,Bifenthrin
their growth or development Cypermethrin
Deters mammals from
Mammal
approaching or feeding on crops Ziram,Trimethacarb
repellents
or stored products.
Molluscicides Used to kill slugs and snails Copper sulfate.
Nematicides Used to control Nematicides Abamectin,
Plant growth Which alter the expected growth 2,4-D,chlormequat,

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regulators and flowering/fruiting or Paclobutrazol.

reproduction rate of plants.
Rodenticides To kill rats and related animals Zinc phosphide,Bromadiolone.
Fumigant Use to control rodents Aluminium Phosphide

[Link]. Classification on the basis of theChemistry

Pesticides classified on the basis of the chemistry are as follows, refer Table1.2
Table 1.2: Classification on the basis of chemistry

A) Insecticides
1 Organo halogen 5 Neonicotinoids 9 Pyrroles
Organophosphorus
2 6 Spinosyns 10 Benzoylureas
3 Carbamates 7 Neriestoxin 11 Quinazolines
Fiproles/
4 Pyrethroids 8 12 Antibiotics
Phenylpyrazoles
B) Fungicides
1 Aliphatic nitrogen 7 Morpholine 13 Sulphur compounds
2 Amide 8 Aromatic 14 Dicarboximide
Conazole/
3 Acylamino acid 9 15 Dithiolane
Triazoles
4 Anilide 10 Copper 16 Dinitrophenol
Carbamate / Methoxyacrylat Dichlorophenyldicarboxi
5 11 17
benzimidazole e strobilurin mide
6 Antibiotic 12 Dithiocarbamate

C) Herbicides

1 Anilide 7 Imidazolinone 13 Quaternary ammonium

Chloroacetanili Organophosphor
2 8 14 Pyrimidinyloxybenzoic acid
de us
3 Triazolone 9 Urea 15 Aryoxyphenoxypropionic
4 Benzothiazole 10 Phenoxyacetic 16 Halogenated aliphatic
5 Dinitroaniline 11 Chlorotriazine 17 Phenylurea
Nitrophenyl
6 12 Sulfonylurea
ether
D) Rodenticides
1 Inorganic 2 Organic

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[Link]. Classification bassed on Chemical Composition

Category Chemical Composition Examples Characteristics

Derived from natural Neem oil, Pyrethrin,
Often less toxic to humans
Organic substances such as Bacillus thuringiensis
and animals, biodegradable,
Pesticides plants, animals, or (Bt), Diatomaceous
and more eco-friendly
microorganisms earth
Made from inorganic Borax, Copper
Generally more toxic, less
Inorganic compounds such as sulfate, Sodium
selective, persistent in the
Pesticides minerals, metals, and cyanide, Lead
environment
salts arsenate
More effective, often highly
Glyphosate, DDT,
Synthetic Chemically synthesized toxic, persistent, and
Chlorpyrifos,
Organic from organic chemicals sometimes more resistant to
Malathion,
Pesticides or petroleum derivatives breakdown in the
Pyrethroids
environment
Derived from natural Bacillus thuringiensis
Environmentally friendly,
organisms such as (Bt), Spinosad,
Biopesticides typically less toxic, and used
bacteria, fungi, or plant Trichoderma,
in organic farming
extracts Pyrethrum
Usually less toxic,
Neem oil, Eucalyptus
Plant-Based Derived from plant biodegradable, and have
oil, Citronella,
Pesticides extracts or essential oils natural insect-repelling
Tobacco extract
properties
[Link] Classification bassed onMode of Action and Chemical Class

Chemical Class Mode of Action Examples Comments

Highly persistent, toxic,
Interfere with nerve DDT, Lindane,
and banned in many
Organochlorines function, causing Chlordane,
countries due to
paralysis in pests Heptachlor
environmental impact
Inhibit the enzyme Malathion,
Highly toxic to humans
acetylcholinesterase, Chlorpyrifos,
Organophosphates and wildlife, but effective
affecting the nervous Diazinon,
for pest control
system Parathion
Similar to
Also inhibit Carbaryl,
organophosphates but less
Carbamates acetylcholinesterase in the Methomyl,
toxic to humans, though
nervous system Propoxur
still hazardous
Synthetic analogs of
Affect the nervous system Permethrin,
pyrethrins, more stable
Pyrethroids by disrupting nerve Cypermethrin,
and effective in pest
impulses Deltamethrin
control
Effective against insects,
Mimic nicotine, Imidacloprid,
but concern over
Neonicotinoids disrupting nerve function Thiamethoxam,
environmental impact,
in insects Acetamiprid
especially on pollinators

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Chemical Class Mode of Action Examples Comments
Inhibit plant enzyme Clodinafop Targeted at plants, with
processes, especially propargyl ,Glyphos varying toxicity levels
Herbicides
involved in amino acid ate, Atrazine, depending on the
synthesis Dicamba compound
Target fungi that cause
Mancozeb,
Inhibit fungal cell growth plant diseases, varying
Fungicides Chlorothalonil,
or reproduction toxicity to humans and
Tebuconazole
animals
Often target coagulation Warfarin, Used for controlling
Rodenticides or inhibit metabolic Bromadiolone, rodents, usually toxic and
processes Zinc phosphide require careful application
Target bacterial
pathogens in plants and
Inhibit bacterial cell Copper sulfate,
Bactericides animals, some are used as
function or growth Streptomycin
medicinal treatments for
animals
[Link] Classification bassed on Environmental Persistence
Category Chemical Composition Examples Characteristics
Remain in the Can accumulate in the food
Persistent DDT, Aldrin,
environment for chain and cause long-term
Pesticides Chlordane
extended periods environmental damage
Malathion, Break down within a few days
Non-Persistent Break down quickly in
Pyrethroids, to weeks, considered more
Pesticides the environment
Chlorpyrifos eco-friendly
Degrade naturally in the Bacillus Environmentally friendly,
Biodegradable
environment through thuringiensis (Bt), break down into non-toxic
Pesticides
microbial activity Spinosad components

[Link] Classification bassed on Chemical Structure

Chemical
Type of Pesticide Examples Mode of Action
Structure
Effective against a wide
Aromatic Organic chemicals with an
DDT, Lindane range of pests, persistent
Pesticides aromatic ring structure
in the environment
Organic chemicals without Used primarily for insect
Aliphatic Malathion,
an aromatic ring (alkanes, control, decompose more
Pesticides Pyrethroids
alkenes) easily than aromatic types
Organic chemicals with a Target specific pests by
Imidacloprid
Heterocyclic ring structure containing interfering with their
(neonicotinoid),
Pesticides atoms other than carbon nervous system or
Fipronil
(nitrogen, oxygen) metabolism

[Link] Classification bassed on Toxicity Level (Chemically Induced)

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Chemical
Toxicity Level Examples Characteristics
Composition
Made with chemicals Can cause severe damage or
Highly Toxic Cyanide, Methyl
that have a high degree death to humans, animals, and
Pesticides bromide, Aldrin
of toxicity the environment
Malathion, Toxic to pests but less so to
Moderately Chemicals with
Chlorpyrifos, humans and animals with
Toxic Pesticides moderate toxicity
Diazinon proper handling
Chemicals with low Neem oil, Generally safer for humans
Low Toxicity
toxicity to humans and Diatomaceous earth, and animals, and can be used
Pesticides
animals Insecticidal soap in organic farming
WHO recommended classification of pesticides on basis of hazard/Toxicity?
Table 1.3: Classification of pesticides according to toxicity, (expressed as LD50)

Class Toxicity LD50 for the rat (mg/kg)

Oral Dermal
Class IA Extremely dangerous <5 < 50
Class IB Highly dangerous 5–50 50–200
Class II Moderately hazardous 50–2000 200–2000
Class III Slightly hazardous Over 2000 Over 2000
Class U Unlikely to present acute hazard 5000 or higher

1.2 INTRODUCTION TOCHIRAL MOLECULES

A chiral molecule is defined as a molecule that is not superimposable on its mirror
[Link] two mirror images forms of a chiral molecule are called [Link]
have identical physical and chemical properties. Except for the direction in which they rotate
the plane of polarized light. Achiral molecule has one or more stereogenic centre which are
typically [Link] stereiogenic carbon attached to four different substituents.

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The two stereoisomers are refered to as (+) and (-) or dextro and levo isomers respectively
based on their direction of optical rotation under certin defined conditions. Chiral molecules
can rotate the plane of polarized light, a phenomenon known as optical activity. The direction
and magnitude of this rotation depend on the specific enantiomer. One enantiomer may rotate
light in a clockwise direction (dextrorotatory), while the other rotates light counterclockwise
(levorotatory).The actual configuration in space at the steregenic (chiral) center is designated
as R or S. The R means Rectus in Latin (means right) and S means Sinister in Latin (means
Left). Molecules that rotate the plane polarised light to right is said as R isomer. Molecule
that rotate the plane polarised light to left is said as S isomer.

Chirality plays an essential role in the design and function of agrochemicals, which include
pesticides, herbicides, fungicides, and other chemicals used in agriculture. In agrochemicals,
chirality can significantly influence the effectiveness, toxicity, and environmental impact of a
compound. A molecule is chiral if it has a non-superimposable mirror image, which can lead
to the existence of two distinct enantiomers (R and S isomers), each with potentially different
properties.

Many agrochemicals are chiral, meaning they contain one or more chiral centers. The
specific configuration (R or S) of these chiral centers can influence how the molecule
interacts with biological targets such as enzymes, receptors, and other molecules in plants,
pests, or microbes.

Chirality is an important factor in agrochemical design and application. By focusing on the

specific enantiomer of a chiral compound, agrochemicals can become more selective, potent,
and environmentally friendly. This can lead to better pest control, reduced toxicity, and a
smaller ecological footprint. As research advances, more chiral agrochemicals may be
developed with optimized enantiomeric forms for improved agricultural sustainability.

Selectivity and Efficiency: One enantiomer of a chiral agrochemical may be more effective
in controlling a pest or plant disease than the other. In some cases, one enantiomer may even
be inactive, while the other is highly active. This means that understanding and controlling
chirality in agrochemical molecules can improve the selectivity and efficiency of these
chemicals, reducing the amount needed for effective action.

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Reduced Environmental Impact: By using the most effective enantiomer, farmers can
minimize the overall use of chemicals in agriculture, which reduces the environmental
impact. The less-active enantiomer might not only be ineffective but could also be more toxic
to non-target organisms. Therefore, using a chiral agrochemical in its optimal form can
decrease the environmental footprint.

Increased Potency: Using a single, active enantiomer can make a pesticide or herbicide
more effective at lower doses, which can help reduce the overall chemical load on the
environment.

Reduced Toxicity to Non-Targets: By selecting or designing the most effective enantiomer,

agrochemical companies can help minimize the toxicity of their products to humans, animals,
and beneficial insects like bees.

Better Environmental Persistence: One enantiomer may degrade more rapidly in the
environment than the other, so the proper use of the correct enantiomer can help ensure that
chemicals break down quickly and safely, reducing long-term environmental impact.

Chirality plays a significant role in the effectiveness and environmental impact of

agrochemicals across different classes of chemicals:

Classifcation of Chiral molecule

 Fungicides: Examples include tebuconazole and fluconazole, where different

enantiomers can show varying levels of antifungal activity.
 Herbicides: Clodinafop prpargyl Glyphosate and dicamba are chiral herbicides that
rely on specific enantiomers to control weeds effectively.

Insecticides: Pyrethroids (like Chirality plays a significant role in the effectiveness

environmental impact of agrochemicals across different classes of chemicals

 Fungicides: Examples include tebuconazole and fluconazole, where different

enantiomers can show varying levels of antifungal activity.
 Herbicides: Glyphosate and dicamba are chiral herbicides that rely on specific
enantiomers to control weeds effectively.

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 Insecticides: Pyrethroids (like permethrin) and fipronil are chiral insecticides, with
enantiomers affecting their insecticidal potency) and fipronil are chiral insecticides,
with enantiomers affecting their insecticidal potency.

Pyrethroids: Pyrethroids are a class of synthetic insecticides that are structurally related to
pyrethrins, natural insecticides derived from chrysanthemums. Many pyrethroids are chiral,
and their insecticidal activity is highly dependent on the specific enantiomer used. For
instance, some enantiomers may be more toxic to insects but less harmful to mammals,
which makes the selective use of one enantiomer advantageous.

Glyphosate: Glyphosate, one of the most widely used herbicides, is an example of a chiral
molecule. It is a racemic mixture, containing equal parts of both enantiomers. The active
ingredient in glyphosate (the enantiomer that interacts with the target enzyme in plants) is
only one of the two enantiomers, while the other may have lesser or no herbicidal activity.

Clodinafop-propargyl: Clodinafop propargyl is most widely used herbicide andchiral

molecule, meaning it has a specific 3D configuration that can exist in two enantiomeric forms
(R and S). These enantiomers are non-superimposable mirror images of each other. In the
case of clodinafop-propargyl, the R-enantiomer is typically the more active form responsible
for the herbicidal action.. It effectively targets and inhibits the ACCase enzyme in grassy
weeds, making it a selective herbicide. The S-enantiomer, on the other hand, may not exhibit
the same level of activity or could even be inactive in terms of herbicidal [Link]
herbicide is selective for grasses and does not affect broadleaf plants, which makes it useful
for weed control in cereal crops.

Prothioconazole:Prothioconazole is a used primarily as a fungicide in agriculture. It belongs

to the class of triazole fungicideschiral moleculeand is effective against a wide range of
fungal pathogens, including those that cause diseases in crops like wheat, barley, corn, and
[Link] is a chiral triazole fungicide used to protect crops from a variety of

fungal diseases. It contains chiral centers in its structure, resulting in the potential for
different enantiomers to have varying levels of biological activity. The fungicide works by
inhibiting ergosterol biosynthesis in fungi, disrupting their cell membranes. The specific
enantiomeric composition of prothioconazole in formulations can influence its effectiveness

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and environmental impact, making the study of chirality important in its development and
use.

Fungicides like tebuconazole and fluconazole are often chiral compounds. The different
enantiomers can have varying levels of activity against fungal species, and, as with other
agrochemicals, selecting the correct enantiomer for the intended target organism can improve
effectiveness.

1.1.2. PESTICIDE FORMULATIONS

Formulation, in a general sense, refers to the process of creating a specific mixture or

combination of substances to achieve a desired outcome. In the context of pesticides,
formulation specifically refers to the mixture of an active ingredient with other chemicals
or substances to make the product suitable for [Link] process includes adding
carriers, solvent s, stabilizers, surfactants, and other additives to the active ingredient to
improve

Pesticide formulations refer to the physical form in which a pesticide is prepared and
presented for use. The formulation determines how the pesticide is applied, how it
behaves during application, and its effectiveness. There are various types of pesticide
formulations, each designed to optimize performance, ease of use, safety, and
environmental impact.

A pesticide formulation typically consists of several components, each serving a specific

purpose to ensure the pesticide's effectiveness, stability, and safe application. These
components may include:

1. Active Ingredient (AI):

The chemical compound or substance responsible for controlling or eliminating

the pest. This is the primary function of the pesticide.

In pesticide formulations, other ingredients (also referred to as inert ingredients,

additives, or excipients) play a crucial role in ensuring the product’s stability,
efficacy, ease of application, and safety. These ingredients don't have direct pesticidal
activity but are vital for enhancing the performance, handling, and delivery of the
active ingredient (AI).
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Here’s a breakdown of the main types of other ingredients found in pesticide
formulations:

1. Solvents

 Purpose: Solvents are used to dissolve or disperse the active ingredient in the
pesticide formulation, making it easier to apply. They help to ensure that the pesticide
can be evenly distributed during spraying or other application methods.
 Common Types:
o Water (for aqueous formulations like emulsifiable concentrates).
o Organic solvents such as acetone, methanol, xylene, or toluene.
 Example: In an emulsifiable concentrate (EC), the solvent helps to dissolve the active
ingredient and enables it to mix with water during application.

2. Surfactants (Emulsifiers or Wetting Agents)

 Purpose: Surfactants help to improve the spreading, wetting, and adhesion of the
pesticide on the target surface (e.g., plant leaves or soil). They also enable the active
ingredient to mix properly with water in formulations like emulsions and suspensions.
 Types:
o Non-ionic surfactants (e.g., alkylpolyglycosides).
o Anionic surfactants (e.g., sodium lauryl sulfate).
o Cationic surfactants (e.g., cetyltrimethylammonium bromide).
 Example: Surfactants in herbicide formulations help the chemical stick to plant
leaves and improve the herbicide's penetration.

3. Stabilizers

 Purpose: Stabilizers are added to pesticide formulations to prevent degradation of the

active ingredient over time. They help to preserve the shelf life and ensure that the
pesticide maintains its potency.
 Types:
o Antioxidants: Prevent degradation due to oxidation (e.g., ascorbic acid,
butylated hydroxyToluene).

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o UV stabilizers: Protect the active ingredient from breaking down when
exposed to sunlight.
o pH stabilizers: Maintain an appropriate pH for the formulation.
 Example: UV stabilizers in pesticide sprays help protect the active ingredient from
light-induced degradation, ensuring it remains effective.

4. Fillers and Bulking Agents

 Purpose: Fillers and bulking agents are used to increase the volume of the
formulation, making it easier to handle, apply, and store. These ingredients may also
help to dilute highly concentrated pesticides to ensure safe application.
 Types:

Talc: Commonly used in wettable powders (WP) as a filler.

Clay: Used in formulations like dusts or granules for bulkiness and ease of
application.

Silica: Often used in wettable powders and as a carrier in some granule

formulations.

 Example: Talc or kaolin clay can be used in fungicide wettable powder formulations.

5. Dispersing Agents

 Purpose: Dispersing agents help the active ingredient to evenly disperse in liquids,
preventing the pesticide from settling or clumping together. These are particularly
important in suspension concentrates (SC) and emulsifiable concentrates (EC).
 Example: Polyvinyl alcohol (PVA) is commonly used in suspension concentrates to
ensure even dispersion of solid particles.

6. Preservatives

 Purpose: Preservatives are added to prevent microbial growth (fungi, bacteria, etc.)
in liquid formulations. They help to extend the product's shelf life, especially for
water-based formulations.

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 Example: Methylparaben and propylparaben are common preservatives used to
prevent bacterial or fungal contamination in aqueous pesticide formulations.

7. Thickeners

 Purpose: Thickeners or gelling agents are used to increase the viscosity of liquid
formulations, making them easier to apply and less likely to drip. They can also
improve the stability of emulsions and suspensions.
 Types:
o Xanthan gum: A natural polysaccharide that thickens liquid formulations.
o Cellulose derivatives: Used to modify the viscosity of liquid formulations.
 Example: Thickeners in gel formulations (like for termite control or spot treatment)
help the product stay in place on vertical surfaces.

8. Antifoaming Agents

 Purpose: Antifoaming agents are added to prevent excessive foam formation during
the mixing and application of pesticide formulations. Foam can interfere with the
proper application of the pesticide and reduce its effectiveness.
 Types:
o Silicone-based antifoams: Very effective at controlling foam.
o Organic oils: Also used to reduce foam in pesticide formulations.
 Example: Silicone-based antifoams in pesticide sprayers ensure smooth,
uninterrupted application.

9. Colorants and Dyes

 Purpose: Colorants are often added to make the pesticide visually distinctive,
allowing users to see where the pesticide has been applied and ensuring proper
coverage. They also help with product identification and regulatory compliance.
 Example: Fluorescent dyes or food-grade colorants like brilliant blue or green
dyes used in pesticide formulations for easier identification.

10. Aromatic Compounds

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 Purpose: Some formulations include aromatic compounds to mask the strong or
unpleasant odors of the active ingredients, making the pesticide easier and more
pleasant to use.
 Example: Lemon or pine scents added to insecticides to improve user experience,
especially in household pest control products.

11. Buffering Agents

 Purpose: Buffering agents are added to adjust and maintain the pH of the pesticide
formulation to optimize the stability of the active ingredient and enhance its
performance.
 Example: Citric acid or sodium carbonate to maintain the pH for certain
pesticide formulations.

Pesticide active ingredients have a range of solubility’s. Some dissolve readily

available in water, others only in oils. Some active ingredients may be relatively
insoluble in either water or in oils. Solubility properties and use of the pesticide
generally define which formulations were best deliver the active ingredient.
The basic chemical terminology is helpful in understanding difference between
various types of formulations.

 Solution: When a substance dissolved in a liquid known as a solution. A True

solution cannot be separated mechanically into the individual components
present in it. Once mixed, a solution does not require any type agitation to keep
into its various parts from settling. Frequently, Solution having transparent in
appearance.
 Suspension: A mixture of finely divided, solid particles dispersed in a liquid
known as Suspension. The solid particles do not dissolve in the liquid, and the
mixture must be agitated (continuously or time to time) to keep the particles
distributed evenly. Most of the times suspension has a cloudy, murky in
appearance.

 Emulsion: When one liquid is dispersed (as droplets) in another liquid is known
as Emulsion. Each liquid retains its own identity. To keep emulsion from
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separating agitation is required to some extent. Usually Emulsions have milky
appearance. When product is mixed with water, an emulsion (oil in water) is
formed. An emulsifying agent (emulsifier) formulated into the product which
helps to prevent the emulsion fromseparating.

[Link] Types of Pesticideformulations

Types of formulations are available in market – Liquid formulations, Dry or
Solid formulations and otherformulations.

A) LIQUIDFORMULATIONS
These are generally mixed with water, but in some times crop oil, diesel,
kerosene or some other light oil used as carrier.
a) Emulsifiable Concentrates (EC orE):
These formulations usually contain a liquid active ingredient, one or more
petroleum-based solvents which give EC formulation their strong odour, and an
emulsifying agent that allows formulation to be mixed with water to form emulsion.
b) Solutions(S):
Some pesticide active ingredients dissolve readily in a liquid carrier such as
water or petroleum-based solvent and they form a solution which does not settle out
or separate into layers.

c) InvertEmulsions:
It contains a water-soluble pesticide dispersed in oil as carrier. Invert
emulsion require a special kind of emulsifier that allows the pesticide to mix with a
large volume of petroleum-based carrier as fuel oil.
d) Flowables (F)/ Liquids(L):
These formulations combine many of the emulsifiable concentrates and
wettable powders. When the A.I. is a solid powder and that does not dissolve in
either of water or oil, then the A.I. is impregnated on a substance such as clay, is
ground to a very fine powder. This powder is then suspended in a small amount of
liquid carrier.
e) Aerosols(A):
These formulations contain one or more A.I. and a solvent. Most aerosols
contain a low percentage of A.I.
f) LiquidBaits:
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Liquid baits consist of insecticides and rodenticides which are being
formulated as baits.

B) DRY OR SOLIDFORMULATIONS
These formulations can be divided into two types: ready-to-use and concentrates
that must be mixed with water to be applied as spray.
a) Dusts(D):
These formulation are ready to use and contain a low percentage of A.I.
(10% or less by weight), and a very fine, dry inert carrier usually talc, chalk, clay,
nut bulls or sometimes volcanic ash. A few dust formulations contain a high
percentage of active ingredients which are used in seedtreatments.
b) Tracking powder:
Tracking powders are special dust used for control and monitoring rodent
and insect.
c) Baits (B):
When an A.I. is mixed with food or another attractive substance is known as
baits. They either attract the pest or placed where the pest will find it. These baits
when eaten by pests they got killed as it contains pesticides.

d) Pastes, Gels and Other injectablebaits:

These are mainly used in the pest-control industry for control and killing
ants and cockroaches. They are designed to be injected or placed as dot inside small
cracks and crevices of building where insects tend tohide.
e) Granules(G):
These formulations are like dust formulations, except granular particles are
larger and heavier than dust particles. Clay, corncobs or walnut shells are used to
make coarse particles. The A.I. either coats on the outer wall of the granules or is
readily absorbed into them. Usually about 1 to 15% by weight A.I. is present. Once
applied, granules release the A.I. slowly.
f) Pellets (P orPS):
These formulations are very similar to granular formulations: the terms are
used interchangeably. In this type of formulations, all the particles are of the same
dimension (weight and shape).

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g) Wettable Powders (WP orW):
WP is dry, finely ground formulations that look like as dusts formulations.
Usually they are mix with water for application as a spray. WP contains about 5 to
95 % A.I. by weight: usually 50% or more.
h) Soluble Powders (SP orWSP):
SP formulation looks like WP. However, when mixed with water, SP
dissolve readily and form a true solution. Usually 15 to 95 % by weight A.I. is
present.
i) Water-dispersible Granules (WDG) or Dry Flowables(DF):
WDG also known as DF, are like WP, except instead of being dust like, they
are formulated as small, easily measured granules. WDG must be mixed with water
to be applied. Once in the water, the granules break apart into fine particles similar
to WP. Constant agitation is required to keep suspended in water. High percentage
of A.I. often as much as 90% by weight ispresent.
C) OTHERFORMULATIONS
They include chemicals that cannot be clearly classified either as liquid or as
dry/solid pesticide formulations.

a) Microencapsulatedmaterials:
Liquid or dry pesticide particles covered in a polymer coating to produce
microencapsulated formulations.
b) Water solublepackets:
They reduce the mixing and handling hazards of highly toxic pesticides.
These bags when drop into a filled spray tank, they readily dissolve and release their
contents to mix with the water.
c) Attractants:
Attractants includes pheromones, a chemical which is secreted by an
animal, especially an insect, which influence the behaviour or developments of
others of the same species. Attractants were used in sticky traps and capturebags.
d) Repellents:
Various types of insect repellents are available market in the form of
aerosols and lotion formulations. These can apply on skin or clothing or to plant
foliage to repelinsects.

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e) Fumigant:
Fumigant forms a gas when applied. Some liquid A.I. is packed under high
pressure and changes to gases when they are released. Used for structural pest
control, in food facilities, in soil, greenhouse.

Table 1.4. Codes for pesticide formulations

AE Aerosol dispenser GA Gas
BR Briquette GL Emulsifiable gel
CB Bait Concentrate GR Granules
CP Contact Powder GS Grease
CS Capsule Suspension GW Water soluble gel
DC Dispersal Concentrate LS Solution for seed treatment
DP Dustable powder MC Mosquito coil
DS Powder for dry seed treatment ME Microemulsion
EC Emulsifiable concentrate OF Oil miscible flowable
EG Emulsifiable granule OL Oil miscible liquid
EO Emulsion, water in oil OP Oil dispersible powder
EP Emulsifiable powder PA Paste
ES Emulsion for seed treatment PS Seed coated with pesticides
EW Emulsion, oil in water RB Bait (ready to use)
SE Suspo-emulsion TC Technical material
FS Flowable concentrate for seed WS Water dispersible powder for
treatment slurry treatment
SG Water soluble granule TK Technical concentrate
SL Soluble concentrate UL Ultra-low volume (ULV) liquid
SP Water soluble powder WG Water dispersible granule
ST Water soluble tablets WP Wettable powder
SC Suspension concentrate WT Water dispersible tablet

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1.2. CHROMATOGRAPHY

1.2.1. HISTORY OFCHROMATOGRAPHY

Chromatography is a fundamental technique in scientific research and industrial

applications. With various types of chromatography available, it's a powerful tool
for separating, identifying, and quantifying components in complex mixtures.

In chemical analysis some methods are truly specific to a particular analyte only. The
analyte of interest must be separated from the sample matrix into individual
components that may be present in sample. Chromatographic technique is single step
processes which simultaneously separates a mixture into its individual components
and provide a quantitative amount of each constituent. Samples may be in gaseous,
liquid or solid nature and can be in a simple mixture to a multi component mixture of
differing [Link] allows for high-resolution separation of complex
[Link]’s versatile and can be used with many types of samples, from liquids and
gases to solids.
Mikhail Tsvet1903 Russian botanist. He developed the first chromatography
method to separate plant pigments (like chlorophyll) in a column of calcium
carbonate. He referred to this technique as "chromatography," from the Greek words
"chroma" (color) and "grapho" (to write), as the pigments would form distinct color
bands as they traveled through the column. This was the first demonstration of the
principle of separating substances based on their physical or chemical
[Link] colour has little to do with modern chromatography, the name
has persisted and is still used for all separation techniques, which consist of a mobile
phase and a stationaryphase.

1930s-1940s: After Tsvet's pioneering work, chromatography largely remained

limited to the field of plant biology. However, during this time, several researchers
made advancements in the technique, including improvements in the preparation of
stationary phases and new types of solvents, which expanded its use for separating
more complex [Link] their research paper, they recommended replacing the
liquid mobile phase by a suitable carrier gas, as the transfer of sample between the
two phases will become faster, and thus provide more efficient separations

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1940s: Richard Synge and Archibald Martin made significant contributions by

developing the partition chromatography method, where a liquid stationary phase is
used in conjunction with a mobile phase. Their work earned them a Nobel Prize in
1952.
1950s: The invention of gas chromatography (GC) by A.J.P. Martin and R.L.
Miles helped expand chromatography's role in analytical chemistry, particularly for
volatile substances. It was a breakthrough for separating gases and vapors. it was
nearly after four decades later this concept was taken seriously and the introduced
high efficiency liquid chromatography columns became a reality. Today,
chromatography is become an extremely versatile technique; it can separate gases,
and volatile substances by GC, Non-volatile chemicals and materials which having
extremely high molecular weight (including biopolymers) by LC and very
inexpensively by TLC. All these three techniques, GC, LC and TLC having common
features which classify them as chromatography systems.

1960s: The introduction of high-performance liquid chromatography (HPLC)

dramatically increased the resolution and efficiency of liquid chromatography.
HPLC replaced traditional gravity-fed column chromatography with more
sophisticated systems using high pressure to force solvents through a packed column,
allowing for faster and more reproducible results.

1980s-1990s: Technological advancements in detectors (such as mass spectrometry)

allowed for the combination of chromatography with other analytical techniques,
improving sensitivity and specificity for identifying components in complex
mixtures.
Chromatography has been defined as follows,Chromatography is a separation
technique used to separate and analyze mixtures of substances. It involves passing a
mixture through a stationary phase (a solid or liquid) and a mobile phase (a liquid or
gas). Different components of the mixture move at different rates due to their
varying interactions with the stationary and mobile phases, leading to the separation
of the [Link] simple terms, chromatography is a method for separating the
individual components of a mixture based on their physical or chemical properties.
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Those components held preferentially in the stationary phase are retained longer in
the system than those that are distributed selectively in the mobile phase. As a
consequence, solutes are eluted from the system as local concentrations in the
mobile phase in the order of their increasing distribution coefficients with respect to
the stationary phase; ipso facto a separation is achieved

1.2.2. CLASSIFICATION OFCHROMATOGRAPHY

Chromatography can be classified in many ways based on the mobile phase (liquid,
gas, or supercritical fluid), the stationary phase (solid, liquid, or gel), the mechanism of
separation (adsorption, partition, ion-exchange, etc.), and the detection method. The
method chosen depends on the sample's properties and the specific separation
[Link] can be classified in several ways based on the nature of
the stationary phase, the mobile phase, and the mechanism of separation

Summary Table of Types of Chromatography

Type of Stationary Mobile

Principle Examples/Applications
Chromatography Phase Phase
Solid (e.g.,
Adsorption Differential Column chromatography,
silica gel, Liquid or gas
Chromatography adsorption TLC
alumina)
Liquid
Partition Differential phase on Liquid Paper chromatography,
Chromatography partitioning solid solvent HPLC
support
Ion- Separation of proteins,
Ion-Exchange Separation by Liquid
exchange peptides, and inorganic
Chromatography ion exchange solvent
resin ions
Porous
Size-Exclusion Separation by material
Liquid Gel filtration, protein, and
Chromatography size (molecular (e.g.,
solvent polymer separation
(SEC) weight) polystyrene
beads)
Ligand
Specific Protein purification,
Affinity attached to a Liquid
molecular immunoaffinity
Chromatography solid solvent
interactions chromatography
support
Solid
High-pressure Pesticide ,Pharmaceutical
particles Liquid
HPLC liquid , environmental, and food
packed in a solvent
chromatography analysis
column

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Type of Stationary Mobile
Principle Examples/Applications
Chromatography Phase Phase
Solid or
Gas Separation Volatile compounds,
liquid phase Inert gas
Chromatography based on essential oils, food, and
(e.g., silica (e.g., helium)
(GC) volatility environmental analysis
gel)
Supercritical Solid or Separation of lipids,
Separation with Supercritical
Fluid liquid phase pharmaceuticals, and
supercritical CO₂ +
Chromatography (e.g., silica high-molecular
fluids modifiers
(SFC) gel) compounds
Capillary
Capillary Separation by Protein, peptide, DNA
column Electrolyte
Electrophoresis size-to-charge analysis, small-scale
(uncoated or solution
(CE) ratio separation
coated)
Separation
Thin Layer Thin layer Solvent or Qualitative analysis of
based on
Chromatography of silica gel solvent organic compounds,
differential
(TLC) or alumina mixture monitoring reactions
affinity

Chromatography can be classified by various types as follows

[Link] On the basis of interaction of solute to the stationary phase
[Link] On the basis of chromatographic bed shape
[Link] Techniques by physical state of mobilephase
[Link] Special Chromatographic Techniques.

[Link]. On the basis of interaction of solute to stationaryphase

1) AdsorptionChromatography

Adsorption Chromatography is based on the principle that some solid substances,

which are known as adsorbent, have the power to hold molecules at their surface.
This holding force is due to weak, non-ionic attractive forces of the van der Waals'
and hydrogen bonding, which only occur at specific adsorption bed. The
equilibrium between the mobile phase and stationary phase for the separation of
differentsolutes.

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Figure 1.1: Adsorption phenomenon in chromatography

2) PartitionChromatography

Partition chromatography is based on differential partitioning of components of a

sample mixture between two phases – stationary phase and mobile phase. Most
commonly it is used in paper chromatography which is a type of partition
chromatography. In paper chromatography a paper is used which is called
chromatography paper, and works as a stationary phase. This paper is made up of
cellulose which is a polar substance. Mobile phase is liquid which runs on
chromatography paper. . Solute distributes themselves between the mobile phase
and the stationary liquid. Solutes which is more soluble in mobile phase, elutes
first than less

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Figure 1.2: Partition phenomenon in chromatography

3) Ion ExchangeChromatography

This method relies on the ionic interactions between the charged stationary phase and
the ions in the sample [Link] exchange chromatography is similar to partition
chromatography which has coated solid as stationary phase. A resin which acts as the
stationary solid phase is used to covalently attach anions or cations onto it. The
opposite charge solute ions in the mobile liquid phase are attracted to the resin by
electrostatic forces.

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Figure 1.3: Ion Exchange phenomenon in chromatography

4)Molecular ExclusionChromatography

Separation is based on the molecular size and the exclusion of larger molecules from the
pores of the stationary phase. Size-exclusion chromatography, also known as molecular sieve
chromatography. Also known as gel permeation or gel filtration chromatography, this type of
chromatography no equilibrium state is established between the stationary phase and solute.
A porous gel separates the molecules according to its size when the liquid or gaseous phase
passes through it. The pores of gel are normally small size and exclude the larger size solute
molecules, but allow smaller size molecules to enter the gel, causing them to flow through a
larger volume. This causes the larger size molecules to pass through the column at a faster
rate than the smaller ones. Thus, separation occurs according to particle size ofmolecules.
Size exclusion chromatography (SEC) separates molecules based on their size by filtration
through a gel. The gel consists of spherical beads containing pores of a specific size
distribution. Separation occurs when molecules of different sizes are included or excluded
from the pores within the matrix. Small molecules diffuse into the pores and their flow through the
column is retarded according to their size, while large molecules do not enter the pores and are
eluted in the column's void volume. Consequently, molecules separate based on their size as they
pass through the column and are eluted in order of decreasing molecular weight.

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Figure 1.4: Molecular Exchange phenomenon in chromatography

5) Affinity Chromatography

In affinity chromatography, the stationary phase contains a ligand that specifically binds
to a target molecule (such as an antibody, enzyme, or protein). This allows for highly
specific separations, often used for purifying proteins and [Link] stationary
phase consists of a support medium, on which the substrate (ligand) is bound covalently,
in such a way that the reactive groups that are essential for binding of the target molecule
are [Link] the crude mixture of the substances is passed through the chromatography
column, substances with binding site for the immobilized substrate bind to the stationary
phase, while all other substances is eluted in the void volume of the [Link] the
other substances are eluted, the bound target molecules can be eluted by methods such as
including a competing ligand in the mobile phase.

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Figure 1.5: Affinity Chromatography phenomenon in chromatograph

[Link]. On the basis of stationary bed shape

1) ColumnChromatography

Column chromatography process involves the uniform percolation of a liquid solute

through a column packed with finely divided material. It involves adsorption, partition or ion
exchange phenomenon. The adsorption column chromatography is based on the retention of
solute by surface adsorption. This technique is useful in the separation of nonpolar
substances and constituents of low volatility.

The sample mixture is set on the top of the column and is made to absorb on the top of the
stationary phase. Eventually, the mobile phase which is applied to the column is used to
remove the mixture via the stationary phase. Separation of compounds in column
chromatography by utilizing the molecule’s polarity. The rate at which molecules move in
the column during the separation process depends on their polarity. As the mobile phase
comes out of the column it is collected in small fractions in the test tube. Then it is allowed to
isolate and purify. Later, the removal of salt is done by a rotary evaporator to obtain the
separated compound. Column chromatography is the most widely used technique for the
purification of compounds due to its versatility. Also, it is a very convenient process.

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Figure 1.6: Column chromatography

2) PaperChromatography

Paper chromatography works on the principle of partitioning between a mobile

phase (solvent) and a stationary phase (the paper). When a mixture is applied to the
paper, the different components of the mixture move up the paper at different
speeds, based on their solubility in the solvent and their affinity for the paper. The
resulting separation is a result of the differing interactions between the substances
and the [Link] involves applying a small dot or line of sample solution onto
strips of chromatographic paper. Then the paper is placed in a jar containing a
shallow layer of solvent and covered. As the solvent moves through the paper, the
sample mixture starts to travel up the paper with the solvent. This chromatographic
paper is made of cellulose, which is a polar substance and if the compounds non-
polar within the mixture travel farther. More polar components bond with the
cellulose paper more quickly and therefore do not moves as far with moving mobile
phase. Paper chromatography works on the partition of solutes between water
adhered on the paper fibres (stationary phase) and the moving solvent (mobile
phase). Pentane, propanone and ethanol these common solvents used as mobile
phase. Mixtures of solvents with varying proportions are also can be used, including
aqueous solutions with differentpolarities.

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Paper chromatography remains a valuable, simple, and low-cost technique for
separating and analyzing mixtures, especially when high resolution or large
quantities are not required. It is particularly useful in educational settings,
preliminary analysis, and qualitative assessments of various substances. Despite its
limitations in terms of resolution and capacity, its simplicity makes it an accessible
tool for many different applications in chemistry, biology, and environmental
sciences.

Figure 1.7: Separation phenomenon in Paper chromatography

3) Thin Layer Chromatography(TLC)

TLC is a widely used chromatographic technique for separating and

analyzing compounds based on their different affinities for the stationary phase and
the mobile phase. TLC is similar to paper chromatography but offers a more efficient
and faster separation process with greater resolution. It is commonly used for
analyzing organic compounds, checking the purity of substances, and monitoring the
progress of [Link].,it involves a thin layer of adsorbent like silica gel,
alumina, or cellulose on a flat, inert substrate such as glass, aluminium foil or
insoluble plastic is used as stationary phase. It has the advantage over to paper that
of faster runs, better separations, and the choice between different adsorbents. For
better resolution and for quantification, high- performance TLC can be applied.
TLC works on the principle of adsorption chromatography, where compounds in a
mixture are separated as they move through a thin layer of stationary phase (adsorbent)
on a solid support. The compounds interact with the stationary phase to varying
degrees, and the mobile phase (solvent) moves them up the plate, resulting in
separation based on differences in their polarity or affinity for the phases. TLC
remains a highly popular and accessible method for separating and analyzing mixtures

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in various fields of research and industry. Its simplicity, speed, and effectiveness for
qualitative analysis make it an essential tool in organic chemistry, biochemistry,
pharmaceuticals, and environmental science.

F
igure 1.8: Separation phenomenon in Thin Layer Chromatography

[Link] On the basis of physical state of mobilephase

1) Gas Chromatography(GC)

Gas-Liquid Chromatography (GLC), commonly referred to as Gas

Chromatography (GC), is a chromatographic technique used for separating and
analyzing volatile compounds in a mixture. . The mobile phase is typically an inert gas
(such as helium, nitrogen, or hydrogen) that moves the sample through the column.
The gas is chemically inert to avoid reactions with the sample [Link]
chromatography is always carried out using a column, which is "packed" or capillary".
This technique is based on partition equilibrium of analyte between a solid stationary
phase (often a liquid silicone-based material) and a mobile gas which is carrier gas,

The stationary phase is adhered to the inside of a glass tube having very small
diameter known as capillary column or a solid matrix inside a larger metal tube
known as packed column. The stationary phase is a liquid(such as silicone oil,
polyethyleneglycol, or other organic liquids) coated on the surface of a solid support
(like silica geloralumina). The column is typically a capillary column made of a thin,
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flexible material, often stainless steel or glass. The column can be packed with
stationary phase or be capillary (thin tubes coated with stationary phase). Capillary
columns generally offer better separation efficiency.

Gas-Liquid Chromatography (GLC) is an essential and widely used

analytical tool in various fields, such as environmental monitoring, pharmaceuticals,
food safety, and forensic science. Its ability to provide highly sensitive and rapid
separation of volatile compounds makes it invaluable for detecting trace amounts of
substances in complex mixtures

The choice of stationary phase depends on the nature of the sample to be

[Link] is widely used in analytical chemical analysis; This technique is not
suitable for high molecular weight biopolymers or proteins decomposes as high
temperature in GC which is frequently occurs in biochemistry analysis.

2) Liquid Chromatography(LC)

Liquid Chromatography (LC) is a widely used technique for separating,

identifying, and quantifying components in a mixture. It involves a liquid
mobilephase that moves the sample through a column packed with a stationary
phase. Liquid chromatography can separate a variety of compounds, including non-
volatile and thermally unstable compounds.

Liquid chromatography works on the principle of partitioning or adsorption

between the stationary and mobile phases. The sample components interact with the
stationary phase based on their chemical properties (like polarity, size, or charge),
causing them to separate as they move through the column with the mobile phase.
Components with different affinities for the stationary phase will be carried at
different rates, leading to their [Link] stationary phase is a solid or a liquid
coating on a solid support inside the [Link] stationary phases include
silica gel, alumina, and specially modified materials that allow for different modes
of separation. The choice of stationary phase is crucial and depends on the nature of
the sample and the type of chromatography being used.

Liquid chromatography is a versatile and essential technique for separating and

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analyzing complex mixtures in many industries and research fields. Whether used in
Agrochemical pharmaceutical analysis, environmental monitoring, or chemical
synthesis, LC allows for high precision, excellent resolution, and the capability to
analyze a broad range of substances. Techniques like HPLC, with its speed and
sensitivity, have revolutionized analytical chemistry and continue to be integral to
modern scientific and industrial practices.

3) Supercritical Fluid Chromatography(SFC)

Supercritical Fluid Chromatography (SFC) is an advanced chromatographic

technique that uses a supercritical fluid as the mobile phase to separate
components in a mixture. Supercritical fluids (SF) exhibit unique properties of both
liquids and gases, making them particularly effective for separation tasks that
require enhanced performance over traditional chromatographic methods. SFC
combines the benefits of both liquid chromatography (LC) and gas chromatography
(GC), providing versatility for separating a wide range of [Link] operates
based on the principle of differential interactions between sample components and
the stationary phase, similar to other chromatographic techniques. However, what
sets SFC apart is the use of a supercritical fluid as the mobile phase.

A supercritical fluid occurs when a substance is heated above its critical temperature
and pressurized beyond its critical pressure. At this point, the substance exhibits
properties of both gases and liquids, making it an ideal solvent for many separation
processes. Common supercritical fluids used in SFC include carbon dioxide
(CO₂), nitrous oxide (N₂O), andethanol.

Supercritical Fluid Chromatography (SFC) is an emerging and powerful technique

that combines the best aspects of liquid and gas chromatography. By using
supercritical fluids as the mobile phase, it offers fast, efficient, and environmentally
friendly separation for a wide range of compounds, While SFC requires specialized
equipment and is not suitable for all types of compounds, its ability to handle
complex mixtures with excellent resolution makes

4) Capillary Electrophoresis (CE)

Capillary electrophoresis is an analytical technique that separates ions based on

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their electrophoretic mobility with the use of an applied voltage. The electrophoretic
mobility is dependent upon the charge of the molecule, the viscosity, and the atom's
radius. The rate at which the particle moves is directly proportional to the applied
electric field--the greater the field strength, the faster the mobility. Neutral species
are not affected, only ions move with the electric field. If two ions are the same size,
the one with greater charge will move the fastest. For ions of the same charge, the
smaller particle has less friction and overall faster migration rate. Capillary
electrophoresis is used most predominately because it gives faster results and
provides high resolution separation. It is a useful technique because there is a large
range of detection methods available.

[Link] Few special chromatographictechnique

1) ChiralChromatography

Chiral chromatography is a specialized type of chromatography used to separate and

analyze chiral compounds molecules that have non-superimposable mirror images,
like left and right hands (enantiomers). Since many biologically active compounds
This is the most common form of chiral chromatography and the sample is passed
through a column packed with a chiral stationary phase, and the enantiomers are
separated based on their different interactions with the stationary phase.
Chiral chromatography plays an important role in the pesticide industry,
particularly when it comes to analyzing the behavior, safety, and effectiveness of
chiral pesticides. Many pesticides, like other chemicals, exist as racemic mixtures
(both enantiomers of a compound), and these enantiomers can have significantly
different toxicological [Link] pesticides are chiral molecules, meaning they
can exist in two mirror-image forms (enantiomers). These enantiomers can have
different levels of biological activity, toxicity, and environmental behavior. In some
cases, one enantiomer may be highly effective at pest control, while the other could
be less effective.

Chiral chromatography involves the separation of stereoisomers.

Enantiomers have no chemical or physical differences apart from being three-
dimensional mirror images of each other. Traditional chromatography or any other
separation processes are incapable of separating them. For effective chiral
separations, either the stationary phase or the mobile phase must be made chiral,
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giving differing affinities between the analytes. In both normal and reversed phase
chromatography columns with chiral stationary phase are commerciallyavailable.
2) Pyrolysis GasChromatography
Pyrolysis Gas Chromatography (Py-GC) is a specialized analytical technique
that combines pyrolysis (thermal decomposition) with gas chromatography (GC) for
the analysis of complex materials. This technique is particularly useful for studying
substances that are difficult to analyze directly, such as polymers, plastics, and
complex natural materials.
Pyrolysis refers to the process of heating a sample to a high temperature (typically
400°C to 1000°C) in the absence of oxygen. This causes the sample to decompose into
smaller volatile fragments, typically in the form of gases, which can then be
[Link] pyrolysis process breaks down large, complex molecules like polymers,
rubber, oils, and fossil fuels into smaller, more volatile compounds that are easier to
analyze using GC.

Pyrolysis Gas Chromatography (Py-GC) is a valuable tool for analyzing complex

materials, particularly in fields like polymer science, environmental monitoring,
forensic science, and petrochemical analysis. By breaking down large, non-volatile
compounds into smaller, volatile ones through pyrolysis, and then separating these
compounds via GC, [Link] technique is useful for the identification of
synthetic polymeric media, such as acrylics or alkyds, and synthetic varnishes, also
can be used for environmental samples, including fossils [Link]-GC allows for
the analysis of materials that are hard to analyze directly due to their size or
complexity (e.g., polymers or complex organic materials).While the pyrolysis
process breaks down the sample into smaller components, the overall approach is
non-destructive, which is important for preserving the [Link] with GC
detection methods, Py-GC can provide very sensitive detection of trace components
in complex matrices
Most Known Chromatographic equipment’s are, [19]

(1) High Performance Liquid Chromatography(HPLC)

(2) Ultra Performance Liquid Chromatography
(3) High Performance Thin Layer Chromatography(HPTLC)
(4) Ion Chromatography(IC)
(5) Gas Chromatography(GC)

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(6) FlashChromatography
(7) CapillaryChromatography.
(8) Capillary Electrophoresis (CE)
(9) Supercritical Fluid Chromatograph
(10)Preparative High-Performance Liquid Chromatography ([Link])
1.3. INTRODUCTION TO HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY (HPLC)

1.3.1. PRINCIPLE

High-Performance Liquid Chromatography (HPLC) is an analytical technique widely

used to separate, identify, and quantify compounds in a liquid sample. It is a powerful
tool used in various fields such as chemistry, biochemistry, pharmaceuticals,
environmental analysis, and food science. HPLC operates on the principle of
partitioning, where different compounds in a mixture are separated based on their
different interactions with a stationary phase (usually a column packed with solid
material) and a mobile phase (a liquid solvent).

Some of the major components of chromatographs include devices specialised in

molecule separation known columns and high-performance pumps for supplying
solvent at a steady flow rate, among the different technologies created for
chromatography. The system once known as High Performance Liquid
Chromatography became known simply as “LC” as associated technologies got more
advanced which is capable of high-speed analysis, is becoming increasingly popular
and is being widely used nowadays.

HPLC is recognized for its precision, speed, and efficiency in separating complex
mixtures and is an essential tool in various industries, including Agrochemical,
pharmaceuticals, environmental testing, food and beverage analysis, and forensic
science clinical and medical laboratories ,cosmetic industry

Chromatography works on the principle that different components of a mixture will

interact differently with two phases: the stationary phase and the mobile phase.
These interactions lead to the separation of components as they pass through the
system. Chromatography is the technique in which the components of a mixture are
separated based upon the rates at which they are distributed themselves between two
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phases, one of which does not move i.e. stationary phase and the other that moving
mobile phase’. When mobile phase is liquid, this technique is known as ‘liquid
chromatography’. Principle of separation is based on the differences in the
distribution of species between two immiscible phases, in which the mobile phase is
a liquid which passes through a stationary phase packed in a column. It is mainly
based on mechanisms of adsorption, mass distribution, ion exchange, size exclusion
or stereo-chemicalinteraction.
Earlier liquid chromatography is carried out in long glass columns with wide
diameter. Nowadays with the help of latest technology, the particle diameters were
reduced as small as to below 10 μm (generally 3-5 µm) and replacement of glass
columns to steel ones which can sustain high pressure. The performance is improved
by applying high flow rate to mobile phase by using high pressure pumps. Due to
this development, mostly called as ‘high-performance liquid chromatography’ or
‘high-pressure liquid chromatography’(HPLC).It is employed to separate, identify,
and quantify the components of a mixture in liquid form. HPLC is an advanced
version of traditional liquid chromatography (LC), offering higher resolution and
speed in separation due to the use of high pressure to push the mobile phase through
the column.

HPLCtechnique is more popular because it is non-destructive and may be applied to

thermally liable compounds which was not analyzed by GC technique. HPLC is mostly
suitable for the separation of macromolecules and ionic species of biomedical interest,
liable natural products, and diverse less stable and high molecular weight compounds.
Short, small-bore columns containing densely packed particles (3-5 µm) of stationary
phase provide the rapid exchange of compounds between the mobile and stationary
phases. A large variety of unique column packing provide a wide range of selectivity
to separation through HPLC. HPLC also offers wide choice of detection methods as
number of unique detectors [Link] is a powerful and highly precise
analytical technique used to separate, identify, and quantify components of a mixture.
It is a form of liquid chromatography that employs a high-pressure pump to pass a
liquid mobile phase through a column packed with a stationary phase.

1.3.2. HPLCINSTRUMENTATION

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HPLC instrument generally constitute of solvent reserviour autosampler,
pumps, and a detector. The autosampler injects specific volume of sample mixture
into the mobile phase stream which carries it into the column. The pumps deliver the
desired flow (isocratic or gradient) of the mobile phase through the column. The
detector generates a signal proportional to the amount of sample component
emerging from the column, The heart of the system is the column where separation
occurs. The information that can be obtained using HPLC includes identification,
quantification, and resolution of a compound.A digital microprocessor
(Communication Bus Module) and user software (LC Solution) control the HPLC
instrument and provide data analysis.
Due to automatic instrumentation and calculation which is carried out by
integrator itself in modern HPLC that offers saving of manual labour. All the
operations are carried out by a computer, which provides long periods of unattended
operation. All these advantages make HPLC more efficient over the all
chromatographic techniques in terms of separation, speed, sensitivity, easy sample
recovery, automation, integration, handling and maintenance. The wide applicability
of HPLC makes it as a most important separation tool in the field of analysis.

Figure 1.9: HPLC instrumentation

A liquid chromatograph instrument consists of a mobile phase reservoir, a
high pressure pump to forcefully push the mobile phase through the system at high
pressure, an injector to introduce the sample, a chromatographic column on
whichseparation to be achieved, detector to detect analyte response, and a data
collection device such as a computer, integrator, or recorder. Further, in some

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modern HPLC instrument, degasser with vacuum pump and pre-column facility is
available.

A brief introduction of HPLC components is given as under:

(a) Pumpingsystems
HPLC pump is required to deliver a flow of mobile phase at constant pressure and
flow rate. In simple language, the HPLC pump has to have ruggedness and at the
same time should be able to provide reproducible flow characteristics run after run.
The operational pressure limits have a vast range depending upon analysis
requirements. In normal analytical operation the pressure can vary between 2000 –
5000 psi. or higher, with maximum delivery rates up to about 10 ml/min. is
[Link] HPLC systems include at least one pump to force the mobile phase
through whose packing is fairly compact. The result of this is a pressure increase at
the injector which can attain 20 000 kPa (200 bars) depending upon the flow rate
imposed upon the mobile phase, its viscosity, and the size of the particles of the
stationar

Pumps used for quantitative analysis should be constructed of materials that

inert to corrosive mobile phase components and be capable of delivering the
mobile phase at a constant rate with minimal fluctuations over extended periods of
[Link] systems consist of microprocessor controlled metering pumps that
can be programmed to deliver either constant isocratic flow of mobile phase or
vary the ratio of mobile phase components, as is required for gradient run.
Advanced pumping system is equipped with a degasser to remove dissolved air
and other gases from the solvent through solvent delivery system.
(b) Injectors
HPLC, the injection of a precise volume of sample onto the head of the
column must be made as fast as possible in order to cause the minimum disturbance
to the dynamic regime of the mobile phase whose flow must be stable from column
to detector. Needle wash between samples will prevent carry-over between
injections. Before the start and at end of analysis .After dissolution of sample in
mobile phase or suitable diluents, compounds to be are injected, either manually by
syringe/loop injectors or automatically by auto-sampler. Auto-sampler consist of a
carousel or rack to hold sample vials with tops that have a pierce-able septum vials
to a loop from which it is loaded into the chromatograph. Auto-sampler can be
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programmed to control to change desired sample volume range from 0.1µ to 100µl,
the number of injections i.e. replication, the interval between injections and rinse
cycles.

(c) Columns

HPLC columns are mostly made up of smooth bore polished stainless steel, are
between 50 and 300 mm long, and have an internal diameter of between 2 and 5 mm.
They are commonly filled with a stationary phase with a particle size of 3–10 µm.
Columns with internal diameters of less than 2 mm are often referred to as microbore
columns. Ideally, the temperature of the mobile phase and the column should be kept
constant during an analysis. Considered the “heart of the chromatograph” the column’s
stationary phase separates the sample components of interest using various physical
and chemical parameters

Particles may range up to 50 μm or more for preparative columns. Stationary

phases for reverse-phase liquid chromatography mostly consist of an organic phase
chemically bound to silica or other materials. Small particles are thinly coated with
organic phase which provide low mass transfer resistance and, hence, rapid transfer
of components between the stationary and mobile phases occurs. Columns may
beheated to achieve more efficient separations, but at temperatures above 60 °C are
used rarely for analysis. Unmodified silica, polar chemically modified silica are used
as stationary phase, e.g. Silica Si 5, cyano-propyl or diol, in normal-phase liquid
chromatographycolumns.
Most of separations for reversed-phase liquid chromatography are based on
partition type mechanisms that utilize chemically modified silica as the stationary
phase and polar solvents as the mobile phase. The surface of the support, e.g. various
silane reagents are reacted with silanol group of silica to produce covalently bound
silyl derivatives covering a number of active sites present on the surface of the
support. To determining the separation properties of the chromatographic system, the
nature of the bonded phase is important parameter.

Table 1.5: Commonly used bonded Stationary phases

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Stationary
BondedPhase Characteristics Applications
Phase
- Reverse phase chromatography
Slica- - C18
- Non-polar (most common for general separations
based (Octadecylsilane)
of organic compounds).

- Reverse phase chromatography.

- C8 (Octylsilane) - Moderate polarity
Less hydrophobic than C18.

- Reverse phase for smaller molecules

- C4 (Butylsilane) - Moderate polarity
or peptides.

- Normal phase chromatography,

- Cyanopropyl (CN) - Polar
especially for polar compounds.

- Polystyrene- - Used for highly hydrophobic

Polymer-
divinylbenzene (PS- - Non-polar compounds and in selectivity
based
DVB) adjustments.

- Poly(ethylene - Separation of polar compounds or

- Polar
glycol) (PEG) biological molecules.

- Normal phase chromatography for

Amino - Amino (NH2) - Polar, basic polar, neutral compounds. Often used
for amino acids and sugars.

- Reverse phase separations,

- Slightly non-
Phenyl - Phenyl (C6H5) especially for aromatic compounds or
polar, aromatic
those with π-π interactions.

- Used for separating polar

Diol
- Diol (Silica gel) - Polar compounds, especially for
(silica)
glycoproteins and lipids.

- Used for ion-exchange separations,

Cation-
- Sulfopropyl (SP) - Polar, anionic especially for cationic compounds
exchange
like proteins.

- Used for ion-exchange separations

Anion- - Quaternary amine
- Polar, cationic of anionic compounds (e.g., nucleic
exchange (Q)
acids).

In ion-exchange chromatography (IC), retention is based on the attraction

between solute ions and charged sites bound to the stationary phase. Solute ions of
the same charge as the charged sites on the column are excluded from binding, while
solute ions of the opposite charge of the charged sites of the column are retained on

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the [Link] is a chromatographic technique used to separate charged particles
(ions) based on their charge properties. It utilizes a stationary phase (usually a
charged resin or material) to attract and bind ions in a sample, allowing for their
separation as they are displaced by other ions in the mobile phase.
In ion-exchange chromatography, the stationary phases are usually synthetic
organic resins; cation-exchange resins contains negatively charged active sites which
separates positively charged substances, while anion-exchange resins have positively
charged active sites which separates compounds with negatively charged groups.
Water-soluble ionic or ionisable compounds are attracted to the resins. The
chromatographic separation occurs on differences in affinity of [Link]
exchange chromatography is a versatile and powerful tool, particularly valuable in
the separation of ions, proteins, and nucleic acids.
Size Exclusion Chromatography (SEC), also known asGel Filtration
Chromatography (GFC), is a chromatographic technique used to separate molecules
based on their size (or molecular weight). It is a non-interactive separation method,
meaning that the separation process relies on the physical size of the molecules
rather than on their chemical properties. This makes SEC particularly useful for
purifying proteins, polymers, and other [Link] size-exclusion
chromatographic columns are packed with a porous stationary phase gel. Smaller
size molecules enter the pores and are increasingly retained as molecular size
decreases, i.e. large molecules elutes faster than small [Link] Filtration
Chromatography Which uses hydrophilic packing to separate polar species and uses
mostly aqueous mobile phases. This technique is mostly used to identify the
molecular weights of large-sized proteins & bio-molecules. Gel Permeation
Chromatography: Which uses hydrophobic packing to separate non-polar species
and uses non-polar organic solvents. This technique is used to identify the molecular
weights of polymers
For chiral chromatography, special chemically modified polar stationary
phases, e.g. cellulose or amylose derivatives, proteins or peptides, cyclo-dextrin etc.,
use for the separation of enantiomers are used.
Table 1.6: Commonly used chiral column Stationary phases

Chiral Stationary Phase Type of Mechanism of

Common Applications
(CSP) Chirality Separation
Chiralpak® Polysaccharide Interaction via Separation of chiral
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Chiral Stationary Phase Type of Mechanism of
Common Applications
(CSP) Chirality Separation
helical structure, Pesticides molecule
Derivatives
hydrogen pharmaceutical
(polysaccharide-based) (Amylose or
bonding, steric compounds, amino acids,
Cellulose)
interactions peptides
Chiral Separation of chiral
Polysaccharide
recognition Pesticides molecule
Chiracel® (polysaccharide- Derivatives
through helical pharmaceutical
based) (Amylose or
structure, van compounds, amino acids,
Cellulose)
der Waals forces peptides
Steric
interactions,
Separation of chiral
Chiralcel® Polysaccharide hydrogen
pharmaceuticals,
(polysaccharide-based) Derivatives bonding, and
steroids, amino acids
hydrophobic
interactions
Formation of
inclusion Separations of small
Cyclodextrin
complexes, chiral molecules like
Cyclodextrin-based CSPs Derivatives (e.g.,
differential sugars, drugs, and
β-CD, γ-CD)
binding of organic compounds
enantiomers
Interaction
Separation of chiral
through
Amylose- acids, amines, alcohols,
Chiralpak® AD hydrogen
Derived CSP and biologically active
bonding and
compounds
steric hindrance
Steric and
Separation of drugs,
Cellulose- hydrogen
Chiralpak® AS amino acids, and organic
Derived CSP bonding
molecules
interactions
Used in complex organic
Pirkle-type Interaction via
molecule separations,
Lux® (Pirkle-Type CSP) (Chiral Selective chiral ligand
such as pesticides and
Ligands) interactions
natural products
Molecular
Protein and peptide
BSA (Bovine Serum Protein-Based recognition and
separations,
Albumin) CSP binding of
biomolecular studies
enantiomers
Formation of
Separation of drugs,
Chiral-Dex® (cyclodextrin- Cyclodextrin inclusion
environmental pollutants,
based) Derivatives complexes for
and food additives
chiral separation
Crown Ether-based CSPs Crown Ether Coordination Separation of metal
Ligands with metal ions complexes, ions, and
or other chiral ligands
components,
differential
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Chiral Stationary Phase Type of
Mechanism of
Common Applications
(CSP) Chirality
Separation
interactions
Interaction via Separation of
Polysaccharide hydrogen pharmaceutical
Cellulose Tris(3,5-
with a chiral bonding, steric compounds, amino acids,
dimethylphenylcarbamate)
ligand and electrostatic and chiral organic
forces molecules
Interaction based
Separation of hydrophilic
on ionic
Ionic Liquid and hydrophobic chiral
Chiralpak® IB exchange and
Stationary Phase molecules and
chiral
biomolecules
recognition

(d) Detectors
The most common HPLC detectors used are UV detectors because of the fact
that most of the compounds absorb in UV or visible region. They give specific
response to the class of compounds or particular compounds depending upon the
functional group of eluting molecules .The basis of working for optical detectors is the
change in intensity when a beam of electromagnetic radiation passes through the
detector flow cell. These detectors are of three different types: Fixed wave length
detectors, variable wavelength detectors and diode array detectors.

UV/Visible spectrophotometers, including diode array detectors are the most

commonly used detectors. Fluorescence spectrophotometers, differential
refractometers, electrochemical detectors, mass spectrometers, light scattering
detectors, radioactivity detectors or other special detectors may also be used
depending upon the separation goal and properties of compound.
Detector consists of a flow cell placed at the end of the column through
which eluent passes. A beam of UV light passes through the flow cell and
transmitted into the detector. As compounds elute from the column, they pass
through the cell and absorb the radiation, resulting in energy level changes. Fixed
Wave Length Detectors: Such type of detectors does not allow change in wavelength
of the radiation. They are difficult to find nowadays due to limitation of working
wavelength. Low pressure mercury lamp is used for very intense light at 253.7nm or
[Link] wavelength detectors can be adjusted to work on any wavelength
over full UV- visible region. The wavelength can be selected at 3nm or [Link] diode

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array detector, the sample is subjected to light of all wavelengths generated by the
lamp at once. The lights from emission source when collimated by an achromatic
lens system, the total light passes through the detector cell onto a holographic grating
and then falls onto a diode array which contains 100s of diodes. The chromatogram
can be produced using the UV wavelength that was falling on that particular diode at
the end of the run. DAD helps to see the response of the analyte at different
wavelengths in only single run and thus saves time and energy.. These detectors
acquire absorbance data over the entire UV-visible range, thus providing the analyst
with chromatograms at multiple, selectable wavelengths, spectra of the eluting peaks
and peakpurity.
Fluorescent detectors are the most selective, sensitive and specific than all
other HPLC detectors. Specific wavelength is used to excite and then emit light
signal in analyte atoms. They intensity of light is monitored continuously to quantify
the analyte concentration. Fluorescence is 10-1000 times sensitive than UV detector
for strong UV absorbing compounds. Even a single analyte in the cell can be
detected by the fluorescence detector. The system is easy to operate and relatively
stable.
A differential refractometer detector (DRI), commonly known as a refractive
index detector (RID), is a universal detector in High-Performance Liquid
Chromatography (HPLC) systems. It measures the refractive index of an analyte
relative to the solvent Differential refractometer detectors measure the difference
between the refractive index of the mobile phase alone and that of the mobile phase
containing compounds as it elutes from the column. Refractive index detectors are
used to detect non-UV absorbing compounds. Fluro-metric detectors are sensitive to
compounds that are inherently fluorescent or that can be converted to fluorescent
derivatives either by chemical transformation of the compound or by coupling with
fluorescent reagents at specific functionalgroups.
Electrochemical detectors in High-Performance Liquid Chromatography
(HPLC) offer high sensitivity and selectivity for compounds with electroactive
properties. Measure the potential difference between a reference electrode and an
indicator electrode immersed in the eluent. The potential change correlates with the
concentration of specific ions or [Link]-metric, or
Polarographic electrochemical detectors are useful for the quantitation of species that
can be oxidized or reduced at a working electrode. These detectors are selective,
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sensitive, and reliable, but require conducting mobile phases free of dissolved
oxygen and reducible metal ions. Electrochemical detectors with carbon-paste
electrodes may be usedadvantageouslyto measure nano-gram (ng) quantities of
easily oxidized compounds, notably phenolsand catechol’s.

(e) Data collectiondevices

Signals from the detector may be collected on chart recorders or electronic
integrators that vary in complexity and in their ability to process, store and re-
process chromatographic data. The computer integrates the response of the detector
to each component and places it into a chromatograph that is easy to read and
interpret. Frequently called the data system, the computer not only controls all the
modules of the HPLC instrument but it takes the signal from the detector and uses it
to determine the time of elution (retention time) of the sample components
(qualitative analysis) and the amount of sample (quantitative [Link] PC
coordinates the identifier’s reaction with each component and records it in
achromatograph that is simple to read and [Link] data collectors
receive and store detector output and print out chromatograms complete with peak
heights, peak areas, sample identification, and method variables. They are also used
to program the liquid chromatograph, controlling most of variables and providing
for long periods of unattended operation.

1.3.3. MODES OFHPLC

Various modes of HPLC utilized to separate compounds are classified as follows:

1) Normal-phasechromatography
In HPLC, if stationary phase is more polar than the mobile phase, it is termed
as normal-phase liquid chromatography. Polar bonded phases that have a diol,
cyano, diethyl-amino, amino, or di-amino functional groups are used as stationary
phase in normal-phase chromatography. Due to lower affinity of nonpolar
compounds to the stationary phases used, nonpolar compounds are elute first while
polar compounds are retained for longer time. Normal-phase chromatography is
widely applied for [Link] Phase Chromatography is a valuable
technique for separating polar compounds, offering high resolution and specificity

2) Reversed-phase chromatography
In HPLC, if stationary phase is less polar than the mobile phase, it is termed
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as reversed-phase liquid chromatography. In this technique, C18, C8, Phenyl, and
cyano-propyl functional groups that chemically bonded to microporous silica
particles are used as stationary phase. Retention in reversed-phase chromatography
occurs by nonspecific hydrophobic interactions of the solute with stationary phase.
The ubiquitous application of reversed-phase chromatography arises from the fact
that practically all organic molecules have hydrophobic regions in their structures
and effectively interact with the stationary phase. It is estimated that over 65%
(possibly as high as 90%) of all HPLC separations are executed in the reversed-
phase mode. The rationale for this includes the simplicity, versatility, and scope of
the reversed-phase method. Reverse Phase Chromatography is a powerful and
versatile technique widely used in analytical chemistry for the separation and
analysis of a broad spectrum of compounds.

3) Ion-pairchromatography

Ion-Pair Chromatography (IPC) is a specialized technique in liquid

chromatography that enhances the separation of charged analytes by introducing
ion-pairing reagents into the mobile phase. This approach modifies the polarity of
ionic compounds, facilitating their retention on non-polar stationary phases, such as
those used in reversed-phase [Link] or partially ionic compounds
can be chromatographed on reversed- phase columns by using ion-pairing reagents.
These reagents are typically long-chain alkyl anions or cations that, when used in
dilute concentrations, can increase the retention of analyte ions. C-5 to C-10 alkyl-
sulfonates is commonly used for cationic compounds while C-5 to C-8 alkyl
ammonium salts are generally used in the cases of anionicsolutes.

1.3.4. HPLC METHODDEVELOPMENT

High-Performance Liquid Chromatography (HPLC) method development is a

systematic process aimed at creating a reliable and efficient analytical method for
separating, identifying, and quantifying compounds in complex mixtures.A many
pathways have been used to develop analytical method for separations. Conventional
chromatographic approaches are discussed here. HPLC method development should
be based knowledge of chromatographic [Link] approaches are advanced and
often based on judgment and the knowledge of the chromatographer. Whereas
individual approaches may be exhibit considerable diversity, method development

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often follows vary, method development often follows the series of steps
summarized in figure

Figure 1.10: Steps in HPLC Method Nature ofSample:

Before beginning the actual method development, it is very important to

review what is known about the sample. The aim of method development should
also define at separation [Link] goals of the separation should also be defined. A
compounds structure, activity and physicochemical characteristics are determined by
the arrangement of its constituent atoms and the bonds between them. The functional
groups often determine whether the molecule is polar or non-polar. Organic
molecules are divided into classes according to the principal functional group(s). The
chemical composition of the sample can provide valuable clues for the best choice of
initial condition of for an HPLC [Link] relative chromatographic retention
of different kinds of molecules is largely determined by the nature and position of
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these functional groups. As shown in Table 1.6, classes of molecules can be ordered
by their relative retention into a range or spectrum of chromatographic polarity from
highly polar to highly non-polar.
Water is a small molecule with a high dipole moment, it is a polar compound.
Benzene is an aromatic hydrocarbon, it is a non-polar compound. Molecules with
similar chromatographic polarity tend to be attracted to each other; those with
dissimilar polarity exhibit much weaker attraction, or may even repel each another.

This the basic principle of chromatographic separation modes based on polarity. IN

another way: oil (non-polar) and water (polar). The stronger attraction between likes
and the weaker attraction between opposites, “Like attracts like” is polarity-based
chromatography.

Table 1.7: Compound Chromatographic Polarity Index

Polarity Functional group Molecular Structure

Low Benzene C6H6
Methylene R-(CH2)2-
Phenyl R-Ph.
Halide R-F, Cl, Br, I
Ether R-O-R
Nitro R-(N+=O)-O-
Ester R-(O)-O-R
Aldehyde R-(C=O)-H
Ketone R-(C=O)-R
Amine R-NH2
Hydroxyl R-OH
Carboxylic Acid R-(C=O)-OH
Nitrile R-CN
High Water H-OH

Apart from chemical structure, number of compounds present in sample other kinds
of sample-related information that can be also important that includes molecular
weight of the analytes, pKa values of the compounds, sample solubility and sample
type (ionic or non-ionic). The choice of the mode of HPLC method should be made
principally from the properties of the sample that has been determined about
molecular weight (MW). On the basis of the solubility of the sample in polar or non-
polar solvents, mode of HPLC can be select as per below flow-chart (Figure: 1.11)

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Figure 1.11: Flowchart for selection of HPLC mode

a) Selection of Stationaryphase:
The selection of the stationary phase in HPLC (High-Performance Liquid
Chromatography) is crucial for achieving optimal separation of the components in a
sample. The stationary phase is the material that is packed into the column, and its
properties (such as polarity, surface area, and particle size) directly influence the
retention and separation of [Link] selecting mode of HPLC, the choice of
the column (stationary phase) should be made after consideration of mode of
chromatography, column-to-column variability, and several other considerations.
The silica particles may be acquired in a variety of sizes, shapes, and degrees of
porosity. The activity or polarity of the silica surface may be modified selectively by
chemically bonding to it less polar functional groups (bonded phase) for the
application to any specific HPLC method. Polarity of different types stationary
phases. Sufficient time is also allowed for column equilibration before starting the
analysis.

Some other characteristics of stationary phase like particle shape, surface area,
particle size, bonding type, pore size and carbon load, surface chemistry shall also
consider at the time of method development which have considerably effect on
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separation

Table 1.8 Polarity spectrum of different stationary phases

Type of
Stationary Phase Stationary Polarity Application/Use
Phase

Most commonly used for a wide variety of

C18 Reverse-phase
Non-polar samples, especially non-polar and moderately
(Octadecylsilane) (RP)
polar compounds.

Used for less non-polar compounds than C18.

Reverse-phase
C8 (Octylsilane) Non-polar Suitable for smaller or more polar compounds
(RP)
than C18.

Reverse-phase Less non-polar than C18, used for more

C4 (Butylsilane) Non-polar
(RP) hydrophobic analytes.

Phenyl Reverse-phase Moderate Used for aromatic compounds; provides different

(Phenylsilane) (RP) polarity interactions due to π-π stacking.

Reverse-phase Moderate Often used for semi-polar compounds and short-

C2 (Ethylsilane)
(RP) polarity chain analytes.

Normal-phase Most common for polar analytes, especially in

Silica (SiO₂) Polar
(NP) normal-phase HPLC.

Normal-phase Used for polar compounds; generally used in

Alumina (Al₂O₃) Polar
(NP) adsorption chromatography.

Normal-phase Moderate Used for intermediate polarity compounds. It’s

Cyanopropyl (CN)
(NP) polarity less polar than silica but more polar than C18.

Used for interactions like hydrogen bonding;

Normal-phase
Amino (NH₂) Polar suitable for polar compounds like amines and
(NP)
alcohols.

Normal-phase Used for polar compounds that might also form

Diol Polar
(NP) hydrogen bonds or interact with water.

Normal-phase Used for compounds with strong hydrogen

NH₂-Silica Polar
(NP) bonding or polar interactions.

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Surface Area:

The surface area of the stationary phase influences the number of adsorption sites
available for interaction with the [Link] surface area stationary phases provide
more surface interactions, improving separation efficiency and [Link] is
particularly important for small-particle stationary phases (e.g., <5 μm) which are
commonly used in modern HPLC for better separation.

Particle shape:
These particles offer a large surface area for interactions between the sample
and the stationary phase, promoting efficient [Link] of stationary
phase are either spherical or irregular. Irregular particles have provide higher surface
areas and higher carbon loads. Spherical particles provide higher efficiency, better
column stability, fast stabilization and lower back pressure compared to irregularly
shaped [Link] particle size, in combination with the shape, plays a major role
in efficiency, resolution, and pressure buildup in the [Link] shape of the
particles impacts the flow dynamics, surface area for interactions, and the efficiency
of separation Refer Figure 1.12

Spherical Irregular

Figure 1.12: Shape of spherical and irregular particles

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Particle size:
The particle size of the stationary phase determines the efficiency of the separation,
as well as the backpressure during the [Link] particles (e.g., 3–5 μm) provide
higher efficiency and better resolution, but they result in higher [Link]
particles (e.g., 10 μm) offer lower backpressure but may reduce [Link] column
back pressure and column efficiency (performance) are inversely proportional to the
square o the particle [Link] size affects back-pressure and the separation
efficiency of [Link] particle sizes for HPLC/UPLC column packing are
represented in Figure 1.13.

Figure 1.13: Different particle sizes of column packing

Bonding type:

Bonding refers to the way the stationary phase is chemically modified or treated to
improve selectivity and [Link]: Used in normal-phase HPLC for
separating highly polar [Link] stationary phases (e.g., C18, C8) are used
in reverse-phase HPLC. The bonding of hydrocarbon chains (such as C18) to silica
reduces the interaction of the stationary phase with water, making it suitable for
reverse-phase separations.

Pore size:

The pore size of the stationary phase is an important characteristic for separating large
molecules or [Link] pore sizes (e.g., 100–300 Å) are typically used for
separating small to medium-sized [Link] pore sizes (e.g., >300 Å) are used for
the separation of large molecules like proteins, polymers, and other [Link]
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size of 150 Å or less is chosen for samples with molecular weight less than 2000 and pore
size 300Å or greater for samples with molecular weight greaterthan 2000. Larger pores
allow larger solute molecules to be retained through maximum exposure to the surface
area of particles. Refer Figure 1.14

Figure 1.14: A representative diagram of pore sizes of particles

Carbon load:
Higher carbon load means more carbon chains are present on the column,
which generally leads to higher retention of hydrophobic compounds. This can
improve the resolution .A lower carbon load can lead to shorter retention times but
may reduce the column's ability to separate hydrophobic compounds [Link]
carbon load is the amount of bonded phase attached to the base material, expressed
in percentage of carbon. Refer Figure 1.14.

Figure 1.15: A representative diagram of carbon load

Surface Chemistry

The surface chemistry of the stationary phase affects how it interacts with
analytes. For example:Hydrophobic phases (like C18) interact primarily through van
der Waals forcesandhydrophobic [Link] phases (like silica) interact
through hydrogen bonding, dipole-dipole interactions, and polar
[Link], such as endcapping (the chemical modification of the
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stationary phase surface to reduce residual silanol groups), can improve stability and
reduce unwanted interactions.

Efficiency and Resolution

The efficiency and resolution of the stationary phase are determined by how well the
analytes are separated based on their affinity for the stationary [Link]-efficiency
stationary phases are characterized by narrow particle size distributions and minimal
tailing in [Link]-resolution stationary phases enable the separation of closely eluting
compounds by maximizing differences in interaction with the stationary phase.

The stationary phase choice should be aligned with the sample’s chemical properties
and the desired separation goals for achieving optimal separation, efficiency, and
resolution in HPLC analysis

Summary of Key Stationary Phase Characteristics:

Characteristic Explanation
Polarity Determines the interaction with analytes (non-polar vs polar)
Surface Area Affects the capacity and efficiency of the column
Influences resolution and backpressure (smaller particles =
Particle Size
higher resolution)
Alters the selectivity and stability (e.g., C18, Cyanopropyl,
Bonding Type
Silica)
Pore Size Affects the separation of large vs small molecules
Resistance to degradation under various conditions (pH,
Chemical Stability
temperature)
Determines how analytes interact with the stationary phase
Surface Chemistry
(hydrophobic, hydrogen bonding)
Ability to distinguish between analytes based on their
Selectivity
properties
Efficiency and
Affects the sharpness and separation between peaks
Resolution
Compatibility with
Ensures optimal performance with chosen solvents
Mobile Phase

Effect of variables on column efficiency

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A mathematical calculation of the behaviour of chromatographic column efficiency is
obtained from the Van Deemter equation. he Van Deemterequation is a mathematical model
that describes the relationship between column efficiency (measured as the number of
theoretical plates, NNN) and various factors such as the flow rate of the mobile phase and
properties of the column. It is widely used in chromatography to optimize the conditions for
best separation.

Van Deemter Equation

HETP = A + (B / u) + Cu
Where:
A Eddy diffusion
B Longitudinal diffusion
C Resistance to mass transfer
u Average mobile phase velocity

Figure 1.16: Van Deemter Plot

Eddy diffusion occurs due to irregularities in the packing of the stationary phase,
leading to multiple flow paths and peak broadening in chromatography. It is a key factor in
reducing column efficiency, and minimizing it involves using smaller particles and ensuring
uniform packing. Reducing eddy diffusion improves chromatographic resolution and leads to
sharper peaks, enhancing the overall separation [Link] eddy diffusion (A),
indicates the multitude of pathways by which a component finds its way through the column.
In a poorly packed column, the retention times of molecules of the same components can
drastically depending on numerous flows that could be taken. This affects leads in band
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broadening. The mobile phase at moderate to high flow rate the zone broadening is higher. At
low flow rate, the molecules of a component are not significantly dispersed by multi-
channelling. The longitudinal diffusion (B/v) indicates band-broadening process that is
inversely related to mobile phase velocity. At high flow rate analyte spend shorter time on
column hence diffusion is less. The mass transfer coefficient Cv indicates equilibrium of an
anlyte established between mobile and stationary phase. Eddy diffusion is more significant in
columns with larger stationary phase particles because these particles create larger gaps
between them, increasing the likelihood of multiple flow paths. Smaller particles reduce the
effect of eddy diffusion because they create a more uniform packing and more direct paths
for the solute molecules. Independence from Flow Rate: The A term (eddy diffusion) in the
Van Deemter equation is independent of the mobile phase flow rate uuu. This means that,
unlike longitudinal diffusion or mass transfer, the influence of eddy diffusion doesn't change
with flow rate. At high flow rate, there is less time for equilibrium to take place and
contribution to be 8broadening effect is observed. Van Deemter plot shown in Figure1.15

b) Selection of mobilephase

The selection of the mobile phase in chromatography is a critical factor that

influences the separation process, resolution, retention times, and overall efficiency of the
chromatographic system. The mobile phase is the solvent or mixture of solvents that moves
the analyte(s) through the stationary phase (column) and enables separation based on
differences in their interactions with both phases. The selection of the mobile phase is a
balance between the chemical nature of the analytes, the type of stationary phase, and the
desired chromatographic conditions. By optimizing the mobile phase composition, the
chromatographer can enhance separation, improve resolution, and minimize issues such as
tailing or broadening of peaks. Proper solvent selection is critical for obtaining accurate and
reproducible chromatographic [Link] very important parameter in HPLC method
development as the selectivity is altered by changing the mobile phase. When selecting
organic solvents for use in mobile phases, various physical and chemical properties of the
solvent should be considered. A scale, such as that shown in Figure: 1.17, upon which some
common solvents are placed in order of relative chromatographic polarity is called an
eluotropic series. Mobile phase molecules compete with the analyte molecules for the active
sites of stationary phase and displace these analytes, causing them to move faster through
thecolumn. Water is at the extreme polar end of mobile phase solvent
scale,whilehexanewhichisanaliphatichydrocarbon,isattheextremenon-polarIn between these,
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single solvents, as well as mixtures of miscible solvent can be placed in order of elution
strength.

Figure 1.17: Mobile Phase Chromatographic Polarity Spectrum

Selected solvent should have low viscosity, be compatible with the detection
system, be easily available in pure form, and, if possible, have low flammability and
toxicity. UV cut-off values of solvents are also important consideration from the
standpoint of detection. The term polarity concerns the ability of a sample or solvent
molecule to interact by combination of dispersion, dipole, hydrogen boding, and
dielectric interactions. The combination of these four intermolecular attractive forces
constitutes the solvent polarity. Polarity is measure strength of solvent that affected
selectivity.
The changes in selectivity may be affected by making use of the following solvent
properties:
 Proton acceptors: amines, ethers, sulfoxides, amides, esters, andalcohols.
 Proton donors: alcohols, carboxylic acids, phenols, andchloroform.
 Large dipole solvents: methylene chloride, nitrites, sulfoxides, and ketones.
Application of reagents such as buffers, ion-pairing reagents, or other modifiers
(such as triethylamine) to the mobile phase is carried out to improve reproducibility,
selectivity, or peak shape. Buffers are used primarily to regulate the pH and the acid-
base equilibrium of the solute in the mobile phase. They may also be utilized to
affect the retention times of ionisable compounds. The buffer capacity should be at
maximum and should not vary in the pH range of 2 to 8 commonly used in HPLC.
The buffers should be soluble, stable, inert to analytes and compatible to the
detector.
By employing gradient elution mode, required % of the organic phase can be
estimated rapidly. For aqueous sample, the best way to start is with gradient reversed
phase chromatography. Gradient run can be started with 5 to 10% organic phase
(acetonitrile or methanol) in the mobile phase and can be increased up to 90% within
30 to 40 minutes. Separation can be optimized by changing the initial mobile phase

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composition and the slope of the gradient according to the chromatogram obtained
from the previous run. The initial mobile phase composition can be estimated on the
basis of where the compounds of interest were eluted at what proportion of organic
and aqueous component of the mobile phase.

Mobile Phase Selection Guidelines:

1. Consider the polarity of the analyte and stationary phase to determine whether
normal-phase or reversed-phase chromatography is more suitable.
2. Choose solvents that are compatible with the nature of the analytes and their
solubility in the chosen mobile phase.
3. Adjust the pH if necessary, especially for ionizable analytes.
4. Use a gradient elution method when dealing with complex mixtures to improve
separation and reduce elution times.
5. Test compatibility with the detection system (e.g., UV transparency, low volatility
for mass spectrometry).

c) Selection of mode and wavelength fordetection:

The selection of the detection modeandwavelength plays a pivotal role in obtaining

accurate and precise chromatographic results. The detection mode should be chosen
based on the nature of the analyte, while the wavelength should be selected based on the
analyte's absorption characteristics. By carefully considering both factors, you can
optimize the chromatographic analysis for your specific [Link] each sample,
good analytical results will be obtained by careful selection of the wavelength used for
detection. This choice requires known UV spectra of the sample. If analyte standards are
available, their UV spectra can be taken prior to HPLC method development. PDA
detector permits the acquisition of UV spectra for all sample components during method
[Link] detector allows for the measurement of absorbance at multiple
wavelengths simultaneously. This enables the identification of the λmax and can help
with the selectivity of the analysis (by comparing different wavelengths).PDA is useful
when you need spectral information about the analyte and when the analyte absorbs at
multiple wavelengths. The wavelength chosen for detection must provide acceptable
response by the various analytes in the sample and there is no interference of baseline
noise. In most cases HPLC method development is carried out with UV detector.

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Maximize sensitivity by selecting the wavelength corresponding to the analyte's
absorption maxima (λmax). For complex mixtures, choose a wavelength where the
analyte absorbs strongly and minimize interference from other components. For trace-
level detection, use a narrow wavelength to reduce noise and enhance selectivity
Alterative detectors are required when sample have low or no UV absorbance.

d) Diluentselection:
Diluent selection is an important aspect of chromatography, as it plays a
significant role in ensuring that the sample is appropriately prepared for injection
into the chromatographic system. The diluent should be chosen carefully to avoid
interference with the analyte, minimize sample degradation, and ensure compatibility
with the mobile phase and detection system. Diluent for sample preparation shall be
selected at initial stage of development on the basis of solubility of the sample.
However, optimization of the diluent is based on its extraction efficiency, effect on
peak symmetry, peak interference in estimation and stability of analyte in diluent.

e) Concentration and Injection volume:

There is often a need to adjust the concentration and injection volume
together to achieve the best results. For instance, if the concentration of the sample is
very high, a smaller injection volume may be required to prevent column overload. If
the concentration is very low, a larger injection volume might be necessary to ensure
the analyte is detected [Link] concentration and injection volume shall be
set according to suitability with extraction process and detector response.
Wherever it is necessary to filter the sample preparation, filter compatibility shall be
check for selection of proper type of filter. To finalize HPLC method development,
optimization can be started only after obtaining preliminary chromatogram that
means with symmetrical peaks of all analytes detected in the chromatogram. By
slight change of the mobile phase composition, the position of the peaks can be
predicted within the range of investigation.
A finalized chromatographic condition is that one in which all the peaks are
well separated in less run time in the produced chromatogram. With application of
more specific column, good symmetry and resolution can be achieved. The

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parameters that are considered for good chromatographic condition and being
optimized are:
All above terms are explained according to Figure 1.18 and Figure 1.19 as below:

Figure 1.18: Explanation for Resolution, Theoretical plates, Capacity factor and
Selectivity

1. Resolution (R)

Resolutionis a critical factor in HPLC that determines how well two analytes can
be separated. Higher resolution is essential for accurate and precise analysis,
particularly in complex mixtures. By adjusting various chromatographic conditions,
resolution can be optimized to achieve the best possible separation.

Resolution expresses the separation of two components in a mixture that

determined by the following equation:

Where, t2 and t1 are the retention times of the two components, and W 2 and
W1 are the corresponding widths at the bases of the peaks obtained by extrapolating
the relatively sides of the peaks to horizontal the baseline. Where electronic
integrators are used, it may be convenient to determine the resolution, R, by the
following equation.

Where by W1h/2 and W2h/2 the width at half-height of corresponding [Link] better
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separation, the ideal value of R is 1.

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2. Theoretical plates(N)
The number of theoretical plates N is a measure of the column efficiency. It
is expressed by followingequation.

Where t is retention time of the peak and W is the width for the peak. W h/2 is width
at half-height of the peak. If the value of theoretical plates is higher than 4000 is
indicating good column [Link] more theoretical plates a column has, the
better its ability to separate compounds. A higher number of theoretical plates
indicates greater resolution and efficiency of the chromatographic process.

3. Tailing factor(T)
The tailing factor (T) is a measure of peak symmetry. It is unity for perfectly
symmetrical peak to value of 1.0 and its value increases as tailing becomes more
pronounced. It is determined by following formula.

T=a+b
2a

Figure 1.19: Explanation for tailing factor

W0.05 is peak width at 5% height from the baseline and f is leading edge of the peak
In general, value of tailing factor should be less than 1.5

Peak Asymmetry

A =b
a

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Figure 1.20: Explanation for peak asymmetry

4. Capacity Factor(k′)
The capacity factor (k′) in chromatography is a measure of how well a
compound is retained in a chromatographic column relative to the mobile phase. It is
used to evaluate the interaction between a solute (analyte) and the stationary phase.
Essentially, the capacity factor tells you how much longer an analyte spends
interacting with the stationary phase compared to the mobile phase.

Capacity factor is the ratio of the reduced retention volume to the dead
volume, which is a measure of how well the peak of sample molecule is retained by
a column during an isocratic separation. It is determined by using followingformula.

The capacity factoris a key parameter for evaluating the retention characteristics of
analytes in chromatography. It helps assess the interaction between the analyte and the
stationary phase and is essential for optimizing separation, retention time, and overall
efficiency of the chromatographic process.

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Where by t0 is the void volume of the column and t1 is the retention time of the
corresponding peak. The ideal value of k′ ranges from 2-10.

5. Selectivity(α)

Selectivity (α) is a term commonly used in chemistry, particularly in areas like

chromatography and catalysis, to describe the ability of a substance or material to
preferentially interact with one component over others in a mixture. In a general sense,
selectivity measures the degree to which a process, such as a reaction or separation, targets
a specific compound or element.

The selectivity is a measure of relative retention of two components in a mixture.

Selectivity is the ratio of the capacity factors of both corresponding peaks. It can be
calculated by following formula.

Where by t0 is the void volume of the column and t 1 and t2 are the retention times of the
corresponding peak. The ideal value of should not be less than 1.
Selectivity is a measure of how well a chromatographic method can separate different
components in a mixture. A high selectivity means that the method can distinguish
between compounds effectively, leading to better separation.

1.4. FORCED DEGRADATION STUDY (STRESS STUDY)

1.4.1. CONCEPT
Forced degradation is the process of subjecting pesticides compounds to extreme
chemical and environmental condition to determine product breakdown levels and
preliminary degradation kinetics. They are used to facilitate the development of
analytical methodology, to gain a better understanding of active ingredient (AI) and
finished product (FP) stability, and to provide information about degradation pathways
and degradation products.

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The purpose of forced degradation testing studies is to evaluate the overall

stability of the material for method development purpose and / or degradation pathway
elucidation. This testing may involve the compound alone and/ or in simple solution/
suspension to validate the analytical procedures. Under forcing condition,
decomposition products may be observed that are unlikely to be formed under
conditions used for confirmatory studies. This information may be useful in developing
and validating suitable analytical methods. The forced degradation studies are performed
to,

 Develop and validate a stability developed method.

 Develop a method to separate and quantify the principle peak from the
degradation impurities and excipient peaks (placebo).
 Interpret possible degradationpath-ways.

1.4.2 STRESSCONDITIONS
Thermal, hydrolysis (acid and base), oxidation and photo-degradation are
commonly used in stress study mechanisms. Degradation level can be achieved by
selecting suitable concentration of acid, base and oxidizing agent, applying combination
of stress (e.g., degradation media along with temperature) and exposure time. The
generally recommended degradation varies between 5-20% degradation.

Hydrolysis (Acid and Base degradation)

Acid and base solutions are used to perform hydrolysis at wide range of pH (e.g.
2 to 12). For acid hydrolysis hydrochloric acid or sulphuric acid and for base
hydrolysis sodium hydroxide or potassium hydroxides are commonly used.
Concentration of acid or base selected on the basis of the stability of molecule. Initially
stress study should be performed at room temperature and with low concentration of
acid or base (0.1Molar) also with appropriate exposure time (generally 30 minutes.). If
desired degradation is not achieved then higher concentration (1 M, 2M), temperature
(40°C, 60°C) and exposure time (1 hr, 2 hrs,1 day, 3 days and 5 days) can be applied or
if degradation is more than desired then lower concentration and less exposure time (10-
30 minutes). Acid or base degradation samples should be neutralized with same
concentration of base or acid respectively and to avoid further degradation reaction and

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injected after filter through syringe filter to save the column silica from damage.
Alcoholic acid agent or alcoholic base agent can also be used for compounds, which are
insoluble inwater.

Oxidation (Oxidative degradation)

Commonly Hydrogen peroxide is used for oxidation degradation. Other
oxidizing agents can also be use such as metal ions, oxygen and radical initiators (e.g.
azobisisobutyronitrile, AIBN). Selection of an oxidizing agent, its concentration (3%,
5%, and 10% with appropriate exposure time) and condition depends on the
[Link](elevatedtemperature)shouldnotbe used
with hydrogen peroxide, as hydrogen peroxide decomposes at ambient temperature.
Heating solution with hydrogen peroxide leads to hydrolysis instead of oxidation.
Heat (Thermal degradation)
Thermal degradation test should be performed on active ingredient and
formulated sample with or without humidity. Solid substance, placebo and product
samples should be exposed to heat with or without humidity, whereas liquid substances
and products can be exposed to heat without humidity. As per CIPAC guidelines
pesticide active substance and products are exposed to heat at 54°C for 2 weeks this
study is known as Accelerated Heat Study (AHS)
Photo stability (Photo-degradation)
This is very important degradation step for light sensitive molecules, but
irrespective to light sensitive evaluated all molecules to identify any unacceptable
change due to light exposure. As per ICH (Q1B) guideline active substance and
formulated product samples should be exposed to UV-VIS light providing exposure of
not less than 1.2 million lux hours and UV energy not less than 200-watt hours/square
meter. It is preferred to expose same samples to white fluorescent and near ultraviolet
lamp. If specified instrument not available, then sample should expose directly to
sunlight for desired days.

1.4.3 EVALUTION OFRESULTS

These the forced degradation samples are injected on developed method. All
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component peak and all unknown impurities should be well separated from known
impurities. Therefore, the analytical method which has capability to resolve known and
unknown impurities from main principle peak of component is known as stability
indicatingmethod.

Peak purity analysis

Peak purity parameters are likely to be used to prove spectral uniqueness of
compound. Peak purity of main component and known impurities in control and
degradation samples should be established using PDA (Photo diode array detector) refer
Figure 1.21.

Figure 1.21: Peak Purity evaluation

1.5 ANALYTICAL METHODVALIDATION

1.5.1 CONCEPT
Evaluation of a method is to ensure that its performance is suitable for the analysis being
carried out is known as method [Link] Method Validation is a process
that ensures an analytical method (used for testing or analysis in laboratories) is reliable,
accurate, and consistent for its intended purpose. It involves confirming that the method
is fit for its intended use in terms of accuracy, precision, sensitivity, specificity, and

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other important performance characteristics.

Validation of an analytical method will ensure that the results of an analysis are reliable,
consist and perhaps more importantly that there is degree of confidence in the results.
Method validation provides the proof that a method is “fit for intended purpose”.
The procedure which provides assurance for the same by the means of laboratory studies
is defined as method validation. “Method validation is the process of demonstrating that
analytical procedures are suitable for their intended use and that they support the
identity, strength and quality, for the quantification of the technical and final products.
Method validation has given considerable attention in the literature and regulatory
agencies. ISO/IEC 17025 includes a chapter on the validation of methods [46] with list
validation parameters. The ICH [47] and SANCO [48] has developed a consensus text
on the validation of analytical procedures. CIPAC [49] and USP [50] also developed
guidance with detailed methodology. The AOAC [51], the EPA, APVMA and other
scientific organizations provide methods that are validated through multi-
laboratorystudies.

1.5.2 PARAMETERS FOR METHODVALIDATION

Method validation is critical to ensuring that an analytical method is reliable and
suitable for its intended purpose. Validating a method involves a thorough examination
of its accuracy, precision, specificity, linearity, sensitivity, and other performance
characteristics. These parameters collectively help ensure that the method can provide
consistent, accurate, and reproducible results, meeting the necessary regulatory
standards and requirements.

Different working groups of national and international committees defined

parameters for method validation and proposed guidelines for same. The defined
validation parameters by the ICH, APVMA and SANCO [53] and other regulatory
bodies and references [54] are summarized as under.
1) Specificitystudy
2) System suitability
3) Precisionstudy
4) Limit of detection and Limit of quantitationstudy

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5) Linearity and rangestudy

6) Accuracystudy
7) Robustness study
8) Solution stabilitystudy

A brief introduction of above parameters is as below:

1) Specificitystudy
Selectivity of an analytical method is its ability to measure accurately an analyte in
the presence of interferences that may be expected to be present in the sample matrix.

Specificity refers to the ability of an analytical method to measure the analyte of

interest in the presence of other components that may be present in the sample, such as
impurities, degradation products, excipients, and matrix components. The term
specificity is also referring to selectivity when several chemical entities that may or may
not be distinguished from each other.
The Specificity of analytical methods must be demonstrated by providing data to shows
that:
 Known degradation products and synthetic impurities do not interfere with the
determination of the active constituents in bulk actives;and
 Known degradation products, synthetic impurities and excipients present in the
formulated product do not interfere with the determination of the active constituents in
pesticideproducts.
In order to check the interference of degradation product from chemical (acid, alkali and
oxidative) and physical (thermal and photolytic) degradation, peak purity of the analyte
peak is evaluated.
2) System suitability study
ASystem Suitability Studyis a set of tests that ensures an analytical system is
performing correctly before actual sample analysis. It is typically conducted before
starting the routine sample analysis to verify that the instrument and method are
operating as expected. The system suitability parameters are designed to confirm the,
consistency, reliabilityof the analytical system for accurate and reproducible results.

Some type of system suitability must be done which demonstrate that the analytical

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system is performing properly. System suitability should be determined byreplicate

analysis of the standard or reference solution. System suitability is considered
appropriate when the % RSD calculated on the results obtained at different time
intervals, does not exceed more than of specified limit of the corresponding value of the
system precision.
3) Precisionstudy
The precision of an analytical method expresses the closeness of agreement (degree
of scatter) between a series of measurements obtained from multiple sampling of the
homogeneous sample under the prescribed conditions. The measurement of precision of
an analytical method is performed on replicate standard preparations and replicate
sample preparations. The results for the same are usually expressed as the variance,
standard deviation or confidence level of a series of measurements. Precision is
performed by means of Repeatability (intra-day), Intermediate precision
(ruggedness/inter-day) and Reproducibility. Repeatability expresses the precision under
the same operating conditions over a short interval of time.
a) Repeatability of an analytical method is determined by analysing 5replicates
injection of same sample preparation on same day by the same operator in the same
laboratory. The mean, % Relative standard deviation (RSD) and number of
determinations should bereported.
b) Intermediate Precision of an analytical method is determined by analysing 5
preparations of same sample. Intermediate precision expresses within- laboratories
variations; different days, different analysts, different equipment, etc. The mean, %
RSD and number of determinations should be reported. The main objective of
intermediate precision is to provide proof that method will provide same results in
the samelaboratory.
Table 1.9: Suggested maximum% RSD as a function of analyte conc.

Analyte (%) RSD (%)

100 ≤1
>10 ≤2
1 to 10 ≤5
0.1 to 1.0 ≤10

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< 0.1 ≤20

c) Reproducibility is a measure of a methods ability to perform a routine analysis and
deliver the same results using a method irrespective of laboratory, equipment and
operator changes. Confirmation of reproducibility is important if the method is to be
used in different laboratories for routine analysis. Reproducibility is expressed in
terms of relative standard deviation. The unmodified Horwitz equation is used as
criteria of acceptability for measuredreproducibility.

The objective is also extent to verify that the method will provide the same results in
different laboratories (ruggedness).

4) Limit of detection (LOD) and Limit of quantitation (LOQ) study[55]

The Limit of Detection (LOD)and Limit of Quantitation (LOQ) are both important
concepts in analytical chemistry and laboratory testing, particularly when assessing the
sensitivity of a method to detect and measure very low concentrations of a substance.

Limit of Detection also known as Detection Limit (DL) is the point at which a measured
value is larger than the uncertainty associated with [Link] detection limit of an analytical
method is the lowest concentration of an analyte (substance) that can be reliably
detected but not necessarily quantified measured accurately
Limit of Quantitation also known as Quantitation Limit (QL) of an analytical method is
the lowest amount of analyte in a sample which can be quantitated with suitable
precision and accuracy. The quantitation limit is the minimum injected amount that
produces quantitative measurements in the target matrix with acceptable precision.
Some common methods for the estimation of LOD and LOQ are:

a) Visualevaluation
b) Signal-to-noiseratio
c) Standard deviation method based on calibration curve
d) Method of least square regression analysis
e) Blank or background measurement method
f) Spike sample method approach

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a) Visual evaluation: This method may be used for non-instrumental methods but may
also be used with instrumental methods. LOD is determined by the analysis of samples
with known concentration of analyte and by establishing the minimum level at which the
analyte can be reliably detected, and for LOQ the minimum concentration level at which
analyte can reliably quantified with acceptable accuracy andprecision.
b) Signal-to-noise Ratio: This approach can only be applied to analytical procedures that
exhibit baseline noise. Determination of Signal-to-noise ratio is performed by comparing
measured signal from samples to with the low concentration of analyte with those of
blank samples establishing the minimum concentration at which the analyte can be
reliably detected is the LOD and the minimum concentration at which the analyte can be
reliably quantified is the [Link], the LOD is defined as the concentration at
which the signal-to-noise ratio (S/N) is 3:1. The LOQ is defined as the concentration at
which the signal-to-noise ratio is 10:1. . Refer Figure1.22

Figure 1.22: Evaluation of LOD-LOQ by Signal to Noise ratio

Steps

 Prepare a series of blank samples and measure their responses.

 Measure the noise level (baseline fluctuation) from the blank samples.
 Prepare low concentration standard samples and measure the signal.
 Calculate the signal-to-noise ratio for each standard.

c) Standard deviation method based on calibration curve:

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This method involves calculating the standard deviation of the response (signal) and the slope of
the calibration curve.

LOD is generally calculated as:

Where:

σ is the standard deviation of the response (noise or blank).

s is the slope of the calibration curve.

LOQ is generally calculated as

Where:

σ is the standard deviation of the response (noise or blank).

s is the slope of the calibration curve.

Steps:

 Construct a calibration curve by plotting the analyte concentration versus the signal.
 Calculate the standard deviation of the response from several blank samples (or low-
concentration standards).
 Calculate the slope of the calibration curve.
 Apply the formulas for LOD and LOQ.

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Calibration Curve Method

This method involves preparing a series of standard solutions with known concentrations of
the analyte and constructing a calibration curve.

The LOD and LOQ are estimated based on the extrapolation of this curve and the noise of
the system.

Steps:

 Prepare multiple standard solutions of varying concentrations of the analyte.

 Measure their responses and plot the calibration curve.
 Determine the slope of the calibration curve.
 Calculate the standard deviation of the blank or low-concentration samples.
 Apply the formulas for LOD and LOQ based on the slope and standard deviation.

d) Method of least square regression analysis

This method involves fitting a regression model (typically a linear regression) to the data
from a series of standards of varying [Link] the regression model is
established, you can determine the noise level and calculate the LOD and LOQ based on
the regression parameters (slope and intercept).

Steps:

 Prepare and analyze a set of standard solutions to generate data points.

 Fit a linear regression model to the data.
 Determine the standard deviation of the residuals (errors between the observed and
predicted values).
 Use the regression slope and the standard deviation of the residuals to calculate the LOD
and LOQ.

e) Blank or background measurement method

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This method estimates the LOD and LOQ by measuring the blank (background noise)
response in the absence of analyte. The LOD and LOQ are then determined based on the
number of standard deviations above the blank.

Steps:

 Measure the signal of the blank samples multiple times to estimate the baseline noise
(standard deviation of the blank).
 Calculate the LOD and LOQ based on the standard deviation of the blank using the
following formulas:

LOD = Mean blank signal + 3 × (Standard deviation of blank)

LOQ = Mean blank signal + 10 × (Standard deviation of blank)

These formulas assume that the analyte signal is distinguishable from the blank noise.

f) Spike sample method approach

In this approach, known amounts of analyte are spiked into blank samples (or samples
with a known analyte concentration) to simulate real-world [Link] LOD and LOQ
are determined by observing how well the method detects and quantifies the spiked
analyte at low concentrations.

Steps:

 Spike the sample with low concentrations of the analyte.

 Analyze the sample and determine the lowest spiked concentration at which the analyte
can still be detected (LOD) or accurately quantified (LOQ).

5) Linearity and rangestudy

The Linearity of an analytical method is its ability (within given range) to obtain
test results, which are directly proportional to the concentration (amount) of analyte in
sample. Linearity may be demonstrated directly by dilution of a standard stock solution

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and/or by using separate weighing’s of physical mixtures of the standard components,

using the proposed procedure. Linearity is determined by replicate injections of 5 or
more concentrations level within the range of 50–150 % and also additional LOQ level.
(i.e. levels are LOQ, 50, 80, 100, 120 and 150%). The response should be directly
proportional to the concentrations of the analytes or proportional by means of a well-
defined mathematical calculation. Linearity is evaluated graphically by plotting a graph
of the relative responses on the y-axis and the corresponding concentrations on the x-
axis. A linear regression equation is
appliedtotheresultstoevaluatecorrelationcoefficient(r>0.99closeto1).In addition, y-
intercept, slope of the regression line and residual sum of squares should also
calculate.A linear response is crucial for ensuring accurate quantification of an analyte at
various concentration levels.
The Range of an analytical method is the interval between the upper and lower
concentrations (amounts) of analyte in the sample (including these concentrations) for
which it has been demonstrated that the analytical procedure has a suitable level of
precision, accuracy and linearity. The range study helps determine the limits within
which the method can be used for reliable quantification without falling outside the
method's ability to measure accuratelyThe range is expressed in the same units as the
test results (e.g., %=percentage, parts per million=ppm) calculated by the analytical
method. Refer Figure 1.23.

Figure 1.23: Linearity, Range and LOD, LOQ

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Both linearity and range studies are critical for ensuring the validity and reliability of an
analytical method. Linearity confirms that the method produces a proportional response
to changes in concentration, while the rangestudy ensures that the method is accurate and
precise over the concentration span expected in real-world samples. Together, these
studies ensure that the analytical method can be confidently used for quantitative analysis
in a wide variety of applications.

6) Accuracystudy
The accuracy of the analytical method is the closeness of agreement between the
value which is accepted either as a conventional true value or an accepted reference
value, and the value found. It is essential to ensure that the method is capable of
providing reliable, truthful results. The accuracy of an analytical method is the extent to
which test results obtained by the method and the true value agree. Refer Figure 1.24

Figure 1.24: Precision and Accuracy

The true value for accuracy assessment can be assessed by following ways:

a) Analysing a sample with known concentrations (certified reference material) and

comparing the measured value with the true value as supplied with thematerial.

b) Spiked / Assay – placebo method: A known amount of pure active constituents is

added to formulation blank (placebo), its recovery can be determined by comparing the
response of the extract with the response of the reference material dissolved in a pure
solvent (withoutmatrix).

c) Standard addition method: a sample is assayed, a known amount of pure active

constituents is added, and the sample is again assayed. The difference between the

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results of the two assays is compared with the expectedvalue.

The accuracy should cover at least 3 concentrations (80, 100 and 120%) and also
additional LOQ level in the expected range. (I.e. four concentrations/ three replicates
each). Accuracy should be reported as percent recovery by the assay of known added
amount of analyte in the sample or as the difference between the mean and the accepted
true value. The % mean recovery should be within the following ranges shown in Table
1.10

Table 1.10: Acceptable Mean Recovery Values

% Active/ impurity content Acceptable mean recovery
> = 10 98 – 102%
>=1 90 – 110%
0.1 – 1 80 – 120%
< 0.1 75-125%

7) Robustnessstudy
The robustness of an analytical method is a measure of its capacity to remain
unaffected by small, but deliberate, variations in method parameters and provides an
indication of its reliability during normal usage.
Robustness study examines the effect of operational parameters on the analysis
results. For the determination of a method’s robustness, method parameters like pH,
flow rate, column temperature, column lot or mobile phase composition, are varied
within a realistic range, and the quantitative influence of the variables is determined. If
the results of this parameter are within a previously specified tolerance, the parameter is
said to be within the method’s robustness range.

8) Solution stabilitystudy
Many solutes readily decompose prior to chromatographic investigations, for
example, during the preparation of the sample solutions, extraction, clean-up, phase
transfer or storage of prepared vials (in refrigerators or in an auto-sampler). Under these
circumstances, method validation should investigate the stability of the analytes and
standards in solution form (in analytical preparations). The standard and sample
preparations are stored up to specified period at specified temperature and its stability is

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evaluated by comparing solution preparations at different time intervals to that ofinitial.

Asolution stability study is critical for ensuring that analytical solutions, reagents, or
standards remain stable under normal storage and handling conditions over time. By
systematically assessing changes in concentration, composition, and physical properties,
it is possible to determine the best storage conditions and optimize the shelf life of
solutions, ensuring that analytical results remain consistent and reliable.
1.5.3 PRIOR STEPS OFVALIDATION

Before starting the formal validation of an analytical method, there are several
prior steps that need to be performed to ensure the method is suitable for its intended
purpose and can provide reliable and reproducible results. These preliminary steps
the groundwork for a successful method validation [Link] define the
purpose of the method and the specific requirements for validation. This includes
understanding the intended use of the method (e.g., quantification, detection, and
identification, the regulatory requirements, and the acceptable performance
criteria.

Prior to start method validation, validation aim should be a well-planned

according to scientific soundness and completeness with pre-defined acceptance
criteria. Because the type of analysis and the other information of a sample have so
much influence on the validation, the objective and scope of the method should
always be defined as the first step of any method validation.
Subsequent to the execution of the validation, results, conclusions and deviations
should present in report. Provided the pre-defined acceptance criteria are met, and
the deviations (if any) do not affect the scientific interpretation of the data, then the
developed analytical method can be considered as valid.

1.6 INTRODUCTION TOKarl Fischer Titration (KF)

Karl Fischer Titration (KFT) is a highly precise and widely used analytical technique for
determining the water content in a wide range of materials, including liquids, solids,
gases, and pharmaceuticals.

History of Karl Fischer Titration:

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The history of Karl Fischer Titration (KFT) is closely tied to the work of Karl Fischer, a
German chemist who developed the method in the early 20th century. His work
revolutionized the analysis of moisture content in various materials and set the stage for a
widely used and precise technique still in use today.

Early Life and Career of Karl Fischer:

Karl Fischer was born in 1901 in Germany and pursued studies in chemistry. He
eventually worked in various industrial and academic settings, where his work focused on
analytical chemistry.

Fischer's interest in moisture analysis was sparked by the need for a more precise method
to determine water content in substances, which was a critical parameter for a variety of
industries.

Development of the Method:

In 1935, Fischer, while working at the Dr. Paul Fischer & Co. (a company in Berlin that
focused on analytical chemistry), developed a chemical titration method for determining
moisture content in substances. This method was based on the reaction of iodine with
waterin a solvent, which allowed for the quantitative measurement of water in a sample.

The key to Fischer's titration method was a two-step reaction where iodine (I₂) reacted
with water (H₂O), producing hydroiodic acid (HI) and iodic acid (HIO₃). Fischer
carefully designed the method to prevent interference from other substances, ensuring
that it was both sensitiveand specific to water.

The initial paper published by Karl Fischer in 1935 described this titration method, which
became known as Karl Fischer Titration. The method was initially applied to substances
like oils and petroleum products to measure their moisture content.

The KF titration was first devised by the German chemist, Karl Fischer, to find the water
content of liquid sulfur dioxide. Since other commonly-used methods were not suitable,
he used the Bunsen reaction shown below.

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SO2 + I2 + 2H2O <-> H2SO4 + 2HI (1)

He used a new reagent including sulfur dioxide, iodine and pyridine, in a methanol
solution, giving rise to the following equation:

SO2 + I2 + 2H2O*Py <-> H2SO4 + 2HI (2)

At end point the color changed from yellow to brown. Thus he performed the first
volumetric KF titration. This was refined by American researchers later in 1939 and the
two steps were modified by changing the molar ratio of water to iodine:

H2O + SO2*Py + Py*I2

+ Py + MeOH <-> 2HI*Py + Py*SO3 (3a)

Py*SO3 + MeOH <-> Py*MeSO3H (3b)

The final version came in 1984 when it was known that pyridine was not part of the
reactants, but only a base (represented as B):

SO2 + HO-R + B <-> R-SO3- + BH+ (4a)

R-SO3- + I2 + H2O + 2B <-> R-SO4- + 2I- + 2BH+ (4b)

The reaction converts the reagent into an alkyl sulfite which is then oxidized by the
iodine, if water is present. The alcohol is essential for proper stoichiometric ratios to be
preserved, at about 50%, but other bases can be used instead of the pyridine – which is
important because of its pungent smell and toxicity which has led to its replacement by
non-toxic imidazole and other bases.

This has another advantage, in that these are generally more basic and so increase the rate
of the reaction leading to a quicker reaction and increased stability of the end point.

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Principles of Karl Fischer titration

The KF reaction is based upon the oxidation of sulfur dioxide by iodine with the
consumption of water in a buffered solution:

I2 + 2H2O + SO2 -> 2HI + H2SO4

The titration reaches its endpoint when the titrating agent has achieved a volume
sufficient to react the total amount of water in the sample. Water and iodine are used up
in an equimolar ratio.

The color change that signals the end point of the reaction is caused by the detection of
excess iodine at the double platinum electrode which acts as the indicator, following the
consumption of all the water in the sample which leads to the cessation of the reaction.
The water content is then calculated from the amount of reagent added.

The titration cell must be kept from atmospheric moisture and the sample must not react
with the KF reagent. For instance, ketones and aldehydes must be titrated in methanol-
free solvents because otherwise they react with methanol, forming water, which results in
a falsely high water concentration and a vanishing end point.

The reaction is sensitive to pH and therefore a buffer must be used if strongly acidic or
basic samples are to be titrated.

Alcohol (methanol, ethanol or a propylene-glycol mixture) is the solvent usually used

both for the sample and in the titration cell. When the iodine is in excess the reaction has
reached its end point. The most advanced KF titration technology uses a double platinum
electrode for electrochemical indication of the end point, but visual and photometric
indicators are also used.

Types of Karl Fischer Titration:

1. Volumetric Karl Fischer Titration:

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The original method, known as volumetric Karl Fischer titration, used a solution of
iodine in methanol and sulfur dioxide. Involves the addition of a liquid reagent (titrant) to
the sample until all the water has reacted. The endpoint is typically detected using a
change in electrical conductivity or a color change, indicating that the reaction is
[Link] amount of titrant consumed corresponds to the water content in the sample.

2. Coulometric Karl Fischer Titration:

In the 1950s, the coulometric Karl Fischer titration method was introduced. This
version was especially useful for measuring small quantities of [Link] this
technique, the iodine is generated electrochemically by applying a current to the
electrolyte solution, where the iodine is produced in situ. The amount of electricity
required to generate iodine corresponds to the water content.

Coulometric titration is typically used for lower concentrations of water, typically in

the range of micrograms to a few percent of water.

3. Automation and Instrumentation:

By the 1960s, advancements in instrumentation allowed for automated Karl Fischer

titrators that improved the reproducibility, accuracy, and ease of the method. These
modern titrators are now equipped with electrodes, automatic endpoint detection, and
digital displays that have made Karl Fischer Titration more accessible to

Applications of Karl Fischer Titration:

Karl Fischer Titration is an essential tool in various industries for moisture analysis
due to its precision, reliability, and sensitivity. Common applications include:

1. Pharmaceuticals:

Determining water content in drug formulations and active pharmaceutical

ingredients (APIs), which is crucial for stability, efficacy, and shelf life.

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2. Food and Beverage:

Moisture content is an important quality parameter for food and beverages,

affecting texture, preservation, and taste.

3. Petroleum and Petrochemical:

Water content in oils, fuels, and other petrochemical products is critical for
quality control and to prevent corrosion, microbial growth, or unwanted chemical
reactions.

4. Cosmetics and Personal Care Products:

Monitoring water levels in cosmetics and personal care formulations to ensure the
quality and shelf life of the products.

5. Chemical Industryand Pesticide Industry

Analyzing moisture levels in raw materials, intermediates, and finished products

to ensure proper processing and quality control.

6. Environmental Analysis:

Water content in environmental samples, such as soil or air, can be quantified

using Karl Fischer Titration.

Advantages of Karl Fischer Titration:

1. High Accuracy: Provides highly accurate and reproducible results for moisture
determination.
2. Wide Range of Samples: Suitable for liquids, solids, gases, and even viscous
substances.
3. Sensitivity: Able to measure very low amounts of water content, from a few
micrograms to several percent.

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4. Specificity: The method is selective for water, meaning that it does not interfere with
other substances in the sample

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