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A Training Manual

ICAR Sponsored Winter School

(December 07–27, 2017)

New Generation Smart Agrochemical Oriented


Approaches for Crop and Human Health Management

Division of Agricultural Chemicals


ICAR-Indian Agricultural Research Institute
New Delhi – 110 012
Training Manual

ICAR Sponsored Winter School


"New Generation Smart Agrochemical Oriented
Approaches for Crop and Human Health
Management"
December 07-27, 2017

Course Director
Dr. Anupama Singh
Course Coordinators
Dr. Aditi Kundu
Dr. Anirban Dutta
Dr. Indu Chopra
Dr. Suman Manna

Division of Agricultural Chemicals


ICAR-Indian Agricultural Research Institute
New Delhi- 110 012
Citation:
A Training Manual on "New Generation Smart Agrochemical Oriented Approaches for Crop and
Human Health Management", Division of Agricultural Chemicals, ICAR-Indian Agricultural
Research Institute, New Delhi 110 012, India, 2017, 331p

Compiled by:

Dr. Aditi Kundu


Dr. Anirban Dutta
Dr. Indu Chopra
Dr. Suman Manna
Dr. Anupama Singh

Cover page designed by:


Dr. Anirban Dutta

Divisional Publication Number: DP/C-30

December, 2017

Available on request from:

The Head, Division of Agricultural Chemicals, ICAR-Indian Agricultural Research Institute,


New Delhi 110 012, India, Tel: 91-11-25841390, E-mail: head_chem@iari.res.in

© Division of Agricultural Chemicals, ICAR-Indian Agricultural Research Institute, New Delhi


110 012, India

Sponsored by:

Human Resource Department, Indian Council of Agricultural Research, New Delhi 110 012
CONTENT
S. No. Lecture Topic Page No.
1 Agrochemicals for crop protection: New Paradigms 1-15
-Aditi Kundu, Rajesh Kumar, Anupama Singh, and Neeraj Patanjali
2 Safety operations in chemical laboratory 16-19
-Aditi Kundu and Mukta Chakrabarty
3 Agrochemicals for crop improvement: An overview 20-30
-Aditi Kundu and Devakumar C.
4 Designing of synthetic molecules: conventional approaches 31-35
-Rajesh Kumar
5 Molecularly imprinted polymers-artificial receptors 36-45
-Indu Chopra and Suman Gupta
6 Cutting edge approach for molecule designing: Molecular docking 46-56
-Aditi Kundu and Ashish Khandelwal
7 Synthesis of Molecularly Imprinted Polymer 57-58
-Indu Chopra and Suman Gupta
8 Bioactive molecules from nature to protect crop health 59-65
-V.S. Rana, N.A Shakil and Parshant Kaushik
9 Isolation of essential oil from the leaves of Eucalyptus spp., by 66-67
hydrodistillation method and determination of its chemical composition
-V.S. Rana and Ashish Khandelwal
10 Nano science in pest management 68-71
-Rajesh Kumar and Abhishek Mandal
11 Recent advancements in seed coating technology in pest management 72-80
-Sudipta Basu, S.K. Lal, Debjani Dey, Rajesh Kumar Sharma and
Vinod Gulabrao Deshmukh
12 Extraction and estimation of azadirachtin from neem 81-83
-Supradip Saha
13 Advancements in pesticide formulation technology 84-98
-Anupama Singh and Neeraj Patanjali
14 Controlled Release Formulations for Pesticide Delivery 99-105
-Jitendra Kumar and Anirban Dutta
15 Emulgels- A new dimension to gels as carrier 106-113
-Ashish Khandelwal
16 Synthesis of hydrogel 114
-Suman Manna and Anupama Singh
17 Biopolymer chemistry in relation to formulation 115-124
-Anupama Singh and Neeraj Patanjali
18 Amphiphilic polymers as carriers for pesticide delivery 125-135
-Najam Akhtar Shakil, Parshant Kaushik and Jitendra Kumar
19 Synthesis of amphiphilic polymers 136-138
-Parshant Kaushik and NA Shakil
20 Recent advances in polymer composites for pesticide delivery 139-146
-Anirban Dutta and Anupama Singh
21 Innovative controlled nutrient delivery devices 147-154
-Suman Manna and Anupama Singh
22 Advances in the development, regulation and use of biopesticides 155-169
-Opender Koul
23 Major characterization techniques for nano formulations 170-183
-Pradip Roy, Nabanita Mukherjee, Satakshi Basu, Samarendra Barik
and Arunava Goswami
24 Nanosizing of molecules and their applications 184-185
-Rajesh Kumar and Indu Chopra
25 Overview of chromatographic techniques 186-198
-Tirthankar Banerjee and Mukta Chakrabarty
26 Application of infra-red spectroscopy as a characterization tool in material 199-210
chemistry
-Suman Gupta, Neethu Narayanan, Tirthankar Banerjee and Anupama
27 Application of NMR for molecule identification 211-222
-Irani Mukherjee, V.S. Rana and Mukta Chakraborty
28 Techniques for pesticide decontamination from fruits and vegetables 223-230
-Shashi Bala Singh, Neera Singh, Indu Chopra and Birendra Kumar
29 Agriwaste adsorbent chemistry to decontaminate environmental matrices 231-243
-Neera Singh, Abhishek Mandal and S.B. Singh
30 Nutraceuticals: Role in human health improvement 244-259
-Suresh Walia and Supradip Saha
31 Recent techniques in isolation of nutraceuticals from nature 260-263
-Supradip Saha
32 Estimation of nutraceuticals from plant sources 264-266
-Supradip Saha and Aditi Kundu
33 Nano- and micro-particle based delivery systems for improved bioactivity of 267-275
nutraceuticals
-Anirban Dutta
34 In-vitro bioavailbility studies of nutraceuticals (Anthocyanin) 276-277
-Supradip Saha and Anirban Dutta
35 Fortification of food with nutraceuticals for value addition 278-287
-Shalini Gaur Rudra and Gajanan Gundewadi
36 Sensors for detection of pesticides in environmental matrices 288-303
-Abhishek Mandal, Tirthankar Banerjee and Neethu Narayanan
37 Analysis of trace level pesticides through highly precise techniques 304-320
-Tirthankar Banerjee, Suman Gupta and Neethu Narayanan

38 Quantitative estimation of molecules by LC-MS and GC-MS 321-324


-Neethu Narayanan and Tirthankar Banerjee
Agrochemicals for crop protection: New Paradigms

Aditi Kundu, Rajesh Kumar, Anupama Singh, and Neeraj Patanjali

Division of Agricultural Chemicals


ICAR-Indian Agricultural Research Institute, New Delhi 110 012

Agrochemicals have been playing a major role in the crop protection. These are vital tools for
protecting crops from pest and subsequently providing enough food for the world. The
development of new agrochemicals for crop protection will continue to be an important task for
the research-based agrochemical organizations for sustainable agriculture to ensure quality food
and fibre for everyone.
Usage of agrochemicals have witnessed huge change from inorganic salts to target specific
organic chemicals; shrinking application rates (10 – 20 g/ha as compared to kg amounts per
hectare several decades earlier); increased potency and spectrum; improved safety to consumers
and the environment and; increasing research and development costs which have to be invested.
To address the concerns due to rising global population growth, resistance development, pest
shifts and abiotic stresses, the development of an ideal agrochemical with following
characteristics is vital.
• Efficacious
• Safe
• Affordable
• Fulfill regulatory requirements

Insect pest management


Organochlorine compounds have DDT, Dieldrin, Aldrin, and BHC in this group. The use of
mentioned insecticides has been banned in most countries because of their long persistence in the
environment and the accumulation of their residues in the fatty tissue of man and other
vertebrates.
Organophosphorous compounds can kill by contact, systemic or fumigant action or a
combination of the three. They affect the nervous system by disrupting an enzyme that regulates
acetylcholine, a neurotransmitter. Being a nerve poison they can cause acute toxic reactions in
humans.
Carbamates are insecticides that affect the nervous system by disrupting an enzyme that
regulates acetylcholine, a neurotransmitter. They do not persist in the environment or in the fatty
tissue of animals. Some such as carbofuran are persistent, systemic pesticides with high toxicity.
Pyrethroids are synthetic analogs of pyrethrin, the natural insecticides produced by
Chrysanthemum species. The bioactive natural compounds were mixtures of esters of
cyclopropane carboxylic acids, primarily consisting of pyrethrins I and II, on which molecules of
all synthetic pyrethroids are based. However, natural pyrethrins have a low stability in light and
oxygen, and the applications are mainly limited to the indoors. To solve these problems, there
has been more than half of a century of research on modifying the structures of natural
pyrethrins, and a large number of pyrethroids with a variety of characteristics have been
discovered like deltamethrin, etofenprox, fenvalerate, cypermethrin, metofluthrin etc..
Pyrethroids are neuroactive insecticides, which blocks voltage-gated sodium channels in nerve

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axons. As a result, the symptoms of pyrethroid poisoning are characterized by hyperexcitation,
convulsions, seizures, and finally followed by death.

Esters of Chrysanthemic acid Esters of Pyrethric acid


R1 R2 R1 R2
Pyrethrin I CH3 CH=CH2 Pyrethrin II COOCH3 CH=CH2
Cinerin I CH3 CH3 Cinerin II COOCH3 CH3
Jasmolin I CH3 CH2CH3 Jasmolin II COOCH3 CH2CH3

Etofenprox Metofluthrin

Neonicotinoids are another group of natural product-derived pesticides. This generation of


nicotine-related insecticides possess either a nitromethylene, nitroimine or cyanoimine group.
Nicotine in the form of tobacco extract was for centuries the best available plant-derived agent to
prevent sucking insects from damaging crops. The search for unusual structures and optimization
revealed a new class of potent insecticides, known as neonicotinoids, which are similar to
nicotine in their structure and action as agonists of the nicotinic acetylcholine receptor (nAChR).
Fortunately, neonicotinoids are much more toxic to insects than mammals due in large part to
differences in their binding site interactions at the corresponding nAChRs. Neonicotinoids work
as agonists on nicotinic acetylcholine receptors and show selective toxicity for insects over
vertebrates.

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Bacillus thuringiensis (Bt) is a natural occurring, soil-borne bacteria that has been used since
the 1950s for natural insect control. It consists of a spore, which gives it persistence, and a
protein crystal within the spore, which is toxic. That toxic protein differs, depending on the
subspecies of Bt producing it, yielding a variance of Bt toxic to different insect species (or none
at all). When the bacteria is consumed by certain insects, the toxic crystal is released in the
insects highly alkaline gut, blocking the system which protects the pest’s stomach from its own
digestive juices. The stomach is penetrated, and the insect dies by poisoning from the stomach
contents and the spores themselves. Bt kurstaki (Bt-k) – Bacillus thuringiensis var kurstaki is a
naturally occurring soil bacteria ideal for controlling tent caterpillars, gypsy moth, cabbage
looper, tomato hornworm and other leaf eating caterpillars on trees, shrubs, tomatoes and other
vegetables.
Bt israelensis (Bt-i) – Bacillus thuringiensis var israelensis is a highly specific biological
pesticide for use against mosquito, black fly and fungus gnat larvae. Bacillus thuringiensis var
tenebrionis is toxic to colorado potato beetle and elm leaf beetle and may be used on potatoes,
egg plant, tomatoes and elms.

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Avermectins are a class of macrocyclic lactones derived from mycelia of the soil actinomycete,
Streptomyces avermitilis (soil inhabiting which is ubiquitous in nature). The naturally occurring
avermectins, a group of 16-membered macrocyclic lactones, are fermentation products from
Streptomyces avermitilis. They possess anthelmintic, insecticidal and acaricidal activity. From
the fermentation, eight different avermectins were isolated, which comprise four pairs of
homologues. Each pair contains a major component (the a-component) and a minor one (b-
component). They are usually produced in a ratio between 80:20 and 90:10. One of these pairs,
avermectin B1, that is the mixture of avermectins B1 a (>80%) and B1 b (<20%), is commonly
referred to as avermectin B1 or abamectin 1.
New lead molecules from this group includes N-Formyl-4’’-deoxy-4’’-(S)-methylamino
avermectin B1, 4’-O-Methoxymethyl avermectin B1 monosaccharide, 4’’-Deoxy-4’’-(S)-4’’-
methyl-4’’-acetylamino avermectin B1, 4’’-Deoxy-4’’-(R)-4’’-cyano-4’-methylamino
avermectin B1, 2’’-O,3’’-O,4’’-O-Trimethyl-4’-O-b-L-rhamnopyranosyl avermectin B1
monosaccharide and 4’’-(R)-2’’-(R)-Methyl-3’’-epi-4’’-methyl avermectin B1.

Natural avermectins

Abamectin Emamectin Benzoate

Spinosyns are a family of bacterial natural products with a unique cross-bridged macrocyclic
structure. Fermentation of the actinomycete Saccharopolyspora spinosa produces mixtures of
several analogs with two, known as spinosyn A and D predominating. Spinosyns include
spinosad, which was originally isolated from the fermentation of the soil actinomycete
Saccharopolyspora spinosa. Spinosad is a mixture of at least two major compounds, spinosyn A
and spinosyn D, with spinosyn A being the major constituent.

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Spinosyn A (R = H), and D (R = Me)
Spinetoram, a new chemical, is a semi-synthetic spinosyn discovered in modification studies of
fermenting substances of Saccharopolyspora spinosa. The active ingredient spinetoram is
composed of two chemical compounds spinetoram-J (major component) and-L, both of which
have a macrocyclic lactone structure. Spinetoram has good insecticidal properties such as broad
insecticidal spectrum, rapid action and short preharvest interval. Spinosad and, by extension,
spinetoram appear to be effective by both ingestion and contact and cause excitation of the insect
nervous system, leading to involuntary muscle contractions, prostration with tremors, and
finally paralysis.

Spinetoram-J Spinetoram-L

Ryanodine receptor activators


Diamide insecticides have emerged as one of the most promising classes of insecticide chemistry
owing to their excellent insecticidal efficacy and high margins of mammalian safety. Discovery
of flubendiamide was based on the elucidation of the molecular target site of the complex
alkaloid ryanodine [ryanodyl 3-(pyridine-3-carboxylate)] from the plant Ryania speciosa.
Although the structure of the synthetic compound is not based on that of the natural
insecticide, it was discovered with in vitro bioassays of the Ca2+ -ryanodine receptor
complex required for insect muscle function. Insecticides that target this complex are
considered very safe for mammals. The journey continues culminating in the discovery of the
chlorantraniliprole and its analogue cyantraniliprole.

Ryanodine Flubendiamide

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Chlorantraniliprole Cyantraniliprole

Neem bioactives

Biological activity of neem seeds, leaves and bark is well-established as feeding deterrent and
growth disrupter against insects and other arthropods. Its constituents manifest acute biological
activity, reflected by insect growth disruption, feeding and oviposition deterrence, repellence and
mortality inflicted on a variety of pests infesting agricultural and horticultural crops. Neem is a
valuable natural pesticide with low toxicity for vertebrates. Azadirachtin-A, a meliacin obtained
from this tree, has been rated as a potential botanical pesticide. The vulnerability of azadirachtins
to various natural environmental factors such as light, heat, humidity and others has raised
concerns about the viability of neem materials in pest control. Catalytic hydrogenation of
azadirachtin concentrates yielded the corresponding dihydro and tetrahydroazadirachtin
concentrates.
Neem plant constitutes a vast array of chemically diverse and structurally complex bioactive
compounds of which tetranortriterpenoids group of compounds commonly referred to as C-
secomeliacins or more specifically called limonoids, are found in abundance in neem seed
extracts. Chemical constituents are found in the leaves of neem as nimbin, nimbanene, 6-
desacetylnimbinene, nimbandiol, nimbolide, ascorbic acid, n-hexacosanol and amino acid, 7-
desacetyl-7-benzoylazadiradione, 7-desacetyl-7-benzoylgedunin, 17-hydroxyazadiradione and
nimbiol etc. Azadirachtin, a tetranortriterpenoid (C26 - terpenoid) has received the utmost
attention as bioactive molecule because of its relative abundance in neem seed kernel. It can
control 413 insect species from several orders (Singh & Saxena, 1999). Besides, Klocke (1987)
prepared eight azadirachtin-A derivatives, namely, 3–deacetylazadirachtin-A, 11– `O –
acetylazadirachtin-A, 11– O – methylazadirachtin-A, 22,23–dihydroazadirachtin-A, 2´, 3´, 22, 23
tetrahydroazadirachtin-A, 1– detigloyl -22, 23–dihydroazadirachtin-A,2´,3´-dihydroxy - 2´,3´,
22,23 tetrahydroazadirachtin-A, 11, 20 – O, O dicarbmethoxy 22, 23 – dihydroazadirachtin-A,
and compared their IGR activity against Heliothis virescens.
COOCH 3
O COOCH 3 O H
O OH OH O
O 11 O
14
1 20
O 8
22 O O
3
O
O OH
O OH 23 H O
H OH O O OH
O H 3 COOC
H 3 COOC

Azadirachtin-A Azadirachtin-B

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O COOCH 3 O
COOCH 3 O
O OH
O O O

O
AcO AcO O
H OH O
OH
O O

Azadirachtin-D Salannin

COOCH O O
3
O
O

O
O
OAc
H 3 COOC OAc

Nimbin Azadirone HO OH

O OH
OAc
O
O O
O

O
AcO
H OH HO O HO OAc
O

Marangin Meliantriol

Essential oil
Many plant essential oils show a broad spectrum of activity against pest insects, fungi and
nematodes ranging from insecticidal, antifeedant, repellent, oviposition deterrent, growth
regulatory and antivector activities. These oils also have a long tradition of use in the protection
of stored products. Recent investigations indicate that some chemical constituents of these oils
interfere with the octopaminergic nervous system in insects. As this target site is not shared with
mammals, most essential oil chemicals are relatively non-toxic to mammals and fish in
toxicological tests, and meet the criteria for “reduced risk” pesticides. Some of these oils and
their constituent chemicals are widely used as flavoring agents in foods and beverages and are
even exempt from pesticide registration. This special regulatory status combined with the wide
availability of essential oils from the flavor and fragrance industries, has made it possible to fast-
track commercialization of essential oil-based pesticides. Though well received by consumers for
use against home and garden pests, these “green pesticides” can also prove effective in
agricultural situations, particularly for organic food production.

Agrochemicals for disease management


Major classical fungicides are classified based on their chemical nature and mode of action.

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1. Inorganic fungicides include sulfur powder, lime sulfur, copper sulfate, mercuric chloride,
lime Bordeaux mixture, copper hydroxide, cuprous oxide, etc.
2.Organic sulfur fungicides including Mori on behalf of ammonium, enemy Rust sodium, ziram,
zineb, mancozeb, thiram, etc.
3.Organophosphorus fungicides or organic arsenic fungicides inhibiting rice blast Edifenphos
Fosetyl, Hinosan, Kitazin P, methyl tolclofos etc.
4.The substituted benzenes fungicides thiophanate-methyl,chlorothalonil, Fenaminosulf etc.
5. Azole fungicides such as triadimefon, carbendazim, hymexazol, procyclidine azole, etc.
6. Antibiotic fungicides multi-resistant vancomycin, kasugamycin, streptomycin, agricultural
antibiotic, etc.
7.Other fungicides such as metalaxyl,the sclerotia Lee,procymidone,iprodione,folpet, captan,etc.

Strobilurins, (Strobilurins A through H) are natural compounds produced by several


basidiomycete fungi, Strobilurus tenacellus, Xerula spp., and Cyphellopsis anomala. They
inhibit electron transfer in mitochondrial respiration by binding to the ubiquinol site of
cytochrome b. Some of the examples include kresoxim-methyl, trifloxystrobin, azoxystrobin,
famoxadone, and fluoxastrobin. These are used on many crops to protect against a wide range of
plant pathogens.

Strobilurin fungicides

Blasticidin S is an antibiotic that is produced by Streptomyces griseochromogenes, s an inhibitor


of protein synthesis in both prokaryotes and eukaryotes. As Blasticidin S proved to exert a potent
curative effect on rice blast disease, it has been widely used as a practical control agent in eastern
Asia.

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Blasticidin S

Kasugamycin was first isolated from the soil actinomycete, Streptomyces kasugaensis. It is
sold as a hydrochloride salt form and inhibits binding of aminoacyl-tRNA to the mRNA-
30S and -70S ribosomal subunit complexes, thereby inhibiting protein synthesis. It is a
systemic fungicide that can be used to treat infected plants, as well as prevent the infection of
crops.

Kasugamycin

Pyrrolnitrin is an antifungal antibiotic. Pseudomonas pyrrocinia and


other Pseudomonas species produce pyrrolnitrin from tryptophan as secondary
metabolite. The fungicides fenpiclonil and fludioxonil are chemically synthesized with
basic skeleton of pyrrolnitrin.

Polyoxins are a group of nucleoside antibiotics composed of heterocyclic moieties


containing nitrogen. The polyoxins are produced by Streptomyces cacao and the nikkomycins
by Streptomyces tendae. The former compounds were discovered during a search for new
agricultural fungicides and pesticides. Both polyoxins and nikkomycins are pyrimidine

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nucleosides that inhibit the enzyme chitin synthase, which leads to the depletion of chitin in the
fungal cell wall. These molecules are transported into the cell via peptide permeases.

Saponins are constitutive triterpenoid, steroid, or steroidal glycoalkaloid molecules with one or
more sugar chains, have detergent-like properties, and are important in plant defense against
microbial infection. Saponins, such as CAY-1, are believed to be fungicidal due to interaction
with fungal membrane components such as sterols. CAY-I is a gitogenin-based, steroidal saponin
with four glucose and one galactose sugars attached to the number three carbon of the steroid
group. It is significantly lethal in vitro against a number of medical and plant pathogens at
concentrations less than 20 µM but is not cytotoxic to mammalian cells below 80 µM.

Natural products have been playing an important role in agriculture either as crop protection
agents or as leads for development of greener pest management agents. The successful products
like spinosyn, neonicotinoids, pyrethroid insecticides, and strobilurin fungicides ensure the role
of natural products in future crop protection strategies.

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CAY-1, a fungicidal saponin from Capsicum frutescens

Agrochemicals for nematode management

The elimination of nematodes from some crops is essential particularly of high-value


horticultural products. Chemical treatment with fumigants or nematicides may be the only
technique widely available.The use of chemicals in protected cropping may still be preferable to
other techniques such as steam treatment for economic and practical reasons. The use of soil-less
growing media in some north European countries has resulted in a decreased demand for
chemical treatments. Many horticultural and salad crops are grown in soil under polythene and
soil treatment with methyl bromide, dazomet or non-fumigant nematicides is widely practised.

Chemical name Trade name Formulation


Fumigants
Methyl bromide Dowfume Gas
1,3 dichloropropene Telone/DD-95 Liquid
Ethylene dibromide1 Dowfume W-85 Liquid
Metam-sodium Vapam Liquid
Dazomet Basamid Dust (prill)
Methyl isothiocyanate Di-Trapex Liquid
Chloropicrin1 Larvacide Liquid
Organophosphates
Thionazin Nemafos Granular or emulsifiable liquid
Ethoprophos Mocap Granular or emulsifiable liquid
Fenamiphos Nemacur Granular or emulsifiable liquid
Fensulfothion Dasanit Granular
Terbufos Counter Granular
Isazofos Miral Granular or emulsifiable liquid
Ebufos Rugby Granular or emulsifiable liquid

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Carbamates
Aldicarb Temik Granular
Aldoxycarb Standak Flowable
OxamyI Vydate Granular or emulsifiable liquid
Carbofuran Furadan/Curaterr Granular or flowable
Cleothocarb Lance Granular

Several natural nematicides are known. An environmentally benign garlic-


derived polysulfide product is approved for use in the European Union (under Annex 1 of
91/414) and the UK as a nematicide. Another common natural nematicide is obtained from neem
cake, the residue obtained after cold-pressing the fruit and kernels of the neem tree. Known by
several names in the world, the tree was first cultivated in India in ancient times and is now
widely distributed throughout the world. The root exudate of marigold (Tagetes) is also found to
have nematicidal action. Alpha-terthienyl, a naturally occurring secondary plant metabolite is
found in abundance in the roots of Tagetes species proved to be highly effective against plant
parasitic nematodes. Nematophagous fungi, a type of carnivorous fungi, can be useful in
controlling nematodes, Paecilomyces being one example.

α-Terthienyl α-Terthienyl-methanol

Agrochemicals for weed management

The different types of herbicides are all designed to kill plant tissue. However, they accomplish it
by two basic methods. They are known as contact herbicides and systemic herbicides. Contact
herbicides are popular because they work quickly by killing the tissue in as fast as one day. Some
herbicides will combine contact with systemic chemicals for a faster effect. The systemic
herbiciides travels with the sap so it usually doesn't have the quick "knockdown" effect. The
greatest benefit of a systemic type of herbicide is that it will kill the entire plant, roots and
all. Based on chemical composition herbicides are classified as

S. No. Group Examples


1 Phenoxy 2, 4-D (2, 4-Dicholoro Phenoxy acetic acid)
Compounds MCPA (2 Methyl, 4-Chlorophenoxy acetic acid)
Sesane (Sodium 2, 4-Dichlorophenoxyethl sulphate)
2, 4-D EP or TRIS (2.4 Dichlorophenxy acetic acid)
2, 4, 5-T (2, 4, 5 – Trichlorophenoxy acetic acid)
2, 4 TP or Silvex (2, 4,5 Trichloro Phenoxy proplanci acid)
2 Phenyl Acetic Fenac or 2, 3, 6 Trichloro phenyl acetic acid
Acid
3 Benzoic Acid 2,3,6 Trichloro Benzoic acid
2, amino 2, 5 Dichlorobenzoic acid
4 Pthalic Acid Endothal, DCPA
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5 Aliphatic Acid TCA – Trichloro acetic acid.
Dalapon
6 Substituted Pentachlorophenol,
Pnenols DNBP or Dintrophenols
7 Heterocyclic Simazine
Nitrogen Atrazine
Derivatives Maleic hydrazide
Amitrol
8 Aliphatic Organic Fenuron
Nitrogen Monuran
Derivatives Diuron
Neburon
9 Carbamates Isopropyl N phenylcarbamate.
CIPC
10 Other Amides Diphenamid
CDAA or Rendexn
11 Sulfonylureas Amidosulfuron, Azimsulfuron,Bensulfuron-
methyl, Chlorimuron-ethyl, cinosulfuron,
Chlorsulfuron, Ethoxysulfuron, Flazasulfuron, Flupyrsulfuron-
methyl-sodium, Imazosulfuron, Metsulfuron-
methyl, Nicosulfuron, Oxasulfuron

There are very limited examples of successful bioherbicides. L-phosphinothricin [homoalanin-4-


yl(methyl)phosphinate], a natural peptide produced by several Streptomyces spp. L-
Phosphinothricin is the active ingredient of glufosinate (sold under several trade names,
including Basta, Ignite, and Liberty. Glufosinate also contains an equivalent amount of the
inactive D-enantiomer. Glufosinate is the only commercial herbicide with the mode of action to
inhibit Gln synthetase which catalyzes the ATP-dependent condensation of Glu with ammonia to
yield Gln in amino acid biosynthesis. Streptomyces hygroscopicus and Streptomyces
viridochromogenes also synthesize the tripeptide L-alanyl-L-alanyl-phosphinothricin (bialaphos,
also known as bilanofos), a proherbicide that releases L-phosphinothricin in planta.

Alternaria alternata ssp. lycopersici (AAL)-toxin and tentoxin, are active at lower
concentrations than many commercial herbicides. Orn carbamoyl transferase, a key enzyme in
the urea cycle that converts Orn and carbamoyl phosphate to citrulline, is the target site of
phaseolotoxin, a sulfodiaminophosphinyl peptide produced by P. syringae. A number of
phytotoxic natural products such as gostatin (5-amino-2-carboxy-4-oxo-1,4,5,6-tetrahydro-
pyridine-3-acetic acid), produced by S. sumanensis and cornexistin, produced by P.
variotii targeted Asp transaminase, also called Asp aminotransferase, is an important pyridoxal
phosphate-dependent enzyme in amino acid metabolism. Besides, Thiolactomycin, a metabolite
from Norcardia and Streptomyces spp., and cerulenin, a metabolite from C. cerulens, are potent
inhibitors of the plant enzyme involved in de novo fatty acid synthesis. AAL-toxins and
fumonisins, produced by A. alternata and Fusarium spp., respectively, inhibit ceramide
synthases at submicromolar concentrations.

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Glufosinate Bialaphos

Thiolactom
ycin

Herbicide safeners are compounds of diverse chemical families which selectively protect crop
plants from herbicide damage without reducing activity in target weed species. They are applied
with herbicides to protect crops against their injury. Using chemical safeners offer practical,
efficient and simple method of improving herbicide selectivity. This method has been applied
successfully in cereal crops such as maize, rice and sorghum, against pre-emergence
thiocarbamate and chloroacetanilide herbicides. Various hypotheses were proposed explaining
mechanisms of action of herbicide safeners: interference with uptake and translocation of the
herbicide, alteration in herbicide metabolism, and competition at site of action of the herbicide.
This produced the first commercial herbicide safener, 1,8-naphthalic anhydride (NA), which was
patented by the Gulf Oil Company in 1971, for use as a seed-treatment to protect maize from
injury by thiocarbamate herbicides.

References

1. Cantrell CL, Dayan FE and Duke SO (2012) Natural products as sources for new pesticides.
Journal of Natural Products 75: 1231−1242.
2. Casida JE and Durkin KA (2013) Neuroactive insecticides: targets, selectivity, resistance,
and secondary effects. Annual Review of Entomology 6: 99–117.
3. Copping LG and Duke SO (2007) Natural products that have been used commercially as crop
protection agents. Pest Management Science 63:524–554.
4. Dayan FE, Cantrell CL and Duke SO (2009) Natural products in crop protection. Bioorganic
& Medicinal Chemistry 17: 4022–4034.
5. Duke SO, Romagni JG and Dayan FE (2000) Natural products as sources for new
mechanisms of herbicidal action. Crop Protection 19: 583-589.
6. Gullino ML, Leroux P and Smith CM (2000) Uses and challenges of novel compounds for
plant disease control. Crop Protection 19, 1–11.
7. Hertlein MB, Thompson GD, Subramanyam B, Athanassiou CG (2011) Spinosad: A new
natural product for stored grain protection. Journal of Stored Products Research 47 (3): 131-
146.

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and human health management", 07-27 Dec, 2017
8. Isman MB (2008) Perspective Botanical insecticides: for richer, for poorer. Pest
Management Science 64:8–11.
9. Pitterna T, Cassayre J, Hüter OF, Jung Pierre MJ, Maienfisch P, Kessabi FM, Quaranta L and
Tobler H (2009) New ventures in the chemistry of avermectins. Bioorganic & Medicinal
Chemistry 17 4085–4095.
10. Popp J, Pető K, and Nagy J (2013) Pesticide productivity and food security - A review.
Agronomy for Sustainable Development 33 (1): 243-255.
11. Isman, M. B. (2008). Botanical insecticides: for richer, for poorer. Pest management
science, 64(1), 8-11.
12. Höfte, H., & Whiteley, H. R. (1989). Insecticidal crystal proteins of Bacillus
thuringiensis. Microbiological reviews, 53(2), 242-255.
13. Knowles, B. H. (1994). Mechanism of action of Bacillus thuringiensis insecticidal δ-
endotoxins. Advances in insect physiology, 24, 275-308.
14. Munhoz, V. M., Baida, F. C., Lopes, G. C., Santiago, D. C., de Souza, J. R. P., & de Mello, J.
C. P. (2017). Extracts and semi-purified fractions of Tagetes patula flowers in the control of
root-knot nematodes. Semina: Ciências Agrárias, 38(6), 3529-3538.
15. Sharma, A., Sharma, S., & Dalela, M. (2014). Nematicidal activity of Paecilomyces lilacinus
6029 cultured on Karanja cake medium. Microbial pathogenesis, 75, 16-20.
16. Duke, S. O., Owens, D. K., & Dayan, F. E. (2014). The growing need for biochemical
bioherbicides. In Biopesticides: State of the Art and Future Opportunities (pp. 31-43).
American Chemical Society.
17. Isman, M. B., & Grieneisen, M. L. (2014). Botanical insecticide research: many publications,
limited useful data. Trends in plant science, 19(3), 140-145.
18. Dhingra, S., Walia, S., Kumar, J., Singh, S., Singh, G., & Parmar, B. S. (2008). Field efficacy
of azadirachtin‐A, tetrahydroazadirachtin‐A, NeemAzal® and endosulfan against key pests
of okra (Abelmoschus esculentus). Pest management science, 64(11), 1187-1194.

15 Winter School on "New generation smart agrochemical oriented approaches for crop
and human health management", 07-27 Dec, 2017
Safety Operations in Chemical Laboratory
Aditi Kundu and Mukta Chakrabarty

Division of Agricultural Chemicals


ICAR-Indian Agricultural Research Institute, New Delhi 110 012
Safety in a chemical laboratory is a foremost requirement. Chemistry wet laboratories contain
certain inherent dangers and hazards. As a chemistry student working in a laboratory, you must
learn how to work safely with these hazards in order to prevent injury to yourself and others
around you. You must make a constant effort to think about the potential hazards associated with
what you are doing, and to think about how to work safely to prevent or minimize these hazards
as much as possible. The following guidelines are here to help you. Please understand and follow
these guidelines and act according to the principles behind them to help everybody to be as safe
as possible. Ultimately, your own safety is your own responsibility. Please make sure you are
familiar with the safety precautions, hazard warnings and procedures of the experiment you are
performing on a given day before you start any work. Therefore, it is necessary to go through the
given instructions thoroughly before planning of any experiment in a chemical laboratory. Any
experiments should not be performed without an instructor in attendance and must not be left
unattended while in progress. No unauthorized experiments are allowed in a chemical laboratory.
No modification of the already developed protocol is allowed alone. No work in a chemical
laboratory outside of regular hours is allowed, except under exceptional circumstances.
Safety rules

❖ Make sure you are familiar with all the safety information given to you about each
experiment before starting the experiment. This includes your manual, safety guidelines,
any required information or any other information provided by the supervisor.
❖ Always wear safety glasses (including during check-in and check-out), except when their
removal has been specifically authorized by the advisor prior to their removal. Contact
lenses are forbidden. You must also wear a face shield or mask to cover nose for the
protection and smell of hazardous chemicals.
❖ You must wear a lab coat always in all chemistry laboratory. Footwear must completely
cover the foot and heel. You must wear long pants to cover maximum body parts. If you
arrive at your chemistry laboratory and do not have the required clothing, you will be
directed to purchase missing items (glasses, lab coats, disposable foot coverings and long
pants) before you will be allowed to participate in the laboratory. Loose hair must be tied
back so as to be out of the way. Dangling jewellery must be removed.
❖ Do not eat or drink in the laboratory. Do not use ear buds or earphones while in the lab.
Visitors are not allowed to be present in the lab.
❖ Please keep your work area and the common work areas neat and tidy. Also, please make
sure the aisles, safety showers, eyewash stations and doorways are unobstructed.
❖ Please keep all glass wares, plastic wares as clean and in drawers with proper marking.
Equipments should be kept along the side wall so that routine activity should not be
hampered. Solvents and reagent bottles should be marked and arranged alphabetically. A
register should be maintained where all the available reagents and chemicals should be

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written alphabetically. Solvents should be kept in 1L or 500 mL bottle on the working
rack. Big reagent and solvent bottles should be kept separately in a solvent chamber.
Hazardous reagents should be kept inside the fume hood. Always use fume hood to
conduct any reaction procedure.
❖ Please clean up spills immediately based on the nature of the spillage. If the spill is large
or is of a hazardous material, inform the technical advisor immediately. Use spill mix to
absorb solvent or caustic liquids.
❖ Please dispose of wastes properly and in a timely manner and according to the
instructions provided in your laboratory manual. Follow the instructions given to dispose
the chemicals and glass wares separately. Do not pour solvents or any liquid chemicals in
wash basins. Certain chemical needs pretreatment before disposed off.
❖ Wash your hands thoroughly when work is over. Check the instruments like oven,
weighing balance, water bath and confirm all these instruments are switched off before
leaving the lab.

Chemical safety

• The vapours of many organic solvents are flammable or combustible. Do not expose
electric sparks, open flames and heating elements to organic solvent vapours. Unless
otherwise stated, assume all organic solvents are flammable.
• Many chemicals (solid, liquid or vapour) are poisonous. Do not taste chemicals. If it is
necessary to smell a chemical, do so by fanning the vapours towards your nose. Never
inhale directly. Avoid inhaling dust or fine powders. Use fume hoods and personal
protective equipment when necessary.
• Do not pipette with your mouth. Be extremely careful when transferring, distilling or
refluxing volatile liquids. Do not return used chemicals back to the stock container. Do
not tap flasks under vacuum.
• Do not heat, measure or mix any chemicals in front of your face. Never heat a closed
system, it will act as a bomb
• Never pour water into concentrated acid. Pour acid slowly into water, stirring constantly.
Mixing acid with water is often exothermic.
• Concentrated acids and bases are stored in the fume hood. Do not carry them to your
bench.
• Make sure test tubes containing reactions are pointed away from people, especially when
they are being heated. Pressurized gas cylinders must only be operated carefully in
presence of trained supervisor.

Emergency procedures

Become familiar with the location of the safety showers, eyewash stations, first aid kits and fire
extinguishers. Remember, every sink with a hose can act as an eyewash station. Know the route
you are supposed to take in case of an evacuation. In a potentially life threatening emergency
situation, notify supervisor and remember the emergency numbers of security and medical etc.

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First aid
• Burns represent the most common injury in the chemistry laboratory. They are generally
of either the thermal or chemical type. First aid for surface burns of the thermal type
involves immersing the burned part in cool water or applying an ice pack to relieve pain
and prevent swelling and blistering. The burn is then covered with a clean, sterile, lint-
free dressing. Do not apply lotions, ointments or oily dressings. For more serious burns
involving deeper layers of skin and tissue, arrange for immediate medical aid.
• To minimize injury due to chemical burns, the chemical must be removed from the skin
immediately. Flush liquid chemicals away with water; continue to flush for 20 minutes.
Continue first aid as for a thermal burn (preceding paragraph). Medical attention should
always be sought in the case of chemical burns, especially as delayed reactions are not
uncommon.
• If chemical spill on cloth then quickly remove all contaminated clothing while using the
safety shower to flush the chemical from the skin. Time is of the essence here and there is
no place for modesty. Continue to flush the affected area with water for at least 20
minutes. Do not use chemical neutralizers.
• If any chemicals splashed in to the eyes immediately flood the eyes with water so as to
dilute and eliminate the chemical. Hold the eyelids open to facilitate the process. Flush
the eyes for at least 20 minutes. Apply clean dressings over both eyes and arrange for
immediate medical aid, regardless of the severity of the injury.

Rules specific for the organic chemistry laboratories.

▪ Be careful when packing melting point tubes or using Pasteur pipettes: glass capillaries
and pipettes are very fragile and can result in cuts to the hand.
▪ Broken glassware should be cleaned and then disposed off in the yellow ‘Broken Glass’
containers.
▪ Do not force glass tubing through corks or stoppers. Use lubricant if necessary.
▪ Chemical reagents should always be stored in labeled bottles. Read the label carefully
before using the reagent. Do not take more than the required amount of chemical and do
not return any unused chemical back to the reagent bottle.
▪ Solid waste should be disposed off in the ‘Solid Waste’ container in the fume hood.
▪ Organic liquid waste should be disposed of in the ‘Organic Liquid’ waste container
located in the fume hood.
▪ Chemically contaminated paper (e.g. filter paper, weigh paper) should be disposed off in
the contaminated paper waste container.
▪ Paper waste is discarded in the waste paper basket found under the central sink.
▪ Silica gel and TLC plates should be disposed of in the silica waste container.
▪ Mercury spills from broken thermometers must be cleaned up immediately.
▪ Dilute aqueous inorganic solutions, acids, and bases can be disposed off down the drain
with large volumes of water.
▪ Do not pour any organic liquids (including acetone) down the drain.

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Instructions need to be followed

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Agrochemicals for Crop Improvement: An Overview
Aditi Kundu and Devakumar C.

Division of Agricultural Chemicals,


ICAR-Indian Agricultural Research Institute, New Delhi – 110 012

Introduction

As the global population is reaching 9 billion the need for plant-derived food and feed is
dramatically increasing. Modern agriculture is challenged to produce more efficient and disease
resistant crop plants. Next to molecular breeding technologies, agrochemicals and biologicals
can assist to improve crop yield. Crop yields increased dramatically in the 20th century. The vast
majority of that increase has occurred since the last world war and has been powered by changes
in the genetic potential of the crop and in the way in which it has been managed. Nevertheless,
the challenge to feed a world population that is likely to rise to 8 billion is formidable,
particularly since recent analyses suggest that the rate of increase in yields of several crops may
have dropped over the last decade. More sophisticated crop protection chemicals designed on the
basis of vastly increased screening potentials and possibilities of rational design will be
supplemented by a battery of decision support systems to aid management choices which can be
precisely implemented. Genetic improvement is the area in which to look for the major
breakthroughs. Improvements may be obtained by re-assorting what has been achieved through
enhanced breeding technologies, by randomly induced change, and by generation of totally new
possibilities through the use of specialty chemicals like chemical hybridizing agents etc.

The failure of a plant to produce functional gametes is known as sterility. Sterility induced by
application of certain chemicals like, auxins and antiauxins (NM, TlBA, 2, 4-0, MH, etc.),
halogenated aliphatic acid (FW-450, daIapon, etc.), gibberelic acid (GAJ, etheophon, OPX-3718,
arsenicals (MSMA, OM, ZMA, etc.), RH-531, RH-532 etc. These chemicals are called
gametocides since they lead to pollen abortion and thereby cause male sterility, sometimes it
results in female sterility also. So the term chemical hybridizing agents (CHA) are used since
after the entire primary objective is to produce a hybrid. A number of CHAs have been reported
to cause male sterility for production of hybrids. Therefore, no need to developed maintainer or
restorer lines and save time, labor and money. In the present review, different aspects of CHA
viz., characteristic, mode of action, stage of treatment, application doses, etc. for different CHAs
and also for different crop wise.

Sterility inducing hybridizing agents


Chemical induction of sterility in plants has been of interest since 1950 when the potential for
selective male sterility was first demonstrated (Moore 1950; Naylor 1950). Though production of
hybrids was successfully achievable via cytoplasmic male sterility, interest began to develop in
the use of chemical compounds that could easily induce selective male sterility in crop. Male
sterile plants are effective females for a crossing programme; and their employment makes the
laborious procedure of emasculation superfluous. The application of male-sterility may permit
the production of hybrid seeds and the commercial exploitation of heterosis in crops where
emasculation on a large scale is not feasible. Chemicals used to induce male sterility include
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chemical hybridizing agents (CHAs), male gametocides and pollen suppressants. Treatment with
maleic hydrazide, tri-iodobenzoic acid, a-naphthalene acetic acid, 2.4-dichlorophenoxiacetic
acid, gibberelic acid and α,β-dicholoroisobutyrate have been successfully investigated to induce
male sterility in plants. However some of these compound induced undesirable effects on
morphological traits. Chemical type, duration of dose, mode of treatment and plant development
stage at which chemicals should be applied were reported as important factors in optimization
(Kaul, 2012). It was recognized that while there may be disadvantages with chemicals there
could also be advantages, especially in terms of the time required to discover economically
viable hybrids. The chemical method for inducing sterility can obviate the often lengthy time
period required to obtain male-sterile and restorer lines, which usually must precede evaluation
of hybrid performance. Consequently, chemicals became of interest both for use as breeding
tools and as a means to produce hybrids on a commercial scale.

Over the years investigators have used various terms to name these compounds such as male
sterilant, selective male sterilant, pollen suppressant, pollenocide, androcide etc. Rohm and Haas
Company, a leader in the field of plant male sterilant chemicals, has attempted to avoid use of
terms that imply knowledge of the mode and/or site of action of a chemical. The reason was the
precise mode or site of action was not known until well after a chemical begins to be investigated
as a hybridizing agent. Further, Rohm and Haas preferred to adopt terminology that is broad
enough to embrace all conceivable modes or sites of action. Since the ultimate goal is to produce
hybrids, the term chemical hybridizing agent has been established.

Chemical hybridizing agents (CHAs)

Male sterility induced through CHAs is an important tool for the exploitation of hybrid vigor in
field crops. Accurate CHA dosages at critical stages of head development could induce complete
male sterility. Selective CHAs have been exploited in many breeding lines, eliminating lengthy
procedures involved in cytoplasmic male sterility (CMS) and maintenance of fertility restoration,
and mitigating the negative impact on performance of inbred lines due to induction of CMS from
other species (Cisar and Cooper, 2003). CHAs can also be utilized for assessing large numbers of
genotypes for general and specific combining ability during the early evaluation phase of the
candidate inbred lines, and can be exploited as a substitute to hand emasculation in interspecific
and inter varietal crosses, as well as recurrent back crosses. A number of approaches have been
proposed to avoid self-pollination for the commercial production of hybrid wheat seeds, which
include genetic male sterility (GMS), cytoplasmic male sterility (CMS), photo-thermo-sensitive
male sterility (PTMS) and chemical hybridizing agents (CHAs). Of these, CHA-induced male
sterility can provide rapid, flexible and high performance seed-producing female parents for F1
hybrid production; they simultaneously avoid fluctuations of genotype and environmental factors
in maintaining male-sterility and/or male-fertility restoration.
While a range of CHAs have been used in various crops, few have attained commercial
success. Major drawbacks of CHA use include reductions in hybrid seed yield by reducing
female fertility, undesirable morphological changes, and difficulties of application. A majority of
chemical compounds developed as CHAs have been reported as phytotoxic. CHAs increased the
production cost and had lower effectiveness to kill the male gametes of wheat (Triticum aestivum
L.). Potential effectiveness of CHAs in inducing male sterility and their use have been reported
in many crops. Some indicated variability in effectiveness of different CHAs for sunflower

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pollen sterility; of the different chemicals used, a synthetic detergent and benzotriazol (C 6H5N3)
could successfully induce pollen sterility. Cytological analysis indicated that application of
CHAs resulted in abnormal plastids in pollen mother and tapetal cells.
In general, easy, cheap and reliable induction of male sterility will certainly allow in several
crops a more economical production of hybrid seeds. Moreover it might offer new possibilities
for the breeding of crops in which effective hereditary male sterility has not yet been explored.

Hybridizing chemicals
i) Maleic Hydrazide (MH)
Maleic hydrazide (1,2-dihydro-3,6-pyridazinedione), classified as an antiauxin, initiated
interest in the chemical control of sterility. After the initial finding of the potential for MH on
maize, established that MH did not prevent pollen production in the field. Nevertheless,
enthusiasm for testing MH on many crop species was generated and it became one of the most
studied sterilants. In spite of this, MH has not proved useful as a CHA on any crop, primarily
because it is not adequately male/female selective. MH could be used as a breeding tool for
Secale cereale L. (rye).

ii) Triiodobenzoic Acid (TIBA)


TIBA, also classified as an antiauxin, was first shown to induce at least some degree of male
sterility in Citrullus lanatus (Thumb.) (watermelon) and Lycopersicon esculentum Mill.
(tomato) . At least partial female fertility was also obtained. In a subsequent study (Hensz and
Mohr 1959), TIBA induced male sterility in watermelon. TIBA-induced sterility was obtained
in Helianthus annuus L. (sunflower) (Kiermayer 1959) and in Vitis vinifera L. (grape). In spite
of these examples of preliminary success, TIBA has not been reported on extensively and has
not found use as a CHA.
A substantial number of auxins have been investigated following preliminary indications that
2,4-dichlorophenoxyacetic acid (2,4-D) induced at least partial sterility with some female
fertility in watermelon. Additionally, naphthalene acetic acid (NAA) induced male sterility in
tomato.

iii) Halogenated Aliphatic Acids


a) FW-450 (sodium p-dichloro isobutyrate): could induce male sterility in cotton without
significant adverse effect on female sterility, it was evaluated extensively on many plant
species. The earliest studies have been reviewed. Initially, FW-450 showed promise that it
might be useful as a commercial CHA for crops such as cotton, and Beta uulgaris L. (sugar
beet). However, it ultimately became clear that FW-450 was often too phytotoxic, was not
adequately male/female selective, or adversely affected germination of the hybrid seed. For
reasons such as these Rohm and Haas Company discontinued research and development in
the early 1960s. FW-450 continued to be investigated through the 1960s and 1970s with the
latest report as recent as 1980.

b) Dalapon: Dalapon (sodium 2, 2-dichloropropionate, Dowpon) is chemically quite closely


related to FW-450, and its use on cotton, tomato, Pisum satiuum L. (pea), and maize was first
reported. Although subsequently it received less attention than FW-450, it was studied
sometimes by itself as well as in comparison with FW-450 and other halogenated aliphatic

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acids. In general, dalapon exhibits the same weaknesses as FW-450. The two agents differ
primarily in degree and not in kind. In comparative studies, FW-450 is usually considered
superior to dalapon but there are exceptions. Dalapon’s shortcomings are such that it has no
potential for production of hybrid seed in commercial quantities. It might be used for
breeding purposes in rye.

b) Other Halogenated Aliphatic Acids: six halogenated aliphatic acids, including FW-450 and
dalapon, and found dichloroacetic acid best for Antirrhinum (snapdragon). Seventeen
halogenated aliphatic acids, including FW-450 and dalapon, were investigated in Capsicum
annuum L. (pepper), and dalapon and sodium a-chloropropionate were the most effective.
Although related acids were found to be superior to FW-450 and dalapon in specific
situations, there is no evidence they have found significant use as CHAs.

iv) Gibberellins
Interest in gibberellins (GA) as male sterilants developed as a consequence of studies in maize,
who found that under appropriate conditions potassium gibberellate induced 100% male sterility
without damage to female fertility. In spite of this encouraging finding, there is no evidence in
the literature to indicate active pursuit of GA as a CHA for maize for over a decade. Complete
sterility when GA was applied at a fairly precise premeiosis growth stage. However, they
concluded that the heterogeneity of growth stages found in large-scale maize plantings precluded
its use as a practical CHA. If the treatment growth stage was too early, adequate male sterility
could not be obtained; if too late, weakening of stems resulted. Successful induction of male
sterility in sunflower by GA was obtained. Early studies on sunflower were reviewed, who also
presented results of their own investigations. By 1970, it had been shown that 100% male
sterility with about 50% female fertility was achievable. Such results clearly established the
potential of GA as a breeding tool for sunflower. GA can be used in practice in sunflower. GA
alone and in combination with TIBA on a range of sunflower genotypes. High levels of male
sterility were obtained but there were significant interactions between weather, site, and
genotype.
Adverse effects on female fertility were variable but significant. Gibberellins have been
studied quite intensively on Allium cepa (onion). GA can be used as a breeding tool in onion but
that relatively poor seed set in male-sterile plants together with high cost precluded its use on a
large scale. Preliminary encouraging results have been obtained with GA on Lactuca satiua L.
(lettuce). Eenick reported that acceptable levels of female fertility were obtained in male-sterile
plants and concluded there is a possibility of using GA for large-scale breeding in lettuce.
Similarly, initial results with GA on Brassica spp. (Brussels sprouts, cabbage, cauliflower, and
kale) were promising.

v) Ethephon
Ethephon [(2-chloroethyl)phosphonic acid, Ethrel, Camposan has been investigated extensively
on many crop species since the initial discovery (Rowell and Miller 1971) that it could induce
selective male sterility in Triticum aestiuum L. (wheat). Such crops include Hordeum uulgare L.
(barley), Auena satiua L. (oats), Oqza satiua L. (rice), X Triticosecale Wittmack (triticale),
Pennisetum spp.(proso), Linurn usitatissimum L. (flax), Solanum melongena L. (eggplant),
maize, rye, lettuce, and sugar beet. Varying degrees of male sterility together with either

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adequate or inadequate female fertility have been obtained in rice, barley; eggplant, proso, and
flax.
Wheat has been the primary crop for ethephon investigations. Ethephon has been shown to
induce adequate male sterility in both winter and spring wheats when applied at the appropriate
premeiosis growth stage. Adequate female fertility can also be maintained. Ethephon has been
used in small-scale crossing blocks to produce sufficient hybrid seed for yield trials, but
numerous disadvantages preclude its use for commercial production of hybrid wheat. Ethephon
cannot sterilize all tillers in populations in which tillers are at variable development stages at
treatment time. This deficiency can be alleviated to some degree by using high seeding rates,
which tend to improve tiller stage uniformity. Another weakness of ethephon is that it inhibits
culm elongation, so spikes may not fully emerge from the leaf sheath. This problem can be
minimized by combining ethephon with GA, but this adds to the application cost, already high
because of the high dosage requirement (about 10 kg/ha).

vi) DPX 3778

The announcement was made in 1973 that DPX 3778 [3-(p-chlorophenyl)-6-methoxy-s-triazine-


2,4-(1H, 3H) dionetriethanol amine] had potential as a CHA for maize (Long et al. 1973) and
this potential was confirmed by Laible (1974). Subsequently, few investigations were reported
on DPX 3778; apparently Du Pont, which initially developed it, elected to discontinue
development at an early stage due to unavoidable weaknesses. This CHA has been studied in the
People’s Republic of China where it is known by the designations KMS-1 and Gametocide No.
2.

vii) Other synthetic CHA:


Rohm and Haas and Shell Chemical Company (Shell) have been patented few CHA which are
being used successfully as CHAs under field conditions.

RH-531: At about the time the selective male sterilant properties of ethephon became known,
Rohm and Haas renewed interest in CHA with the discovery that RH-531 [sodium l-(p-
chlorophenyl)-l,2-dihydro- 4,6-dimethyl-2-oxonicotinate induced male sterility in barley (Yih et
al. 1971). Subsequently some degree of female fertility was retained under conditions of
essentially complete male sterility. Additional studies, however, quite clearly established that
RH-531 was not adequately male/female selective in barley or in wheat.
RH-532: RH-532 is another Rohm and Hass chemical and it differs slightly form RH-531. This
chemical initially was not distributed to public (or) private investigators to any extent better than
RH-531. It could induce 100% male sterility in a broad spectrum of wheat genotype at
application ratio of 1-3 kg/ha. However, like RH-531, it too was inhibitory and was not
adequately male/female selective for large scale use. Nevertheless, many wheat hybrids in the
range of 85-100% purity were obtained in USA and France. Thus, RH-531 was a significant
improvement from Ethephon and DPX-3778.

RH-2956 and RH-4667: These chemicals were developed by Rohm and Haas between 1976 and
1981. Their important feature is that they are significantly less inhibitory to growth than RH-532,
indicating that inhibition is not obligatorily associated with male sterility. Spike length inhibition

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is minimal and male sterile florets have good flowering opening characteristics with these two
compounds. Further they are better than RH-532, with regard to female fertility. The major
disadvantage is the relatively high application rate (about 10 kglha).
Hybrid (RH-0007): This is a Rohm and Haas chemical with a common name Fenridaxon-
potassium and a trade mark HYBRED. This induces 95-100% male sterility in a very broad
spectrum of hexaploid winter and spring wheat. Genotypes responded to dosages as low as 0.5 to
2.0 kglha. This is very slightly inhibitory, and florets on sterile plants have good opening and
closing characteristics. Though fenridodaxon-potassiu appears to be close to ideal gametocide,
improper treatment timing can have unfavorable impact.
Chemicals that affect pollen viability have potential value to plant breeders for production of
F1hybrids and seed. The non-protein amino acid [1R- (1α, 2β, 5α) ]-3-azabicyclo[3.1.0]-hexane-
2-carboxylic acid, otherwise known as cis-3,4-methano-L-proline, originally isolated from the
seeds of Aesculus parviflora, was found to be highly effective chemical hybridizing agent. Novel
convenient method capable of yielding multigram amounts of both cis- and trans- ethanoproline
isomers as racemic hydrochloride salts has already been devised and evaluated.
Advantages and disadvantages of gametocides
The long and cumbersome process of developing male-sterile lines and their maintenance
through B-lines can be overcome. The danger of narrow genetic base for cytoplasmic male-
sterility being experienced in 3 line hybrid breeding programme would cases to be a problem.
Any two parents which are heterotic in a cross can be directly used for hybrid production.
Unstable but otherwise best combining CMS lines can be made use of complete sterility through
the use of gametocides. Gametocides are very useful in cases where induction of EGMS is
incomplete due to deviations in temperature and/or' photoperiod as the sensitive phase of panicle
development for EGMS and gametocides. If synchronization of flowering or' consecutive rainy
days proves a limitation to the use of gametocides, hybrids seeds may not be produced. However,
there will not be heavy yield losses as the yield of female parent would compensate it to a greater
extent.
The gametocide spray should be taken up at the most responsive stage during plant growth for
maximum sterility to be caused. Only certain specific developmental stages are sensitive. The
need for repeated application increases the cost of hybrid seeds. In cereals since there is
difference in the development of spikelets within and between panicles, single treatment of
gametocide even at the most responsive stage would not ensure sterility in flowers. In some
crops the flowering in tillers does not coincide with that of the main stem flowering and such
cases warrants repeated spraying. High doses of these chemicals have been found to be
phytotoxic. In many cases it has been found to cause some amount of female sterile. to be a
problem. The responses to the plants have been found to be genotype dependent. This also is
dependent on environment, if heavy rainfall occurs immediately after the spray, the gametocidal
action would be nullified. Carry over effect of some chemicals has also been reported e.g. in
triticale treated with ethrel, F1 seed germination reduced from 93 % in control to 73 % when the
concentration was 500 ppm. Higher concentration of 1000, 2000 and 4000 ppm further reduced
seed germination to 57,42 and 30% respectively. In cases where it causes incomplete sterility,
pollen shedders may result. Cost involved is more especially when repeated spraying is done. In

25 Winter School on "New generation smart agrochemical oriented approaches for crop
and human health management", 07-27 Dec, 2017
plants having c1eistogamous flower, where selfing is the rule, gametocide usage in limited only
for academic purpose. These chemical are teratogenic and must be handled with utmost care.
Future Perspective

Considering the relative advantageous and disadvantageous effects of high concentrations of


gametocide on the one hand and differential phase of development of spikelets within and
between panicles on the other, there is great scope for gametocides possessing stable and
systemic action. The systemic nature would facilitate slow and continuous release of the
gametocide at low concentration. As believed by some workers, the granular from of Ethrel
might prove effective. Research efforts directed towards the discovery and extensive use of such
chemical as well as detailed studies on the stage specificity, environmental and genotype
influence on the efficiency of the gametocide would enhance the prospects of hybrid seed
programme in self pollinated crops like wheat and Rice.
Many plants produce exudates which have suppressing action on other plants. Such
chemicals can be tested for gametocide potential thereby producing cheap alternative for the
growth regulation and also the risk of their toxic effects on human beings can be negated e.g.
plants of Brassica sp. Produce brasinosteroids which have been identified as having growth
regulator potential. Similar other substances can be studied for gametocidal action.
The concept of using chemicals to restore fertility in genetic male sterile has been around
for about as long as there has been interested in chemical hybridizating agents. The advantages
orchemical restoration of genetic male sterility is at least three fold. Firstly the chemical need not
be 100% effective since its use is only to increase supplies of female parent seed. But if it is
more effective more efficiently seeds could be multiplied. Secondly, all seeds produced is
exactly what is wanted. It is a fail safe method. Thirdly since the chemical dose not have to be
used in the hybrid seed production, a lower volume is required compared with the amount of
gametocides required. This in turn has favourable impact on crop and environmental residues.
Once an adequate male sterile female parent seed supply is available, hybrid seed production
using the current production techniques can be followed.
In crops that produce tillers, breeding work should be carried out to produce lines in
which tillers do not deviate from main stem for days to flowering.

Plant growth promotimg chemicals


Natural waxes as plant growth regulator

A large number of naturally occurring PGR have been reported. Some of the popular natural
hormones include auxins, gibberellins, cytokinins, abscisic acid, brassinolides, and polyamines
which find wide use in agriculture. Triacontanol, a naturally occurring plant growth regulator
obtained from plant cuticular waxes (rice bran wax, sugarcane wax) or insect cuticular waxes
(beeswax). Seaweed extract and protein hydrolysate are other group of natural PGRs which find
wide acceptability by the farmers. Natural waxes derived from rice bran wax, sugar cane wax
and bees wax contain a number of long chain aliphatic alcohols of which triacontanol is well
known for its plant growth regulatory activity. Many triacontanol based PGRs are commercially
available in the market. Triacontanol has been particularly found to increase the growth and yield
of rice (Oryza sativa), wheat (Triticum aestivum), tomato (Lycopersicon esculentum) and maize
(Zea mays). It increased the leaf area, dry weight, water-soluble protein, reducing sugars and free

26 Winter School on "New generation smart agrochemical oriented approaches for crop
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aminoacids in rice and maize seedlings. Natural occurring lac insect wax is also a good source of
long chain aliphatic alcohols. Such waxes and products derived from them can find use as PGR
or as seed coating material.

Triacontanol (TRIA)

The plant growth regulatoryactivityof triacontanol (TRIA) was first discovered in alfalfa
(Medicago sativa L.). TRIA is a secondaryplant growth substance and cannot be considered as a
phytohormone. Such types of growth regulators enhance the physiological efficiencyof the cells
and, thus, exploit the plant genetic potential to a large extent. TRIA is considered to be the only
primaryalcohol found in the wax of the rice leaves. The callus tissue contained a homologous
series of alkanes, namely, n-C23H48, n-C25H52, and n-C27H56 compared to n-C27H56, n-C29H60, and
n-C31H64 found in the rice leaves.

Triacontanol (TRIA) is a natural plant growth regulator found in epicuticular waxes. It is


used to enhance thecrop production in millions of hectares, particularlyin Asia. Quite a number
of researchers have reported the TRIA-mediated improvement in growth, yield, photosynthesis,
protein synthesis, uptake of water and nutrients, nitrogen-fixation, enzymes activities and
contents of free amino acids, reducing sugars, soluble protein, and active constituents of essential
oil in various crops. Expectedly, TRIA enhances the physiological efficiency of the cells and,
thus, exploits the genetic potential of plant to a large extent. In fact, TRIA increased free amino
acids, reducing sugars, and soluble protein of rice (Oryza sativa L.) and maize (Zea mays L.)
within 5 min. TRIA elicited the appearance of L(-)-adenosine within 1 min in the roots of plants,
the shoots of which were sprayed with nanomolar concentrations of TRIA. TRIA and
octacosanol (OCTA), the primaryalcohols, are ubiquitous in theenvironment. OCTA was
reported to inhibit the activityof TRIA in the seedlings of rice (Oryza sativa L.) at equimolar
concentrations; and both TRIA and OCTA elicited a second messenger, known as OCTAM and
triacontanol second messenger (TRIM), respectively.

Hexacosanol

Octacosanol

Dotriacontanol

Tetrattriacontanol

TRIA rapidly increases the ratio of L(-)- to D(-)-adenosine, probablyat the tonoplast.
However, it is to be resolved as to how TRIA elicits L(-)-adenosine and what is the source of L(-

27 Winter School on "New generation smart agrochemical oriented approaches for crop
and human health management", 07-27 Dec, 2017
)-adenosine in plants. Based on known metabolic processes, de novo synthesis of L(-)-adenosine
is unlikely, because of the rapidity of the response. TRIA-mediated increase in dry matter
production could influence the inter-relationship between primaryand secondary metabolism,
leading to increased biosynthesis of secondary products. Various studies present strong
evidences that application of TRIA applied either to the root medium or to leaves enhanced the
growth and yield of vegetables and other crops, including agronomic and horticultural crops as
well as medicinal and aromatic crop plants under normal and adverse conditions. However,
further investigations are required to elucidate the possible role of TRIA on plant growth
regulation, physiological activities and secondary metabolite biosynthesis regarding medicinal
and aromatic plants subjected to abiotic stress. The present review covers the pivotal role of
TRIA in plant growth and development, its mode of action and its significance in improving the
crop productivity and quality of agricultural crops.

Nitrification Inhibitors

Nitrification Inhibitors The nitrification inhibitors (NIs) decrease the availability of nitrate and
consequently its vulnerability to escape mechanisms. A lot of work has been reported on the
ways to retard/inhibit the rate of nitrification not only to reduce fertilizer N losses but also to
prolong the persistence of fertilizer N in ammoniacal form. Since ammonia or ammonium-
producing compounds are the main source of fertilizer N, maintenance of the applied N in the
ammonium form should mean that less N is lost by denitrification. Numerous substances have
been tested for their ability to inhibit nitrification. These include 2-chloro-6-(trichloromethyl)
pyridine (nitrapyrin), sulfathiazole, dicyandiamide, 2-amino-4-chloro- 6-methyl pyrimidine, 2-
mercaptobenzothiazole, thiourea, and 5-ethoxy-3-trichloromethyl-1,2,4- thiadiazole (terrazole).
Unfortunately, most of these compounds have limited usefulness. For example, the most
commonly used nitrification inhibitor, nitrapyrin, is seldom effective because of sorption on soil
colloids, hydrolysis to 6-chloropicolinic acid, and loss by volatilization.

Dow Chemical Company. It is marketed under the trade name “N-Serve 24 nitrogen stabilizer”
(a.i. 240 g/L) and “N-Serve 24E nitrogen stabilizer” (a.i. 240 g/L). The rates of application
advised by Dow Chemical Company for band and row placement are 1.125–1.25 L/ ha of N-
Serve 24E for cotton, maize, sugar beet, sorghum, and wheat and 4.50–6.75 L /ha for potatoes
before or after planting or sowing. For broadcasting, the rate of application has to be increased
considerably. When granulated fertilizer is used, it can be applied at 0.2–1.0 % of the amount of
fertilizer N. Because of its high vapor pressure, nitrapyrin cannot be granulated with solid-N
fertilizer like urea without loss of the inhibitor during processing, storing, and handling.
Nitrapyrin sometimes shows poor activity due to sorption on soil colloids, hydrolysis to 6-
picolinic acid, and loss by volatilization. AM 2-Amino-4-chloro-6-methylpyrimidine is another
well-known nitrification inhibitor developed by Toyo Kaotsu Industries Inc. (now Mitsui Toatsu)
of Japan. Pure AM is a white crystalline substance and is soluble in water but unlike nitrapyrin; it
is relatively insoluble in organic solvents. AM is less volatile and less effective than nitrapyrin.
AM is effective when applied at 5–6 kg/ha. Etridiazole 5-Ethoxy-3-trichloromethyl-1, 2, 4-
thiadiazole (Terrazole, Etridiazole, Dwell) is an effective nitrification inhibitor developed by
Olin Corporation, Baltimore, USA. This product is available as a wettable powder or technical
grade liquid with 35 % and 95 % a.i., respectively. As a coating on ammonium sulfate and urea,
terrazole 95 % a.i. is used up to 1.5 % by weight. The recommended rates of compound for crops

28 Winter School on "New generation smart agrochemical oriented approaches for crop
and human health management", 07-27 Dec, 2017
like potatoes, sugar beet, lettuce, and onions are 0.6–1 kg/ha. Dicyandiamide (DCD, IV) It has
been developed both as a slow-release nitrogen source as well as nitrification inhibitor. In Japan,
it is added to mixed fertilizer and a product urea-form plus containing 10 % by weight of DCD is
produced. A fertilizer containing urea and DCD in a 4:1 ratio is commercially available in West
Germany. DCD is toxic to plants, but the effect differs with plant species. This compound is
effective over a period of 1–3 months.
Synthetic nitrification inhibitors, though expensive, can efficiently inhibit nitrification. Certain
allelochemicals released by plants are also reported to have an inhibitory effect. Rice postulated
that because inhibition of nitrification results in conservation of both energy and nitrogen,
vegetation in late succession or climax ecosystems contains plants that release allelochemicals
that inhibit nitrification in soil. Some natural products from neem (Azadirachta indica, A. Juss),
karanja (Pongamia glabra, Vent.), mint (Mentha spicata, Mentha arvensis L.), and mahua
(Madhuca longifolia, L.) are reported to inhibit the activity of nitrifiers.

Conclusion

Allusions have already been made to the fact that Gametocide can be valuable breeding tools.
Perhaps their greatest advantage in this context is the relative ease with which kilogram
quantities of hybrids seed can be produced with chemical hybridizing agent techniques in
contrast to the gram quantities normally obtained by conventional hand crossing producers. From
the afore-stated literature it is vivid that, in spite of its innate advantages research on
development of gametocides is very limited and also there is a virtual vacuum for gametocidal
research during the past decade. However, in recent years, many foreign firms have tested and
developed proprietary sterility inducing hybridizing agents and have either not made them
available (or) made them available on a very restricted basis to public and seed company
breeders. Hence, there is a paucity of published information on the most recent chemicals. Only
limited quantities of hybrid seed are required for breeding purposes. Thus, even though a CHA
may have some adverse affect on female fertility, it may still be possible to produce seed in
quantities sufficient for breeding programs. Thus, a CHA that may not have potential for large-
scale commercial seed production may have potential for use in breeding programs. Research
works needs to be intensified to develop efficient gametocides and their potential needs to be
exploited.
While application of chemical fertilizers to agricultural crops has resulted in tremendous increase
in yield, problems arising due to escape to the environment of different nitrogen species,
especially N2O, nitrite, and nitrate, have raised serious economic and environmental concerns.
Of the different processes responsible for these, nitrification and denitrification are of prime
importance. Hence, efforts have to be made to regulate the process of nitrification (major source
of different N species) as a means to enhancing the use efficiency of N, decreasing
environmental/ economic concerns, and optimizing the functioning of agroecosystems. Use of
nitrification inhibitors has been helpful in mitigating the negative effects of fertilizer application.
However, continued efforts need to be made for finding more efficient and environment-friendly
products to suit the ever-changing agroclimatic conditions.

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and human health management", 07-27 Dec, 2017
References
1. Cheng, Y., W. Wang, Z. Li, J. Cui, S. Hu, H. Zhao and M. Chen. 2013. Cytological and
comparative proteomic analyses on male sterility in Brassica napus L. induced by the
chemical hybridization agent monosulphuron ester sodium. PLOS ONE. 8: 80191.
2. Kaul, M.L. 2012. Male sterility in higher plants. 10th vol. Springer Science and Business
Media.
3. Sleper, D.A. and J.M. Poehlman. 2006. Breeding field crops. 5th ed. Blackwell Pub
lishing.
4. Cross, J.W. and J.A.R. Ladyman. 1991. Chemical agents that inhibit pollen development:
tools for research. Sex. Plant Reprod. 4: 235–243.
5. Kalidasu, G., C. Sarada, P.V. Reddy and T. Yellam. 2009. Use of male gametocide: an
alternative to cumbersome emasculation in coriander (Coriandrum sativum L.). J. Hortic.
For. 1: 126–132.
6. Sharma, Y. and S.N. Sharma. 2005. Chemical hybridizing agents (CHA)–a tool for
hybrid seed production–a review. Agric. Rev. 26(2): 114–123.
7. Tripathi, S.M. 2008. Abnormal anther development and high sporo pollen in synthesis in
benzotriazole treated male sterile Helianthus annuus L. Ind. J. Exp. Biol. 46:71–78.
8. Tripathi, S.M. and K.P. Singh. 2013. Hybrid seed production in Helianthus annuus L.
through male sterility induced by benzotriazole. Open Access Sci. Rep. doi:10.4172/
scientificreports.645.
9. Wang, M.Y., Y.L. Song, S.X. Zhang, X.L. Zhao, J.W. Wang, N. Niu and G.S. Zhang.
2015. Analysis of skp1 gene expression in physiological male sterility induced by
chemical hybridizing agent SQ-1 in wheat (Triticum aestivum L.). Cereal Res. Comm.
43(2): 204–212.
10. Parodi, P.C. and M.D. Gaju. 2009. Male sterility induction by the chemical hybridizing
agent clofencet on wheat, Triticum aestivum and T. turgidum var. durum. Ciencia Inv.
Agr. 36: 267–276.
11. Li, Z., Y. Cheng, J. Cui, P. Zhang, H. Zhao and S. Hu. 2015. Comparative transcriptome
analysis reveals carbohydrate and lipid metabolism blocks in Brassica napus L. male
sterility induced by the chemical hybridization agent monosulfuron ester sodium. BMC
Genom. 16: 206.
12. Cheng YF, Wang Q, Li ZJ, Cui JM, Hu SW, Zhao HX, et al. Cytological and
comparative proteomic analyses on male sterility in Brassica napus L. induced by the
chemical hybridization agent monosulphuron ester sodium. Plos One. 2013; 8.
13. Willy D. Kollmeyer1, S. K. Barrett, and D. H. Flint. 1987. Synthesis of Racemic cis- and
trans-Methanoproline. Synthesis and Chemistry of Agrochemicals. Chapter 34, pp 401–
408.
14. Naeem, M., Khan, M. M. A., & Moinuddin. (2012). Triacontanol: a potent plant growth
regulator in agriculture. Journal of Plant Interactions, 7(2), 129-142.

30 Winter School on "New generation smart agrochemical oriented approaches for crop
and human health management", 07-27 Dec, 2017
Designing of Synthetic Molecules: Conventional Approaches
Rajesh Kumar

Division of Agricultural Chemicals,


ICAR-Indian Agricultural Research Institute, New Delhi – 110 012

Since the earliest days of agriculture, weeds, diseases, and infestations by insects have
always been major reasons for yield losses. Today modern agrochemicals together with
mechanization and precision farming are contributing in a decisive manner to secure the
sustainable production of quality food, feed and fibre. Agrochemicals, including crop protection
agents, are one of the most critical inputs in modern agriculture for producing food, fibre and
controlling pests. These products has been a driving force in the green revolution by providing
tools to control a myriad of plant diseases, weeds and pest insects, enabling increasing yields of
food and fiber and subsequently play an important role in ensuring food security by improving
both crop quality and quantity.

The judicious usage of agrochemicals is helpful in providing a sustainable production of


food, feed and fuel; in securing the most efficient use of land, water and energy; for protecting
crop yields; for increasing food quality; in enabling an efficient and effective production of food
at the lowest possible costs and in providing sustainable incomes to the farmers.

The continued improvements in crop yields are required to address the following major
concerns. There is enormous pressure on the scientists, researchers working in the field of
agrochemical development to deal with these issues.

31 Winter School on "New generation smart agrochemical oriented approaches for crop
and human health management", 07-27 Dec, 2017
• Sustainable production of quality food for meeting the food demand for increasing
population, rising calorie consumption and changing dietary habits
• Increasing environmental stresses
• Growing more food from decreasing agricultural land
• Changing regulatory guidelines
• Increasing research and development costs
• Global financial instability

Due to the complexities with existing agrochemicals viz. development of resistance, the
desire for products with more favorable environmental and toxicological profiles, shifting pest
spectra, and changing agricultural needs and practices, new agrochemicals with better bio-
efficacy, novel modes of action and better safety profiles are continuously sought.
The desirable characteristics of any ideal agrochemical are given below.
• Rapid action on target pests / site
• Completely harmless to non-target organisms like human, domestic animals, wildlife,
and other aspects of the environment
• No residues after completion of desired effect on target pests
• Inexpensive and readily available in necessary quantities
• Chemically stable
• Non-flammable
• Safe to use around homes or industrial sites
• Easy to prepare and apply
• Non-corrosive and non-staining
• No undesirable odor

The process for bringing new agrochemical to the market involves research, development
and registration. The steps for developing a novel agrochemical include discovery of a bioactive
lead compound through chemical synthesis followed by modification of the chemical structure
and optimization of the biological activity; selection of the commercial candidate after
consideration of cost, efficacy, selectivity, and safety factors. Discovering and developing a new
agrochemical products takes years, almost a decade of effort and dedication. The cohesive efforts
of an integrated multidisciplinary team of chemists, chemical engineers, computational chemists,
toxicologists, and biologists are required to achieve this goal.

Following are the common methods for discovering agrochemical lead compounds.

a) Random Synthesis and Screening: It involves the random synthesis of number of series of
compounds followed by their bioassay against the different pests. Random synthesis and
screening has been considered as one of the most successful approaches for new product
discovery.

b) Modification of Natural Compounds: Natural products play an important role in


discovery and development of novel bioactive products. Natural products embody
inherent chemical structural novelty and complexity as well as attractive biological
activity, which lead to the discovery of new targets, pathways, or modes of action.

32 Winter School on "New generation smart agrochemical oriented approaches for crop
and human health management", 07-27 Dec, 2017
Modification of natural products is a versatile approach to explore their targets, binding
modes and cellular pathways. Among conventional pesticides, about 20% are either
natural products or natural product-derived substances. Therefore the natural products are
an important part of conventional pesticide discovery strategies. Increasing demand for
“greener” pest management products is also fueling discovery and development efforts
for new bioactive products by utilizing this approach for agrochemical discovery.

Cost of bringing a new product to market

c) Me Too Chemistry: This approach utilizes the bioactive molecules with similar structures
to the existing products. The molecular modifications of existing bioactive compounds,
lead to new me-too bioactive compounds. Me Too Chemistry has also been considered as
one of the two successful approaches for new agrochemical discovery.

d) Combinatorial Chemistry: Combinatorial chemistry may be defined as the systematic and


repetitive, covalent connection of a set of different building blocks of varying structures
to each other to yield a large array of diverse molecular entities. Combinatorial chemistry
is technique involving synthesis of compounds in mass instead of a single compound,
which are screened as a whole mixture for particular biological activity. The rapid
synthesis of compounds saves the time and cost associated with the agrochemical
discovery.

e) Intermediate Derivatization Methods (IDMs): This IDM approach involves the


application of a wide variety of synthetic methodology on key intermediates resulting in
an efficient route to innovative chemical structures, which, in conjunction with biological

33 Winter School on "New generation smart agrochemical oriented approaches for crop
and human health management", 07-27 Dec, 2017
screening, become patentable leads or target compounds. There are following three types
of IDMs.

• Common Intermediate Method involves the modification of key intermediates used as


building blocks that possess functionality amenable to preparing a diverse set of
structures.

• Terminal Group Replacement Method uses the modification of key intermediates that
possess functionality amenable to replacing terminal moieties of existing
agrochemicals.

• Active compound Derivatization Method utilizes the modification of known active


compounds possessing functionality amenable to derivatization.

These approaches have been exploited for the discovery of new bioactive agrochemicals.
Approaches like Intermediate Derivatization Methods are more efficient than old traditional
discovery methods. These recent approaches are helpful in reducing the increasing time and cost
of the discovery process by enabling greater efficiency to the discovery process of new
agrochemicals and will enhance the prospects of sustainable development new bioactive
products.

Conclusion

The above mentioned research methodologies provided a sustained availability of bioactive


leads to develop commercial agrochemical products for meeting the need of crop growers.
Though the current arsenal of effective agrochemicals is available to crop growers, the scientists
all over the world need to develop new innovative research tools for discovering agrochemicals
having enhanced toxicological and environmental safety for addressing the grand challenge of
providing food and nutritional security to a world population of nine billion people in the year
2050 after overcoming the hurdles of limited arable land, increasingly frequent extreme weather
events, and weed and pest resistance.

References

1. Cantrell CL, Dayan FE and Duke SO (2012) Natural products as sources for new
pesticides. J Nat Prod 75: 1231–1242.
2. Guan A, Liu C, Yang X, and Dekeyser M (2014) Application of The Intermediate
Derivatization Approach in agrochemical discovery. Chem Rev 114: 7079-7107.
3. Lamberth C, Jeanmart S, Luksch T and Plant A (2013) Current challenges and trends in
the discovery of agrochemicals. Science 341 (6147): 742-746.
4. Loso MR, Garizi N, Hegde VB, Hunter JE and Sparks TC (2017) Lead generation in crop
protection research: A portfolio approach to agrochemical discovery. Pest Manag Sci 73:
678–685.
5. Maienfisch P and Stevenson TM (2015) Discovery and Synthesis of Crop Protection
Products, ACS Symposium Series, American Chemical Society, Washington, DC.

34 Winter School on "New generation smart agrochemical oriented approaches for crop
and human health management", 07-27 Dec, 2017
6. Planche AS, Cordeiro MNDS, Montero LG and Bueno RY (2011) Current computational
approaches towards the rational design of new insecticidal agents. Curr Comput Aided
Drug Des 7: 304-314.
7. Scherkenbeck J and Lindell S (2005) Applications of combinatorial chemistry in the
agrosciences. Comb Chem High Throughput Screen 8(7): 563-576.
8. Sparks TC and Lorsbach BA (2017) Perspectives on the agrochemical industry and
agrochemical discovery. Pest Manag Sci 73: 672–677.
9. Whitford F, Pike D, Burroughs F, Hanger G, Johnson B, Brassard D, and Blessing A. The
Pesticide Marketplace - Discovering and Developing New Products
(https://www.extension.purdue.edu/extmedia/PPP/PPP-71.pdf accessed on 16-10-2017 at
1400 hrs)

35 Winter School on "New generation smart agrochemical oriented approaches for crop
and human health management", 07-27 Dec, 2017
Molecularly Imprinted Polymers-Artificial Receptors
Indu Chopra and Suman Gupta
Division of Agricultural Chemicals
ICAR-Indian Agricultural Research Institute, New Delhi-110 012

To prevent crops from the attack of pests and diseases for ensuring increased production and
quality of food, it becomes inevitable to use pesticides in modern agriculture. But prolonged,
indiscriminate and non-judicious use of these chemicals has not only posed threat to different
environmental matrices but has also been found to be dangerous to the non target organisms
including humans. Due to their potential impact on flora, fauna and non target organisms even at
low concentration, it becomes imperative to develop selective, simple and reliable method for
detection as well as removal of these contaminants from different environmental compartments.
Target specific recognition by natural receptors like antibodies, aptamers, enzymes etc. is
crucial for biochemical processes to take place. Though these interactions are complex but are
highly specific and selective for the target molecule. These natural receptors have been utilized
as recognition units for wide variety of assays and sensor systems 1,2. However, the low stability
and durability of these biorecognition elements along with the high cost and poor performance in
different solvents has limited their widespread applicability and commercialization. Limitations
of using the biological receptors for different applications prompted the researchers to design
stable artificial receptors which can serve as analogue of natural ones. Consequently numerous
efforts have been made to design and synthesize artificial receptors with bio-mimetic properties.
Amongst different methods, molecular imprinting technique offers a facile and straight forward
strategy towards the development of such artificial receptors 3,4,5. As compared to natural
recognition units the molecularly imprinted polymers (MIPs) so prepared are inexpensive, robust
and have better performance at different pH in different solvents 6,7. The technique has wide
applications for selective recognition of different molecular structures ranging from ions to
biomacromolecules 8,9.

1.1 What are Molecularly Imprinted Polymers (MIPs)?

Molecularly Imprinted Polymers (MIPs) are the synthetic polymeric materials which are
obtained by the copolymerization of functional monomer, cross linker and analyte of interest/
template molecule in suitable porogen. Subsequent removal of template from the polymeric
material leaves behind the specific recognition sites/ cavities which are complementary in size,
shape and chemical functionality to that of the template. These cavities help in the selective
recognition of the target analyte amongst different complex moieties (Fig.1). The imprinted
polymers have the ability to selectively recognize and bind with the template molecule amongst
the closely related chemical species. Along with the low cost of production, these materials are
stable to extreme variation in pH, temperature and organic solvents as compared to their
biological counterparts 10.

36 Winter School on "New generation smart agrochemical oriented approaches for crop
and human health management", 07-27 Dec, 2017
Fig 1. Molecular Imprinting Process

The efficiency and performance of MIPs is based on three factors:


(1) Binding affinity (BA) which measures the amount of template molecule adsorbed within the
cross linked polymer,
(2) Imprinting factor (IF) which takes into account the relative binding affinity of template
molecule in MIP and similarly produced NIP without the template and
(3) Separation factor (SF) which relies on the ability of MIP to differentiate between adsorption
of the template molecule and similar non template molecule11.
It has been found that BA is proportional to the binding energy12,13 and different models evaluate
these measures on the basis of following equations

BA= [UMIP] (1)


IF = [UMIP]/[UNIP] (2)
SF = [UMIP]/[Unon-MIP] (3)

where [U] is the average energy of the bound analyte, subscripts MIP, non-MIP, and NIP have
the respective following meaning: template binding in MIP, analyte (not template) binding in
MIP, and template binding in NIP. The BA for the template molecule is expected to be higher
than that of a non-template molecule. IF >>1, suggests that the MIP will recognize the template
molecule selectively as compared to the corresponding NIP.

1.2 Components of MIPs

1. Template: It is the target molecule for which imprinting has to be done or MIP has to be
prepared. Usually the target analyte is used as template for MIP preparation but in few
cases closely related structural analogue is used due to limited availability, high toxicity
or cost of the actual target molecule. This analogue is also known as dummy template or
template mimic. While selecting the template, it should be assured that the template
molecule should be thermo or photo stable, its functional groups should not affect
polymerization kinetics and be soluble in the prepolymerization mixture 14,15. Though

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MIPs can be prepared for different molecules but success rate for preparation of
molecules with molecular weight ranging from 200 to 1200 Da is better16.

2. Functional monomer: The functional monomers are the substances with functional
groups which interact with template molecule resulting in binding sites on the imprinted
polymer17. MIPs with higher selectivity and better binding capacity can be prepared if the
interaction between the template and the functional monomer are stronger. Few
monomers used generally in covalent as well as non-covalent imprinting are shown in Fig
2. The structure of the recognition sites is determined by the choice of functional
monomers18

(I)

Methacrylic acid Acrylamide Itaconic acid Styrene


(II)

4- vinylbenzaldehyde 4-ethenylphenylboronic acid 4-vinylbenzylamine

Fig 2. (I) Functional monomers commonly used in non-covalent imprinting (II)


Functional monomers generally used in covalent molecular imprinting

3. Cross-linker: In the synthesis of MIPs the cross-linker plays a crucial role in controlling
the morphology and imparting mechanical stability to the polymeric network. It also
stabilizes the imprinted binding sites on the polymer. Main function of cross-linker is to
ensure that functional groups which are being used for template binding are fixed in well-
defined positions in the polymeric network. The amount and size of cross-linker affects
the selectivity along with binding capacity of the polymer. Therefore, selection of type
and amount of cross-linker for the polymer synthesis should be done carefully.
Commonly used cross-linkers are presented in Fig 3.

Ethylene glycol dimethacrylate Divinylbenzene N,N'-Methylenebisacrylamide

Fig 3. Cross-linkers commonly used in molecular imprinting

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4. Solvent: It serves to form the monomer-template complex after mixing all the
components in one phase. It drives the reaction either in forward or reverse direction on
the basis of strength of interactions. As it helps to create pores in macroporous polymeric
network so it is also known as porogen19. In the non-covalent imprinting, porogen has
been found to affect the stability of template- monomer complex in the pre-
polymerization mixture. It has been found that the rebinding performance of MIPs is
usually better in the same solvent which has been used in polymerization as it provides
similar solvation conditions20

5. Initiator: Most of the imprinted polymers are synthesized by using conventional free
radical polymerization technique. The initiator helps in the radical generation by
decomposing itself either by thermolysis or photolysis. The initiators that are most
commonly used for free radical polymerization include azo compounds and peroxo
compounds. For eg: AIBN. It is to be noted that if the concentration of the initiator is
increased, it results in higher polymerization rates but the polymer with lower molecular
weight is obtained.

1.3 Fabrication of MIPs

On the basis of binding interactions between monomer and template molecule during the
reaction, MIPS can be fabricated through three different approaches: non-covalent,
covalent and semi-covalent.
I) Non-covalent or self assembling approach: It is the most extensively used method
in which a pre-polymerization mixture is formed with template, functional monomer,
cross-linker and porogen along with the initiator. A stable complex is formed
between template and the monomer by self assembly through hydrogen bonds,
Vander Waals forces, ionic or π-π interactions. The bonds so formed are less specific
and non- directional with binding sites being heterogeneous in nature.

II) Covalent or pre-organized approach: In this approach, the interaction between the
template molecule and the functional monomer is covalent. It results in well defined
and homogeneous binding sites. The bonds resulting from the method are specific for
particular functional group and directional.

III) Semi- covalent: It is a hybrid approach in which covalent bond is formed between
template molecule and the monomer to form the complex. Subsequent rebinding to
the polymer occurs through non-covalent interactions in this approach.

1.3.1 Synthesis
On the basis of final applicability, MIPs can be synthesized in a variety of physical
forms by choosing different methods of polymerization. Commonly used methods for
the purpose include:

I) Bulk Polymerization: The reaction occurs within the monomer itself which is
catalyzed by additives like initiator in the presence of heat/ light. The polymer so
obtained is of non-uniform molecular mass distribution as the reaction is highly

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exothermic which cannot be controlled easily21. This method can be used to obtain
the particles in the size range of micrometers.

II) Suspension Polymerization: The reaction is carried out with monomer, initiator and
modifier being dispersed in water by vigorous stirring. Stirring creates a uniform
suspension keeping the monomer droplets suspended. The reaction can be utilized
with metal ions, drugs or proteins as template. The beads so obtained are of diameter
5-50 micrometers depending on the speed of stirring and the amount of surfactant
used.

III) Emulsion Polymerization: In the commonly used method of oil in water emulsion,
droplets of the functional monomer are emulsified with surfactant in a continuous
phase of water leading to an emulsion. This technique can be used to prepare sub
micrometer sized beads having low poly-dispersity index22.

IV) Precipitation/Dispersion Polymerization: In this method, polymerization takes


place under diluted conditions (generally below 5% w/v monomer-solvent) and after
reaching a critical mass it results in precipitation of highly cross-linked polymeric
network. There is no need of extra stabilizer as the coalescence of the particles is
prevented by the rigidity of the cross linked polymer. The beads so obtained have
well defined size and in the sub- micron scale (0.3-10 µm).

1.3.2 Template removal


For optimum performance of MIPs, efficient removal of template or analyte from
polymerized network is of utmost importance. Non- extracted template from the polymer can
cause bleeding of the analyte under study that can lead to inaccurate results and restrict the
applications of molecularly imprinted polymers. Though, there are several extraction techniques
available for template removal but different factors like cost of the method, ease of application,
effect on different environmental components and the possibility of commercialization are
required to be taken into consideration23. The procedure generally used for template removal
depends on repeated washing cycles of the polymer, under mild conditions, with the solvent
suitable for the purpose. Different extraction techniques which can be used for the purpose are as
following:

➢ Soxhlet Extraction
➢ Washing online
➢ Solid phase extraction
➢ Pressurized solvent extraction
➢ Microwave assisted extraction
➢ Ultrasound assisted extraction
➢ Accelerated solvent extraction
It is important to note that the method of extraction needs to be chosen carefully as changes
taking place in the swelling of MIP network during extraction and subsequent desiccation can
possibly cause either collapse of the cavity which would hinder (sterically) the entrance of target
molecule, or in a distortion of the binding points or the strength of the interactions24,25.

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1.3.3 Optimization of MIP synthesis

Though the method of preparation of MIPs appears to be simple but the optimization of the
method for a specific template by standardizing the compositional variables (monomers, cross-
linkers, porogenic solvents, template to monomer ratios along with operational variables like
temperature, pressure, time etc.) is a cumbersome task. Change in any of the experimental
parameters can influence morphology, properties and performance of the resulting material. To
some extent, the experimental parameters can be optimized by using computational modeling
and simulation studies. Computational modeling based approach utilizes the computer software
to simulate and study the complex system behavior with the help of different principles of
mathematics, physics and computer science. The computational techniques ranging from
statistical treatment to quantum mechanical simulations can be used to study different aspects of
molecular imprinting26.

1.4 Characterization of Molecularly Imprinted Polymers

As the polymers so obtained are not uniform (molecularly as well as structurally) so it becomes
difficult to characterize these macromolecular substances. However there are some techniques
which can be used for their chemical, morphological and surface characterization. The main
techniques used for the purpose are mentioned below:

I) Chemical characterization

a. Elemental microanalysis: It is a destructive method which is used in a routine manner to


measure the elemental composition (carbon, hydrogen, nitrogen, chlorine) within the samples.
The information so obtained can be utilized to calculate the monomer composition of the
polymer. But this method is not sensitive enough to detect trace quantities of template that
remain in MIPs.

b. Fourier Transform Infra red Spectroscopy (FTIR): This technique can be used to get
qualitative information about the polymeric composition. Due to different chemical
environments in sample arising from the functional monomer and cross-linker, diagnostic signals
are received which can be used to know the chemical constitution of the polymer.

c. NMR: This technique can be used to find out the extent of complex formation in the pre-
polymerization mixture. It can also be used to investigate the chemical constitution of the sample
along with the steric configuration. 1H and 13C are the most commonly used nuclei for the
purpose.

II) Morphological characterization


Like other porous material, surface morphology of imprinted polymers can be characterized
using following techniques:

a. Microscopy: Characterization of the polymers like assessing the homogeneity of the


material and surface topography can be done through optical microscopy, Transmission Electron
Microscopy (TEM) and Scanning Electron Microscopy (SEM). Light microscopy can be used to

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get the information on structural integrity of the polymers. It provides information about the
general appearance and structure along with the homogeneity of the material27
b. Nitrogen sorption porosimetry by using BET (Brunauer, Emmett and Teller) analysis is
the main technique used to determine specific surface area, specific pore volume, pore size
distribution and average pore diameter values of the polymer particles while mercury intrusion
porosimetry is sometimes used and is more suitable for larger pores characterization.

1.5 Applications of MIPs


Molecular imprinting, being advantageous in terms of selectivity, stability and cost effectiveness,
has entered many entities including chemistry, biochemistry, biotechnology and many more. The
technique has widened the opportunity to imprint the polymers with different kinds of templates
including drugs, pesticides, sugars, proteins etc from analytical point of view along with its use
in catalysis, synthesis and drug development and screening28,29.
Recently, the development of molecularly imprinting polymers (MIPs) technology based
on the mechanism of molecular recognition and their further application as selective sorbents in
Solid Phase Extraction (MISPE) or as stationary phases in High Performance Liquid
Chromatography (HPLC) has provided a new versatile tool for highly selective quantification of
the toxins in food matrices.
Magnetic molecularly imprinted polymers have additional advantage of easy separation. The
polymer after use can easily be separated conveniently and economically by using a strong
magnet and the tedious steps of centrifugation and filtration can be avoided30. Use of MMIP
makes the method easier, quicker, simpler, and more effective to perform than MIP-SPE with
cartridge mode.
A simple, sensitive and selective method for detection of melamine in milk was successfully
developed using magnetic molecularly imprinted polymer (MMIPs) as sorbent. The MMIPs were
synthesized using melamine as template, methacrylic acid (MAA) as monomer and Fe3O4@ SiO2
as magnetic support. For characterization of the prepared materials FT-IR, XRD, TGA, SEM
and VSM were used. The imprinted polymer layer was coated on the surface of modified Fe3O4
nanomaterials. Prepared MMIPs showed high adsorption, fast binding and high selectivity for
melamine. Recoveries of melamine from real samples ranged from 85.6-104.2%. This work
proposed a, rapid, reliable and convenient approach for the determination of trace melamine in
complicated milk sample31.
A monolithic SPME (solid-phase microextraction) fibre based on a molecularly imprinted
polymer was synthesized and coupled with gas chromatography (GC) for extraction and
determination of chlorpyrifos. It was found that the temperature, pH, ionic strength and time of
extraction were the most significant factors affecting extraction. The extraction efficiency was
found to be very high and the method was used for the detection of chlorpyrifos in apple and
grape fruits 32.
The molecularly imprinted polymers for selective extraction of imidacloprid from rice were
prepared and used as the dispersant in matrix solid-phase dispersion (MSPD) extraction. Co-
extractives from the sample matrix were successfully eliminated and the recoveries obtained
were in the range of 93-92%. The optimized extraction conditions were: 8 min dispersion time,
sample to MIPs ratio as 1:2 and aqueous methanol (20%) as washing solvent33.
MIP technology can also be used as antibody-like materials with high selectivity and
sensitivity, owing to their long-term stability, chemical inertness and insolubility in water and
most organic solvents. To date, MIPs have been successfully used with different types of

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transducers and several methods have been used to achieve a close integration of the transduction
platform with the polymer. In particular, the integration of MIPs with sensors can be realized by
in situ polymerization, using a photochemical or thermal initiator, or by surface grafting with
chemical or UV initiation. The advantage of this latter approach lies in the possibility of
controlled modification of inert electrode surfaces with thin films of specific polymers. Today, a
simple approach to obtain sensor device, is the combination of MIPs with a piezoelectric
transducer (e.g., quartz) to create an acoustic sensor called QCM-MIP sensor. The coating of the
crystal with the MIP can be obtained by in situ polymerization directly onto the surface of the
device or via pre-prepared MIP particles that are immobilized on the sensor surface using a PVC
matrix.
A matrix based on MIP has broad technical applicability in various disciplines like
chemical separation, medical use or in analytical chemistry. In recent years molecularly
imprinted polymers (MIPs) have been applied in wide range of areas such as chemical and
biological sensors, solid phase extraction and drug assays owing to their inherent robustness,
reusability and reproducibility. Furthermore, MIPs can also be used as tools for studies
concerning antibody/receptor binding site mimicry as well as being used as antibody substitutes
for biomedical applications. Viral detection is a rapidly growing field owing to its increasing
prevalence and ongoing evolution of viral variants and drug resistance.

References
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12. Wu, L, Sun, B, Li, Y, and Chang, W (2003). Study properties of molecular imprinting
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22. Markowitz, MA, Kust, P R, Deng, G, Schoen, PE, Dordick, JS, Clark, DS, and Gaber, BP
(2000). Catalytic silica particles via template-directed molecular imprinting. Langmuir,
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23. Lorenzo, RA, Carro, AM, Alvarez-Lorenzo, C, and Concheiro, A (2011). To remove or
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29. Ye, L, and Mosbach, K (2001). Molecularly imprinted microspheres as antibody binding
mimics. React and Funct Polym, 48(1), 149-157.
30. Hu, Y, Liu, R, Zhang, Y, and Li, G (2009) Improvement of extraction capability of
magnetic molecularly imprinted polymer beads in aqueous media via dual-phase solvent
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imprinted polymer for melamine and its application in milk sample analysis by HPLC. J
Biomedical Sci., 5, 2.
32. Moghaddama, HH and Jedia, DJ (2015) Solid-phase microextraction of chlorpyrifos in
fruit samples by synthesised monolithic molecularly imprinted polymer fibres. Internl J
of Environ Anal Chem, 95, 33-44
33. Chen, L and Li, B (2012) Determination of imidacloprid in rice by molecularly
imprinted-matrix solid-phase dispersion with liquid chromatography tandem mass
spectrometry. J Chromatogr B, 897, 32-36.

45 Winter School on "New generation smart agrochemical oriented approaches for crop
and human health management", 07-27 Dec, 2017
Cutting Edge Approach for Molecular Designing: Molecular
Docking
Aditi Kundu and Ashish Khandelwal
Division of Agricultural Chemicals
ICAR-Indian Agricultural Research Institute, New Delhi 110 012
Docking aims to accurately predict the structure of a ligand within the constraints of a receptor
binding site and to correctly estimate the strength of binding. As evidenced by the expansion in
docking literature , the field has come of age. The main developments in docking continued to be
along the expected lines: receptor flexibility, solvation, fragment docking, postprocessing,
docking into homology models, and docking comparisons. Several new, or at least newly
invigorated, advances occurred in areas such as nonlinear scoring functions, using machine-
learning approaches.
The completion of the human genome project has resulted in an increasing number of new
therapeutic targets for drug discovery. At the same time, high-throughput protein purification,
crystallography and nuclear magnetic resonance spectroscopy techniques have been developed
and contributed to many structural details of proteins and protein–ligand complexes. These
advances allow the computational strategies to permeate all aspects of drug discovery today,
such as the virtual screening (VS) techniques for hit identification and methods for lead
optimization. Compared with traditional experimental high-throughput screening (HTS), VS is a
more direct and rational drug discovery approach and has the advantage of low cost and effective
screening. VS can be classified into ligand-based and structure-based methods. When a set of
active ligand molecules is known and little or no structural information is available for targets,
the ligand-based methods, such as pharmacophore modeling and quantitative structure activity
relationship (QSAR) methods can be employed. As to structure-based drug design, molecular
docking is the most common method which has been widely used ever since the early 1980s.
Programs based on different algorithms were developed to perform molecular docking studies,
which have made docking an increasingly important tool in pharmaceutical research. Various
excellent reviews on docking have been published in the past, and many comparison studies
were conducted to evaluate the relative performance of the programs.
The molecular docking approach can be used to model the interaction between a small molecule
and a protein at the atomic level, which allow us to characterize the behavior of small molecules
in the binding site of target proteins as well as to elucidate fundamental biochemical processes.
The docking process involves two basic steps: prediction of the ligand conformation as well as
its position and orientation within these sites (usually referred to as pose) and assessment of the
binding affinity. These two steps are related to sampling methods and scoring schemes,
respectively, which will be discussed in the theory section.
Knowing the location of the binding site before docking processes significantly increases the
docking efficiency. In many cases, the binding site is indeed known before docking ligands into
it. Also, one can obtain information about the sites by comparison of the target protein with a
family of proteins sharing a similar function or with proteins co-crystallized with other ligands.
In the absence of knowledge about the binding sites, cavity detection programs or online servers,

46 Winter School on "New generation smart agrochemical oriented approaches for crop
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e.g. GRID, POCKET, SurfNet, PASS and MMC can be utilized to identify putative active sites
within proteins. Docking without any assumption about the binding site is called blind docking.
The early elucidation for the ligand-receptor binding mechanism is the lock-and-key theory
proposed by Fischer, in which the ligand fits into the receptor like lock and key. The earliest
reported docking methods were based on this theory and both the ligand and receptor were
treated as rigid bodies accordingly. Then the “induced-fit” theory created by Koshland takes the
lock-and-key theory a step further, stating that the active site of the protein is continually
reshaped by interactions with the ligands as the ligands interact with the protein. This theory
suggests that the ligand and receptor should be treated as flexible during docking. Consequently,
it could describe the binding events more accurately than the rigid treatment.
Considering the limitation of computer resources, docking has been performed with a flexible
ligand and a rigid receptor for a long time, and remains the most popular method in use. Recently
many efforts have been made to deal with the flexibility of the receptor, however, flexible
receptor docking, especially backbone flexibility in receptors, still presents a major challenge for
available docking methods. In our study, we propose a Local Move Monte Carlo (LMMC)
approach as a potential solution to flexible receptor docking problems.

Theory of docking
Essentially, the aim of molecular docking is to give a prediction of the ligand-receptor complex
structure using computation methods. Docking can be achieved through two interrelated steps:
first by sampling conformations of the ligand in the active site of the protein; then ranking these
conformations via a scoring function. Ideally, sampling algorithms should be able to reproduce
the experimental binding mode and the scoring function should also rank it highest among all
generated conformations. From these two perspectives, we give a brief overview of basic
docking theory.

Sampling algorithms
With six degrees of translational and rotational freedom as well as the conformational degrees of
freedom of both the ligand and protein, there are a huge number of possible binding modes
between two molecules. Unfortunately, it would be too expensive to computationally generate all
the possible conformations.
Matching algorithms (MA) based on molecular shape map a ligand into an active site of a protein
in terms of shape features and chemical information. The protein and the ligand are represented
as pharmacophores. Each distance of the pharmacophore within the protein and ligand is
calculated for a match; new ligand conformations are governed by the distance matrix between
the pharmacophore and the corresponding ligand atoms. Chemical properties, like hydrogen-
bond donors and acceptors, can be taken into account during the match. Matching algorithms
have the advantage of speed; thus they may be used for the enrichment of active compounds
from large libraries. Matching algorithms for ligand docking are available in DOCK, FLOG,
LibDock and SANDOCK programs.
Incremental construction (IC) methods put the ligand into an active site in a fragmental and
incremental fashion. The ligand is divided into several fragments by breaking its rotatable bonds

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and then one of these fragments is selected to dock into the active site first. This anchor is
usually the largest fragment or the piece which may have significant functional role or
interaction with protein. The remaining fragments can be added incrementally. Different
orientations are generated to fit in the active site, which realizes the flexibility of the ligand. The
incremental construction method has been used in DOCK 4.0, FlexX, Hammerhead, SLIDE and
eHiTS.
In addition to IC, Multiple Copy Simultaneous Search (MCSS) and LUDI are fragment-based
methods for the de novo design of ligands and modifications of known ligands that may enhance
their binding to the target protein. MCSS makes 1,000 to 5,000 copies of a functional group,
which are randomly placed in the binding site of interest and subjected to simultaneous energy
minimization and/or quenched molecular dynamics in the forcefield of the protein. Copies only
interact with the proteins and any interactions among the copies are omitted. Consequently a set
of energetically favorable binding sites and orientations for the functional group is identified
based on the interaction energies. The binding site is mapped by using different functional
groups. New molecules which perfectly match the binding site can be designed through the
linkage of those different functional groups.
LUDI focuses on the hydrogen bonds and hydrophobic contacts which could be formed between
the ligand and protein. Its central concept are interaction sites, which are discrete positions in
space suitable for forming hydrogen bonds or for filling a hydrophobic pocket. A set of
interaction sites is generated either by searching the database or using the rules. The fragment is
then fitted onto the interaction sites and evaluated by distance criteria. The final step is the
connection of some or all of the fitted fragments to a single molecule.
Stochastic methods search the conformational space by randomly modifying a ligand
conformation or a population of ligands. Monte Carlo (MC) and genetic algorithms are two
typical algorithms that belong to the class of stochastic methods.
onte Carlo (MC) methods generate poses of the ligand through bond rotation, rigid-body
translation or rotation. The conformation obtained by this transformation is tested with an
energy- based selection criterion. If it passes the criterion, it will be saved and further modified
to generate next conformation. The iterations will proceed until the pre-defined quantity of
conformations is collected. The main advantage of MC is that the change can be quite large
allowing the ligand to cross the energy barriers on the potential energy surface, a point that isn’t
achieved easily by molecular dynamics based simulation methods. Examples of applying the
Monte Carlo methods include an earlier version of AutoDock, ICM, QXP and Affinity.
Genetic algorithms (GA) form another class of well-known stochastic methods. The idea of the
GA stems from Darwin’s theory of evolution. Degrees of freedom of the ligand are encoded as
binary strings called genes. These genes make up the ‘chromosome’ which actually represents
the pose of the ligand. Mutation and crossover are two kinds of genetic operators in GA.
Mutation makes random changes to the genes; crossover exchanges genes between two
chromosomes. When the genetic operators affect the genes, the result is a new ligand structure.
New structures will be assessed by scoring function, and the ones that survived (i.e., exceeded a
threshold) can be used for the next generation. Genetic algorithms have been used in AutoDock,
GOLD, DIVALI and DARWIN.

48 Winter School on "New generation smart agrochemical oriented approaches for crop
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Molecular dynamics (MD) is widely used as a powerful simulation method in many fields of
molecular modeling. In the context of docking, by moving each atom separately in the field of
the rest atoms, MD simulation represents the flexibility of both the ligand and protein more
effectively than other algorithms. However, the disadvantage of MD simulations is that they
progress in very small steps and thus have difficulties in stepping over high energy
conformational barriers, which may lead to inadequate sampling. On the other hand, MD
simulations are often efficient at local optimization. Thus a current strategy is to use random
search in order to identify the conformation of the ligand, followed by the further subtle MD
simulations.

Scoring functions
The purpose of the scoring function is to delineate the correct poses from incorrect poses,
or binders from inactive compounds in a reasonable computation time. However, scoring
functions involve estimating, rather than calculating the binding affinity between the protein and
ligand and through these functions, adopting various assumptions and simplifications. Scoring
functions can be divided in force-field-based, empirical and knowledge-based scoring functions.
Classical force-field-based scoring functions assess the binding energy by calculating the sum of
the non-bonded (electrostatics and van der Waals) interactions. The electrostatic terms are
calculated by a Coulombic formulation. Since such point charge calculations have problems in
modeling the protein’s real environment a distance-dependent dielectric function is generally
used to modulate the contribution of charge–charge interactions. The van der Waals terms are
described by a Lennard-Jones potential function. Adopting different parameter sets for the
Lennard-Jones potential can vary the “hardness” of the potential which controls how close a
contact between protein and ligand atoms can be acceptable. Force-field-based scoring functions
also have the problem of slow computational speed. Thus cut-off distance is used to handle the
non-bonded interactions. This also results in decreasing the accuracy of long-range effects
involved in binding.
Extensions of force-field-based scoring functions consider the hydrogen bonds, solvations and
entropy contributions. Software programs, such as DOCK, GOLD and AutoDock, offer users
such functions. They have some differences in the treatment of hydrogen bonds, the form of the
energy function etc.. Furthermore, the results of docking with force-field-based functions can be
further refined with other techniques, such as linear interaction energy and free-energy
perturbation methods (FEP) to improve the accuracy in predicting binding energies.
In empirical scoring functions, binding energy decomposes into several energy components, such
as hydrogen bond, ionic interaction, hydrophobic effect and binding entropy. Each component is
multiplied by a coefficient and then summed up to give a final score. Coefficients are obtained
from regression analysis fitted to a test set of ligand-protein complexes with known binding
affinities.
Empirical scoring functions have relatively simple energy terms to evaluate. However, it is
unclear as to how well they are suited for ligand-protein complexes beyond the training set.
Additionally, each term in empirical scoring functions may be treated in a different manner by
different software, and the numbers of the terms included are also different. LUDI, PLP,
ChemScore are examples derived from empirical scoring functions
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Knowledge-based scoring functions use statistical analysis of ligand-protein complexes crystal
structures to obtain the interatomic contact frequencies and/or distances between the ligand and
protein. They are based on the assumption that the more favorable an interaction is, the greater
the frequency of occurrence will be. These frequency distributions are further converted into
pairwise atom-type potentials. The score is calculated by favoring preferred contacts and
penalizing repulsive interactions between each atom in the ligand and protein within a given
cutoff.
The appeal of knowledge-based functions is computational simplicity, which can be exploited to
screen large compound databases. They can also model some uncommon interactions like
sulphur-aromatic or cation-π, which are often poorly handled in empirical approaches. However,
they are still faced with the problem that some interactions are underrepresented in the limited
training sets of crystal structures as well as by the bias inherent in the selection of proteins for
successful structure determination thus the obtained parameters may not be suitable for
widespread use, especially with interactions involving metals or halogens. PMF, DrugScore,
SMoG and Bleep are examples of knowledge-based functions which differ mainly in the size of
training sets, the form of the energy function, the definition of atom types, distance cutoff or
other parameters.
Consensus scoring is a recent strategy that combines several different scores to assess the
docking conformation. A pose of ligand or a potential binder could be accepted when it scores
well under a number of different scoring schemes. Consensus scoring usually substantially
improves enrichments (i.e., the percentage of strong binder among the high-scoring ligands) in
virtual screening, and improves the prediction of bound conformations and poses. However, the
prediction of binding energies might still be inaccurate.
Typical scoring functions face the problem of affinity prediction partly because of the limited
treatment of solvation effect. One of the ways to solve this problem is physics-based scoring, e.g.
MM-PB/SA and MM-GB/SA (MM stands for molecular mechanics, PB and GB for Poisson-
Boltzmann and Generalized Born, respectively, SA for solvent-accessible surface area), which is
involved in rescoring or lead optimization to improve the accuracy of binding affinity prediction.
Promising results were obtained using MM-PB/SA or MM-GB/SA in some studies. However,
recently Guimarães and Mathiowetz reported that the GB/SA model poorly estimated protein
desolvation on certain systems, while incorporating WaterMap into the MM-GB/SA method
instead of GB/SA protein desolvation gave the best ranking result. Singh and Warshel compared
several methods for evaluating the affinity of protein-ligand complexes and suggested that
PDLD/S-LRA/β (protein dipoles Langevin dipoles linear response approximation) appears to
offer an appealing option for the final stages of massive VS and in contrast, PB/SA appears to
provide erroneous estimates of the absolute binding energies because of its incorrect estimation
of entropies and the problematic treatment of electrostatic energies.
Methodology
Rigid ligand and rigid receptor docking
When the ligand and receptor are both treated as rigid bodies, the search space is very limited,
considering only three translational and three rotational degrees of freedom. In this case, ligand
flexibility could be addressed by using a pre-computed a set of ligand conformations, or by
allowing for a degree of atom–atom overlap between the protein and ligand. The early versions
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of DOCK, FLOG and some protein-protein docking programs, such as FTDOCK, adopted such a
method that kept the ligand and receptor rigid during the process of the docking.
DOCK is the first automated procedure for docking a molecule into a receptor site and is being
continuously developed. It characterizes the ligand and receptor as sets of spheres which could
be overlaid by means of a clique detection procedure. Geometrical and chemical matching
algorithms are used, and the ligand-receptor complexes can be scored by accounting for steric fit,
chemical complementation or pharmacophore similarity. Within its improved versions,
incremental construction method and exhaustive search are added to consider the ligand
flexibility. The exhaustive search randomly generates a user-defined number of conformers as a
multiple of the number of rotatable bonds in the ligand. With respect to scoring, the latest version
DOCK 6.4 has included both an AMBER-derived force-field scoring with implicit solvent and
GB/SA, PB/SA solvation scoring.
FLOG generates ligand conformations on the basis of distance geometry and uses a clique-
finding algorithm to calculate the sets of distances. Up to 25 explicit conformations of the ligand
could be used to dock for some flexibility. FLOG allows users to define essential points which
must be paired with a ligand atom. This approach is useful if an important interaction is already
known before docking. Conformations are scored with a function considering van der Waals,
electrostatics, hydrogen bonding and hydrophobic interactions.

Flexible ligand and rigid receptor docking


For systems whose behavior follows the induced fit paradigm, it is of vital importance to
consider the flexibilities of both the ligand and receptor since in that case both the ligand and
receptor change their conformations to form a minimum energy perfect-fit complex. However,
the cost is very high when the receptor is also flexible. Thus the common approach, also a trade-
off between accuracy and computational time, is treating the ligand as flexible while the receptor
is kept rigid during docking. Almost all the docking programs have adopted this methodology,
such as AutoDock, FlexX.
AutoDock 3.0 incorporates Monte Carlo simulated annealing, evolutionary, genetic and
Lamarckian genetic algorithm methods to model the ligand flexibility while keeping the receptor
rigid. The scoring function is based on the AMBER force field, including van der Waals,
hydrogen bonding, electrostatic interactions, conformational entropy and desolvation terms. Each
term is weighted using an empirical scaling factor obtained from experimental data. AutoDock
4.0 is able to model receptor flexibility by allowing side-chains to move. Additionally,
interaction of protein-protein docking could be evaluated in this version of AutoDock. AutoDock
Vina was recently released as the latest version for molecular docking and virtual screening. By
redocking the 190 receptor-ligand complexes that had been used as a training set for the
AutoDock 4, AutoDock Vina simultaneously showed approximately a two orders exponential
improvement of magnitude in speed and a significantly better accuracy of the binding mode
prediction.
FlexX uses an incremental construction algorithm to sample ligand conformations. The base
fragment is first docked into the active site by matching hydrogen bond pairs and metal and
aromatic ring interactions between the ligand and protein. Then the remaining components are

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incrementally built-up in accordance with a set of predefined rotatable torsion angles to account
for ligand flexibility.

Flexible ligand and flexible receptor docking


The intrinsic mobility of proteins has been proved to be closely related to ligand binding
behavior and it has been reviewed by Teague. Incorporating the receptor flexibility is significant
challenge in the field of docking. Ideally, using MD simulations could model all the degrees of
freedom in the ligand-receptor complex. But MD has the problem of inadequate sampling that
we mentioned earlier. Another hurdle is its high computational expense, which prevents this
method from being used in the screening of large chemical database.
In addition to the historic induced fit several theoretical models, conformer selection and
conformational induction, have been proposed to illustrate the flexible ligand-protein binding
process. According to the definition given by Teague, conformer selection refers to a process
when a ligand selectively binds to a favorable conformation from a number of protein
conformations; conformational induction describes a process in which the ligand converts the
protein into a conformation that it would not spontaneously adopt in its unbound state. In some
cases, this conformational conversion can be likened to a partial refolding of the protein.
Various methods are currently available to implement the receptor flexibility. The simplest one is
so-called “soft-docking”, decreases the van der Waals repulsion energy term in the scoring
function to allow for a degree of atom-atom overlap between the receptor and ligand. For
example, the LJ 8-4 potential in GOLD and smooth potential in AutoDock 3.0 belong to this
class. This method may not include adequate flexibility. Nevertheless, it has the advantage of
computational efficiency as the receptor coordinates are fixed, simply by adjusting van der
Waals parameters.
Utilizing rotamer libraries is another approach to modeling receptor flexibility. Rotamer libraries
include a set of side-chain conformations which are usually determined from statistical analysis
of structural experimental data. The advantage of using rotamers is the relative speed in
sampling, and the avoiding of minimization barriers. ICM (Internal Coordinates Mechanics) is a
program using rotamer libraries with the biased probability methodology, coupled with Monte
Carlo search of the ligand conformation.
AutoDock 4 adopts a simultaneous sample method to deal with side chain flexibility. Several
side chains of the receptor can be selected by users and simultaneously sampled with a ligand
using the same methods. Other portions of the receptor are treated rigidly with a grid energy map
during sampling. Grid energy map introduced by Goodford is used to store energy information of
the receptor and simplify interaction energy calculation between ligand and receptor.
Still another way to deal with the protein flexibility is to use an ensemble of protein
conformations, which corresponds to the theory of conformer selection. A ligand is separately
docked into a set of rigid protein conformations rather than a single one, and the results are
merged depending on the method of choice. This method was originally implemented in DOCK,
which generates an average potential energy grid of the ensemble and is extended in many
programs in different ways. For example, FlexE collects multiple crystal structures of a certain
protein, merging the similar parts while marking the dissimilar areas as different alternatives.

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During the incremental construction of a ligand discrete protein conformations are sampled in a
combinatorial fashion. The highest scoring protein structure is selected based on a comparison
between the ligand and each alternative.
Hybrid method is another practical strategy to model receptor flexibility. One example is Glide, a
very popular program in the field of docking. Glide designs a series of hierarchical filters to
search the possible poses and orientations of the ligand within the binding site of the receptor.
Ligand flexibility is handled by an exhaustive search of the ligand torsion angle space. Initial
ligand conformations are selected based on torsion energies and docked into receptor binding
sites with soft potentials. Then a rotamer exploration is used to further model receptor flexibility
IFREDA utilizes a hybrid method that combines soft potential and multiple receptor
conformations, accounting for receptor flexibility. Other programs, like QXP and Affinity,
perform a Monte Carlo search of ligand conformations followed by a minimization step. During
minimization, the user-defined parts of the protein are allowed to move in order to avoid atom
clashes between the ligand and receptor. SLIDE is designed to incorporate flexibility with the
ability to remove clashes by directed, single bond rotation of either the ligand or the side chains
of the protein. An optimization approach based on the mean-field theory is applied to model
induced-fit complementarities between the ligand and protein.
Methods mentioned above either include only side chain flexibility or full flexibility of the
receptor. We have known that loops forming active sites play an important role in ligand
binding. In some cases the loop may undergo dramatic conformational change whereas in other
portions of the receptor there is little change upon ligand binding. For this situation, side chain
flexibility methods fail to sample the correct protein conformation and full flexibility seems to be
a computational waste. The active site of triosephosphate isomerase has an 11-residue loop
which moves 7Å upon ligand binding. However, the rest of the enzyme has no movement in
comparison to their apo and holo structures. Several enzyme families also involve loop
rearrangement within the active site responsible for ligand binding, such as Bromodomain, an
extensive family related to acetyl-lysine binding, or Dihydrofolate reductase, responsible for the
maintenance of the cellular pools of tetrahydrofolate, as well as other kinds of kinases. In the
next section, we present the Local Move Monte Carlo (LMMC) loop sampling method, a new
approach which focuses on sampling ligand conformation within loop-containing active sites.

Application examples of molecular docking for drug discovery


Molecular docking has been the most widely employed technique. Though the main application
lies in structure-based virtual screening for identification of new active compounds towards a
particular target protein, in which it has produced a number of success stories, it is actually not a
stand-alone technique but is normally embedded in a workflow of different in silico as well as
experimental techniques. Several research groups focus on evaluating of the performance of
various docking programs or on making improvements to the scoring functions when
experimental testing has already been done. Such efforts could give meaningful guidance to
choose the methodology for a particular target system. Docking, combined with other
computational techniques and experimental data, also could be involved in analyzing drug
metabolism to obtain some useful information from the cytochrome P450 system, for example.
In the following, three examples of successful applications of docking are presented.

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DNA gyrase is a bacterial enzyme that introduces negative supercoils into bacterial DNA and
unwinds of DNA, thus being studied as antibacterial target. HTS failed to find novel inhibitors of
DNA gyrase. Firstly, 3D complex structures of DNA gyrase with known inhibitors, ciprofloxacin
and novobiocin, were carefully analyzed to get a common binding pattern, in which both
inhibitors donate one hydrogen bond to Asp73 and accept one hydrogen bond from a conserved
water molecule. In addition, some lipophilic fragments should be included in the molecule to
have lipophilic interaction with the receptor. Based on this information, LUDI and CATALYST
were employed to search the Available Chemicals Directory (ACD) and a part of the Roche
compound inventory (RIC), respectively, and collected about 600 compounds. Close analogs of
these compounds were also considered, thus in total 3000 compounds were further tested using
biased screening. Consequently 150 hits were selected and clustered into 14 classes of which 7
classes were proven to be the true and novel inhibitors. Subsequent hit optimization relied
strongly on the knowledge of 3D structures of the binding site and eventually generated a series
of highly potent DNA gyrase inhibitors.
Another example is focused on the validation of docking and scoring applied in cytochromes
P450 and other heme-containing proteins. Docking against heme-containing complexes appears
to be difficult because certain ligands coordinate directly to the heme iron atom and the precise
energetics of this contact for different chelating groups needs to be properly balanced with other
energetic terms, and in the case of the P450s, the environment above the heme group is very
hydrophobic compared to other enzymes and some scoring functions and docking methods
perform poorly on interactions driven entirely by lipophilic contacts. In this study, 45 complexes
from the PDB database comprising heme-containing proteins and ligands were selected. The
native ligands were removed and then docked into the defined active cavities using the GOLD
software which employs genetic algorithms to generate ligand conformations. The scoring
functions used to rank the docking poses were Goldscore and Chemscore. The results show that
the success rates are 64% and 57% for Chemscore and Goldscore respectively, which is
significantly lower than the value of 79% observed with both scoring functions for the full
GOLD validation set. Additionally, it is apparent from the data that the search algorithm was
very unlikely to be responsible for the failure in docking. Further research indicated that re-
parameterization of metal-acceptor interactions and lipophilicity of planar nitrogen atoms in the
scoring functions resulted in a significant increase in the percentage of successful docking poses
against the heme binding proteins (Chemscore 73%, Goldscore 65%), which might be useful in
docking applications on P450 enzymes and other heme-binding proteins.
Both VS and HTS were applied to screen the inhibitors of the protein tyrosine phosphatase-1B
(PTP-1B). For the HTS a library of approximately 400,000 compounds from a corporate
collection were screened. Some 85 compounds were found with IC50 values less than 100 μM,
corresponding to a hit rate of 0.021%. And the most active had an IC 50 value of 4.2 μM. For VS,
235,000 commercially available molecules were docked into the crystal structure of PTP-1B
(PDB code 1pty) using the Northwestern University version of DOCK3.5. After docking, the
top-scoring 1000 molecules (500 for the ACD and 500 for the combined BioSpecs and
Maybridge databases) were considered for further evaluation. A total of 889 molecules were
actually available, and after visual inspection 365 compounds were chosen for testing. Of these,
127 molecules were found to be active with IC50 <100 μM, corresponding to a hit rate of 34.8%.
Structure-based docking therefore enriched the hit rate by 1700-fold over random screening.

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Another point that should be noted is that the hits from VS and HTS are very different from each
other, which implies combination of VS and HTS may be more helpful for lead discovery.
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56 Winter School on "New generation smart agrochemical oriented approaches for crop
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Synthesis of Molecularly Imprinted Polymer
Indu Chopra and Suman Gupta
Division of Agricultural Chemicals
ICAR-Indian Agricultural Research Institute, New Delhi-110 012
Aim

To synthesize molecularly imprinted polymer for the target analyte.

Principle

Molecularly imprinted polymers (MIPs) are specially designed polymers which are synthesized
with functional monomer, cross linker and template molecule in suitable porogen. After the
removal of template molecule from the three dimensional polymeric network, the material is left
behind with the cavities or imprints which are complementary in size, shape and chemical
functionality to that of the template. Now these polymers have the ability to selectively recognize
and rebind with the template molecule amongst the closely related chemical species.
Chemicals required
Template: Atrazine Solvent: Acetonitrile
Monomer: Methacrylic acid Initiator: Azobisisobutyronitrile
Crosslinker: Ethylene glycol dimethacrylate (EGDMA)

Chemical Molecualr weight No of mmoles Quantity


Atrazine 215.68 1 215 mg
Methacrylic acid 86.06 5 430 mg
Ethyleneglycoldimethacrylate 198.22 20 3.964g
(EGDMA)

Method of Synthesis
Synthesis

Weigh the template, monomer, crosslinker and add to flat bottom glass flask of appropriate size.
To the flask, add 100 ml of acetonitrile and shake the components vigorously to ensure that
uniform solution is obtained. To the mixture, initiator (50 mg) was added and the flask was
purged with dry nitrogen for 15 minutes to ensure complete removal of oxygen which can
interfere in the reaction otherwise. Then the flask was sealed. In another flask, all the chemicals
were put except the template and the above mentioned procedure was repeated to synthesize non-
imprinted polymer (NIP). The sealed flasks were placed in preheated water bath at 60 ̊C. The
reaction is carried out for 16-20 hours to ensure that the reactants react completely. The solid
particles so obtained are collected by centrifugation

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Template Removal
For optimum performance of MIPs, efficient removal of template from polymeric network is of
utmost importance. Though there are different techniques available for the purpose but the most
commonly used techniques are soxhlet extraction and sonication. Soxhlet extraction is normally
done with a mixture of methanol/acetic acid (9:1 or 8:2, v/v). Minimum of 8-10 siphoning cycles
should be done for efficient removal of the template from the polymeric network. To ensure the
formation of imprinted cavities, characterization of the complex and the template is done through
different techniques viz. Fourier Transform Infra Red Spectroscopy, Scanning Electron
Microscopy, Transmission Electron Microscopy and X-Ray Diffraction.
The prepared imprinted material can selectively be used for removing template molecule
from the complex environmental matrices.
Result
Based on the relative sorption characteristics of MIP and NIP towards the template and their
closely related analogues binding affinity, imprinting factor and separation factor can be
calculated.

58 Winter School on "New generation smart agrochemical oriented approaches for crop
and human health management", 07-27 Dec, 2017
Bioactive Molecules from Nature to Protect Crop Health
V.S. Rana, N.A Shakil and Parshant Kaushik

Division of Agricultural Chemicals


ICAR-Indian Agricultural Research Institute, New Delhi-12

Introduction

Globally, the use of natural products, specially botanical pesticides for killing of insect-pests and
fishes is known for centuries. Pesticidal plants and molecules of biological origin have showed
the ways for the discovery and development of many groups of synthetic pesticides such as
carbamates, pyrethroids and neonicotinoids currently being used in insect-pests and disease
management in agriculture. The discovery of organochlorine and organophostate pesticides and
their use in agriculture have declined the use of botanical pesticides to great extent. It is well
known that the use of synthetic pesticides has contributed a lot in crop production, yield and food
security but the unscientific use of many synthetic pesticides has led to the problem of pest
resistance to pesticides, environmental pollution and health hazards. Thus, environmental and
health concern has further made public understand to move towards the biopesticides, biocontrol
agents or other safer alternatives of insect-pest and disease management, specially in agriculture,
which are environment and consumer friendly, less or no toxic, target specific and
biodegradable.

The development of crop protection agent is similar to drug development and is presently based
on synthesizing novel molecules that hitt with well-defined targets found in the pests, however
toxicity is the major hurdle that needs to be overcome in the development of novel pesticides or
drugs. Most of the compounds are eliminated due to their toxic effects but analysing non-toxic
plants for activity reduces the risk of discovering toxic biopesticides. Plants have been found to
be best source for providing novel leads for crop protection. The use of pyrethrum
(Chrysanthemum cinerariefolium has been used since 400 BC.

The tobacco leaves has been used dates back to the seventeenth century when it was shown that
nicotine obtained was lethal to plum beetles. Another plant Lonchocarpus was known since
around 1850 as pesticidal and is now known source of rotenone was introduced. Rotenone is a
flavonoid derivative extracted from the roots of was also found in Derris spp. (Fabaceae). Later
on azadirechtin A from neem (Azadirachta indica as antifeedant and insect growth regulator.
Large numbers of plant species have been scientifically evaluated against the insect-pests and
diseases and some of the important molecules have been identified. Currently, many botanical
insecticides are being marketed globally. Interestingly, the active compounds from the botanical
are providing new basic structures for pesticides and thus contributing to the development of new
pesticides. The main use of biopesticide/biocontrol agents is organic agriculture, horticulture,
green houses, parks, gardens, and households. However, organic agriculture could be major
market for biopesticide as organic farmers are not allowed to use conventional agrochemicals.
Thus, it is expected that the biopesticide market will be growing due to consumers’ demand for
nutritious and safe food and problems associated with the use of synthetic pesticides specially
insect-pests management in agriculture provided the availability of the authentic and effective

59 Winter School on "New generation smart agrochemical oriented approaches for crop
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biopesticide products which is the main concern of users. May bioactive compounds has been
identified from bioresources as pest control agents Some of the important pest control molecules
identified are summarized.

1. Nicotine
Nicotine has been reported as the main bioactive component in Nicotiana tabacum L., N. glauca
Graham, and N. rustica L. The amount of nicotine in the leaves of N. tabacum and N. rustica
ranged from 2–6% on dry weight basis. It is used for the management of number of insects,
including aphids, thrips, and whitefly infesting ornamentals and field crops. Nicotine is a non-
systemic insecticide that binds to the cholinergic acetylcholine nicotinic receptor (nAchRs) in the
nerve cells of insects, leading to a continuous firing of this neuroreceptor.15 Nicotine has been
used for many years as a fumigant for the control of many sucking insects. Nicotine is very toxic
to humans by inhalation and by skin contact. It is toxic to birds, fish, and other aquatic
organisms, and is toxic to bees, but has a repellent effect. In the United Kingdom, nicotine is
subject to regulation under the Poisons Act. The use of nicotine as a pesticide is banned in South
Africa, severely restricted in Hungary, canceled in Australia and New Zealand, as well as not
being registered in numerous African, Asian, and European countries.

H
N

N
Figure 1: Structure of nicotine

2.Rotenone
Rotenone, a naturally occurring substance containing a cis-fused tetrahydrochromeno [3,4-b]
chromene nucleus, was discovered from the roots of Derris chinensis (Family: Leguminosae) in
1912. Later on, it was also found in other genus such as Lonchocarpus and Tephrosia. Although,
rotenone is the major constituent in root extract and rotenone products but other active
compounds such as deguelin, rotenolone, and tephrosin are also reported. It is used against soft
bodied insects, red spider, greenfly, caterpillar and wasps. It is a contact and stomach poisons
and acts on mitochondria / electron transport system and cause respiratory depression and
paralysis of insects but has low human toxicity. Rotenone based products are approved for use as
organic insecticides.

H
O O
O

H
O
OMe
OMe
Figure 2: Structure of rotenone

60 Winter School on "New generation smart agrochemical oriented approaches for crop
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3. Cevadine and Veratridine
These compounds are found in the seeds of plant of the genus Schoenocaulon and are mainly
obtained from the sabadilla lily (Schoenocaulon officinale). The biological activity of Sabadilla
products is due to alkaloids cevadine and veratridine which exist in a 2:1 ratio and are
collectively referred to as veratrine. The mode of action of sabadilla alkaloids found to be similar
to that of the pyrethrins as they work on voltage-sensitive sodium channels. Sabadilla based
products are approved for use in the USA as an organic insecticide.

H
H N
N OH
OH
OH
H H OH
OH
H H OH O
O H OH OH
H OH O
OH O O
O O HO
HO O

Figure 3. Structures of cevadine and veratridine

4. Karanjin
The seed oil of Pongamia pinnata (L.) Pierre) is reported to possess pest control property.
Karanjin was identified as a potent deterrent compounds to many genera of insects and mites
infestin a wide range of crops. Karanjin has a dramatic antifeedant/repellent effect, with many
insects infesting number of crops. It suppresses the effects of ecdysteroids and thus acts as an
IGR and antifeedant. There is not reports which shows its any adverse effects on non-target
organisms or on the environment.

O O

OMe
O
Figure 4: Structure of karanjin

6. Pyrethrins
Pyrethrins are refers to the oleoresin extracted from the dried flowers of Chrysanthemum
cinerariaefolium (family: Asteraceae). Its flowers are the main source of the pyrethrins.
pyrethrum contains a mixture of at least six pyrethrin esters and has unusual insecticidal
properties. It has been used safely for the past 160 years as a botanical insecticide around the
world. It shows quick knockdown effect against insects at very low doses, as well as degrads
quickly in the environment due to its instability toward heat, light, and air.

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O
R O
O

R2
Pyrethrin I, R=Me, R2= C2H3 Cinerin I, R =Me, R2 =Me
Pyrethrin II, R =COOMe, R2 = C2H3 Cinerin II, R =COOMe, R2 =Me
Jasmolin I, R =Me, R2 =Et Jasmolin II, R =COOMe, R2 =Et

Figure 6: Structures of the six major compounds of pyrethrum

Pyrethrum products are neurotoxic and approved for use in organic agriculture for
management of more than 40 insects on more than 200 different fruits and vegetables crops. This
group of compounds blocks voltage-gated sodium channels in nerve axons and the symptoms of
pyrethrin poisoning are characterized by hyperexcitation, convulsions, seizures and followed by
death.
7. Azadirachtin A
It is obtained from seed kernel of neem tree, Azadirachta indica L. (Family: Meliaceae).
Neem seeds contain numerous structurally similar compounds belongs to limonoid group besides
oil but the major bioactive compound for pest control has been identified as azadirachtin A. The
remaining minor compounds likely contribute little to overall efficacy of the extracts.

HO
HO
O O O O O
O
O OH O
H O H OH
HO H O HO H
O O
H H
O H O O
O H

O O O O
O O O O

O O
Figure 7: Structure of azadirachtin A and dihydroazadirachtin

Azadirachtin A, well known potent insect antifeedant and insect growth regulator (IGR)
and was found to work by blocking the synthesis and release of molting hormones, ecdysteroids
from the prothoracic gland in insects. Neem oil based products are approved as organic
insecticides for use in organic agriculture. The use of neem oil based products has the additional
advantage to control fungal infections as well as a wide variety of both insect and mite
pathogens. The bioactive compounds of neem seeds, azadirachtin is sold by the number of

62 Winter School on "New generation smart agrochemical oriented approaches for crop
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industries as an emulsifiable concentrate (EC) under with different trade names. Azadirachtin-
based products are commonly used in India and their use is increasing in other countries. It is
considered to be non-toxic to mammals and is also not expected to have any adverse effects on
the environment or non-target organisms. Products containing azadirachtin can be used in a wide
range of crops, including vegetables (like cabbage, tomatoes and potatoes), tobacco, cotton, tea,
and ornamentals and in forests plants.

Similarly, dihydroazadirachtin is a derivative and reduced form of azadirachtin. This


compound is also effective against a wide range of insect pests. Dihydroazadirachtin has both
antifeedant and insect growth regulator (IGR) activities. Products based on dihydroazadirachtin
are not widely used outside the Indian subcontinent but its use is also increasing.
Dihydroazadirachtin possesses low toxicity to mammals and less risk to the environment and is
not persistent in recommended constions and is also relatively short-lived in the environment due
to degradation by microorganisms.

8. Strobilurin-A
The most successful commercial fungicides identified based on natural products are the
strobilurins obtained from Basidiomyces growing on decaying wood (66). Strobilurins gave new
class of fungicides (i.g. trifloxystrobiin, azoxystrobin, and fluoxastrobin) and a new mode of
action by inhibiting respiration at the complex III cytochrome bc1 site. The strobilurin-based
fungicides are the major class of fungicides being used in agriculture.

O O

Figure 8: Structure of Strobilurin A

9. Spinosads
This group of compounds was originally isolated from Saccharopolyspora spinosa
(Actinomycetes). Spinosad is a mixture of at least two major compounds, spinosyn A and
spinosyn D (Figure 2), in which spinosyn A is a major constituent. Spinosad was found to have
both, ingestion and contact toxicities. It causes excitation of the insect nervous system and thus
causing involuntary muscle contractions, prostration with tremors, and finally paralysis. These
effects are consistent with the activation of nicotinic acetylcholine receptors by a mechanism.
Spinosad is approved for use as an organic insecticide and recommended for the management of
different group of insects belonging to caterpillars, leaf miners, thrips and foliage-feeding
beetles. Spinosad products are useful not only for organic farmers but also for conventional
farmers.

63 Winter School on "New generation smart agrochemical oriented approaches for crop
and human health management", 07-27 Dec, 2017
CH3
Me2N
O

O
CH3
O OMe
H H
O OMe
H
Et O H O
O HH OMe
R CH3

Spinosyn A, R = H
Spinosyn D, R = CH3
Figure 9: Structure of Spinosyns

10.Avermectins and Milbemycins


Avermectins and milbemycins of compounds which are structurally similar were
discovered from Streptomyces sp., and are used against worms, ticks, flies and agricultural pests.
Abamectin, a commercial product, is a mixture of >80% avermectin B1a and <20% avermectin
B1b which are obtained from Streptomyces avermitilis, a soil bacterium (23) whereas
milbemectin products, is a mixture of ≥70% milbemycin A4 and ≤30% milbemycin A3 obtained
from another soil bacterium, Streptomyces hygroscopicus subsp. Aureolacrimosus. Milbemycins
and avermectins have the same mode of action which is to potentiate glutamate and GABA gated
chloride-channel opening.

OMe
H O
HO
O R
OMe
H3C O O
H
O O
H3C O O H O

O
OH H
H
R
H
O O
O
OH H
H
O
OH
H
OH Milbemycin A3, R = CH3
Avermectin B1a, R = CH3CH2
Avermectin B1b , R = CH3 Milbemycin A4, R = CH3CH2

Figure 10: Structures of Avermectins and Milbemycin

64 Winter School on "New generation smart agrochemical oriented approaches for crop
and human health management", 07-27 Dec, 2017
Conclusions
It is well known that biopesticides/biocontrol agents are considered to be safe and target
specific than conventional pesticides. Globally, the demand and use of bioresources based
biopesticides/biocontrol agents is increasing because of increasing public awareness towards
benefits of safe edible products, and hazards of the environmental pollution and health concern
due to toxicants especially highly toxic conventional pesticides but the sufficient quantity of
biopesticides/biocontrol agents for the use is not available to farmers. There is urgent need to
establish the large production units for existing and known biopesticide/molecules at commercial
scale for present use and identification of new sources of biopesticide/biocontrol agents for
future come. Farmer’s awareness about the benefits of currently available
biopesticides/biocontrol agents in the market and policy for more investment in future research
and development for the development of new biopesticides/biocontrol agents are essentially
needed to protect the environment and health from future chemical hazards.

References

1. Chang, C.P., Plapp and F.W., Jr. DDT and Synthetic pyrethroids: Mode of action,
selectivity, and mechanism of synergism in the tobacco budworm (Lepidoptera:
Noctuidae) and a predator, Chrysopa carnea Stephens (Neuroptera: Chrysopidae). J.
Econ. Entomol. 1983, 76, 1206–1210.
2. Clark, J.M. Insecticides as tools in probing vital receptors and enzymes in excitable
membranes Pestic. Biochem. Physiol. 1997, 57, 235-254.
3. Barton, D.H.R., Jeger, O., Prelog, V., and Woodward, R.B. The constitutions of cevine
and some related alkaloids. Experientia, 1954, 10, 81-90.
4. Isman, M.B. The role of botanical insecticides, deterrents and repellents in modern
agriculture and an increasingly regulated world. Annu. Rev. Entomol. 2006, 51, 45–66.
5. Mordue, A.J. and Blackwell, A. Azadirachtin: an update. J. Insect Phys. 1993, 39, 903-
924.
6. Kornis, G.I. Avermectins and milbemycins. In Agrochemicals from Natural Products;
Godfrey, C.R.A., Ed.; Marcel Dekker: New York, NY, USA, 1995, pp. 215-255.
7. Arena, J.P., Liu, K.K., Paress, P.S., Frazier, E.G., Cully, D.F., Mrozik, H. and Schaeffer,
J. The mechanism of action of avermectins in Caenorhabditis elegans: Correlation
between activation of glutamate-sensitive chloride current, membrane binding, and
biological Activity. J. Parasitol., 1995, 81, 286-294.
8. Sauter, H., Ammermann, E. and Roehl, F. Strobilurins-from natural products to a new
class of fungicides. In Crop Protection Agents from Nature: Natural Products and
Analogues; Copping, L.G., Ed., Royal Soc. Chem., Cambridge, UK, 1996, pp. 50-81.
9. Jacobsen, M. Pharmacology and Toxicology of Neem. In Focus on Phytochemical
Pesticides, Vol. 1: The Neem Tree; M. Jacobsen, Ed.; CRC Press: Boca Raton, FL, 1989,
pp. 133-153.
10. Campbell, W.C. Ivermectin and Abamectin; Springer-Verlag: New York, NY, USA,
1989, p. 361.

65 Winter School on "New generation smart agrochemical oriented approaches for crop
and human health management", 07-27 Dec, 2017
Isolation of Essential Oil from the Leaves of Eucalyptus spp., by
Hydrodistillation Method and Determination of its Chemical
Composition
V.S. Rana and Ashish Khandelwal
Division of Agricultural Chemicals
ICAR-Indian Agricultural Research Institute, New Delhi 110 012

Introduction

Essential oils are volatile in nature and found in the whole or different parts such as blossoms,
seeds, fruits, leaves, fruits, stem, bark, wood, flowers or roots of the aromatic, spice and flavoring
plant species. Number of plant such as Amyris balsamifera, Pimpinella anisum, Anisum verum,
Pimenta racemosa, Myrocarpus frondosus, Carum carvi, Cedrus spp., Juniperus spp.,
Cinnamomum spp., Cymbopogon spp., Artemisia spp., Eucalyptus spp., Lavendula spp., Litsea
cubeba, Brachyleana hutchinsii, Myristica fragrans, Pogostemon cablin, Pimenta dioica, Aniba
rosaeodora, Santalum album, Ocotea pretiosa, Tagetes spp., Thymus vulgaris, Vetiveria
zizanioides, Pelargonium spp., Cananga odorata, Mentha spp., Citrus spp., Curcuma spp.,
Zingiber spp., etc., are known sources of essential oils and the work on the identification of new
sources is continued. Essential oils are mainly used therapeutic, flavouring and perfumery purpose
and have huge market. Some essential oils are routinely used in the preparation of cosmetic, dhup,
agarbatti, soap and toiletries products. Since many compounds of the essential oils are
thermolible, the composition of the oil also depends on method used for the isolation of essential
oil. In this experiment, essential oil will be isolated from the leaves of one Eucalyptus spp., and
the chemical components of the oil will be identified by gas-chromatography-mass spectrometry.

Objectives
To determine yield and chemical composition of the leave oil

Requirements
1. Heating mantle (3L)
2. Round bottom flask (3L)
3. Clevenger apparatus
4. Eucalyptus leaves
5. Cold water
6. Conical flask (150ml)
7. Separatory funnel (250ml)
8. Diethyl ether
9. Anhydrous sodium sulphate
10. GC/ GC-MS

Procedure
a) Collection of fresh leaves of Eucalyptus
b) Preparation of samples
c) Isolation of essential oil by hydrodistillation using Clevenger type apparatus
66 Winter School on "New generation smart agrochemical oriented approaches for crop
and human health management", 07-27 Dec, 2017
d) Purification of essential oil from distillate
e) GC-MS analysis of the Eucalyptus leave oil
f) Identification of compounds using MS library

Precautions
• Continuous circulation of cold water through condenser during hydrodistillation need to
be maintained.

67 Winter School on "New generation smart agrochemical oriented approaches for crop
and human health management", 07-27 Dec, 2017
Nano Science in Pest Management
Rajesh Kumar and Abhishek Mandal
Division of Agricultural Chemicals
ICAR-Indian Agricultural Research Institute, New Delhi 110 012

Use of synthetic chemicals is one of the important components of pest management. The
injudicious use of plant protection chemicals has caused threat to human and environmental
health. Agricultural traditional plant protection strategies are insufficient; especially in changed
climate scenario and application of persistent chemicals have adverse effects on soil fertility,
animals and human. The present plant protection methodologies have certain limitations as these
are expensive, inefficient, and impose a profound environmental load. These problems can be
addressed by utilizing nanotechnological tools having huge potential in improving agricultural
technologies.
Nanotechnology comprises a set of technologies pertaining to most of the scientific disciplines
and industry sectors. Nanoscience crosses the boundaries between various disciplines viz.
chemistry, physics, mathematics, biology, engineering and information technology.
Agri-nanotechnology has got the potentiality to revolutionize the next stage of development of
genetically modified crops and plant breeding, molecular nanotechnology, plant disease
diagnostics, microbiology, efficient fertilizers and chemical pesticides, post-harvest technology,
soil management, water purification, inputs for animal production, and techniques for precision
farming. The interest in agri-nanotechnology is resulted by the accomplishments of numerous
nanoproducts, biosensors, miniature detection devices and nano sensors in medicine / pharma
sector. Agri-nanotechnology holds promise for diverse applications in agriculture. The examples
include nano-based smart delivery systems of various agrochemicals for their efficient use by
site-specific delivery and / or controlled or slow release. The technique of nano-encapsulation
has helped to reap the benefit of safer handling and more efficient use of plant protection
products with less exposure to the environment. The pesticidal molecules can be nanosized as
nanoemulsion, nanoencapsulation and different nanoparticles. The main advantages of the
nanosizing of pesticidal chemicals are the improved bioefficacy because of increased surface
area and increased solubility, mobility and the less toxicity due to the removal of hazardous
chemicals especially solvents. Nano-chemicals have enormous potential for more efficient and
effective control of agricultural pests. Nanotechnology can support food and agricultural industry
with new tools for the molecular treatment of diseases, improving the ability of crops to absorb
nutrient etc. Nano based delivery systems and sensors will be helping the agricultural industry to
combat pests like insects, weeds, viruses and other crop pathogens. Nonostructured catalysts will
help in increasing the efficiency of plant protection products and consequently lower doses can
be used. Nanotechnological tools will also contribute in protecting the environment indirectly
through the use of renewable energy supplies, and filters or catalysts for reducing the
environmental pollution and cleaning up existing pollutants. The perspectives of nanomaterials
use in pest management programmes seem to be promising with the increasing biological
agriculture after thorough study of its target specific effect.

68 Winter School on "New generation smart agrochemical oriented approaches for crop
and human health management", 07-27 Dec, 2017
Preparation of nano-chemicals
Approaches for preparation of nanomaterial can be divided into two categories: “top-down” and
“bottom-up”. Either of these approaches can be used to prepare nano-chemicals.
Top-down methods
The most straightforward method for obtaining nanoparticles is the top-down disintegration
methods. This includes the reduction of larger particles to smaller one and depends on the input
of mechanical energy and high shear forces required to break the particle to the nanometric size
particles. The methods used for nano-sizing include dry and wet milling, spray drying, different
precipitations and recrystallization techniques, supercritical fluids technologies and high-
pressure homogenization. The main disadvantages of these methods are: the necessity to invest
high energy and shear forces to reach the sub-micron dimensions; the in-process heat generation
because of friction, which could be harmful to the milled material and should be controlled by a
constant cooling; long procedure time (usually 30–60 min), since the shorter milling time can
lead to a higher variance in particle sizes; and the need for special, high-cost equipment. On the
other hand, these methods are simple; in certain systems the particle size polydispersity is very
low; high load of the pesticide in the nanoparticles can be achieved; no auxiliary organic solvents
are required; the methods are applicable to materials that are poorly soluble in both water and
organic media; and the scale-up process is simple with a minimal batch-to-batch variations.
Bottom-up methods
The bottom-up approach is different from the top-down approach. It exploits the chemical
properties of the molecules to cause them to self-assemble in a natural and self-regulating
manner. In this process, nano-sizing can be achieved by growing particles to a desired size. The
common bottom-up approaches are chemical vapor deposition, chemical synthesis, film
deposition and growth.
Neither the bottom-up nor the top-down approach is superior; each one has its own advantages
and disadvantages. However, the bottom-up approach has a better chance of producing
homogenous chemical composition, nanostructures with less defects and better short- and long-
range ordering. Because of the advantages of absolute precision, complete control over the
process, and minimum energy loss compared with that of a top-down approach the bottom-up
approach can produce much smaller sized particles and has the potential to be more cost-
effective in the future.
Various nano-pesticides for insect pest management have been reported and some of the
examples are given below.

Nano-material Active compound Polymer


Capsule Imidacloprid Lignin-polyethylene glycol-ethylcellulose
Neem seed oil Alginate-glutaraldehyde
Gel Cypermethrin Methyl methacrylate and methacrylic acid with
and without 2-hydroxy ethyl methacrylate crosslinkage

69 Winter School on "New generation smart agrochemical oriented approaches for crop
and human health management", 07-27 Dec, 2017
Film Endosulfan Starch-based polyethylene
Fibre Pheromones Polyamide
Micelle Rotenone N-(octadecanol-1-glycidyl ether)-O-sulfate chitosan
octadecanol glycidyl ether
Particle Azadirachtin Carboxymethyl chitosanricinoleic acid
Resin Pheromone Vinylethylene and vinylacetate
Sphere Carbaryl Glyceryl ester of fatty acids
Suspension Carbofuran Poly(methyl methacrylate)- poly(ethylene glycol)

Silver nanoparticles exhibited significant effect on the colony formation of Bipolaris sorokiniana
and Magnaporthe grisea. The antifungal effects of silver nanoparticles, especially on the hyphal
growth of sclerotia-forming phytopathogenic fungal species was observed in the following order,
Rhizoctonia solani > Sclerotinia sclerotiorum >S. minor. A microscopic observation revealed the
severe damage to the hyphae exposed to silver nanoparticles, resulting in the separation of layers
of hyphal wall and collapse of hyphae.
Different concentrations of nano-sized silica-silver inhibited the growth of Pseudomonas
syringae and Xanthomonas compestris pv.vesicatoria, Magnaporthe grisea, Botrytis cinerea,
Colletotrichum gloeosporioides, Pythium ultimum, and R. solani.
The antifungal activity of Zinc oxide nanoparticles against two pathogenic fungi, Fusarium
oxysporum and Penicillium expansum was dose dependent. Hence, maximal inhibition of
mycelial growth corresponded to the highest experimental concentration (12 mg L-1).
Significant antifungal effect of copper nanoparticles against phyto-pathogenic fungi viz., Phoma
destructiva, Curvularia lunata, Alternaria alternata and F. oxysporum was observed. The
bioefficacy of copper nanoparticles against all the above phyto-pathogenic fungi was superior to
that of the commercially available fungicide bavistin.
Nanosulphur fungicide exhibited effective control of Erysiphe cichoracearum of okra in
comparison to control and the germination of conidia of E. cichoracearum was significantly
reduced. Apart from inhibition of conidial germination, disruption in cleistothecial appendages
were also observed in contact with nano-sulphur and the cleistothecia became sterile. Better
antifungal activity with encapsulated nano-sulphur was observed in comparison to commercial
formulations and subsequently lower amount of nano-sulphur could be applied for controlling
powdery mildew disease for its better efficacy. Significantly higher miticidal activity was
observed with nanosulphur as compared to commercial sulphur against red spider mite,
Tetranychus urticae.
Nano-hexaconazole was more effective in controlling phytopathogens. Nanohexaconazole
proved to be a superior ergosterol biosynthesis inhibitor over conventional hexaconazole and
substantial change in ergosterol biosynthesis of the pathogen was observed.
Various studies regarding effect of nanohexaconazole on soil health and beneficial organisms
revealed that it performed equivalent or better than commercial hexaconazole and was safe to
soil health and other beneficial organisms. There was no adverse effect on various soil enzyme
70 Winter School on "New generation smart agrochemical oriented approaches for crop
and human health management", 07-27 Dec, 2017
activities, total soil microbial count, nitrogen fixing bacteria and blue green algae, nitrifying
bacteria viz., Nitrosomonas and Nitrobacter species, germination of mustard seeds and
Trichoderma species in the presence of nanoparticles of nano-hexaconazole.
Nanotechnology has extremely high potentiality in pest management in agriculture. The nano-
agrochemicals comprising synthetic chemicals or natural products have been employed to
control agricultural pests. In order to prepare nano-agrochemicals, several physical and chemical
technologies have been developed to avoid the emergence of new toxicological and
environmental problems. The applications of these new nano-agrochemicals are the new
emerging ways to control pests.
References
1. Ahmed S, Ahmad M, Swami BL and Ikram S (2016) A review on plants extract mediated
synthesis of silver nanoparticles for antimicrobial applications: A green expertise.
Journal of Advanced Research, 7(1): 17-28.
2. Anders GV and Glotzer SC (2012) DNA nanotechnology: The world's smallest assembly
line. Nature Chemistry 4: 79–80.
3. Chaudhary SR, Nair KK, Kumar R, Gogoi R, Srivastava C, Gopal M, Subhramanyam B,
Devakumar C and Goswami A (2010) Nanosulfur: Potent fungicide against food
pathogen, Aspergillus niger. American Institute of Physics Proceedings 1276: 154–157.
4. Gogoi R, Singh PK, Kumar R, Nair KK, Alam I, Srivastava C, Yadav S, Gopal M,
Chaudhary SR and Goswami A (2013) Suitability of nano-sulphur for biorational
management of powdery mildew of okra (Abelmoschus esculentus Moench) caused by
Erysiphe cichoracearum. Journal of Plant Pathology and Microbiology 4: 171.
5. Gopal M, Srivastava C, Gogoi R, Kumar R and Goswami A (2012) Addressing
Environmental Concern with Nano Pesticides for Sustainable Agriculture. The
International Journal of Environmental Sustainability 8(1): 115-130.
6. Hoseinzadeh E, Makhdoumi P, Taha P, Hossini H, Stelling J, Kamal MA and, Ashraf
GM (2017) A Review on Nano-Antimicrobials: Metal Nanoparticles, Methods and
Mechanisms. Curr Drug Metab 18(2):120-128.
7. Khot LR, Sankaran S, Maja JM, Ehsani R and Schuster EW (2012) Applications of
nanomaterials in agricultural production and crop protection: A review. Crop Protection
35: 64-70.

71 Winter School on "New generation smart agrochemical oriented approaches for crop
and human health management", 07-27 Dec, 2017
Seed Coating Technology for Pest Management
Sudipta Basu, S.K. Lal, Debjani Dey 1, Rajesh Kumar Sharma and Vinod Gulabrao
Deshmukh
Division of Seed Science and Technology, 1Divission of Entomology
ICAR-Indian Agricultural Research Institute, New Delhi 110012
Seed is the basic input in agriculture and plays a crucial role in boosting up the
production and economy of the country. To meet the growing needs of the population and
enhancing productivity, timely availability of quality seed at reasonable price to the farming
community is desirable. The quality seed is the key to higher crop yield, income generation,
livelihood security for the farmers.
‘Quality seed’referes to a seed which has high genetic and physical purity, germination
and seed health with optimum moisture content for safe storage. The quality seed needs to be
protected to deliver it as an efficient input. When seed is planted during an environmental
stress; biotic or abiotic its germination, plant establishment and susceptibility to
pest is hampered. Due to global warming, there is significant increase in population of
some pest more than their economic threshold value which directly affects the crop
production in general and yield per unit area in particular. In context of global warming,
ensuring food and livelihood security for the growing Indian population is a major
challenge.

Seed coating, pelleting, encrusting, film-coating apart from seed priming are seed quality
enhancement treatments which aid to deliver materials (eg. pesticides, nutrients, inoculants,
beneficial microorganisms) needed at the time of sowing for assured plant stand and biotic
stress tolerance. There are specific problems/conditions in the different species of crop
which needs to be addressed through incorporation of specific treatments.

Seed enhancement is defined as a post-harvest beneficial treatments performed on


seeds (after harvesting and conditioning) to improve their physical or physiological
performance or facilitate the delivery of seeds and other materials required at the time of
sowing(Copeland and McDonald, 1995).These methods are not mutually exclusive and
many techniques can be combined to obtain additive effects. These treatments have the
potential to alleviate stress effects and improve plant stand, pest and disease control and
thus, improving the planting value of the seed (Taylor et al.,1998).The enhancement
treatments are used most extensively in high-value, horticultural crops such as vegetables
and flowers (Basu, 1976; Heydeckerand Coolbear, 1977). Many agronomic species are now
film coated/pelleted to provide better delivery of high -value crop protection
chemicals and higher cost, stacked genetic traits seed (Asrafand Foolad, 2005). As seed value
increases, commercial seed enhancements are commonly available and requested by the
farmers. Seeds vary greatly in size, shape and colour thus their approach of coating differs based
on needs of their end users. The various seed coating techniques commonly used are:
A. Seed encapsulation: It is the process of applying a coating over the seed by misting with
water or other liquid and gradually adding a fine inert powder after putting seed in a

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and human health management", 07-27 Dec, 2017
rotating pan so as each misted seed becomes the center of an agglomeration of powder that
gradually increases the size and weight but comparatively less material than in pelletting
is applied so that the original seed shape is still apparent.
B. Seed coating: Seed coating is a technique of applying needed inputs such as fertilizers,
organic, inorganic nutrients, plant growth regulators, biofertilizers, and pesticides, moisture
attractive or repulsive agents etc., to seed by adhesive agents to provide a self sustaining seed
unit with an improved micro-environment for germination, seedling development and
tolerance to early seedling diseases. During seed coating the seed shape of the seed is not
obscured and coating material is placed directly onto the seed. Seed coating technology can
also act as carrier for plant protectants which can be applied in the target zone with minimal
disruption to the soil ecology and environment. The application of polymers to seed serves as
an extra exterior shell in order to give the desired seed characteristics (e.g., quick or delayed
water uptake and enhanced germination) that would be beneficial for better emergence and
establishment in certain environments (Taylor et al., 1998). In this technique, a thin and
permeable layer of pesticide is stuck on seed surface to prevent damage caused by seedborn
pathogens. This layer is melted or splited after absorption of moisture and suitable
temperature by seed, and let the radical to exit the seed. In this approach, materials are used
accurately with seed, the adverse effects of some pesticides on seeds are diminished, and
increase the accuracy and performance of pesticide with decreasein their consumption,
environmental pollution and costs.
C. Film coating: Film coating is the process of applying precise amount of active ingredients
along with a liquid material directly onto the surface of seed without obscuring its shape but
total seed weight may increase up to 10%. Most film coatings contain a colorant that helps in
visual monitoring and placing of seeds accurately during automatic seeding. Film forming
formulations consists of a mixture of polymer, plasticizer and colorants (Halmer, 1988).
There are two types of polymers used for film coating, namely; hydrophilic polymer, which
absorbs water uniformly and facilitate early emergence and hydrophobic polymer, which
repel water and cause delayed germination. The germination of most of the crop seeds is not
adversely inhibited by polymer film coating as film coating materials are usually inert and
non phytotoxic to seed. Fungicides and insecticides can also be added to film coating. Film
coatings can help in making seed surface smooth, allowing the seed to flow more evenly
during automatic seeding. Some novel applications of film coating can confer temperature-
sensitive water permeability to seeds or gaseous exchange. Film coating is often used on seed
species that do not require pelleting (e.g. Brassica sp.) for precision planting but the seed
requires some encapsulation due to plant protectant application (Halmer, 1994). The
advantages of filmcoating include; increased flowability in the planter caused by better
"slippage" between individual seeds, increased visibility of seed in the soil and seed
treatment identification by using different colorants. There are some
disadvantages of film coating as well viz.; plant protectants that inhibit germination
may not be used because of inadequate separation between the seed and the active
chemical; seed size and shape and weight are not altered sufficiently to make a dramatic
difference in plantability; and polymers and plasticizers in the film coating may be toxic
or inhibitory to the seed species, seeds of different species can have different sensitivities to
the same film coating chemical. Some novel applications have also been developed using the
film coating method. For example, artificial polymers have been developed that exhibit
temperature-sensitive permeability to water (Landec Corporation, Menlo Park, California).

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These Intelimers are permeable to water at warm temperatures, but not at cool temperatures.
The coatings are also being used to delay germination after planting, such as for timing the
emergence of male parent lines at different times for hybrid seed production.
D. Seed pelleting: It is the process of enclosing a seed with small quantity of inert material to
produce a globular unit of standard size to facilitate precision planting. The inert material
creates natural water holding media and provides small amount of nutrients to young
seedlings. Seed pelleting serves as mechanism of applying needed material in such a way that
they affect the seed or soil at the seed soil interface. Seed pelleting controls the size, shape,
weight and surface properties; there by, improving seed appearance and plantability
(Jamieson, 2008). Pelleting ensures more uniform sowing of seeds, makes the sowing of
small, rough seeds (such as carrot, sugarbeet and parsley seeds) easier, reduces the amount
of work involved in thinning the seedlings, fosters economization onplanting material,
improves the growing conditions of the plants, and increases yield of onions, carrots,
cucumber, parsley, tomatoes, and table beets by 20-25percent. There are two component of a
seed pellet: bulking (or coating) material and binder. The bulking material can be a
mixture of several different minerals and/or organic substances or a single component.
The coating material is the "work-horse" of the duet. Desirable characteristics of a good
coating material include: uniformity of particle-size distribution, availability of active
ingredient, and low phytotoxicity. The binder, holds the coating material together.
Binder concentration is critical as higher and lower concentration of binder delays
germination and causes chipping and cracking of pellets respectively. Many different
compounds have been used as binders, including various starches, sugars, gum arabic,
clay, cellulose, vinyl polymers (Halmer, 1988). The different types of pelleting are
(Ponnuswamy, 1993):
i. Inoculant pelleting – Different biofertilizers such as Rhizobia, Azospirillum or
Azotobacter are used as filler materials which are fixed to the seed with the help of an
adhesive, which helps in improving the activity of microorganisms of rhizosphere and
help in nitrogen fixation.
ii. Protective coating–Seed pelleting is done with fungicides, pesticides, antibiotics and bio-
control agents (Trichoderma spp., Bacillus spp. or Streptomyces spp) and they are added
to the adhesive and coated on the seeds for providing protection/ control of diseases
through pelleting. However, caution should be taken in such a manner that the filler
material should be compatible with the fungicide and pesticides used.
iii. Herbicide coating – Filler antidote or absorbent coatings can be used before sowing.
Herbicide antidote like 1, 8 naphthalic anhydride (NA) is found the best as seed
pelletizer. The absorbent, activated carbon can also be used as filler material which is
found good in protecting the seed from 2, 4-D and Alachor herbicide damage.
iv. Nutrient coating – The nutrient coating with micro and macro nutrients enhances the
germination and seedling growth and also makes it less available to weed species and
also avoid the wastage of nutrients. The micronutrients used are ZnSO4, FeSO4, CuSO4,
KH2PO4, KCl, Borax, etc. the micronutrients are added at required quantity to the
adhesive and are filled with the filler material. The micronutrients are required in less
quantity than soil or foliar application.
v. Hydrophilic coating –Starch graft polymers, polymers that can adsorb upto 1000 times
their own weight of water, and magnesium carbonate are capable of improving
movement of air and water. The increase in germination is due to increase in the rate of

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imbibition where the fine particles in the coating act as wick or moisture attracting
material or perhaps to improve seed soil contact.
vi. Oxygen supplier coating – Seed are coated with peroxides of zinc or calcium which aids
in increased oxygen supply to the germinating seeds. The seeds which have got seed
coats impermeable to oxygen will be highly benefited for enhancing germination.
E. Seed treatment: Seed treatment is a physical, mechanical, chemical or biological process
designed to improve and enhance the quality of seed so that the emerging seedling could be
healthy and vigorous. Diseases and pests affecting crops can have devastating consequences
in agricultural and horticultural production if not properly managed. Breeding is an excellent
tool tobuild resistance against pests and diseases in the plants. However, breeding alone does
not address all of the agronomic challenges, therefore crop protection products are
oftenneeded and used for good crop management. Seed treatments can be used as a primary
tool in a successful integrated pest management program for sustainable agriculture since
they target the pests and diseases with smaller amountsof active ingredients per hectare. Seed
treatment can be applied as seed dressing, seed coating or pelleting with chemical
protectants (Captan, Apron,Vitavax, etc.), microorganisms (Rhizobium), bio formulations
(Trichoderma spp). Seed treatment can also deliver high levels of efficacy for the control of
early season pests and diseases at a reduced usage rate compared to many foliar or soil
applied alternatives. Using a seed treatment reduces the area in contact with a crop protection
product from 10,000 m2 for foliar application or 500 m2for furrow application to only 50 m2.
As an example, for an insecticide in corn, at a plant rate of 100,000seeds per ha, the
application rate is also reduced from 1,350gactive ingredient per hectare for foliar application
or 600g ai/ha for furrow application to 50g ai/ha for a seed treatment. This reduced active
ingredient loading minimizes the impact on the environment significantly by decreasing the
effect on non-target organisms and the movement of the product in the environment. Foqué
et al., (2017) compared different techniques to assess the risk of dust drift during seed
treatments and reported pesticide seed coating was better than dust application for seed
treatment.
Application of coating technology for pest management
Wide range of pesticides are being used for control of lepidoterans, sucking pests (aphids, white
flies, leaf and planthoppers, thrips), coleopterans, mites, fungi and bacteria. Up to 1990s, soil
application of pesticides were commonly used but due to its its non specific action and high
residual toxicity focus shifted to systemic and non systemic pesticides for higher efficacy. The
pesticides are commercially applied as seed dressing, seed coating or pelleting for better
efficiency. Among pesticides, neonicotinoid insecticides which comprise of seven commercially
marketed active ingredients: imidacloprid, acetamiprid, nitenpyram, thiamethoxam, thiacloprid,
clothianidin and dinotefuran are most commonly used seed treatment/coating for pest control due
to their broad spectrum, systemic and translaminar action, and unique mode of action.
Neonicotinoids are widely used for seed coating in cotton (Gossypium spp), sugarbeet (Beta
vulgaris L), oilseed rape (Brassica napus L) corn (Zea mays L) (Elbert et al., 2008). Application
on the seed coating with neonicotinoids, provides effective control of sucking pests; leaf hoppers
and aphids which are vectors of viruses, wireworms (Agriotes spp.) and root worm (Diabrotica
virgifera Le Conte) (Maienfsch et al., 2001). Budge et al.,(2012) reported positive impact of
neonicotinoid seed coating on crop yield and pest control on oilseed rape. Westwood et al.,

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(1998) and Epperlein (2001) compared efficacy of Imidacloprid coated pelleted and untreated
pelleted sugarbeet seeds for aphid control. Results showed higher aphicidal activity and
prolonged control of aphids in Imidacloprid coated pelleted seeds than pelleted seed alone. It
controlled egg laying capacity of aphids and their build up for 13 weeks with reduction of viral
diseases and higher sugar content and yield in sugarbeat. Imidacloprid pelleted seeds had no
adverse effect on soil organisms, i.e. spiders (predominantly Linyphiidae), millipedes (mostly
Brachydesmus sp.), ground beetles (Trechus quadristriatus, Bembidion lampros, Platynus
dorsalis, and Pterostichus cupreus), and rove beetles (Xantholinae and Tachyporinae). Film
coating of leek (Allium porrum L.) seeds with insecticides (Fipronil, Imidacloprid and
Vamidothion) controlled incidence of thrips (Thrips tabaci Lind.). Among insecticides
compared, Fipronil was most effective with least phytotoxity. Onion maggot (Delia antiqua
Meigen), a major pest of onion requires insecticidal application at time of planting. Application
of neonicotinoids; Fipronil, Spinosad and Clothianidin at rates of 25, 25 and 50 g (a.i.)/kg of
seed, respectively as seed coating, significantly controlled damage of onion maggot in onion
(Ester et al., 1997; Taylor et al.,2008). But Yildirim and Hoy (2003) reported non significant
effect of Cyromazine seed treatment for control of onion maggot. Jyoti et al., (2003) also studied
role of film-coating with Chlorpyrifos on seed germination and control of cabbage maggot, Delia
radicum (L) on cabbage transplants. Chlorpyrifos film-coated seed treatments had no adverse
affect on lab germination and provided significant plant protection against cabbage maggot for
several weeks after transplanting. Filmcoating of cauliflower seed (Brassica oleracea L. var.
botrytis L.) with insecticide, Chlorpyrifos (28.8g a.i./kg) and Isofenphos (30 g a.i./kg) is also
reported to provide effective control as a post-planting treatment against cabbage root fly (Delia
radicum) (Ester et al., 1994). Later, Ester et al., (2003) reported film coating of cabbage
(Brassica oleracea L. var.capitata L.) and cauliflower (Brassica oleracea L.var. botrytis L.)
seeds with Imidacloprid and Spinosad provided control against cabbage root fly larvae (Delia
radicum), flea beetle (Phyllotret anemorum and P. undulata) and cabbage aphid (Brevicoryne
brassicae). Among seed coating pesticides; Spinosad was most effective for control of cabbage
root fly larvae and caterpillars @ 24 and 48 g a.i. per 100,000 seeds respectively whereas
Imidacloprid was found effective in controlling flea beetle and cabbage aphids at a rate of 70 g
a.i. per 100,000 seeds.The combined application of Spinosad and Imidacloprid as a filmcoating
on seeds was found to be an environment friendly alternative for protecting Brassica crop. Seed
treatment with insecticide combined with fungicide in different proportions on Canola (Brassica
napus L. and Brassica rapa L.) is reported to reduce feeding of flea beetle and improved
seedling growth, plant density and seed yield (Soroka et al., 2008). Raveton et al., (2007)
reported control of wireworms in sunflower with seed-coated formulation of fipronil and its
metabolites. A comparative study of insecticide seed coatings (Imidacloprid, Fipronil,
Thiamethoxam, Tefluthrin) and soil applied insecticides in-furrow (Chlorpyrifos, Diazinon,
Tefluthrin) both were ineffectiveness for control of western corn rootworm (Diabrotica virgifera
Le Conte) and flea beetle (Phyllotreta spp.) in corn (Furlan et al., 2006) and Brassica sp. (Trdan
et al., 2009).
Many vegetable insect pests are managed through neonicotinoid and pyrethroid
insecticides. Unfortunately, these insecticides are toxic to many bees and natural enemies and
some pests have developed resistance against pesticides. Seed coating with a neonicotinoid
insecticide had been reported to have negative effect on the population of wild bees in corn,
sunflower and seed rape (Rundlöf et al., 2015) whereas is reported to have non significant effect

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on Bumble bee (Bombyx sp.) population, its foraging and homing behaviour in sunflower (Tasei
et al.,2001). In oilseed rape, seed coating with Elado, an insecticide containing a combination of
the neonicotinoid clothianidin and the non-systemic pyrethroid β-cyfluthrin, reduced wild bee
density, solitary bee nesting, and bumblebee colony growth and reproduction under field
conditions. Elbert et al., (2008) reported loss of pollinating bees as colony collapse disorder
(CCD), characterized by a sudden disappearance of worker bees that do not return to the hive
due to neonicotinoids (imidacloprid, clothianidin, and thiamethoxam) poisoning. Girolami et al,
(2012) studied the translocation of neonicotinoid insecticides from coated seeds of maize on Apis
mellifera L.through studies of leaf guttation (excretion of xylem fluid at leaf margins) of maize
plant. It was reported that leaf guttation drops of the corn plants germinated from neonicotinoid-
coated seeds contained higher amount of insecticide (100 mg/l for thiamethoxam and
clothianidin, and up to 200mg/l for imidacloprid) as compared to untreated seeds, depicting
higher translocation and residual effect causing toxicity to bees.
The Anthranilic diamide insecticides provide a promising alternative to neonicotinoids
and pyrethroids due to its effective control of herbivorous arthropod pests with low toxicity to
beneficial arthropods and mammals. Schmidt-Jeffris et al., (2016) compared anthranilic diamides
applied as seed, soil and foliar application significantly reduced O. nubilalis damage. Results
from laboratory bioassays revealed that diamides application as seed and in-furrow treatments
caused high O. nubilalis neonatal mortality up to 44 days after application in snap bean.
Application of chitosan based seed coating could be an appropriate option for pest control
replacing highly toxic pesticides. Soybean seeds coated coated with chitosan showed anti
feedent action against soybean pod borer and soybean aphid, promoted germination, plant
growth and yield in soybean (Zeng et al., 2012).
Plant growth-promoting rhizobacterium (PGPR) are also reported to have good fungicidal
activity in different crops. Seed pelleting of sesame seeds with a PGPR; Paenibacillus polymyxa
strain E681 controlled pre- and post-emergence damping-off and wilt and promoted plant growth
and the grain yield (Ryu et al., 2006).
The pesticides when laden to seed through afiller (clay, matrix, polymer) or polymer like
polyethylene glycol is reported to have better and controlled release with less toxicity. Shakil et
al., (2010) and Adak et al., (2012) have developed and studied efficacy of polyethylene glycol
based novel amphiphilic polymers for controlled release formulations of Carbofuran and
Imidacloprid which could be used as seed coating formulation for pest management.
A liquid bioplastic formulation for film coating had been evaluated for microbial
biocontrol agent for pest management in agricultural and horticultural crops. Achili et al.,
(2016) evaluated bioplastic film-coating on corn and canola and reported to have no effect of
film coating on seed germination of both the species. Bioplastic coatings containing spores of the
plant-growth promoting fungus, Trichoderma harzianum, significantly stimulated the growth,
shoot and root length (by 29% and 44% respectively), than in uncoated seeds. Similarly, in
canola seedlings, shoot and root length increased by 19% and 20%, respectively over control. It
is reported in bioplastic seed coatings; T. harzianum spores prevented damage caused by
imidacloprid and Metalaxyl-M used as seed coating. The adhesive and plastic properties of bio

77 Winter School on "New generation smart agrochemical oriented approaches for crop
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plastic also reduced dust-off from bioplastic-coated seeds by 96% in corn and 99% in canola
compared to seeds coated with a commercial polymer.
Hydrophobic film coating technology that represented the combined synthesis of
knowledge on the use of hydrophobic films, physical particle barriers, and white reflective
surfaces to suppress arthropod pests and diseases of agricultural crops. The hydrophobic particle
film is based on the inert mineral, kaolin, that is surface treated with a water-repelling agent.
Glenn et al.,(1999) reported suppression of important tree fruit arthropod pests and diseases by
altering the plants surface with dust applications of these hydrophobic particles. The film barrier
repelled and suppressed infestation by making the plant visually unrecognizable as a host by the
anthropod. In addition, insect movement, feeding, and other physical activities are reported to be
severely impaired in arthropods due to the film. The fungal diseased could also be prevented by
enveloping the plant with hydrophobic film barrier which prevented disease inoculum or water
from directly contacting the leaf surface. The hydrophobic particle film also reduced heat stress
by reflecting sunlight with its bright white color but not the photosynthesis activity due to its
porous nature. The hydrophobic particle film concept could offer broad spectrum protection
against arthropod pests and diseases in agricultural and horticultural crops.

References

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Extraction and Estimation of Azadirachtin from Neem
Supradip Saha

Division of Agricultural Chemicals


ICAR-Indian Agricultural Research Institute, New Delhi-110012

Azadirachtin-A as well as other derivatives of azadirachtin are the most important active
ingredient in neem. Azadirachtin’s use as a pesticide has been commercialized and a number of
formulations have been introduced worldwide. However application of azadirachtin as a pest
control agent requires a sensitive and reliable method for its quantitation in neem extracts, for
monitoring its efficacy, stability and toxicity. The most acceptable method of analysis of
azadirachtin is the use of HPLC. Reverse phase liquid chromatography is used for separation and
quantitation of azadirachtin.
Sample preparation

Standard solution of azadirachtin (1000 mg/mL) was prepared by dissolving 10 mg of the


compound in 10 ml of HPLC-grade acetonitrile. Serial dilutions were made in the range of 100-
10 mg/mL to plot the calibration curve. The standard solutions were stored at –20°C.
Extraction
One gram of seed kernel powder was taken in a 15 ml centrifuge tube. Distilled ethanol (6 ml)
was added to each tube. The tubes were screw-capped and vortexed for 15 minutes. The tubes
were then centrifuged at 5000 rpm for 10 min. The supernatant was transferred into a new tube
and the residue was re-extracted twice with ethanol (2 X 6 ml). The pooled extracts were
combined and the final volume was made up to 25 ml in a volumetric flask. A part of this sample
(4 ml) was filtered into an autosampler vial through a 0.22 mm membrane filter. Filtered sample
is then ready for HPLC analysis.
Chromatographic conditions

The samples were filtered through a 0.25 um membrane filter before injection and the retention
time (Rt) for each compound was measured. The sample (20 l) was injected into the HPLC for
analysis.

HPLC conditions:
1. Detector: PDA/UV
2. Stationary phase: C18 column (250 X 4.6 mm; 4µm) or phenyl column (250 X 4.6 mm;
4µm)
3. Mobile phase: methanol: water (65:35 v/v) flow rate of or acetonitrile : water (40 : 60
v/v), isocratic condition
4. Flow rate: 0.75 mL min-1 or 1 mL min-1
5. λmax: 217 nm

6. Run time: 15 min

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Quantification of azadirachtin A

Azadirachtin in the samples was quantified by employing standard azadirachtin sample (95%
pure). The value of azadirachtin content was calculated based on calibration and expressed as
ppm (mg/g of the kernel weight).

Azadirachtin content= (A1/ A2) (m1/m2) x P


Where, A1= peak area of azadirachtin in sample
A2= peak area of azadirachtin in reference standard
m1=mass, in grams, of the test sample.
m2=mass, in grams, of the reference standard.
P =Purity of the reference standard sample

0.30

Azadirachtin-A
0.25

Azadirachtin-B
Azadirachtin-H

0.20
AU

0.15

0.10

0.05

0.00
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00
Minutes

Fig. HPLC Chromatogram of neem seed kernel extract using methanol:water (65:35 v/v) with
flow rate of 0.75 ml min-1

References

1. Mínguez-Mosquera, M.I., Hornero-Méndez, D. and Pérez-Gálvez, A. 2008. Carotenoids


and Provitamin A in Functional Foods. In: Methods of analysis for functional foods and
nutraceuticals, Hurst, W.J. (Eds.) Second Edition. CRC Press, 277-336
2. Adsule, P.G. and Dan, A. 1979. Simplified extraction procedure in the rapid
spectrophotometric method for lycopene estimation in tomato. J. Food Sci. Technol. 16,
216-216.
3. Beerh, O.P. and Siddappa, G.S. 1959. A rapid spectrophotometric method for the
detection and estimation of adulterants in tomato ketchup. Food Technol. 13, 414-418.
4. Sadler, G., Davis, J. and Dezman, D. 1990. Rapid extraction of lycopene and b-carotene
from reconstituted tomato paste and pink grapefruit homogenates. J. Food Sci. 55, 1460-
1461.
5. Lockwood, B. 2007. Nutraceuticals. Second edition. Pharmaceutical Press, 39-40

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Advancements in Pesticide Formulation Technology
Anupama Singh1 and Neeraj Patanjali2
1
Division of Agricultural Chemicals, 2Division of Soil Science and Agricultural Chemistry,
ICAR-Indian Agricultural Research Institute, New Delhi 110012

The primary objectives of pesticide formulations is to give a product which is user and
environment friendly and provide optimum biological activity at minimum possible dose.
However, because of huge variation in physico-chemical properties of pesticide active ingredient
(a.i.) it is not possible to prepare a universal formulation which is effective for all types of
applications. Conventional pesticide formulations generally comprising; solvent based
emulsifiable concentrates (EC) and powder based dusts (DP) and wettable powders (WP). The
use of petroleum based solvents in EC formulations and dusty powders in DP and WP
formulations; leading to safety concerns for the applicator and has overall negative impact on the
environment. These drawbacks of conventional formulations, urged the demand for user and
environment friendly pesticide formulations. Recent technological innovations in pesticide
formulation and introduction novel formulation types can ably extend the overall life-cycle of
pesticide a.i. by imparting value addition to formulation product for competitive advantage over
the conventional formulations. Increasing pressure from regulatory bodies and consumer safety
groups; urging formulators to develop user friendly and safer formulations as well as adjuvants
in order to decrease the residues of toxic chemicals on edible crops after harvest. All these
concerns, collectively forced the researchers to look for newer improved formulations and
adjuvant technologies.
Introduction
From the onset of ‘Green Revolution’ in
India, farmers have relied heavily upon
pesticides for crop protection. To meet this
demand, generally conventional
formulations simple dust based powders
and petroleum solvent based spray
formulations have been used in order to
save crops from the attacking pests.
However, since the ‘Green revolution’ the
pesticide manufacturers have fulfilled the
needs of farmers through continual
advancements in agrochemicals. In the Figure 1: Indian Agrochemical Market 2015-161
recent decades, the overall world population has increased tremendously and is projected to
reach about 10 billion by 2040, generating further pressure on food availability for humans. This
further urged the need for newer formulation products, additives, packaging and process
techniques to allow the formulation of agrochemicals with improved physico-chemical
characteristics. In contrast to the world market; insecticides hold largest share of the

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agrochemicals market i.e. 60%, while herbicides presently holding 16% share in the market and
considered as most rapidly growing segment in India.
India is among leading countries producing agrochemicals and stands 4th after the USA, China
and Japan. The agrochemical sector produced a value of $ 4.4 billion in financial year 2015 and
is supposed to grow @ 7.5% per annum to reach $ 6.3 billion by the financial year 20201. About
50% of the produced agrochemical is used by Indian consumers while the remaining goes for
export purpose. The demands in India are expected to rise @ 6.5% annually while exports are
projected to grow @ 9% annually during the similar period.

Figure 2: Indian Agrochemical Market (USD Billion)1

Water soluble a.i. can be formulated as aqueous solution (SL), while oil soluble a.i. are generally
formulated with petroleum solvent like EC. Suspension concentrates (SC) or water dispersible
granules (WG) are the suitable formulations where a.i. is very less soluble in water as well as
petroleum solvents2. Other formulation types such as granules; used for direct application into
the soil or directly to seeds for seed treatment. In the recent decades more and more pressure
from regulatory bodies and consumer safety groups urged the urgent need for products and
formulations which are safer, easy to use and which are effective at lower doses with minimum
toxicity towards non-target organisms and environment. Rapid development in formulation
technologies in the past few decades leading to more sophisticated formulations as a result of
availability of more effective surfactants and other adjuvant. On the other hand improved
knowledge of the colloid and interface science helped in a great way to enhance formulation
quality, stability and bio-efficacy. Processing techniques has also improved very much during
this period generating finer and uniform particles for enhanced stability and bio-efficacy.
Formulation objectives
The key objective of formulation is to develop a product which is convenient, safe, user-friendly,
easy to handle and having long shelf life in order to obtain the maximum inherent activity of a.i.
present in formulation. This requires careful consideration of numerous interacting factors to
choose specific formulation for each a.i. to be formulated. The key factors which govern the
selection of formulation are:
• Physical and chemical characteristics of a.i.
• Toxicity and mode of action
• Method of application
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• Safety towards user
• Formulation cost
• Climatic conditions
• Market preferences and transportation
Upon selection of above mentioned parameters, a suitable formulation can be selected.
Thereafter, inert ingredients including surfactants and other adjuvents are taken in appropriate
ratio in order to produce a stable formulation of required a.i. having shelf life of at least 2 years
during storage under variable climatic conditions.
Formulation types
The most commonly used formulations are still SL for water-soluble a.i., EC for oil-soluble a.i.
and WP and SC for insoluble solids. GR and seed treatment formulations have also been
produced for direct application for many years. In recent times, newer formulations has been
introduced vastly to meet the growing demands of user and environmental concerns or to
enhance the efficacy of the a.i. formulated. As a result, an international coding system was,
developed by GIFAP in 1984 (in 1996 GIFAP was renamed as GCPF - Global Crop Protection
Federation, based in Brussels, Belgium. The major types of formulations and their codes are
shown below:
Table 1: Common pesticide formulations and their codes

Formulation type Code Formulation type Code


Granule GR
Solution concentrate SL
Emulsifiable concentrate EC
Wettable powder WP
Suspension concentrate SC
O/W emulsion EW
Suspoemulsion SE
Microemulsion ME
Water dispersible granule WG
Microcapsule CS
Emulsifiable gels GL
Gels for direct application GD
Seed treatments DS,WS,LS,FS

Trend towards new generation formulations:


The pressure from the regulatory authorities urged the need to develop safer eco-friendly
formulations for the user. The main issues which need to be addressed are:
• safety during manufacturing process
• ease of handling and application suitability as well as safety for the user
• ease in package disposal and its reusability
• minimization of pesticide application dosage
• minimization or elimination of pesticide wastage

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These complex requirements can be addressed by technological advancements in pesticide
formulation through proper understanding of principles of colloid and interface science.
Utilization of advanced surfactants and other formulation adjuvents is the key in formulation
advancement, in particular appropriate use of surfactant blends and effective dispersing agents.
The product free from volatile organic solvents, having minimum or no package disposable
problems and giving maximum biological activity at lowest possible dose without exposure to
applicator would seem to be the ideal one. Based on these requirements current trends in
pesticide formulation are given below:
• use of aqueous emulsions or safer solvents wherever possible to eliminate volatile
organic solvents
• replacement of dusty formulations by aqueous suspensions and WG to reduce exposure
hazards to applicator
• development of multiple active ingredient formulations for synergistic activity and
inhibition of pesticide resistance development
• use of bio-enhancements such as surface wetting agents, stickers for reducing off target
delivery and enhancing biological activity
• to develop novel formulations like tablet or gel with minimum pack disposal problems
• to develop formulations with controlled release rate and target specific delivery by
encapsulation and seed treatment
Research work is being carried out by leading agrochemical companies and Government
organizations on developing WG and WP as water soluble sachets which may be added directly
into the spray tank in order to reduce exposure hazard, but still lot of work need to be done in
this area. There is an increasing trend of shifting from dusty formulations towards dust free
formulations (Figure 3). However, it is not possible to formulate each and every active ingredient
in this way so other formulation and packaging options also need to be evaluated. Aqueous based
formulations (e.g. EW, ME, SE, nanoemulsion provides a possible alternative for the removal of
volatile organic solvents from the formulations (Figure 4). Other possibilities include
emulsifiable gels and tablets.

Fig 3: Gradual improvement from dusty formulations to dust free formulations

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Figure 4: Gradual improvement from solvent based formulations to water based formulations
Microemulsion and Nanoemulsion
The two major types of colloidal dispersions which may be obtained from oil, water and
surfactants, are microemulsions and nanoemulsions. There are numerous resemblances between
these, but there exists some significant distinctions between them which differentiate them from
each other. Both of these can be of oil-in-water (O/W) type or water-in-oil (W/O) type and for
pesticide formulation both may be formulated to encapsulate lipophilic pesticides; so the
discussion is restricted to O/W type dispersions only.
Microemulsions (ME)
Oil-in-water microemulsions are thermodynamically stable transparent to somewhat translucent
emulsions where oil phase is dispersed within an aqueous medium in the form of very fine
droplets of size less than 0.1 µm. They are stable over wide temperature range and usually
consisting of three components:
• liquid or solid a.i. dissolved in organic solvent (oil phase)
• water (aqueous phase)
• Surfactant/cosurfactant blend for solubilisation of both aqueous and oil phase.
These three components form a single phase consisting of relatively large ‘‘swollen-micelles’’ in
which the oil phase of the a.i. and solvent are dissolved or solubilised by the
surfactant/cosurfactant system. ME formulations can be developed for either liquid or semi-solid
a.i. (having low melting point) which are insoluble as well as chemically stable in water. For the
preparation of ME; a.i. is dissolved in minimum quantity of solvent which serves as oil phase.
The water phase is consisting of surfactant blend, co-surfactant and other adjuvents. The two
types of surfactants needed to prepare ME formulation are of different types; one being water
soluble and the other one being oil soluble. The surfactant which is water soluble is typically
anionic or non-ionic type with a very high HLB value, and the hydrophobic part of the molecule
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should match the oil. On the other hand, the cosurfactant should be oil soluble and should
possess very low value of HLB, these are usually alcohols for example butanol or hexanol. The
percent concentration of surfactants in a ME may be as high as 10-30%, in comparison to just
about 5% for a normal o/w emulsion3.
Table 2: Typical composition of microemulsions

Component Percent weight


a.i. 10-35
Surfactant 10-30
Cosurfactant 0-5
Anti-freeze 2-10
Water Upto 100
The concentration of a.i. in ME formulations are relatively low, but the higher surfactant
concentration and smaller micelle size of the emulsion droplets possibly permit better
transportation of the a.i. through cellular membranes in plant as well as insects; thereby resulting
in improved efficacy towards pests4. The only real disadvantage of microemulsions is the
relatively higher level of surfactant required which makes them expensive and non-competitive
with conventional emulsifiable concentrate.
Nanoemulsion
An O/W nanoemulsion is thermodynamically unstable emulsion consisting of two immiscible
liquid phases, with one liquid being dispersed as small spherical droplets (of size less than 100
nm) in the other liquid.
Nanoemulsions are considered as kinetically stable as a result of their extremely small size
resulting in inhibition of droplet flocculation and coalescence. Hence, the process of Ostwald
ripening alone governs the process of destabilization. These are usually formulated by means of
so-called “high-energy” methods through specific devices (like ultra-sound generators or high
pressure homogenizers) which can ably supply enough energy to increase the W/O interfacial
area resulting in the formation of ultrafine droplets.
Similarities among microemulsion and nanoemulsion
Microemulsions and nanoemulsions share a lot of similarities among themselves in their
composition, dimensions, structure and preparation, which gives rise to significant confusion
throughout the scientific literature about the exact nature of the colloidal dispersions being under
consideration. These are some similarities between microemulsions and nanoemulsions:

• Both of them typically require quite similar components for their preparation: an oil
phase containing a.i. dissolved in solvent, an aqueous phase, a surfactant blend and
generally a co-surfactant. Certainly, it is likely to prepare both types of emulsions from
exactly the identical constituents through utilizing them in different ratios.

• A key source of confusion among microemulsions and nanoemulsions is the prefixes


which are used to represent them. The term ‘‘micro’’ typically means 10-6, while the term
‘‘nano’’ typically denote 10-9, which would indicate that particles of nanoemulsions are

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smaller than those of microemulsions. However, in actual situation the particles in ME
are generally smaller as compared to those in the nanoemulsions.

• Both O/W nanoemulsions as well as microemulsions comprise of extremely small


particles (usually spherical in shape) of oil and surfactant molecules usually dispersed
within the aqueous phase. The particles comprise of a hydrophobic core made up of oil
molecules and surfactant tails, and a hydrophilic shell made up of surfactant head groups.
Difference between microemulsion and nanoemulsion

• Thermodynamic stability: The ultimate means of differentiating nanoemulsions and


microemulsions is to study their thermodynamic stability characteristics: nanoemulsions
are thermodynamically unstable; while, microemulsions have high thermodynamic
stability. In case of nanoemulsions, the free energy is higher than the free energy of the
separate oil and water phases, which means that nanoemulsions are thermodynamically
unstable (Figure 5). While, in case of microemulsions, the free energy is lower than the
free energy of the separate oil and water phases, which means that a microemulsions are
thermodynamically stable (Figure 5).

Figure 5: Schematic diagram of the free energy of microemulsion and nanoemulsion systems.
The two states are separated by an activation energy ΔG*5.

• Kinetic stability: Microemulsions are kinetically unstable while nanoemulsions are


kinetically stable

• Shape of particles: Particles in nanoemulsion are usually spherical as a result of very


high interfacial tension and comparatively low particle size, giving rise to high Laplace
pressure which favours the reduction in the total interfacial surface. While, particles in a
ME system may be spherical as well as non-spherical as a result of comparatively low
interfacial tension subjected to the optimum curvature of the surfactant monolayer and
the quantity of oil phase present in the system. Therefore, O/W microemulsions may

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contain particles that are spheroid in shape because this allows the surfactant molecules
to assume their optimum packaging. In case a system has non-spherical particles, then it
is possibly a microemulsion. Whereas, in case a system contains spherical particles it
may be either a ME or a nanoemulsion.

• Surfactant concentration: The concentration of surfactant in ME formulations are much


higher (10-30%) as compared to the much lower in case of nanoemulsions (4-6%). The
higher concentration of surfactant is responsible for high thermodynamic stability of ME
formulations.

• Particle size distribution: ME usually gives rise to single narrow peak for the
distribution of their particle size, while nanoemulsions either have single or multiple
peaks which are either narrow or broad. Hence, in case an emulsion consists of a single
narrow peak in the nanometre range; then, it can be either a ME or nanoemulsion, but if it
gives rise to multiple peaks or broad peaks then it is possibly a nanoemulsion.

• Method of preparation: Nanoemulsions in general are prepared by high energy methods


such as by using ultrasonic generators or high pressure homogenizers. On the other hand,
microemulsions can form spontaneously upon normal stirring upon mixing of all three
phases in an appropriate ratio.
O/W Emulsions (EW)
A fluid heterogeneous formulation consisting of a solution of a.i. in a hydrocarbon solvent,
dispersed as fine spherical globules in a continuous aqueous phase.
In the present time, O/W emulsions are getting significant attention of researchers in order to
overcome the disadvantages related to hydrocarbon solvent based conventional formulations
such as skin-irritation and flammability6. Their capability to reduce the usage of toxic petroleum
based solvents for making eco-friendly formulations makes them further more attractive and user
friendly as compared to conventional formulations. To some extent, they are like a ready to use
formulation except being concentrated. Fundamental thing is that the a.i. must possess very low
water solubility in order to overcome the chances of crystallization. Being water based, EW
formulations are having significant benefits over EC in terms of cost, transportation and
environmental toxicity.
Table 3: Typical composition of O/W emulsion7

Component Percent by weight


a.i. 30-60
Emulsifier 4-8
Anti-freeze 0-10
Anti-foam 0.1-0.2
Thickener (if required) 0.2-2
Water Upto 100

EW formulations have reduced toxicity towards skin and eye than the corresponding EC
formulations as well as higher flash points. Though, they need cautious selection of emulsifiers
in order to rule out the chances of flocculation, creaming and coalescence of the dispersed oil

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droplets. Variety of surfactants like non-ionic surfactants, block co-polymers and other polymer
based surfactants are now being utilized to prepare stable EW formulations. However, while
using non-ionic emulsifiers it is better to combine a low and a high HLB emulsifier which gives
an average HLB of 11–16 for optimum emulsion stability3. The size of droplets may also
indicate the formulation stability and should remain below 2 µm. The EW formulations are
frequently thickened with biopolymers like natural gums in order to rule out the separation of oil
droplets. Sometimes, polymers like polyvinyl alcohol is also used which may serve dual purpose
of both emulsifier as well as thickener/stabiliser.
Suspoemulsions (SE)
A liquid heterogeneous formulation, consist of a stable dispersion of two or more a.i. in the form
of solid particles as well as fine globules in a continuous water phase.
SE consists of a combination of SC and EW technologies, where the two a.i. with altogether
different physic-chemical properties can be combined to form single formulation. The benefits of
SE are that it make it possible to formulate multiple a.i. of different nature together, resulting in
the expansion of the spectrum of biological activity and eliminating the problem of tank-mix
incompatibles. SE formulations are quite challenging to formulate because of the presence of
multiple a.i. with contrasting physic-chemical characteristics. The biggest problem in the
formation of SE is the physical stability of the final product. This stabilization may be
incorporated through cautious selection of adjuvents and process control parameters.
Considerable part of technologies used to develop kinetically stable EW formulations may be
effectively used for the generation of SE formulations. In order to avoid breakdown processes
such as homo- or hetero- flocculation, crystal development and coalescence, specially designed
polymer based surfactants should be used. Stable anchoring and stability of colloidal suspension
is the vital requirement to achieve desired performance of the finished product.

Multiple emulsions
Multiple emulsions consisting of complex poly-dispersed systems in which both O/W and W/O
emulsion exist simultaneously. These systems are stabilized by the careful selection of lipophillic
as well as hydrophilic surfactants respectively in appropriate ratios. The surfactant ratio is critical
in attaining stable multiple emulsions. Among water-in-oil-in-water (w/o/w) and oil-in-water-in-
oil (o/w/o) type multiple emulsions; the former has much more applications and is being
generally used in the pharmaceutical, food and agrochemical sectors. The oil phase of w/o/w
multiple emulsion prevented the a.i. within the inner aqueous core from freely diffusing into the
external continuous aqueous phase. The multiple emulsions are able to cause substantial
reduction in toxicity of the agrochemicals. Stabilization of multiple emulsions may offer precise
osmotic diffusion levels of a.i. in order to achieve customized delivery rates or may provide
multiple compartments for incompatible a.i. within a single formulation. For crop protection
purpose, multiple emulsions are generally w/o/w type in which one or more a.i.- soluble in water
or oil phase may be incorporated. In recent times, significant consideration has been given to
multiple emulsions as probable delivery systems for the controlled release of water or oil soluble
plant protectants. Recent work has not only generated the ability to produce multiple emulsions
but also provide a possible alternative for micro-encapsulation of these emulsions selectively at
either of the interfaces or both as desired in the finished product. The preparation of multiple
emulsions is highly money and time consuming and their exploitation has been obstructed by the
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lack of physical stability attributes upon addition of a.i. in the formulation. Future developments
focused on emulsion systems that are adequately robust to bear storage and handling operations
during intended use.

Water Dispersible Granules (WG)


It is a type of pesticide formulation in granular form which disintegrates and disperses quickly
upon dilution in water and generally used after dilution with water. The granular product has
distinct particles with variable size in the range 0.2 to 4 mm.
WG are popularly known as dry flowables, these are comparatively newer formulations and are
being considered as safer, user-friendly and commercially attractive alternative to WP and SC
formulations. They are almost similar to WP except that, instead of being dusty, they are
formulated as small, easily measurable granules. They gradually become popular because of:
• their convenience in packaging and user safety
• being free from dust
• free flowing nature
• quick and uniform dispersion upon addition into water in the spray tank.
These advantages of WG are providing it technological edge over WP. They can be easily
packed in suitable packaging material without causing much of the contamination and pack
disposal problems as associated with the conventional formulations. The time taken for
dispersion in the water is of prime importance and need proper assurance in this regard. It is
essential for all the granules in WG to disperse uniformly and completely within 2 minutes at
varying degrees of water temperature and hardness. This may be attained by careful optimization
of the formulation adjuvents and process parameters8. Wetting agent and dispersing agent are
added in the WG formulations in a similar manner as added in WP or SC. Typical composition
of water dispersible granule is as follows:
Table 4: Typical composition of Water Dispersible Granule (WG) formulation7

Component Percent by weight


a.i. 50-90
Wetting agent 1-5
Dispersing agent 5-20
Disintegrating agent 0-15
Soluble or insoluble filler Upto 100
The technologies used for the preparation of water WG are somewhat complex as they can be
formulated using several processing techniques, but in every situation the final product must re-
disperse quickly and uniformly in the spray tank in order to give uniform particle size
distribution comparable to that of original powder or suspension from which it is prepared. The
most important processes for producing water dispersible granules are as follows9:
• Pan granulation: this is performed by spraying a liquid, usually water, onto the tumbling
powder to cause agglomeration by the “snow-ball effect”. Being dusty it becomes less
and less popular nowdays.
• High speed mixing agglomeration

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• Compaction – in this process a powder with an included binder is compressed into a
small sphere, disc or obloid.
• Extrusion granulation: resulting in the production of cylindrical pellets and among the
safest, most versatile and economical process and is probably the most favoured process
used by agrochemical companies at the present time.
• Fluid bed spray granulation: it involves the filtration and containment of large volumes
of hot air which makes the plants tend to be rather large and expensive
• Dry compaction: gives a hard product with very poor dispersion properties in water
unless effervescing agents are added to the formulation.
• Spray drying

Multiple factors, like the physico-chemical characteristics of the a.i. and adjuvants, need careful
consideration when deciding the process to be used. These factors and the processing techniques
used to make WG regulate the fate of properties of the finished product in terms of granule
shape, size, degree of dustiness and ease of dispersion into water.

Controlled Release Formulations (CRF)


Controlled release formulations (CRF) are getting more and more popular because of their ability
to minimize mammalian toxicity and extend the time interval of activity, through utilizing
targeted delivery and customized release rates. These formulations can ably reduce the losses
through evaporation, leaching, degradation which ultimately reduces the dose of pesticide for
optimum pest management for longer durations and thereby reducing phytotoxicity and load of
pesticides in the environment. CRFs can be grouped into four main systems:
1) polymer membrane - pesticide reservoir systems
2) matrix systems containing physically trapped pesticides
3) polymer systems containing covalently bound pesticides
4) coated pesticide granule systems.
In polymer membrane - pesticide reservoir systems the rate of diffusion govern the release rates
of a.i. of pesticide. These matrix systems may be further sub-classified into inert or erodible
categories. Diffusion is the key factor which govern the release rates of pesticide a.i. in an inert
matrix system while rate of degradation of the matrix is responsible for the controlled release of
pesticide from an erodible matrix system. The release rates of systems where pesticide is
covalently bound to a polymer is governed by on the rate of cleavage of the specific chemical
linkage which joins the pesticide to the substrate. The release rates of pesticide from a polymer
coated pesticide granule is governed by several factors like the degradation rates of the coating
material and the rate of water permeability through the coating.

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Figure 6: Active pesticide concentrations in conventional and controlled release formulations10

Novel gel technology


Gel formulations can be broadly classified as emulsifiable gels which need to be diluted with
water as emulsion before the intended use and ready to use gels which are directly applied on the
intended surface without dilution.
Emulsifiable gels
Packaging of water insoluble products as gels has become an attractive alternative for
minimizing the packaging waste in selected formulations. Emulsifiable gel formulations are
advanced products, which can be designated as thickened ECs packaged in water soluble sachets
or boxes. Thickeners are used to enhance the viscosity. Viscosity of gel in the finished product is
a compromise between the transport stability in the water soluble sachet or box and its
dispersibility upon dissolution in water. This formulation approach is to resist leakage from the
pinhole imperfections of the water-soluble bags. This formulation provides the agrochemical
market a new form of a product in combination with packaging. The first fungicide formulated as
a gel is propiconazole launched as PRACTIS® in France in 1991. Gel products offer several
advantages that are highly appreciated by users. The pre-measured doses in water soluble bags
offer advantages in ease of handling and enhanced user safety while the outer packaging is
sometimes considered as non-contaminated with product and, therefore, more easily disposed of.
In this method, non-aqueous liquid formulations are gelled and then are suitable for packaging
within polyvinyl alcohol film as water-soluble sachets or bags. Gelation of liquid formulations
can be brought about by a variety of thickening agents like polyacrylic acid, natural gums, silica,
clays, surfactants and combinations thereof. Syngenta found that gelation could be brought on by
mixing a liquid EC formulation with high surface-area silica in combination with an ethoxylated
non-ionic surfactant. They found that gelation occurs at ambient temperature, gel rheology is
highly controllable and the process is versatile. Upon addition of water to such soluble bags
containing pesticidal gels, the soluble bag should release its contents within 1 min, the gel
disperses homogeneously and the sachet film dissolves completely within 3 min. Water-based
gels that have stable formulations of hydrolytically unstable sulfonylurea herbicides have also
been developed. Gels containing sulfonylurea and co-pesticides add a new dimension to delivery
of compounds.

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Ready to use gels
The ready to use gel formulations generally used in two ways; as insecticidal baits and as
repellents on skin. Insecticidal gal baits are used mainly by the household pest control industry
for controlling household pests like termites and ants. They are packaged as ready to use
formulations and comprised of baits which attract the pest. Gel baits have numerous advantages
and are specifically effective against ants and cockroaches. Ants and cockroaches typically
accept and transfer gel baits into their colonies. Gel formulations to be applied on skin usually
contain; a.i. emulsified or dispersed in water and a thickening or gelling agent to generate the
desired viscosity. Other ingredients that may be included are emollients, preservatives, fragrance
and colouring agent.
Microencapsulations
Encapsulation is the entrapment of tiny particles or droplets of an active ingredient inside an
inert coating material to obtain small capsules with varying diameter. Based on the size of
capsules, encapsulated formulations can be classified as:
• Nanoencapsulation 30-100 nm
• Microencapsulation 0.1-1000 µm
• Macroencapsulation >1 mm
Encapsulated pesticides are mixed with water and applied in similar way as other sprayable
formulations. After spraying, the capsule wall ruptures slowly and releases the a.i. at optimum
dose for extended time interval. Microencapsulated formulations have many benefits over
conventional formulations:
• Reduced odour and less skin hazards
• Ease of application as a result of increased safety to applicators towards mixing and
application
• prolonged effectiveness as a result of slow release of the active ingredient enabling lesser
applications
• slowing down the volatilizations and other losses of pesticides resulting in reduced loss of
pesticides from the site of application 11
The price of microencapsulation may differ significantly and is basically dependent upon the
technique employed for preparation. Few techniques need specialized equipment, whereas others
do not. Some techniques using costly chemicals in the process, whereas others use very cheap
ingredients. Processes where high temperature is involved are in general costlier than those
which do not. Elimination of the continuous phase to produce a “dry” product will need an
additional processing step further increasing the cost. Some products, particularly those of high
value or low volume, are better able to absorb such higher cost. In order to economically
encapsulate high volume products or those which produce a low profit margin it is necessary to
employ one of the cheaper techniques if this is possible for the application in question.
Seed coating formulations
Seed treatment formulations consist of pesticide formulations which are used to treat seed with
chemical or biological agents prior to planting to provide protection to seeds against different
pests12. Fungicides predominate the seed treatment formulations with approximately 70% share.
Seed coating helps conserves the health of seeds during storage, transport and germination. This
enables farmers to obtain higher yields through the efficient use of a.i. Seed coating formulations

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are a modification of SC formulations with additional additives for adhesion to the seed surface
and dye as an indication of seed treatment with toxic fungicides. On the basis of their physical
characteristics seed treatment formulations generally classified into 4 categories:
• Powder dry seed treatment (DS)
• Water slurryable powder for seed treatment (WS)
• Non-aqueous solution for seed treatment (LS)
• Flowable suspension for seed treatment (FS)
Tablets
Tablet formulations are almost identical to WG formulations. They are small, easy to handle,
easy to measure formulations. Upon coming in contact with water, the tablets break apart into
fine particles in a similar manner as WP formulations. The percentage of a.i. in the product can
be as high as 95% by weight. The formulations are produced by compaction of homogenous
mixture of ingredients in a tablet press.
Typical formulation composition comprises of tablet:
• active ingredient
• binder
• disintegrant
• dispersant
• anti-caking agent
Tablets share similar advantages and disadvantages as of WG formulations.
Conclusion
Due to immense pressures on product performance, pesticide formulation is gaining more and
more importance day by day. It has become a key technology through which agrochemical
companies can improve their products by imparting substantial value addition. Introduction of
novel products is a key driving factor in product refreshment and new formulation technology
can impact this greatly. This article has described recent advancements in pesticide formulation
technology and the further trends that are driving formulation technologies such as water-based
dispersion formulation technology for EW, ME and SE as well as other formulation types such
as emulsifiable gels and dry product formulations such as WG. New formulation techniques
usually utilize polymers and surfactants in novel ways which results in significant formulation
improvements.
References
1. Next Generation Indian Agriculture - Role of Crop Protection Solutions (2016) A report
on Indian Agrochemical Industry. Federation of Indian Chambers of Commerce &
Industry (FICCI).
2. Valkenburg W van (Ed.) (1973). Pesticide formulations. New York: Marcel Dekker.
3. Tadros TF (1995). Surfactants in agrochemicals, surfactant science series 54 New York:
Marcel Dekker.
4. Izquierdo P, Feng J, Esquena J, Tadros TF, Dederen JC and Garcia MJ (2005) The
influence of surfactant mixing ratio on nanoemulsion formation and stability. Journal of
Colloid and Interface Science 285:388-94.

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and human health management", 07-27 Dec, 2017
5. McClements DJ (2012) Nanoemulsions versus microemulsions: terminology, differences,
and similarities. Soft Matter 8: 1719-1729.
6. Hiromoto B (2007) Pesticide microemulsions and dispersant/ penetrant formulations.
United States Patent, Patent No: US 7297351.
7. Knowles A (2007)Recent developments of safer formulations of agrochemicals.
Environmentalist 28:35–44.
8. Bell G (1990) The structure/physical property relationships of a model water dispersible
granule. Journal of Pesticide Science 29: 467–473.
9. Capes CE. (1980). Particle size enlargement 1. Amsterdam: Elsevier.
10. Roy A, Singh SK, Bajpai J and Bajpai AK (2014) Controlled pesticide release from
biodegradable polymers. Cent Eur J Chem 12(4): 453-469.
11. Fernández-Pérez M (2007) Controlled release systems to prevent the agro- environmental
pollution derived from pesticide use. Journal of Environmental Science and Health B
42:857-62.
12. Hazra DK. (2015) Recent Advancement in Pesticide Formulations for User and
Environment Friendly Pest Management. International Journal of Research & Review
2(2): 35-40.

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Controlled Release Formulations for Pesticide Delivery

Jitendra Kumar1 and Anirban Dutta2

1
Institute of Pesticide Formulation Technology, Gurugram – 122016, Haryana
2
Division of Agricultural Chemicals, ICAR-Indian Agricultural Research Institute, New Delhi –
110012

The world’s scientific communities today face a major challenge in terms of continuous growth
of population and ever-increasing demand for food. The need for more efficient operations in
agricultural production is thus paramount. Methods and processes that afford higher yields and
better quality, require less time and money, and do not pose a threat to the environment are being
sought at an unparalleled pace. Unquestionably a crucial task to meet the goal of ensuring food
security is to efficiently control crop pests. The world’s crop production losses billions of dollars
every year due to inefficient pest control. Though worldwide the large-scale annual average
usage of pesticides presents a contrasting picture. From the beginning of agriculture, use of
pesticides is an integral part for better crop pest management. However, inefficient pest control
strategies made the pesticides one of the major culprits for environmental pollution.
Development of issues like resistance and resurgence, compel the farmers to use more and more
pesticides with repeated applications, which in turn create problems of residue in the food and
environment. One of the way out to combat the problem of emerging pests is to develop new
pesticidal molecules, but this is a very costly affair both in terms of monetary and human
resources. However, use of these novel agents to produce the desired biological response is often
inefficient, primarily because of inabilities to deliver the agents to their targets at the precise time
and in the optimum quantities required. Therefore, the alternative approach i.e. to develop
improved and efficient delivery systems of both older and newer pesticidal molecules is the need
of hour. In this context, the controlled release formulation (CRF) attracts a major focus from the
scientific communities.
Terminologically, a controlled release formulation or delivery system can be defined as a
combination of biologically active agent and other excipients (usually a polymer) arranged to
allow delivery of the agent to the target at controlled rates over a specified period1. For the past
four decades CRFs have taken on added significance in the pesticide industries because of the
realization that this type of formulations can minimize the impact of pesticides on the
environment as well as can reduce the toxicity to the end-users2. Additionally, loss of active
ingredient (a.i.) after application of conventional formulations due to leaching, volatility,
adsorption to clay and organic matter, chemical and microbial degradation can be reduced by use
of CRFs. This further reduces the need of application of more active agents. Since it has been
realized that CRFs can have the ability to improve pesticide selectivity, certain a.i. which would
have been dropped for commercial consideration because of phytotoxicity issues can be further
taken up to the developmental stage. Due to the unique formulation technology, CRFs can also
diminish the physical incompatibility of pesticide mixtures and thus can reduce their biological
antagonism during field application. Owing to its ability to mitigate the undesirable
environmental effects encountered during pesticide applications by conventional methods and to
maintain an effective level of a.i. for a longer period without any undesirable side-effects CRFs

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have been addressed by researchers all over the world as the ultimate technology to combat pest
problems.
Types of controlled release formulations
The simplest way to slow release of an a.i. is to increase the size of a particle, thereby reducing
the specific surface per unit mass and increasing surface per particle. Release of a.i. is slower
from a granule as compared to a powder prepared using same carrier and other excipients. The
advancement in CRF technology recommends the use of customized polymers for developing
such formulations with controlled release properties. Active ingredient can be trapped, coated,
dispersed, dissolved or bound in/on a polymer matrix to get desired products. Depending on the
a.i.-polymer interaction, CRFs can be classified into four major types:
Polymer membrane reservoir
In this case, a.i. is either present in a liquid core surrounded by polymer wall (microcapsule) or
sandwiched as a central reservoir layer between two protective layers of polymer (microstrip)
and the release of a.i. is controlled by Fickian diffusion through the micropores of the polymer
layer.
Matrix containing physically trapped active ingredient
The a.i. is dispersed heterogeneously or dissolved in a polymeric matrix which controls the
release of a.i. through diffusion and/or erosion.
Covalently bound active ingredient
An a.i. is covalently bound to a polymer which on hydrolysis, thermodynamic dissociation,
microbial degradation or some other retrograde chemical reaction releases the a.i.
Coated active ingredient granule system
In this case, the a.i. remains impregnated into clay or any other inert materials which is further
coated with a polymer film that controls the release of it.
Mechanism of controlled release
Controlled release is not synonymous with sustained release, a much older and well recognized
concept from which the new science is emerging, although the two are similar in principle and
sometimes overlap (Fig 1). Sustained release formulations are so called because they contain
several times the normal single application, and they provide for replacement of the agent at
some rate which gives a measurable increase in the duration of activity. The rate may decrease
due to gradual loss of agent, or increase through a maximum due to breakdown of a protective
barrier. A controlled release formulation, in contrast, may exhibit a fast or a slow release, or a
constant or a changing release, depending on the design. The principal difference lies not entirely
in the profiles of release but in the mechanism of release. The distinction is drawn mainly by the
degree of control of both the optimum level and the optimum time of availability of the
biologically active agent.

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Fig 1. Concentration vs time curve for different release pattern

The concept and practice of controlled release encompasses many mechanisms. Although
the earliest formulations were based on the desorption of pesticides from strong sorbents like
silica gel, mica, and activated charcoal, most of the current systems are based on more
controllable mechanisms such as diffusion through rate-controlling media, erosion of
biodegradable barrier materials, and retrograde chemical reactions. Designers of controlled
release formulations or devices usually strive for zero-order (constant) rates of release, but
systems with time dependent release kinetics are proving to be useful for pesticides, especially
when the rate and duration of release are predictable and well controlled. In practice, the rate of
pesticide release may be controlled by several sequential or simultaneous mechanisms which do
not lend themselves to simple analysis, but it is usually possible to determine experimentally an
overall order of release for these complex systems.
Release by membrane-moderated diffusion
Diffusion-controlled membrane devices can be divided into two main categories: reservoir
systems in which the pesticide is totally encapsulated within a rate-controlling membrane, and
monolithic systems in which the pesticide is dispersed or dissolved in a rate-controlling matrix
(Fig 2). It has been demonstrated that the diffusion rates from controlled-release systems follow
Fick's law of diffusion, which states that the rate of diffusion depends on five factors. Two of the
factors involve the geometry or dimensions of the device (surface area and thickness of the
membrane), and three involve pesticide-polymer interactions (diffusion coefficient of the
pesticide in the polymer, saturation solubility of the pesticide in the polymer, and partition
coefficient of the pesticide between the polymer and the medium which surrounds the device).

Fig 2. Reservoir and monolithic diffusion controlled devices


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When applied to reservoir systems, Fick's law predicts that if a pesticide is enclosed
within an inert membrane, and if the concentration is maintained constant within the enclosure,
then a steady state will be established during which the release rate will be zero order, i.e.
constant. On the other hand, membrane-moderated monolithic systems in which the pesticide is
dispersed or dissolved in a rate-controlling polymer matrix are simple to prepare, but they do not
have the zero-order release kinetics of the reservoir systems. The pesticide is released from the
surface layers of a monolithic device first, and the distance the pesticide must diffuse to reach the
surface increases with time. Thus, these systems have slowly declining rates of release.
Release by erosion
When the polymer containing dispersed or dissolved pesticide is water soluble, hydrolytically
unstable, or otherwise biodegradable, the a.i. can be released by diffusion of the pesticide from
the polymer, by erosion of the polymer, or by a combination of both diffusion and erosion.
Release by erosion is a surface area dependent phenomenon, and if the surface area of a device
stays constant while it is eroding, the release of pesticide will be of zero order.
For the flat film or slab geometry, the surface area does not change as erosion occurs, and
consequently, the release of agent is constant until the film ultimately disappears. If the pesticide
is contained as a single reservoir within a spherical bioerodible membrane, as in some
microcapsules, the mechanism of release can be the erosion and rupture of the barrier membrane.
Various delivery patterns, including essentially constant release, can be achieved by blending
microcapsules of appropriate wall thicknesses.
Release by retrograde chemical reaction
As opposed to physical combinations of pesticides dissolved or entrapped in polymers, chemical
combinations have the pesticide firmly attached to the polymeric substrate by a definite
identifiable chemical bond. The active material is released when environmental reactants such as
water, air, sunlight, or microorganisms act to cleave the specific chemical linkages which attach
the pesticide to the substrate. The rate of release of pesticides via retrograde reactions depends on
the properties of the macromolecule and its surrounding medium. When water present in the
environment is used to activate the release of pesticide, the rate of hydrolysis depends on the
strength and chemical nature of the polymer pesticide bond.
Applications
For the past four decades research on CRFs has been reached to a new height. A number of
literatures are available on CRFs for pesticide formulations. With the progress of material
science, polymer chemistry and nanotechnology several new aspects of CRF technology have
been emerged. The earlier concept for developing CRFs was based on simple polymeric
formulations, but in recent times CRFs based on polymer composites, hydrogels, amphiphilic
polymers etc. have been conceptualized.
The primary focus to develop CRFs is to either encapsulate or adsorb pesticide a.i. in/on
some polymeric matrix, so that it can be retained by the matrix for a longer period of time and
slowly released to the environment as per requirement. Earlier attempts were made to develop
such kind of formulations by simply adsorbing the a.i. on clay particles. The idea was to develop

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such formulations by exploiting the property of adsorption of pesticides by clays and organo-
clays to reduce the leaching potential, degradation and volatilization of pesticides3. Clay minerals
being negatively charged, have strong affinity for cationic pesticides. Organo-clays were mainly
designed to promote the adsorption of neutral and hydrophobic pesticides and slow their release.
The adsorption of organic cations on clays modifies the nature of the clay mineral surface,
transforming it from hydrophilic to hydrophobic. The modified clay mineral surface can enhance
affinity for sorbing neutral organic molecules of hydrophobic characteristics. Montmorillonite or
bentonite clay was mostly modified for this purpose with quaternary ammonium cations to get
better adsorption of herbicides. Optimal organo-clay formulations yielded slow release in water,
for example, a 1% (w/w) suspension of a montmorillonite–phenyltrimethyl ammonium (PTMA)
formulation of acetochlor was found to release only 2% of the herbicide after two days4.
With the advancements of polymer science, polymer composites play a major role in
CRFs where pesticides were trapped in polymer matrix and filler materials like clays were used
to create barriers for further controlling the release of a.i. Both kaolinite5 and bentonite6 clays
were used as filler materials for developing polymeric CRFs. It was also found that modified
clays obtained by acid activation, pillared with metal hydroxides, or saturated with organic
cations influenced the release of pesticides from polymer matrix to a greater extent7. With due
course of time, nanoparticles have been introduced as filler materials for such composites.
Halloysite nanotubes, carbon nanotubes, nanoparticles etc. are now experimrntally being used to
get better controlled release properties of pesicides from polymer matrices8,9.
One of the basic methods to prepare CRFs of pesticides is via synthesis of polymer with
pesticides as pendent substituents. The earlier ways to prepare such CRFs is either by reacting
pesticide with polymerizable monomer to give macromolecular combination or by chemical
modification of preformed synthetic or natural polymers with pesticide or their derivatives 10.
More emphasis has been given recently on micro-/nano-encapsulation of pesticide a.i. in polymer
matrices. The simplest method to prepare such CRFs is just to embed pesticide a.i. in natural or
synthetic polymer matrices. A number such formulations have been reported in literature11,12.
Modern day science has shown a number of ways to develop CRFs with desired characteristics
to encapsulate almost all pesticides and control their release rate as per the requirements.
Insecticide formulations with polyethylene glycol (PEG) based amphiphilic copolymers were
studied in a series of experiments13. Release of the a.i. in water was found to be significantly
slower than from commercial formulations for imidacloprid, thiamethoxam, carbofuran, thiram
and β-cyfluthrin14-18. The release rates increased with increasing PEG molecular weight,
potentially allowing the availability to be tuned to the optimum period. Now-a-days the focus has
been shifted to develop CRFs from which the release of a.i. can be controlled by environmental
factors like pH, temperature, moisture, UV light etc. Few such triggered-release CRFs have been
reported recently in literatures where release of chlorpyrifos and thiamethoxam can be controlled
depending on pH of the surrounding environment19,20.
Conclusion
Controlled release formulations are the future of pesticide delivery systems. With the growing
concerns about pesticide contamination, residue problems and resistance development of
conventional pesticides, this is high time to think seriously about formulation technology. Instead
of developing new molecules, existing molecules if used in a scientific way can solve many of

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the emerging problems. CRFs not only can control pest efficiently, but also can reduce the
pesticide load on/in the environment. Efficient pest control strategy lies in the way the pesticides
are applied and if they are applied in a green and safer way, food security can be ensured for
millions of people in this world.
References
1. Lewis DH and Cowsar DR (1977) Principles of controlled release pesticides. In:
Controlled Release Pesticides, H Scher (Ed), ACS Symposium Series, American
Chemical Society, Washington DC, pp. 1-16.
2. Kydonieus AF (1980) Fundamental concepts of controlled release. In: Controlled Release
Technologies: Methods, Theory and Applications, AF Kydonieus (Ed), CRF Press, Boca
Raton, pp. 1-20.
3. Nir S, El-Nahhal Y, Undabeytia T, Rytwo G, Polubesova T, Mishael Y, Rabinovitz U
and Rubin B (2006) Clays and pesticides. In: Handbook of Clay Science, Volume I, F
Bergaya, BKG Theng and G Lagaly (Eds), First Edition, Elsevier, Amsterdam, pp. 677-
691.
4. El-Nahhal Y, Nir S, Serban C, Rabinovitz O and Rubin B (2001) Organo-clay
formulation of acetochlor for reduced movement in soil. J Agric Food Chem, 49, 5364–
5371.
5. Kumar J, Nisar K, Shakil NA, Walia S and Prasad R (2010) Controlled release
formulations of metribuzin: Release kinetics in water and soil. J Environ Sci Health, Part
B, 45(4), 330-335.
6. Sahoo S, Manjaiah KM, Datta SC, Shabeer TPA and Kumar J (2014) Kinetics of
metribuzin release from bentonite-polymer composites in water. J Environ Sci Health,
Part B, 49(8), 591-600.
7. Li J, Li Y and Dong H (2008) Controlled release of herbicide acetochlor from
clay/carboxylmethylcellulose gel formulations. J Agric Food Chem, 56, 1336–1342.
8. Zhong B, Wang S, Dong H, Luo Y, Jia Z, Zhou X, Chen M, Xie D and Jia D (2017)
Halloysite tubes as nanocontainers for herbicide and its controlled release in
biodegradable poly(vinyl alcohol)/starch film. J Agric Food Chem, 65(48), 10445-10451.
9. Bibi S, Nawaz M, Yasin T and Riaz M (2016) Chitosan/CNTs nanocomposite as green
carrier material for pesticides controlled release. J Polym Res, 23,154.
10. Dubey S, Jhelum V and Patanjali PK (2011) Controlled release agrochemicals
formulations: a review. J Sci Ind Res, 70, 105-112.
11. Fernandez-Urrusuno R, Gines JM and Morillo E (2000) Development of controlled
release formulations of alachlor in ethylcellulose. J Microencapsul, 17(3), 331-342.
12. Dowler CC, Dailey OD and Mullinix BG (1999) Polymeric microcapsules of alachlor
and metolachlor:  preparation and evaluation of controlled-release properties. J Agric
Food Chem, 47(7), 2908-2913.
13. Shakil NA, Singh MK, Pandey A, Kumar J, Pankaj, Parmar VS, Pandey RP and
Watterson AC (2010) Development of poly(ethylene glycol) based amphiphilic
copolymers for controlled release delivery of carbofuran. J Macromol Sci, Part A, 47,
241–247.

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14. Adak T, Kumar J, Shakil NA and Walia S (2012) Development of controlled release
formulations of imidacloprid employing novel nano-ranged amphiphilic polymers. J
Environ Sci Health, Part B, 47(3), 217–225.
15. Sarkar DJ, Kumar J, Shakil NA and Walia S (2012) Release kinetics of controlled release
formulations of thiamethoxam employing nanoranged amphiphilic PEG and diacid based
block polymers in soil. J Environ Sci Health, Part A, 47(11), 1701–1712.
16. Pankaj, Shakil NA, Kumar J, Singh MK and Singh K (2012) Bioefficacy evaluation of
controlled release formulations based on amphiphilic nanopolymer of carbofuran against
Meloidogyne incognita infecting tomato. J Environ Sci Health, Part B, 47(6), 520–528.
17. Kaushik P, Shakil NA, Kumar J, Singh MK and Yadav SK (2013) Development of
controlled release formulations of thiram employing amphiphilic polymers and their
bioefficacy evaluation in seed quality enhancement studies. J Environ Sci Health, Part B,
48(8), 677–685.
18. Loha KM, Shakil NA, Kumar J, Singh MK, Adak T and Jain S (2011) Release kinetics of
beta-cyfluthrin from its encapsulated formulations in water. J Environ Sci Health, Part B,
46(3), 201–206.
19. Xiang Y, Zhang G, Chen C, Liu B, Cai D and Wu Z (2017) Fabrication of a pH-
responsively controlled-release pesticide using an attapulgite-based hydrogel. ACS
Sustainable Chem Eng (Published online).
20. Sarkar DJ and Singh A (2017) Base triggered release of insecticide from bentonite
reinforced citric acid crosslinked carboxymethyl cellulose hydrogel composites.
Carbohydr Polym, 156, 303-311.

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Emulgel: A New Dimension to Gels as Carrier
Ashish Khandelwal
Division of Agricultural Chemicals
ICAR-Indian Agricultural Research Institute, New Delhi 110 012

Emulgels are primarily emulsions (oil-in-water or water-in-oil), which are gelled by mixing with
a gelling agent1. Emulsion itself is a controlled release system where entrapped drug particles in
internal phase pass through the external phase to the skin and slowly get absorbed. Internal
phases act as reservoir of drug and slowly release drug in a controlled way through the external
phase to the skin (Fig. 1). Gel forms cross-linked network where it captures small drug particles
and provides its release in a controlled manner (Fig. 2). Topical application of drugs can be
performed in various ways (Fig. 3). Out of various method of application, emulgel is novel
concept for delivery of drugs on to the skin. Due to its mucoadhesive property it prolongs the
contact period of medication over the skin. Since Emulgel possesses the property of both
emulsions and gel it acts as dual control release system2,3. Oil-in-water emulsions are most useful
as water washable drug bases and for general cosmetic purposes, while water-in-oil emulsions
are employed more widely for the treatment of dry skin and emollient applications. It is accepted
that utility of any topical preparation lies on its penetration ability and refers to the disappearance
of product or oiliness from skin. The processes of penetration into skin are simplified, if
emulsion is thixotropic, i.e. if it becomes less viscous during shearing. Thus, to improve
emulsion stability and penetration ability it is incorporated into gel4,5.

Fig 1: Schematic presentation of Emulgel penetration through skin.

Emulgels have proven as most convenient, better and effective delivery system. Due to its non-
greasy, gel like property lacks of oily bases, it provides better release of drugs as compared to
other topical drug delivery system. Incorporation of emulsion into gel makes it a dual control
release system further problem such as phase separation, creaming associated with emulsion gets
resolved and its stability improves. Emulgel loaded with specific drugs has been found effective
in some topical disorders & it is emerging as potential drug delivery system in area of
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dermatology. Further, gels for dermatological use have several favorable properties such as being
thixotropic, greaseless, easily spreadable, easily removable, emollient, nonstaining, compatible
with several excipients, and water-soluble or miscible. The type and concentration of the
polymer that form the gel matrix could influence the stability as well as the release rate of the
incorporated drug. Some inherent limitations of Emulgels that remain are poor absorption of
macroparticle via skin and entrapment of bubble during formulation. Apart from this, Emulgels
possess the previously mentioned advantages of both emulsions and gels, they have good patient
acceptability. Due to its non-greasy nature it can be conveniently applied to the skin as compared
to other topical formulations such as creams, ointments which are very much thick, greasy and
require excess rubbing. Gel–sols–gel behavior imparts stability as well as improves
bioavailability of system. However, stability of system can be affected by many factors like pH,
temperature, polymer concentrations, polymer modification or combinations, addition of cations
or anions 6-8.

Fig 2: Controlled release of drug particle through gel cross linked network
Scientists nowadays are facing problem during development of new drug since many drugs
coming directly from synthesis or from high throughput screening have poor solubility. Based on
in vitro solubility and in vivo permeability data biopharmaceutical classification system divides
drugs into four classes. Among the four classes, class II drugs show poor solubility and high
permeability. It is obvious that for class II drugs the low ability to dissolve is a more important
limitation to their overall rate and extent of absorption then their ability to permeate through the
membrane. Therefore, emulgels may serve as better option for topical delivery of poorly water-
soluble drug9-10.
Emulsified gel has proven a stable one and better vehicle for hydrophobic or poorly water-
soluble drugs. Direct (oil-in-water) systems entrap lipophilic drugs, where the incorporated
hydrophobic drug gets captured in the oil phase and slowly releases to the skin through the
external phase. Therefore, they have been recently used as vehicles to deliver various
hydrophobic drugs (ketoconazole, acyclovir, diclofenac and calcipotriol) to the skin thereby
broadening the spectrum of delivery system11, 12.

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Fig 3: Various approaches used for topical drug delivery

Present Scenario: Emulgels now have been used for treatment of various kinds of skin disorder
such as those infected by viral, bacterial, and fungal species (eczema, Herpes simplex, acne). At
present, emulgel based formulation are mainly using in the pharmacology field. In recent years,
application of emulgel is increased due to its various advantages13. Their application in
agriculture sector is yet to be explored. Some of the marketed emulgel products mainly used for
human health, indicated in Table 1.
Table 1: Marketed Emulgel product

Product Drug Manufacturer


Voltaren emulgel Diclofenac diethyl ammonium Novartis Pharma
Miconaz-H-emulgel Miconazole nitrate, Medical union Pharmaceuticals
Hydrocortisone
Excex gel Clindamycin, Adapalene Zee laboratories
Pernox gel Benzoyl peroxide Cosme Remedies Ltd
Lupigyl gel Metronidazole Lupin Pharma
Clinagel Clindamycin phosphate Stiefel Pharma
Allantoin
Topinate gel Clobetasol propionate Systopic Pharma
Kojivit gel Kojic acid, Dipalmitate Micro Gratia Pharma
Arbutin, Octinoxate
Acent gel Aceclofenac, Methyl Intra labs India Pvt Ltd
salisylate, Capsaicin
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Avindo gel Azithromycin Cosme Pharma laboratories
Nadicin cream Nadifloxacin Psychoremedies
Zorotene gel Tezarotene Elder Pharmaceuticals
Cloben gel Clotrimazole, Indoco Remedies
Beclomethasone,
Dipropionate, Neomycin
General method of synthesis: Aqueous phase of emulsion is prepared by dissolving
surfactant/penetration enhancer/formulants (Table 2) based on requirement in purified water;
Oily phase of emulsion is prepared by dissolving surfactant in oil. Then both the phases are
mixed at constant/different temperature with constant stirring until cooled to room temperature.
Gel phase of Emulgel is prepared by dispersing gelling agent in water. When both the
components both emulsions and gel get ready then the Emulgel is prepared by mixing emulsion
with gel in suitable proportion with gentle stirring13.

Fig 4:Flow chart for emulgel synthesis

Table 2: Formulants used in emulgel

Formulants Ingredient Quantity


Aqueous material Water, alcohol 40-90%
Oil Light liquid paraffin, isopropyl myristate, isopropyl stearate, 1-30%
Isopropyl palmitate, Propylene glycol, castor oil, Olive oil,
Wheat germ oil, Wool wax, Thyme oil, Balsum oil, Myrrh oil,
Birch oil, Rose hip oil
Gelling agent Carbopol 934, Carbopol 940, HPMC, Sodium CMC, Pluronic® 1-5%
F127,
Penetration Oleic acid, Lecithine, Urea, Linoleic acid, Clove oil, Menthol 1-10%

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enhancer
Emulsifiers PEG 40 stearate, Sorbitan mono-oleate (Span 80), 0.1-10%
Polyoxyethylene sorbitan monooleate (Tween 80), Stearic acid,
sodium stearate
Formulation considerations: The selection of oily phase, emulsifying agent and gelling agent
mainly depends on desired property of the product. Therefore, it is necessary to keep various
concepts, while selecting different solvents12.

A. Selection of oil phase: The use of oil phase in emulsion primarily depends on the ultimate
use of product. The consistency of these lipids may range from mobile liquids to high solids. The
selection of oil phase also affects other parameters such as viscosity, permeability, and stability
in emulgel. Few Oils or extract of plant material (Geranium and jojoba oil) also have medicinal,
insecticidal and anti-bacterial properties and Some of medicinal oils also have synergistic
combination with drug molecules such as olive oil, castor oil, birch oil can also be used in
suitable proportion. Paraffin wax, a crystallizable oil phase, used as phase change material
(PCM), paraffin wax has several advantages including its relatively large latent heat, low vapor
pressure and chemical inertness. When paraffin is dispersed in aqueous medium the
paraffin/water emulsion is formed which is more suitable for many applications, due to its
greatly accelerated heat-exchange and negligible thermal resistance. Pseudoternary phase
diagrams can be constructed to get desire release and permeability through skin by optimizing
different concentration of oil, surfactant, co-surfactant, and water for micro-emulsion.

B. Selection of emulsifying agent: Emulsions are thermodynamically unstable systems. The


stability of this system can be significantly increased by using appropriate emulsifying agents
(desired HLB value) which results decrease interfacial tension between two different solvents
(Fig 5). Nonionic surfactants such as spans, tweens have HLB values greater than 8 and are used
in the formulation of o/w emulsions whereas mineral oils such as liquid paraffin have HLB
values less than 8 & therefore are employed in the formulation of water in oil emulsions.

Fig 5. Enhancement of stabilization of dispersed globules with emulsifying agent.

Emulgel was developed using tween 20 as emulsifier in its aqueous phase & span 20 in its oily
phase. Both surfactants are sorbitan lauric acid esters with the same cyclic structure. However,

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Tween 20 contains additional polyoxyethylene units. Tween surfactants are polysorbate
molecules each attached to a hydrophilic head group of oligo (ethyleneglycol) (OEG) chains and
a hydrophobic tail of fatty acid ester moiety. Span 20/Tween 20 mixtures contribute towards
greater stability of the emulsions as compared with pure Tween or Span systems. Polymeric
emulsifier pemulen are hydrophobically-modified copolymers of acrylic acid (Acrylates/C10–
C30 alkyl acrylates) that could act both, as primary emulsifiers for o/w emulsions and viscosity
enhancing agents. They are able to stabilize o/w emulsions because their short lipophilic part
integrates into the oil droplets while their long hydrophilic part forms a micro gel around the
droplet. Oil-in-water (o/w) emulsion was prepared by incorporating the oily phase (liquid
Vaseline, span 60, and erythromycin ethyl succinate) to the aqueous phase (Tween 60, water, and
kanamycin sulphate) at 70 °C. Surfactants generally employed, are toxic in nature. Therefore,
bio-surfactants may serve as alternative. Because of their short fatty acid tail and polar head
groups, bio-surfactants are highly sticky and both hydrophilic and hydrophobic. The features that
make them commercially promising alternatives to chemically synthesized surfactants are their
lower toxicity, higher biodegradability and, hence greater environmental compatibility, better
foaming properties and stable activity and at extremes of pH and temperature. So these may
serve as better option as emulsifier for disperse system (emulsions) and one could effectively
take the advantage of its property.

C. Selection of gelling agent: Incorporation of gelling agent, natural or synthetic, makes product
thixotropic. Thixotropy is the phenomenon of the fluid which shows a reversible structural
transition (i.e., gel–sol–gel conversion) due to the time-dependent changes in the viscosity
induced by temperature, pH or other components without any changes in the volume of the
system (Fig 6). Gel–sols–gel behavior imparts stability as well as improves bioavailability of
system. However, stability of system can be affected by many factors like pH, temperature,
polymer concentrations, polymer modification or combinations, addition of cations or anions.

Fig 6. Thixotropic behavior of gel (gel–sol–gel property).

Carbopol polymers are polymers of acrylic acid cross-linked with polyalkenyl ethers or divinyl
glycol. Carbomers readily absorb water, get hydrated and swell. Besides its hydrophilic nature,
its cross-linked structure and it's insolubility in water makes carbopol a potential candidate for
use in controlled release drug delivery system. There is an inverse correlation between the
concentration of gelling agent and the extent of drug released. The prepared Emulgel showed
Non-Newtonian shear thinning behavior with little or no thixotropy and variable viscosity
dependent on both the concentration and type of gelling agent. Stability testing under several
conditions (centrifugation, temperature cycle test or storage for 1 year) showed that formulation
containing low level of Carbopol or combination of two gelling agents has better stability
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compared to other formulations. Hydroxypropyl methylcellulose can be used as thickening
agent, tablet binding, modified release and film coating. HPMC based Emulgel was found to be
better than Carbopol based Emulgel since it showed better drug release rate. HEC based Emulgel
showed good drug release profiles and good rheological characteristics but low mucoadhesion as
compare to NaCMC which shows high mucoadhesion. The major drawback of natural gelling
agents is that they are prone to microbial degradation. Various kinds of synthetic and
semisynthetic gelling agents are replacing natural gelling agent now these days. Cellulose
derivatives are now been commonly using as gelling agent. One of the popular cellulose
derivatives is sodium carboxymethyl cellulose. Sodium carboxymethyl cellulose is a suitable
vehicle for sterile jellies because it can withstand autoclaving without serious deterioration.

Future Scope: In agriculture, most of newly developing molecules/pesticides are hydrophobic in


nature and their target delivery is very challenging task. This behavior is responsible for poor
water solubility and bioavailability of drugs. For topical delivery of drugs different delivery
systems such as ointments, lotion, creams and pastes are applied. These topical formulations
generally include large number of oleaginous bases such as petrolatum, bees wax or vegetable
oils that themselves are hydrophobic in nature that do not allow the inclusion of water or aqueous
phase. It makes them an excellent emollient but retards the release of drugs and makes the
product thick & greasy. Whereas gel provides aqueous environment to drug, favors its
dissolution and provides quicker release of drug as compared to other topical delivery systems.
Emulsion based gel provides a suitable medium for delivery of such hydrophobic drugs where
such drugs can be incorporated in to its oily phase and delivered to skin. All such advantages of
Emulgel over other topical delivery systems make them more efficient & productive. In future
these properties will be used to deliver more number of topical drugs or contact pesticide in the
form of Emulgel12,13.

References
1. Mohamed MI (2004) Optimization of chlorphenesin emulgel formulation. AAPS J 6(3):
article 26.
2. Alexander A, Ajazuddin, Tripathi DK, Verma ST, Maurya J, Patel S (2011) Mechanism
responsible for mucoadhesion of mucoadhesive drug delivery system: a review. Int J
Appl Biol Pharm Technol 2(1): 434-445.
3. Jain A, Deveda P, Vyas N, Chauhan J (2011) Development of antifungal emulsion based
gel for topical fungal infection. Int J Pharma Res Dev 2: 18–25.
4. Panwar AS, Upadhyay N, Bairagi M, Gujar S, Darwhekar GN, Jain DK (2011) Emulgel:
a review. Asian J Pharm Life Sci 1(3): 333-343.
5. Sarisozen C, Vural I, Levchenko T, Hincal AA, Torchilin VP (2012) PEG-PE-based
micelles co-loaded with paclitaxel and cyclosporine A or loaded with paclitaxel and
targeted by anticancer antibody overcome drug resistance in cancer cells. Drug Deliv 19:
169–176.
6. Kumar NPM, Patel MR, Patel KR, Patel NM (2013) Emulgels: a novel approach to
topical drug delivery. Int J Univ Pharm Bio Sci 2: 134–148.
7. Hu L, Yang J, Liu W, Li L (2011) Preparation and evaluation of ibuprofen-loaded
microemulsion for improvement of oral bioavailability. Drug Deliv 18: 90–95.

111 Winter School on "New generation smart agrochemical oriented approaches for crop
and human health management", 07-27 Dec, 2017
8. Kang SN, Lee E, Lee MK, Lim SJ (2011) Preparation and evaluation of tributyrin
emulsion as a potent anti-cancer agent against melanoma. Drug Deliv 18: 143–149.
9. Shahin M, Hady SA, Hammad M, Mortada N (2011) Novel jojoba oil-based emulsion gel
formulations for clotrimazole delivery. AAPS Pharm Sci Tech 12: 239–247.
10. Giri TK, Choudhary C, Ajazuddin, Alexander A, Badwaik H, Tripathi DK (2013)
Prospects of pharmaceuticals and biopharmaceuticals loaded microparticles prepared by
double emulsion technique for controlled delivery. Saudi Pharm J 21(2): 125-141.
11. Zhang J, Wang C, Wang J, Qu Y, Liu G (2012) In vivo drug release and antibacterial
properties of vancomycin loaded hydroxyapatite/chitosan composite. Drug Deliv 19:
264–269.
12. Ajazuddin, Alexander A, Khichariya A, Gupta S, Patel RJ, Giri TK, Tripathi DK (2013)
Recent expansions in an emergent novel drug delivery 
 technology: Emulgel. J Control
Release 171(2): 122-132.
13. Singla V, Saini S, Joshi B, Rana AC (2012) Emulgel: A New Platform for topical
drug delivery. Int J Pharma and Bio Sciences 3(1): 485-498.

112 Winter School on "New generation smart agrochemical oriented approaches for crop
and human health management", 07-27 Dec, 2017
Synthesis of Hydrogels
Suman Manna and Anupama Singh

Division of Agricultural Chemicals


ICAR-Indian Agricultural Research Institute, New Delhi

Introduction
Hydrogel is a network of polymer chains that are water-insoluble, sometimes found as a colloidal
gel in which water is the dispersion medium. Hydrogels are polyacrylate and it is formed by free
radical polymerization reaction.

Objectives
To synthesize polyacrylate hydrogel from acrylamide by free radical polymerization technique

Materials: a. Acrylamide,
b. Methylenebisacrylamide,
c. Persulphate,
d. Distilled water
e. Methanol
Procedure:
1. 10 ml distilled water taken in beaker.
2. 2g acrylamide and 0.2g methylenebisacrylamide added till dissolution.
3. 0.1g persulphate added with continuous stirring.
4. Mixture kept in oven at 70 °C for 2h till gel point is observed.
5. Remove from oven.
6. Dry with methanol.
7. Kept till constant weight.
8. Determine water absorbency.

Water absorbency: 0.1g hydrogel was immersed in the excess of distilled water and kept till
equilibration. Free water was filtered through a nylon sieve, gel allowed to drain on sieve for 10
min, and finally weighed. The water absorbency (QH2O) was calculated using the following
equation:

QH2O (g/g) =W2−W1/W1

where W1 is the weight of xerogel (dry hydrogel) and W2 is the weight of equilibrated swollen
gel.

Result
The water absorbency of prepared hydrogel is …….. g/g.

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Biopolymer Chemistry in Relation to Formulation
Anupama Singh1 and Neeraj Patanjali2
1
Division of Agricultural Chemicals, 2Division of Soil Science and Agricultural Chemistry,
ICAR-Indian Agricultural Research Institute, New Delhi 110012, India

Biopolymers comprise of a diverse class of materials which are either produced by biological
systems or synthesized from biological source materials. Biopolymer chemistry play substantial
part in formulation and biopolymers especially polysaccharides are extensively used in
formulation technology as functional ingredients in order to control the release rates of active
ingredient (a.i.) and to impart desired characteristics such as emulsification, thickening, gelling
etc. to the finished product. Biopolymers are also extensively used in the synthesis of Hydrogels
(HGs) which found numerous applications in agriculture and allied areas. There is an emphasis
on further research for exhaustive exploration of biopolymer chemistry to produce designed
biopolymers for imparting desired effects to the finished formulation product.
Introduction
The term 'biopolymers' represents diverse and highly versatile class of macromolecules that are
directly or indirectly related to living systems. However, biopolymers primarily falls into two
categories: 1) polymers that are produced by biological systems such as microorganisms, plants,
and animals; and 2) polymers that are synthesized chemically but are derived from biological
starting materials such as amino acids, sugars, natural fats, or oils1. Cellulose, starch, chitin,
proteins, peptides, DNA, and RNA are all examples of biopolymers in which the monomeric
units, respectively, are sugars, amino acids, and nucleotides. Synthetic polymers are non-
biodegradable and their synthesis too involves the use of toxic compounds or generation of toxic
by-products. Therefore, these synthetic polymers have caused a concern from an environmental
perspective and also from a health point of view. One such alternative approach is the use of
polymers which are of direct biological origin, or which are synthesized using biological
precursors. In this regard, biopolymers being biocompatible, biodegradable, and versatile,
permeating their use in all sectors of human endeavour and offering positive attributes in terms
of green chemistry.
One such sector is formulation technology; where, industries like agrochemical, food,
pharmaceutical, cosmetics, and so forth are exhaustively exploring biopolymers. The chemistry
of natural biopolymers is extensively utilized for the preparation of controlled release
formulation systems and for the formulation of HGs. Apart from that; biopolymers incorporating
the desired effect to the formulation product such as gelling, thickening, stabilizing to the desired
product. In the present article, we explore the possibilities of biopolymer chemistry in relation to
the formulation especially on slow release formulation and HG synthesis.
Sources of biopolymers
Novel families of biopolymers can be produced in significant quantities from lower and higher
plants, microbes and animals, and also the readily available renewable agricultural waste and
feed stocks. Plants are the most abundant sources of biopolymers such as starch, proteins,
polysaccharides, and lipids. Microbial biopolymers are produced by a range of microorganisms

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cultivated under various growth and nutrient conditions. Biopolymers such as
polyhydroxybutyrate (PHB) granules were first observed by Lemoigne in Bacillus megaterium (a
gram positive bacterium) in 1926. The most common biopolymers derived from animals are
chitin and chitosan. Chitin is a macromolecule found in the shells of crabs, lobsters and insects.
The primary unit in the chitin polymer is 2-deoxy-2-(acetylamino) glucose. Chitin is insoluble in
its native form but chitosan, the partly deacetylated form, is water soluble. The materials are
biocompatible and have antimicrobial activities. Agricultural wastes also form a rich source of
biopolymers.
Commonly used biopolymers for agrochemical delivery systems / formulations
Biopolymers have numerous advantages such as low cost and readily availability, which
facilitates the large-scale production of products derived from them. One of the applications of
biopolymers is in controlled release formulations that are used in a variety of areas, including
agriculture. Some of the main biopolymers employed as carriers in agriculture are described
below. Natural biopolymers such as proteins and polysaccharides are extensively used in
formulation technology as functional ingredients in industries including agrochemical, food,
pharmaceutical, cosmetics, and so forth. Biopolymers are commonly used as adjuvants in
formulation; they may or may not be chemically active but, impart important physical properties
to finished product. They are used extensively in products to customize the formulation to
specific needs and compensate for local conditions. The use of appropriate biopolymer to the
formulation may reduce or even eliminate the problems associated with the formulation product
and thereby improving overall performance. Some of the main biopolymers employed as carriers
in agriculture are described below:
Alginate
Obtained from brown macroalgae, is a linear polysaccharide composed of 1-4 bonds of β-D-
manuronic acid (M) and α-L-guluronic acid (G), with variations in the composition and
sequential structure along the chain.

Alginic acid
Starch
It is a homopolysaccharide consisting of chains of amylose and amylopectin. Amylose is
constituted of glucose units connected by α-(1,4) bonds, forming a linear chain, while
amylopectin forms branched structures between the glucose units by means of α-(1,4) and α-
(1,6) bonds.

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Starch
Cellulose
Most abundant polysaccharide in nature, and its useful properties include biodegradability,
biocompatibility, low toxicity, and low cost. The cellulose molecule is composed of sequences of
β-D-glucopyranose units linked by β-(1,4) glycosidic chemical bonds. Cellulose and its
derivatives are widely used as delivery systems for bioactive compounds.

Cellulose
Guar gum
Neutral polysaccharide composed of a main chain of D-mannopyranose residues connected by β-
(1,4) glycosidic bonds, linked to D-galactopyranose residues by α-(1,6) glycosidic bonds.

Guar gum
Chitosan
Partially deacetylated compound obtained by the deacetylation of chitin in an alkaline medium.
Chitin is composed of D-glucosamine and N-acetyl-D-glucosamine monomers, linked by β-(1,4)
glycosidic bonds. The extent of deacetylation, the content of impurities, and the distribution of
the molar mass of chitosan depends on the natural source of the primary material as well as the
preparation method.

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Chitosan
Dextrans and pectins
Dextrans are polysaccharides derived from bacteria, composed of glucose monomers connected
by α-(1,6) bonds in the main chain and by α-(1,4), α-(1,3), and α-(1,2) bonds in the branches.
Pectins are a family of polysaccharides present in the cell walls of higher plants. Their structures
consist of D-galacturonic acid units connected by α-(1,4) bonds, forming a linear polysaccharide
interrupted by highly branched regions.

Biopolymer based HGs


HGs are swollen, cross-linked polymeric structures consisting of covalent bonds which are
produced by the reaction of one or more monomer units through physical cross-links as a result
of chain entanglements. Bonds in HGs are hydrogen bonds or strong van der Waals interactions
between the chains or crystallites; bringing together two or more macromolecular chains. The
application of HGs dates back to 1960s, when hydrophilic networks of cross-linked poly(2-
hydroxyethyl methacrylate) as soft contact lens material2. HGs may be classified into different
categories depending on various parameters including the preparation method, the overall
charge, and the mechanical and structural characteristics. On the basis of the preparation method,
homopolymer and copolymer HGs can be distinguished. Alternatively, HGs can also be
classified as neutral, anionic, or cationic depending on the charges of the building blocks.
Finally, classification can be made according to the physical structure: amorphous, semi-
crystalline, hydrogen-bonded, supramolecular, or hydrocolloidal.
Polysaccharide based HGs
In recent decades, superabsorbent HGs incorporating biopolymers such as polysaccharides have
been widely investigated because of their potential as ecologically as well as economically viable
alternatives to other materials. Major characteristics which allowed their use in polysaccharide
based superabsorbent HGs are their diversity in chemical structure, presence of free chemically
active groups for substitution non-toxicity, eco-friendly nature, availability, and efficiency of
application. The polysaccharides are used in HGs field as a constituent-key, serving as a support
on polymer network and allowing other properties such as bio-degradability. In other words, if
the polysaccharide chains break, the HG unmakes. This characteristic makes the polysaccharide-
based HGs appropriate for uses in soils as a fully biodegradable system for controlled release of
nutrients, because the polysaccharides are susceptible to bio-degradation by microorganisms3 or
chemical or physical stimulus4.
The polysaccharides in their native form are not able to produce HGs with good stability, which
is an essential condition for uses as controlled system of nutrients into soil. Polysaccharides
based HGs can be prepared using chemical as well as physical cross-linking5-8. Chemical based
crosslinking through covalent bonds has an advantage; that the 3-dimensional matrix remains
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stable over the period of time and helps in preserving the gel properties. To overcome the
challenges linked with the lack to ability to produce mechanically stable HGs from not
chemically modified polysaccharides, an approach based on a chemical reaction that involves
linking carbon-carbon double bonds to polysaccharide chains has been widely used9.
Chemical HGs based on different vinyl-modified polysaccharides through addition of
methacrylate groups
Pectin: The chemical modification of pectin has been shown to be an efficient strategy using
polysaccharides as starting poly-functional moieties to create a superabsorbent hydrogel10. The
swelling profiles of modified pectin-based superabsorbent hydrogels in saline solutions were in
same order magnitude of those found in distilled water, i.e., they interestingly do not lose their
capacity of water super absorption in the presence of salt in certain conditions. Those HGs also
showed controlled release characteristics of urea, phosphate and potassium10. The modification
of polysaccharides with the use of the glycidyl methacrylate occurs by trans-esterification and/or
epoxide ring-opening reaction mechanisms11,12. The occurrence of one or both of the reaction
mechanisms depends on pH and chemical nature of polymer and solvent. At acidic medium,
glycidyl methacrylate reacts with both carboxylic and hydroxyl groups by epoxide ring-opening
mechanism in a forward and irreversible reaction route11. The reaction resultant products of
epoxide ring-opening reaction are two isomers: 3-methacryloyl-1-glyceryl and methacryloyl-2-
glyceryl esters of polysaccharide. At basic condition, glycidyl methacrylate reacts with hydroxyl
groups by both the transterification, which occurs in the forward and reverse direction, and the
epoxide ring-opening. Under this condition, the following isomers are formed: vinyl
methacrylate of polysaccharide, 3-methacryloyl-1-glyceryl ether of polysaccharide, and 3-
methacryloyl-2-glyceryl ether of polysaccharide13.
Cashew gum: An effective superabsorbent hydrogel was prepared using chemically modified
cashew gum copolymerized with acrylamide and submitted to further hydrolysis14. The cashew
gum was firstly modified with glycidyl methacrylate using a mixture of water and dimethyl
sulfoxide (DMSO), as solvent, and N,N,N’,N’-tetramethylethylenediamine (TEMED), as
catalyst. Depending on the reaction time used for acrylamide hydrolysis, the cashew gum HG
can swell in water up to about 1,500 times its own dry weight, without significant loose the
geometrical form as compared to original material (dry material). That HG is a high performance
water absorbent, which makes it attractive for soil conditioning 14, but the problem was the use of
DMSO as solvent for chemical modifying the polysaccharide.
Starch: Starch is among the most widely used polysaccharides in superabsorbent hydrogel
production, and has been the target of several studies including industrial and academic sectors15
. It is the second most abundant carbohydrate polymer in nature (next to cellulose) and stands out
as one of the most important natural polymers. Beyond being obtained from renewable sources,
this polysaccharide offers other important advantages such as low cost, ease of chemical
modification, ability to replace some synthetic polymers, good mechanical resistance and
plasticity, and so on. The modification of starch with glycidyl methacrylate has been commonly
processed in the presence of catalytic agents such as 4-(N,N-dimethylamino)pyridine (DMAP)
and N,N,N´,N´-tetramethylethylenediamine (TEMED). The product resultant from that reaction
was able to undergo radical reaction that leads to HGation. Chemical starch HGs of high water
absorption capacity were prepared through ultrasound-assisted radical
crosslinking/polymerization reaction in the presence of acrylamide and acrylic acid. The

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asobtained material was able to absorb approximately 150 g of water per gram of dry HG after
200 min of immersion16.
Arabic gum: Arabic gum was also successfully modified with glycidyl methacrylate 17. From
then on, the use of potentially toxic reactants and of organic solvents in the modification reaction
was discontinued. Vinylated Arabic gum was obtained by an adapted approach that uses water as
solvent without catalytic agent. However, when only water is used as solvent, a further problem
had to be overcomed: glycidyl methacrylate is insoluble in water. To address the challenges
related to insolubility of the glycidyl methacrylate in the reaction medium, a heterogeneous
phase system composed of water-soluble Arabic gum and water-insoluble glycidyl methacrylate
was provided using high stirring speed at a temperature range of 60-65 °C. Under these
conditions, the polysaccharide is able to be vinyl-modified at the interface of the glycidyl
methacrylate -water system. The product was easily crosslinked through reaction with
acrylamide and sodium acrylate monomers, forming SH. It absorbs ca. 500 g of water per gram
of dry HG in 60 min, without its mechanical stability being seriously affected17.
Chemical HGs based on polysaccharides vinyl-modified by redox reaction
Thermal treatment of the material in the presence of peroxides, which leads to formation of
radical ions on hydroxyl or carboxyl groups, is also an efficient approach to transform uncross-
linked polysaccharides into a HG. In such a case, covalent cross-links can be introduced by
adding vinyl monomers that reacts with the radical hydroxyl groups of the polysaccharides. For
instance, Diao et al.18 modified peanut hull cellulose using potassium
persufate at 50 °C and the product from that reaction was further polymerized with acrylic acid,
acrylamide, 2-acrylamide-2-methyl-1-propanesulfonic acid producing SH. The process to
convert the uncrosslinkable polysaccharide into a functionalized/crosslinked polymer starts off
with the thermal decomposition of the potassium persulfate. At temperatures close to 85 °C or
above, the potassium persulfate undergoes homolytic cleavage forming sulfate radical ions. After
the breakdown of the peroxide, the radical reacts with oxygen of sulfate or hydroxyl groups in
carbohydrate. Then the new formed radical reacts with vinyl monomers in the same manner as
the sulfate radical ions did. It follows that there is an addition of more and more monomers to the
ever-increasing chain. Equivalent approaches have been used to modify kappacarrageenan,
carboxymethyl cellulose, sodium alginate, xanthan gum, carboxymethyl chitosan , chitosan and
so forth. The production of SH based on chitosan has significantly increased in recent years.
Chitosan can be modified through reaction with vinyl monomers, compounding with
functionalized components, and complexation with polyelectrolytes.

Physically (non covalent) HGs based on polysaccharides


Another approach that has been explored involves introducing physical crosslinks to prepare
physical (or noncovalent) HGs. In this direction, carboxymethyl cellulose was obtained through
the carboxylation reaction of cellulose with monochloroacetic acid in hydroxide solution. The
product from that reaction is a polyelectrolyte that can be physically (ionically) crosslinked with
multivalent metal ions such as aluminum. There are natural anionic polysaccharides with ability
to form strong HGs with divalent and trivalent cations, although divalent ions cause contraction
of polymer network (during crosslinking process) affecting the water absorption capacity. On the
other hand, these HGs can be readily transformed to aerogel by scCO2-assisted drying19. The

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aerogel possesses highly porous polymeric network with connected pathways throughout the
material. This type of architecture allows fast water diffusion into and through the HG.
Polysaccharide-based HGs prepared by emulsion technology
Particulate SH can also be produced through crosslinking/polymerization emulsion process.
Although the polysaccharides have no good sphere-forming capacity, because of complexity of
their macromolecular fragments and structural-conformational characteristics, this approach
provides particles with spherical shapes, as a result of minimum single-particle surface energy20.
Emulsion polymerization offers advantages for simple, easy and quick preparation; products with
high molecular weight21; and well-defined preparation stages. The stability of an emulsion is
related both to chemical nature of interfacial film and to attraction/repulsion balance force
occurring among the particles suspended in the liquid, which are important in the prevention of
the droplets coalescence20. An efficient strategy to prepare crosslinked polysaccharide particles
with controlled size is the crosslinking/polymerization in a water-in-oil emulsion under vigorous
stirring22. In such a case, the spherical structures results of a random movement of water droplets
inwards the oil phase under vigorous stirring. Under these conditions, stable emulsion of oil
phase-surrounded water droplets with controlled size within a certain range is formed. The
reaction occurs within the small water droplets due to hydrophilicity of the reactants. The water-
in-oil can be stabilized by amphiphilic substances such as poly(vinyl alcohol) (PVA), an
emulsifying agent. The adsorbed PVA on interface reduces the interfacial tension. Ultrasound-
assisted polymerization has also been an excellent tool in the production of HG particles with
defined outlines23. The main advantage of such an approach is that the crosslinked
polysaccharide particles are formed in few seconds. On the other hand, reactions that require
polymerization times longer than 3-5 minutes can cause thermal degradation of the
polysaccharide, owing to an overheating of the emulsion23. In such a case, polymerizations at
room temperature under the protection of N2 atmosphere should be considered, if possible.
Polysaccharide based HGs prepared by supercritical CO2 assisted drying of emulsions
The water absorption capacity of HG particles can be improved by increasing their porosity. A
way of doing this is to dry the particles in supercritical CO2. Beyond providing porous particles,
supercritical drying also is an efficient approach to prepare small particles with narrow particle
size distribution, as compared to traditional solvent evaporation of emulsions used in the
production of polymeric nanoparticles 24. In supercritical CO2, the original configurations of the
polymer chains are kept owing to a fast removal of solvents from emulsion by the solubilization
in the supercritical CO2 phase. In the most elementary form, CO2 dissolves into the liquid
droplets generated by the collapse of stream of the liquid and the solvent evaporates in the
supercritical CO2 phase. In the supercritical environment, the pressure is equal in all directions
around a particle, providing more defined shapes.
Polysaccharide-based controlled-release formulation in the form of microspheres and
nanoparticles
A variety of polymeric nano- and microparticles can be used as carriers for bioactive molecules.
Depending on the materials and methods used, it is possible to obtain nano- and microspheres, or
nano- and microcapsules, which differ both structurally and in terms of their composition.
Nanocapsules are systems composed of a polymeric shell and an oily nucleus, where the active
principle can be dissolved in the nucleus or adsorbed on the polymeric wall. Nanospheres do not

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contain oil in their composition, and consist only of a polymeric matrix. In this case, there is no
distinct nucleus, and the active principle is adsorbed or retained by the polymeric matrix25.
Spheres and microspheres
Fernández-Pérez et al. studied the mobility in the soil column of isoproturon released from
alginate–Bentonite spheres26. Simulations were made of typical soil structures, using columns
consisting of fractions of sand, peat, altered soil, and native soil. Sorption and desorption tests
were performed for all the fractions. It was found that use of the alginate–bentonite formulations
resulted in reductions in the vertical mobility of isoproturon, compared to columns containing the
herbicide alone. The greatest sorption capacity was obtained for the peat layer. The use of
alginate–Bentonite could therefore provide an effective system for reducing the amount of
herbicide leached through the soil, hence diminishing possible risks of contamination of
underground water bodies. Flores Céspedes et al. described the synthesis of alginate spheres
containing different adsorbents (bentonite, anthracite, and activated carbon), and the influence of
the adsorbents on the encapsulation efficiency of two herbicides (metribuzin and chloridazon), as
well as on the release rates of the active principles in aqueous media27. The release rates of
systems prepared with metribuzim were faster than those containing chloridazon (which is more
hydrophobic than metribuzim). The formulations containing the adsorbents provided slower
release of both herbicides, compared to the use of the alginate spheres alone. In terms of
reducing the release rate, the most efficient adsorbent was activated carbon.
Nanoparticles
dos Silva et al. studied the release profile and soil sorption of alginate/chitosan nanoparticles
used as carriers of the herbicide paraquat28. The average size of the particles was 635±12 nm,
and the encapsulation efficiency was 74.2 %. Encapsulation of the herbicide in the nanoparticles
resulted in a significant change in the release profile of the herbicide, with the release being
slower, compared to free paraquat. In soil sorption experiments, it was found that sorption of
both the encapsulated and free forms was dependent on the content of organic matter in the soil,
although the sorption profile was reduced when paraquat was associated with the nanoparticles,
hence improving the herbicidal action. Celis et al. used different methods to prepare a
bionanocomposite material based on chitosan and clay (montmorillonite), used as an adsorbent
for the herbicide clopyralid present in an aqueous solution or in a mixture of water and soil 29.
The nanoparticles were characterized using FTIR, X-ray spectroscopy, and SEM. The
bionanocomposites showed good herbicide adsorption capacity at pH levels at which the anionic
form of the active principle and the cationic form of chitosan predominated. Removal of the
herbicide from aqueous solution was more effective when a higher concentration of chitosan was
used in the bionanocomposite. At slightly acid pH, the composites effectively adsorbed
clopyralid from soil. The use of this type of formulation could help to limit the mobility of
anionic pesticides in the environment, reducing risks of contamination of surface and
subterranean water bodies.
Polysaccharide-based controlled-release formulation in the form of beads
Alginates-based controlled-release formulations have been investigated with various herbicides
such as monolinuron, desmetryn, chloridazon, atrazine, simazine, and chloroxuron as active
ingredients. Release rate from these formulations showed sufficient retardation of herbicide
release30. Alginate formulations have decreased the rate of release of metribuzin herbicide

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compared to the conventional formulation. The reaction parameters for the synthesis of beads
such as concentrations of crosslinker, pesticide, alginate to gelatin ratio, and temperature affect
the release profiles of pesticide from the controlled-release systems31. The presence of the
crosslinker in the formulations affects release of active compound from the formulations. In one
study, it has been observed that carbaryl insecticide release from Ni-alginate beads was faster
than that of Ca-alginate beads32.
Prospects
Polysaccharides have been used as a substitute as individual or in combination to the synthetic
moieties for the synthesis of ordinary as well as superabsorbant HGs and other controlled
delivery formulation systems. These biopolymer based formulation systems may found very
important technological applications in variety of areas such as as in agriculture (as soil
conditioners, controlled pesticide delivery systems and nutrient carriers), in environment for
waste water treatment and numerous other fields This is due mainly to the relatively low-cost,
abundance, renewability, and biodegradability, among others advantages for using biopolymers.
Also, important technologies have been developed mostly in the last two decades for chemically
modifying biopolymers such as polysaccharides enabling them to the synthesis of HG based on
biopolymers. Few examples were given in this chapter, but a very vast window in this context
still remains opened. As a trend for this field, some highlights can be given, among others:
• preparation of HGs of low cost, that can show, at same time, superabsoption
characteristics and good mechanical properties. This remains as a challenge to be
confronted, despite some works showing that the incorporation of nanofillers either from
polysaccharides, such as nanowhiskers, nanfibrils or from inorganic sources, such as
kaolin, montmorillonite, and attapulgite etc., may improve the swelling ratio, the swelling
rate and mechanical properties
• Synthesizing eco-friendly methods for chemically modification of polysaccharides in
order to allow chemical cross-linking or for providing permanent electrical charges in
polymer backbone for physical HG formation (through polyelectrolyte complexation).
• Synthesizing superabsorbent hydrogels in nanoscale (nanogels) with very high water
holding capability (e.g. thousand folds). Their properties can be improved by
incorporating CNCs into formulations, prepared mainly by inverse mini emulsion
methods. In addition, technologies associated to nanoparticles may reduce the sensitivity
of superabsorbent hydrogels to saline conditions.
• Minimizing the final cost of synthesis remained a challenge for applications of
superabsorbent hydrogels in agriculture, in a large scale. Even though the environmental
necessities such as no toxicity to soil or plant, biodegradability and so on has been
fulfille; the final price will be an mandatory parameter for spreading more and more the
use of superabsorbent hydrogels as soil conditioner and/or nutrient carriers.
Conclusion
This chapter attempted to update and discuss some important aspects of synthesis,
characterization and application of superabsorbent hydrogels, mainly those based on
polysaccharides, as soil conditioners and as carriers for nutrient release. Some methods for
chemically modifying polysaccharides were given and some directions for HGs preparation as

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well. It was reinforced that superabsorbent hydrogels remain as very hot issue in materials
science due to both intensive academic researches and technological applications. Of course,
more comprehensive studies will expand the understanding of structure properties- applications
relationship in superabsorbent hydrogels, which will, certainly, increase even more the
importance of the class of soft materials. Controlled release formulation specially based on
polysaccharides. These formulations not only release the active compound in a slow manner but
also after degradation increase the crop output besides proving the water-holding capacity of the
soil. Release systems decrease the amount of active ingredient available for leaching and
volatilization. These formulations will control the environment, ecosystem and health hazards
caused by the conventional pesticide formulations. Hence, these polysaccharide based
formulations could be utilized for the safe management of agrochemicals which will decrease
their toxic effects and helpful for their better delivery to the field.

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Amphiphilic Polymers as Carriers for Pesticide Delivery
Najam Akhtar Shakil, Parshant Kaushik and Jitendra Kumar

Division of Agricultural Chemicals


ICAR-Indian Agricultural Research Institute, New Delhi – 110 012

The advent of agrochemicals has increased the production of food substantially.


However, the non-judicious use of these indispensable chemicals has led to serious ecological
problems due to their leaching and volatilization. About 90% of applied agrochemicals never
reach their target in a definite time in the precise quantities. Their impact on surface water and
groundwater is also a major concern along with their potential health hazards to the general
population.
Controlled release formulation products increase pest control efficiency through
utilization of reduced quantity of toxicants, reduced leaching and extended residual activity.
Numerous examples in literature highlight such products to effectively combat pests (1-4).
Over the past few decades, there has been considerable interest in developing
biodegradable nanoparticles as effective drug delivery devices. The advantages of using
nanoparticles for drug delivery result from their two main basic properties. First, nanoparticles,
because of their small size, can penetrate through smaller capillaries and are taken up by cells,
which allow efficient drug accumulation at the target sites. Second, the use of biodegradable
materials for nanoparticle preparation allows sustained drug release within the target site over a
period of days or even weeks. The development of nanoparticulate delivery systems for targeted
drug delivery has been reviewed recently.
Scientific publications and patents on nanomaterials (NM) used in plant
protection or fertilizer products have exponentially increased since the millennium shift. While
the United States and Germany have published the highest number of patents, Asian countries
released most scientific articles. About 40% of all contributions deal with carbon-based NM
followed by titanium dioxide, silver, silica, and alumina. Nanomaterials come in many diverse
forms (surprisingly often >> 100 nm), from solid doped particles to (often non-persistent)
polymer and oil-water based structures. Nanomaterials serve equally as additives (mostly for
controlled release), or active constituents.

Therefore, the importance and development of polymers for crop protection is well
recognized. The polymers can either physically adsorb the drug or active ingredients inside the
insoluble polymer matrix, or, solubilize the drug carriers and bound them to the polymer
backbone leading to release of active ingredients in controlled release manner. However,
amphiphilic block copolymers with hydrophilic and hydrophobic segments have been
investigated extensively because of their unique self-organization characteristics and wide range
of potential applications, such as in drug delivery and separation technology systems.
Amphiphilic polymers have very high solubilization power, low critical micelle concentration
(CMC) and high stability (5). The self-assembling of amphiphilic polymers into nano micellar
aggregates in aqueous media enables these nanocarriers to encapsulate hydrophobic drugs in
their core, thus improving the drug’s water solubility. The micellar characteristics of amphiphilic

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diblock copolymers depend on the nature of each block and the surface properties of self-
organized micelles are highly dependent on the structures of the hydrophilic blocks.

Large amphiphilic molecules represent a new frontier in the field of surfactant science
and technology. Their macromolecular nature provides a range of architectures, length scales,
time scales and levels of interactions much wider than those offered by small amphiphilic
molecules. Constant efforts are being made to design new surfactants with an improved
performance, since it is well known that the efficiency of a surfactant increases with its
amphiphilicity. While the small amphiphilic molecules can be restricted to AB, ABA and BAB
types, the large amphiphiles having high molecular weights, can have much more diverse
molecular architectures such as A-B-A triblock copolymers and graft copolymers. The large
amphiphilic molecules organize themselves in the form of micelles when dissolved in selective
solvents and also get adsorbed on the surfaces, as the small amphiphilic molecules do. But the
micelle formation in both the cases differs in terms of size and degree of segregation between the
blocks that form the micelle. However, most of the functional properties are common in micelles
formed by both small and large amphiphilic molecules.

The unique properties of poly(ethylene glycol) (PEG), including a wide range of


solubility, lack of toxicity, noninterference with enzymatic activities and conformations of
polypeptides and ease of excretion from living organisms, make them ideal drug carriers.
Further, the two hydroxyl end groups of PEG can be suitably functionalized prior to coupling
with ligands of biological relevance, although the hydroxyl groups themselves can be used as
well. Because the number of terminal groups of PEGs (only two) to attach with drug limits their
drug loading capacity, extensive work has been done to functionalize them by copolymerizing
PEGs with various functional monomers.

The application of nanotechnology in pesticide delivery is relatively new and in the early
stages of development. This technology aims to reduce the indiscriminate use of conventional
pesticides and ensure their safe application. The nano-scale particles are being exploited rapidly
for their new chemical and/or physical properties with application in fields of medicine,
biotechnology, electronics, material science and energy sectors, among others. These promising
nanoparticles also eye on the agricultural sector, which strongly requires continuous innovation
to meet the increasing global food security and climate change challenges. Research on
agricultural nanotechnology applications has addressed several agricultural and environmental
challenges, such as sustainability, improved varieties and increased productivity with an
enormous number of both scientific publications and patents in agricultural nanotechnology,
especially for disease management and crop protection. Nanomaterials in agriculture aims, in
particular, to reduce the amount of sprayed chemical products by smart delivery of active
ingredients, minimize nutrient losses in fertilization and increase yields through optimized water
and nutrient management. In addition, bio-nanocomposites can also be generated from the
traditionally harvested agricultural materials like wheat straw and soy hulls. In terms of
agrochemical (pesticides, fertilizers, growth hormones, etc.) delivery, nanoscale particles have
novel properties which can increase the agrochemicals’ efficiency and make the delivery system
‘smart’. Through a smart delivery system, chemicals can be delivered in a controlled and
targeted manner that is similar to nano-drug delivery to humans.

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Interest in synthesis and characterization of amphiphilic polymers has increased in recent
times. This owes to their exclusive molecular structure which consists of two antagonistic parts
one a hydrophobic block that is insoluble in water and another water-soluble hydrophilic block,
constituting an amphiphilic (amphi: of both kinds; philic: having an affinity for) character.

The presence of two counter parts in the molecular structure of amphiphilic molecules
leads to particular characteristic properties in solution, such as adsorption at interfaces and
surfaces, self-assembly into micellar aggregates with a wide variety of geometries. Polymeric
micelles composed of amphiphilic block copolymers demonstrate a series of attractive properties
in drug and pesticide delivery systems, such as good biocompatibility and high stability in vitro
and in vivo, and can be successfully used for the encapsulation of various poorly soluble agents
in aqueous solution. These polymers are obtained by the polymerization of more than one type of
monomer, typically one hydrophobic and one hydrophilic, so that the resulting molecule is
composed of regions that have opposite affinities for an aqueous solvent.

The micellar characteristics of amphiphilic block copolymers depend on the nature of


each block and surface properties of self-organised micelles are highly dependent on the
structures of the hydrophilic blocks. These polymers have attracted a great deal of attention in
terms of their ability to form various types of nanoparticles.

Amphiphilic polymers are the most promising basic building blocks for mounting
complex and simple hierarchical nanosystems. The applications of these polymers are extremely
broad and polymer-based nanotechnologies are fast emerging. These polymers can be classified
based on the number of dimensions in the nanometer range; non self assembled structures and
self-assembled structures. Polymeric nanoparticles present high stability when in contact with
biological fluids and their polymeric nature allows controlled drug release. Nanoparticles
represent drug delivery systems suitable for most of the administration routes, even if a rapid
recognition by the immune system limits their use as injectable carriers. Nanoparticles may be
prepared either from pre-formed polymers, such as polyesters (i.e. polylactic acid), or from a
monomer during its polymerization, as in the case of alkylcyanoacrylates. Nanostructured
polymers are growing rapidly because of their size coupled properties. Combination of self-
assembly at different length scales leads to structural hierarchies. It offers rich possibilities to
construct nanostructured matter, nanoscale parts and switching (responsive) properties based on
the phase transitions of the self-assembled structures. In recent years, nanomaterials such as
polymer–drug conjugates, liposomes, nanoparticles and polymeric micelles have been
considered as potential carriers for hydrophobic drug delivery that may resolve the
aforementioned problems. Polymeric micelles composed of amphiphilic block copolymers
demonstrate a series of attractive properties in drug delivery systems, such as good
biocompatibility and high stability in vitro and in vivo, and can be successfully used for the
encapsulation of various poorly soluble agents. These nano sized micelles consist of a
hydrophilic outer shell and a hydrophobic inner core that can be used to incorporate lipophilic
drugs in aqueous solution.

Poly(ethylene glycols) (PEG)-based (co)polymers have been utilized as one of the


primary components of DDS, not only because PEGs have been shown to be nontoxic,
biocompatible and durable enough for biological systems, but also because of their good

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solubility, low cost, easy availability, and ease with which these can be chemically modified or
linked with other polymers. A key property of PEG is its attachment to other molecules and
surfaces to provide a biocompatible, protective coating. This protective coating slows the
rejection of materials in biological systems (such as human body ) and greatly reduces bacterial
adsorption. Also, it can be attached to other molecules without effecting their chemistry, but can
control their solubility and increase their sizes. These novel properties can be further enhanced
by chemical modification of the molecular structure.

The presence of just two terminal groups in PEG limits its utility to attach drugs which in
turn effects the drug loading capacity. In order to make the drug delivery systems more versatile,
researchers the world over have attempted to synthesise copolymers based on PEGs by the
synthesis of PEG-based polymers/ copolymers by taking the PEGs of different molecular
weights and polymerizing them with a moiety having a hydrophobic segment so that the polymer
formed is amphiphilic in nature. It is desirable that in order to design a micelle that is able to
physically encapsulate small active reagents (i.e. the drugs, pesticides), the polymers should have
amphiphilic structures. By attaching the hydrophilic PEG segment with one or more
hydrophobic ligand(s) or segment(s), an amphiphilic polymer can be prepared. The amphiphilic
polymer should be able to self-assemble into micelles in a specific solvent system, such as water.

Amphiphilic block copolymers with hydrophilic and hydrophobic segments have been
investigated extensively not only because of their unique self-organization characteristics but
also for their wide range of potential applications, such as in drug delivery and separation
technology systems. The micellar characteristics of amphiphilic block copolymers depend on the
nature of each block and surface properties of self-organized micelles are highly dependent on
the structures of the hydrophilic blocks. The formation of micelles by the self assembly of
amphiphilic copolymers in aqueous media as shown in Figure - 1:

Figure -1

As shown above, by changing the type of solvents, i.e. from organic to an aqueous
system, the polymer does self-assemble into a stable aggregation or micelle. The hydrophobic

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side chains were most likely in the core of the micelle. The radius of gyration of these micelles
fall within the nano range and hence it is possible to develop slow release nano formulations of
bioactive molecules based on PEG-based polymers.

The chemistry of PEGs and aromatic di-acids and their di-esters has been explored for the
synthesis and self assembly of copolymers. The amphiphilic polymers used in the self-assembly
are based on poly(ethylene glycol) and various diesters, synthesized by chemical and enzymatic
methods. The chemoenzymatic condensation of Dimethyl-5-hydroxyisophthalate with
polyethylene glycols of varying molecular weights catalyzed by Novozyme-435 (immobilized
Candida antartica lipase B) has been developed for bulk synthesis of copolymers (6). Also,
amphiphilic copolymers have been synthesized by transesterification–polycondensation of
hydrophilic PEGs and hydrophobic functionalized Dimethyl-5-hydroxyisophthalate monomers
(7). The newly synthesized copolymers, thus obtained, can encapsulate both hydrophilic and
hydrophobic drugs and work in drug delivery systems (8,9). The design of the system and
synthetic strategy is very flexible and provides a high degree of control over the polymer
structures, thus, allowing tuning of the properties of the micelle disruption, the critical micelle
concentration and the size of micelles. Using these copolymers of poly(ethylene glycols) and
various dimethyl esters, which self assemble into nano micellar aggregates in aqueous media,
controlled release (CR) formulations of azadirachtin-A, a bioactive constituent derived from the
seed of Azadirachta indica A. Juss (Meliaceae), have been prepared (10). Release from the
commercial polyethylene glycol (PEG) formulation was found to be faster than the other CR
formulations. Also, the rate of release of encapsulated azadirachtin-A from nano micellar
aggregates reduced on increasing the molecular weight of PEG. The diffusion exponent (n value)
of azadirachtin-A in water ranged from 0.47 to 1.18 in the tested formulations. The release was
diffusion controlled with a half release time (t1/2) of 3.05 to 42.80 days in water from different
matrices, thus, suggesting that depending upon the polymer matrix used, the application rate of
azadirachtin-A can be optimized to achieve insect control at the desired level and period.
Amphiphilic copolymers, synthesized from poly (ethylene glycols) and various aliphatic diacids,
have also been used to develop CR formulations of imidacloprid using encapsulation technique
(11). High solubilisation power and low critical micelle concentration of these amphiphilic
polymers increased the efficacy of formulations. Release from the commercial formulation was
faster than the CR formulations. The diffusion exponent (n value) of imidacloprid, in water
ranged from 0.22 to 0.37 in the tested formulations. While the time taken for release of 50 % of
imidacloprid ranged from 2.32 to 9.31 days for the CR formulations. The bioefficacy of the
prepared CR formulations was evaluated against major pests of soybean, namely stem fly,
Melanagromyza sojae Zehntmer and white fly, Bemisia tabaci Gennadius along with a
commercial formulation at an experimental farm of Indian Agricultural Research Institute
(IARI), New Delhi during kharif 2009 and 2010 (12). Most of the CR formulations of
imidacloprid gave significantly better control of both stem fly incidence and Yellow Mosaic
Virus (YMV) infestation transmitted by white fly as compared to its commercial formulations.
Some of the developed CR formulations recorded higher yield over commercial formulation and
control. Nodulation pattern of soybean was not affected due to treatment of CR of imidacloprid.
Also, the residues of imidacloprid in seed and soil at harvest were not detectable for both CR and
commercial formulations.

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Further amphiphilic copolymers, useful for encapsulation of bioactive compounds such
as β-carotene and curcumin, have also been synthesized by attachment of saturated and
unsaturated long chain acid chlorides to the copolymer of PEG and Dimethyl-5-
aminoisophthalate via an amide linkage (13). Similarly, synthetic polyesters have been
synthesized using poly(ethylene glycols) (PEGs) of different molecular weights, viz. 300, 600
and 1000 as hydrophilic block and aliphatic diacids namely glutaric acid, adipic acid, pimelic
acid and suberic acid as hydrophobic block in presence of catalyst Conc. H2SO4 for delivery of
pesticides (14-16). The resulting polymers have been further alkylated by attaching heptyl and
tetradecyl chains to phenolic hydroxyl group (17,18). The synthesis of these amphiphilic
polymers has been further extended to encapsulate β-cyfluthrin in nanospheres (19). Release
from the commercial formulation was faster than with the developed CR formulations. The rate
of release of encapsulated β-cyfluthrin from nano micellar aggregates reduced on increasing the
molecular weight of PEG. The diffusion exponent (n value) of β-cyfluthrin in water ranged from
0.427 to 0.622 in the tested formulations. The release was diffusion controlled with a half-release
time (t1/2) of 3.92 to 7.9 days in water from different formulations, and the period of optimum
availability (POA) of β-cyfluthrin ranged from 1.4 to 20.5 days. Further, bioefficacy of
developed β-cyfluthrin formulations has been evaluated against Callosobruchus maculatus
(Coleoptera: Bruchidae) (20). The mean EC50 of the commercial formulation against C.
maculatus was quite high as compared to those of developed formulations. The results provide a
comparison of developed formulations with the commercial one showing an earlier degradation
of β-cyfluthrin in the latter and relatively prolonged activity in the former. According to the
results obtained from this research, the increased residual toxicity of β-cyfluthrin can be
exploited with advantages for insect control. Therefore, one-time application of these
formulations can be sufficient for effective control of the target insect/pest.

The nano micelles formed by amphiphilic copolymers have been used further for the
encapsulation of carbofuran, [2,3–dihydro-2,2-dimethylbenzofuran-7-yl methylcarbamate], a
systemic insecticide-nematicide, for the development of controlled release formulation (21). The
bioefficacy of the developed carbofuran formulations along with commercial formulation of
carbofuran 3G (CP0) has been evaluated against the root-knot nematode, Meloidogyne incognita
infecting tomato (cv. Pusa Ruby) in pot and field conditions (22). The bioefficacy data indicated
that the formulations developed by utilizing polymers having PEG – 900 as hydrophilic segment
were effective even at 14 days post inoculation (dpi) as evident from shoot and root length. Both
the CR formulations (CP1 and CP2) in general significantly reduced the number of galls when
compared to CP0. The developed CR formulations of carbofuran have the potential for effective
management of M. incognita in tomato under the field conditions.

CR formulations of Thiamethoxam (3-(2-chloro-1,3-thiazol-5-ylmethyl)-5-methyl-1,3,5-


oxadiazinan-4-ylidene(nitro)amine) developed using encapsulation techniques showed slower
release than the commercial formulations (23). The time taken for release of 50 % of
thiamethoxam ranged from 3.56 to 6.07 days for the CR formulations. The diffusion exponent (n
value) of thiamethoxam in soil ranged from 0.532 to 0.881 in the tested formulations showing
non-Fickian transport. These CR formulations may be used in safer, effective and economic crop
protection. Further, these thiamethoxam formulations have been used for coating soyabean seeds
and improving the seed quality (24). The percent thiamethoxam recovery ranged from 86.1-
93.2% from different CR formulation seed coats. Better thiamethoxam retention was observed on

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soybean seed coats treated with CR formulations. The seeds coated with different
nanoformulations had better seed quality over control and commercial formulation. Similarly,
Controlled release formulations of Thiram (Dimethylcarbamothioylsulfanyl-N,N-
dimethylcarbamodithioate), a contact fungicide, have been prepared using laboratory synthesized
poly(ethylene glycol) (PEG) based functionalized amphiphilic copolymers (25). Suitable
polymers that could reduce the seed deterioration during storage and act as an effective carrier of
fungicide thiram were identified. The results demonstrate that the seeds coated with the different
formulations deteriorated at a slower pace as manifested in high germination percentage over
control. Apart from the fungicidal effect of thiram, the polymers acted as barriers to moisture
reducing the rate of seed deterioration and checked the degradation of thiram. The CR
formulation, with PEG 2000, was found to be most effective as seed coat. The diffusion
exponent (n) of thiram in water ranged from 0.356 to 0.545 in the tested formulations. The half-
release (t1/2) values ranged between 14.78 to 22.1 days, and the Period of Optimum Availability
(POA) of thiram ranged from 7.79 to 25.15 days. The controlled release nanoformulations of
carbendazim have also been developed employing amphiphilic polymers and their bioefficacy
evaluated against Rhizoctonia solani (26). The range of carbendazim release was found to be
between 10th to 35th day as compared to commercial formulation which was up to 7th day. The
diffusion exponent (n value) of carbendazim in water ranged from 0.37 to 0.52 in the tested
formulations. The half-release (t1/2) values ranged between 9.47 and 24.20 days, and the period
of optimum availability (POA) of carbendazim ranged from 9.15 to 26.63 days. Also, ED50
values of the developed formulations vary from 0.40 to 0.74 mg L-1. Slow release formulations
of β-carotene have been developed employing poly(ethylene glycols) (PEGs) based
functionalized amphiphilic polymers (27). Encapsulation efficiency and loading capacity of the
developed formulations were determined which ranged from 22.60 to 28.08 % and 2.2 to 2.8 %
respectively. The release kinetics of β-carotene from developed formulations in water revealed
increased solubility and prolonged stability of β-carotene. The diffusion exponent (n values)
ranged from 0.1540 to 0.2342 for developed formulation. The results showed that developed
slow release formulations were unaffected by the highly acidic conditions referring to the gastric
environment of human body. However, the release of β-carotene was high at pH 7.8 and slightly
higher at pH 6.8.

Controlled release (CR) nanoformulations of Mancozeb (manganese-zinc double salt of


N, N-bisdithiocarbamic acid), a protective fungicide, have been prepared using laboratory
synthesized poly(ethylene glycols) (PEGs) based functionalized amphiphilic copolymers without
using any surfactants or external additives. The release kinetics of the developed Mancozeb CR
formulations were studied and compared with that of the commercially available 42%
Suspension Concentrate (SC) and 75% Wettable Powder (WP). Maximum amount of Mancozeb
was released on 42nd day for PEG-600 & octyl chain, PEG-1000 & octyl chain and PEG-600 &
hexadecyl chain, 35th day for PEG-1000 & hexadecyl chain, 28th day for PEG-1500 & octyl
chain, PEG-2000 & octyl chain, PEG-1500 & hexadecyl chain, and PEG-2000 & hexadecyl
chain in comparison to both the commercial formulations (15th day). The diffusion exponent (n
value) of Mancozeb in water ranged from 0.42 to 0.62 in the tested formulations. The half-
release (t1/2) values ranged from 17.35 to 35.14 days, and the period of optimum availability
(POA) of Mancozeb ranged from 18.54 to 35.42 days. Further, the in vitro bioefficacy evaluation
of developed formulations was done against plant pathogenic fungi Alternaria solani and
Sclerotium rolfsii by poison food technique. ED50 values of the developed formulations varied

130 Winter School on "New generation smart agrochemical oriented approaches for crop
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from 1.31 to 2.79 mg L-1 for A. solani and 1.60 to 3.14 mg L-1 for S. rolfsii. The present
methodology is simple, economical and ecofriendly for the development of environment friendly
CR formulations of Mancozeb. These formulations can be used to optimize the release of
Mancozeb to achieve disease control for the desired period depending upon the matrix of the
polymer used. Importantly, the maximum amount of the active ingredient remains available for a
reasonable period of time after application. Also, the developed CR formulations were found to
be suitable for fungicidal application allowing use of Mancozeb in lower doses (28-29).

Controlled release nanoformulations of carbendazim (Methyl 1H-benzimidazol-2-


ylcarbamate), a systemic fungicide, have been prepared using laboratory synthesized
poly(ethylene glycols) (PEGs) based functionalized amphiphilic copolymers. The release
kinetics of carbendazim from developed controlled release (CR) formulations was studied and
compared with that of the commercially available 50% Wettable Powder (WP). Further, the bio
efficacy evaluation of developed formulations was done against plant pathogenic fungi
Rhizoctonia solani by the poison food technique method. The release of maximum amount of
carbendazim from developed formulations was dependent on the molecular weight of PEGs and
was found to increase with increasing molecular weights. The range of carbendazim release was
found to be between 10th to 35th day as compared to commercial formulation which was up to 7th
day. The diffusion exponent (n value) of carbendazim in water ranged from 0.37 to 0.52 in the
tested formulations. The half-release (t1/2) values ranged between 9.47 to 24.20 days, and the
period of optimum availability (POA) of carbendazim ranged from 9.15 to 26.63 days. Also,
ED50 values of the developed formulations vary from 0.40 to 0.74 mg L-1. These formulations
can be used to optimize the release of carbendazim to achieve disease control for the desired
period depending on the matrix of the polymer used (30).
Several controlled release/ slow-release formulations of pesticides have been developed
and their behavior has been recorded. The studies show that the controlled release formulations
provide better pest management than commercial formulations without leaving any terminal
residues in crops and soil. Hence, these formulations can minimize the impact of pesticides on
the environment by decreasing their rapid losses after application. Also, less active ingredient
needs to be used to achieve bioefficacy similar to the currently available commercial
formulations. Therefore, development of CR formulations and their use can be further explored
for efficient pest management. There are, however, very few available data about the behavior of
the nanopesticides after their application in the field, and it is not known whether nanopesticides
can be adequately evaluated within the current pesticide regulatory framework. An adequate
exposure assessment of nanopesticides is essential for assessing the new risks and new benefits
associated with these new products. Currently, it is not possible to detect or quantify polymer
nanocarriers in the soil matrix, because of the similarity of the elemental composition. Further
efforts will thus be needed to develop suitable analytical techniques that support the design of
more strategic nanoenabled delivery systems for pesticides and other bioactive substances, while
ensuring a robust assessment of the new risks and benefits (31).
References
1. Nisar, K., Kumar, J., Shakil, N.A., Pankaj, Walia, S. and Parmar, B.S. (2009) Controlled
Release Formulations of Acephate: Water and Soil Release Kinetics. J Environ Sc
Health, Part B, 44 (6), 520–525.

131 Winter School on "New generation smart agrochemical oriented approaches for crop
and human health management", 07-27 Dec, 2017
2. Kumar, J., Nisar, K., Shakil, N.A., Walia, S., and Prasad, R. (2010) Controlled release
formulations of metribuzin: Release kinetics in water and soil. J Environ Sc Health, Part
B, 45(4), 330-335.
3. Kumar, J., Nisar, K., Shakil, N.A., and Sharma, R. (2010) Residue and bioefficacy
evaluation of controlled release formulations of metribuzin against weeds in wheat. Bull
Environ Contam Toxicol, 85(3), 357–361.
4. Kumar, J., Shakil, N.A., Khan, M.A., Malik, K. and Walia, S. (2011) Development of
controlled release formulations of carbofuran and imidacloprid and their bioefficacy
evaluation against aphid, Aphis gossypii and leafhopper, Amrasca biguttula biguttula
Ishida on potato crop. J Environ Sc Health, Part B, 46(8), 678-682.
5. Watterson, A.C.; Parmar, V.S.; Kumar, R.; Sharma, S.K.; Shakil, N.A.; Tyagi, R.;
Sharma, A.K.; Samuelson, L.A.; Kumar, J.; Nicolosi, R.; Shea T. (2005) Pure Appl.
Chem., 77(1), 201–208.
6. Kumar, R., Shakil, N.A., Chen, M.H., Parmar, V.S., Samuelson, L.A., Kumar, J., and
Watterson, A.C. (2002) Chemo-enzymatic synthesis and characterization of novel
functionalised amphiphilic polymers. J. Macromol. Sci., Pure & Appl. Chem. Part A, 39,
1137–1149.
7. Danprasert, K., Kumar, R., Chen, M.H., Gupta, P., Shakil, N.A., Prasad A.K., Parmar,
V.S., Kumar, J., Samuelson, L.A., and Watterson, A.C. (2003) Synthesis of novel
poly(ethylene glycol)based amphiphilic polymers. Eur. Polymer J., 39 (10), 1983–1990.
8. Sharma, S.K., Kumar, R., Kumar, S., Mosurkal, R., Parmar, V.S., Samuelson, L.A.,
Watterson, A.C., and Kumar, J. (2004) Chem. Commun., 23, 2689–2691.
9. Kumar, R., Tyagi, R., Shakil, N.A., Parmar, V.S., Kumar, J., and Watterson, A.C. (2005)
Self-assembly of PEG and diester copolymers: Effect of PEG length, linker,
concentration and temperature. J. Macromol. Sci. Pure & Appl. Chem. Part A., 42, 1523–
1528.
10. Kumar, J., Shakil, N.A., Singh, M.K., Pankaj, Singh, M.K., Pandey, A. and Pandey, R.P.
(2010) Development of Controlled Release Formulations of Azadirachtin A employing
Poly(ethylene glycol) Based Amphiphilic Copolymers. J Environ Sc Health, Part B,
45(4), 310-314.
11. Adak, T., Kumar, J., Shakil, N.A. and Walia, S. (2012) Development of controlled
release formulations of imidacloprid employing novel nano-ranged amphiphilic
polymers. J Environ Sc Health, Part B, 47(3), 217-225.
12. Adak, T., Kumar, J., Dey, D., Shakil, N.A. and Walia, S. (2012) Residue and bio-efficacy
evaluation of controlled release formulations of imidacloprid against pests in soybean
(Glycine max). J Environ Sc Health, Part B, 47(3), 226-231.
13. Gupta, B., Kumar, V., Kumar, G., Khan, A., Shakil, N.A., Dhawan, A., Parmar, V.S.,
Kumar, J., and Watterson, A.C. (2011) Amphiphilic Copolymers having Saturated and
Unsaturated Aliphatic Side Chains as Nano Carriers for Drug Delivery Applications. J.
Macromol. Sci. Pure & Appl. Chem. Part A., 48(12), 1009–1015.
14. Adak, T., Kumar, J., Shakil, N.A., Walia, S., Kumar, A., and Watterson, A.C. (2011)
Synthesis and Characterization of Novel Surfactant Molecules Based on Amphiphilic
Polymers. J. Macromol. Sci. Pure & Appl. Chem. Part A., 48(10), 767–775.
15. Sarkar, D.J., Kumar, J., Shakil, N.A., Adak, T., and Watterson, A.C. (2012) Synthesis
and characterization of amphiphilic PEG based aliphatic and aromatic polymers and their
self assembling behavior. J. Macromol. Sci. Pure & Appl. Chem. Part A., 49(6), 455-465.

132 Winter School on "New generation smart agrochemical oriented approaches for crop
and human health management", 07-27 Dec, 2017
16. Kumari, A., Kumar, J., Shakil, N.A., Singh, B.B., and Watterson, A.C. (2015) Synthesis
and Characterization of Amphiphilic PEG Based Polymers and Their Self Assembling
Behaviour. J. Macromol. Sci. Pure & Appl. Chem. Part A., 52(6), 417-424.
17. Koli, P., Shakil, N.A., Kumar, J., Singh, B.B., and Watterson, A.C. (2014) Synthesis and
Characterization of Novel Encapsulating Materials based on Functionalized Amphiphilic
Block Copolymers J Environ Sc Health, Part B, 51(9), 729-736.
18. Kaushik, P., Shakil, N.A., Kumar, J., and Watterson, A.C. (2012) Synthesis and
characterisation of novel poly(ethylene glycol) based amphiphilic polymers. J Environ Sc
Health, Part B, 49(1), 111-115.
19. Loha, K.M., Shakil, N.A., Kumar, J., Singh, M.K., Adak, T. and Jain, S. (2011) Release
kinetics of β-Cyfluthrin from its encapsulated formulations in water. J Environ Sc Health,
Part B, 46(3), 201-206.
20. Loha, K.M., Shakil, N.A., Kumar, J., Singh, M.K., and Srivastava, C. (2012) Bio-efficacy
evaluation of nanoformulations of β-Cyfluthrin against Callosobruchus maculatus
(Coleoptera: Bruchidae). J Environ Sc Health, Part B, 47(7), 687-691.
21. Shakil, N.A., Singh, M.K., Pandey, A., Kumar, J., Pankaj, Parmar, V.S., Pandey, R.P.
and Watterson, A.C. (2010) Development of Poly(ethylene glycol) Based Amphiphilic
Copolymers for Controlled Release Delivery of Carbofuran. J. Macromol. Sci. Pure &
Appl. Chem. Part A., 47, 241-247.
22. Pankaj, Shakil, N.A., Kumar, J., Singh, M.K. and Singh, K. (2012) Bioefficacy
evaluation of controlled release formulations based on amphiphilic nano-polymer of
carbofuran against Meloidogyne incognita infecting tomato. J Environ Sc Health, Part B,
47(6), 520-528.
23. Sarkar, D.J., Shakil, N.A., Kumar, J., and Walia, S. (2012) Release kinetics of controlled
release formulations of thiamethoxam employing nano-ranged amphiphilic PEG and
diacid based block polymers in soil. J Environment Science Health, Part B, 47(11), 1701-
1712.
24. Sarkar, D.J., Shakil, N.A., Kumar, J., and Walia, S. (2012) Quality enhancement of of
soybean seed coated with nano-formulated Thiamethoxam and its retention study. Pestic
Res J, 24(1), 55-64.
25. Kaushik, P., Shakil, N.A., Kumar, J., Singh, M.K., Singh, M.K., and Yadav, S.K. (2013)
Development of controlled release formulations of thiram employing amphiphilic
polymers and their bioefficacy evaluation in seed quality enhancement studies. J Environ
Sc Health, Part B, 48(8), 677-685.
26. Koli, P., Singh, B.B., Shakil, N.A., Kumar, J., and Kamil, D. (2014) Development of
controlled release nanoformulations of carbendazim employing amphiphilic polymers
and their bio efficacy evaluation against Rhizoctonia solani. J Environ Sc Health, Part B,
50(9), 674-681.
27. Singh, B.B., Shakil, N.A., Kumar, J., Walia, S. and Kar, A. (2015) Development of slow
release formulations of β-carotene employing amphiphilic polymers and their release
kinetics study in water and different pH conditions. J Food Sci & Technol, 52(12), 8068-
8076.
28. Sujan Majumder, Najam A. Shakil, Braj B. Singh and Arthur C. Watterson (2016)
Synthesis and characterization of functionalized amphiphilic polymers for utilization as
surfactants. J. Macromol. Sci. Pure & Appl. Chem. Part A., 53(2), 75–81.

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and human health management", 07-27 Dec, 2017
29. Sujan Majumder, Najam A. Shakil, Jitendra Kumar, Tirthankar Banerjee, Parimal Sinha,
Braj B. Singh and Parul Garg (2016) Ecofriendly PEG-based controlled release
nanoformulations of Mancozeb: Synthesis and bioefficacy evaluation against
phytopathogenic fungi Alternaria solani and Sclerotium rolfsii. J Environ Sc Health, Part
B.
30. Pushpendra Koli, Braj B. Singh, Najam A. Shakil, Jitendra Kumar and Deeba Kamil
(2015) Development of controlled release nanoformulations of carbendazim employing
amphiphilic polymers and their bio efficacy evaluation against Rhizoctonia solani. J
Environ Sc Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes
50:9, 674-681.
31. Melanie Kah and Thilo Hofmann (2014) Nanopesticides research: current trends and
future priorities. Environment International, 63, 224-235.

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Synthesis of Amphiphilic Polymers
Parshant Kaushik and NA Shakil
Division of Agricultural Chemicals
ICAR-Indian Agricultural Research Institute, New Delhi

Aim
To synthesize poly (ethylene glycol) (PEG) based amphiphilic copolymers
Principle

Amphiphilic polymers have been used in drug delivery for giving more stable formulations
because of their high solubilisation power and low critical micelle concentration (CMC) value
(Torchilin, 2001). Amphiphilic polymers have been used in delivery systems for encapsulating
both hydrophilic and hydrophobic compound (Shakil et. al., 2010). This approach is based on the
formation of nano-micelles by the self assembly of amphiphilic copolymers in aqueous media.
The micellar size is mainly determined by the hydrophobic forces which sequester the
hydrophobic chains in the core and by the excluded volume repulsion between the chains which
limits their size, a difference in the balance of these two forces copolymers may account for their
different size. Encapsulation process will provide controlled release formulations of pesticides.
Controlled release (CR) formulations permit safer, efficient and economical crop protection.
These also reduce phytotoxicity, degradation, leaching and chemical load in the environment,
enable convenient handling and distribution and an extended release period of chemical.

The unique properties of poly(ethylene glycol) (PEG), including a wide range of solubility, lack
of toxicity, non-interference with enzymatic activities and conformations of polypeptides and
ease of excretion from living organisms, make them ideal drug carrier .

General method of polymerization

Take the monomers, dimethyl 5-hydroxyisophthalate and poly(ethylene glycols) of different


molecular weights, viz 600, 1000, 1500 and 2000 in two necked round bottom flask and keep on
silicon oil bath at 80- 90oC. The reaction is performed under vacuum with constant stirring. After
135 Winter School on "New generation smart agrochemical oriented approaches for crop
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proper mixing of both the reagents, add O O
one drop of concentrated H2SO4 (1% with
O
respect to monomers). Monitor the reaction MeO OMe + HO n OH
by thin layer chromatography (TLC). After
2a-2d
completion of reaction, quench the reaction
by adding chloroform and neutralize H SO
2 4
OH 80-90 0C
unreacted H2SO4 by using NaOH solution. 1
Then evaporate organic solvent vacuum
and dialyze the residue using membrane O O
(MWCO 10000). Freeze-dry the product 7 10
O β
polymers and characterize with the help of MeO O 9 O
OH

1 3 8
H and 13C Nuclear Magnetic Resonance 1 n-1
α
(NMR) (Bruker 400MHz) spectroscopy
5
and determine the particle size by Dynamic 3a-3d m
OH
Light Scattering (DLS) using zetasizer.
General method of alkylation of polymers

Take polymers and bromododecane in equimolar quantities and dissolve in dry acetone. To this
resultant solution, add equimolar amount of anhydrous potassium carbonate. Reflux the reaction
mixture at 60 0C and monitor the reaction by TLC using ethyl acetate in petroleum ether (30%).
After completion, remove potassium carbonate by filtration and solvent under vacuum to get the
products.
O O
Sample preparation for particle size 2 7 10
β
O OH
MeO O 9 O
TM 3 8
Particle size analyzer (Zetatrac ) is based 1 n-1 α
on Dynamic Light Scattering (DLS) which 4 6
5 3a-3d
detects the fluctuation of the scattering m
OH
intensity due to the Brownian motion of
macromolecules or particles in suspension. K2CO3 Acetone / Br y CH3
Reflux
DLS measurements were performed at 25oC y=9
and light scattering is detected at a fixed
angle. Prepare 200 mg L-1 sample solution in
distilled water. To 5 ml of the sample O O
2 7 10 β
solution add minimum quantity (50µL) of O OH
MeO O O
chloroform and sonicate for 5 minutes to 3 1 8 9 n-1 α
4 6
form a proper emulsion. Dual optical probe 5
4a-4d m
technology is used for particle size analysis. O
11
Optical light sources are dual solid-state
y
laser diodes in 780 nm (near-infrared)
21
wavelength.
22
CH3

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Encapsulation of bioactive molecules in Nanosphere

Check the solubility of the synthesized polymers and bioactive molecule in different solvents
then dissolve amphiphilic polymers and bioactive molecule separately and mix together in round
bottom flask at room temperature. In a typical procedure for the encapsulation, stir the solution
for 2 hour. After removal of the solvent, dissolve the residue in water and stir again for the
formation of nano-spheres. In this step, some percentage of bioactive molecule gets encapsulated
in amphiphilic polymer and un-encapsulated/ non-incorporated one precipitates out of the water.
Separate the non-incorporated pesticide from the aqueous layer by filtration. The encapsulated
material is obtained by freeze - drying and lyophilizing (Shakil et. al., 2010).

References

1. Torchilin V.P. (2001) Polymeric Micelles as Targetable Pharmaceutical Carriers.


J.Control. Rel., 73:137.
2. Shakil N.A., Singh M.K., Pandey A., Kumar J., Pankaj., Parmar V.S., Pandey R.P. and
Watterson A.C. (2010) Development of poly(ethylene glycol) based amphiphilic
copolymers for controlled release delivery of carbofuran. J. Macromolecular Science,
Part A: Pure & Applied Chemistry, 47: 241-247.

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Recent Advances in Polymer Composites for Pesticide Delivery

Anirban Dutta and Anupama Singh

Division of Agricultural Chemicals,


ICAR-Indian Agricultural Research Institute, New Delhi – 110012

Target specific delivery of pesticides at the right dose with minimum contamination to the
environment is always a challenge to the formulation chemists. Though drug delivery strategies
applied in medical sciences give some leads in the area of agrochemicals, but one has to
remember that the agricultural system, being complex and nature dependent, is completely
different from that of the arena of medical science. Today control of pests not only depends on
the choice of pesticides, but also on their formulation strategy, delivery and application
techniques. Environmentally safe but effective pesticide formulations are the demand of recent
times. With the advancement of polymer chemistry, material science and nanotechnology,
science has now many options to fulfill the contemporary need. Polymers and polymer
composites are now being widely used for every aspect of life. A plethora of naturally occurring
polymers and synthetic polymers gave mankind the flexibility to choose the right kind of
material for the right purpose. This ever-emerging branch of science thus gives an alternative
option to the formulation chemists for preparation of targeted delivery systems for pesticides
with all necessary qualities which can be customized depending upon the need. This chapter
highlights about the polymer composites, their properties and their possible applications in the
field of pesticide formulations.
Polymer composites: overview
A composite is a material made from two or more constituents with significantly different
physical or chemical properties that, when combined, produce a material with characteristics
different from the individual components1. The individual components or constituents remain
separate and distinct within the finished structure. The different systems are combined
judiciously to achieve a system with more useful structural or functional properties non-
attainable by any of the constituent alone. There are two main categories of constituent materials:
matrix and reinforcement or filler (Fig. 1). At least one portion of each type is required to make
composite material. The matrix material surrounds and supports the reinforcement materials by
maintaining their relative positions. The reinforcements impart their special mechanical and
physical properties to enhance the matrix properties being embedded in the matrix in a
discontinuous form. The basic difference between blends and composites is that the two main
constituents in the composites remain recognizable while these may not be recognizable in
blends.

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Fig 1. Constituents of composites – matrix and reinforcement

Polymers are most common matrices used for polymer composites, while different
materials like fibers (e.g. cellulose, carbon fibers, glass fibers), clays, carbon nano-tubes etc. can
be used as reinforcements. Generally, for the polymer composites polymer matrix acts as a
continuous phase which binds the reinforcement and shares a load with it and nature, texture,
shape, orientation, rate etc. of the reinforcement (dispersed phase) determines the quality of the
composite. The interfacial compatibility between the reinforcement and the matrix ultimately
controls the properties of need based composites.
Properties of different polymers will determine the application to which it is appropriate.
The chief advantages of polymers as matrix are low cost, easy processability, good chemical
resistance, and low specific gravity. On the other hand, low strength, low modulus, and low
operating temperatures limit their use2. Varieties of polymers for composites are thermoplastic
polymers, thermosetting polymers, elastomers, and their blends (Fig. 2).

Fig 2. Types of polymers: thermoplastics, thermosets and elestomers

Thermoplastics consists of linear or branched chain molecules having strong


intramolecular bonds but weak intermolecular bonds. They can be reshaped by application of
heat and pressure and are either semi-crystalline or amorphous in structure. Examples include
polyethylene, polypropylene, polystyrene, nylons, polycarbonate, polyacetals, polyamide-imides,
polyether ether ketone, poly- sulfone, polyphenylene sulfide, polyether imide, and so on. On the

139 Winter School on "New generation smart agrochemical oriented approaches for crop
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other hand, thermosets have cross-linked or network structures with covalent bonds with all
molecules. They do not soften but decompose on heating. Once solidified by cross-linking
process they cannot be reshaped. Common examples are epoxies, polyesters, phenolics, ureas,
melamine, silicone, and polyimides. An elastomer is a polymer with the property of
viscoelasticity, generally having notably low Young’s modulus and high yield strain compared
with other materials. They are amorphous polymers existing above their glass transition
temperature, so that considerable segmental motion is possible. At ambient temperatures, they
are relatively soft and deformable. Natural rubber, synthetic polyisoprene, polybutadiene,
chloroprene rubber, butyl rubber, ethylene propylene rubber, epichlorohydrin rubber, silicone
rubber, fluoroelastomers, polysulfide rubber, and so on are some of the examples of elastomers.
The behavior of a polymer composite can be explained not only on the basis of the
polymer matrix but its combined behavior with the reinforcing element, and the fiber/matrix
interface (also known as the mesophase). To attain superior mechanical properties the interfacial
adhesion should be strong. Matrix molecules can be anchored to the fiber surface by chemical
reaction or adsorption, which determine the extent of interfacial adhesion. The developments in
atomic force microscopy (AFM) and nano indentation devices have facilitated the investigation
of the interface. Shape, size and orientation of the reinforcing materials play major roles in
developing composite materials. The shape of the reinforcing particles can be spherical, cubic,
platelet, or of regular or irregular geometry. Based on the form of reinforcement composites can
be named as, fibrous composites where reinforcement material is filamentous with very high
length to diameter ratio; particulate composites with particles with small dimensions are used as
reinforcement. Whiskers which are short, discontinuous, nearly perfect single crystals with
polygonal cross-sections or flakes which are laminar, layered, two-dimensional sheets are also
being used as reinforcement in different need based polymer composites.
Polymer nanocomposites
Nanocomposites are composite materials, which contain at least one component of nanometric
scale (10-9 m). In case of polymer nanocomposites this component is usually filler, called then
nanofiller. Nanofillers might be classified according to their chemical nature, type of physical
structure, but usually they are classified based on the shape of the particles. There are following
types of nanofillers: 1D - linear (e.g. carbon nanotubes), 2D - layered (e.g. montmorillonite) and
3D - powder (e.g. silver nanoparticles)3. The attractiveness of the nanocomposites is a result of
the fact that polymer matrix and nanofiller interact with each other on molecular level. Due to
that, nanofiller of dimensions below 100 nm, added in small amount to matrix (usually few
percent) might greatly change selected properties of composite material. Production of
nanocomposites might be carried out using the same methods that are usually used for typical
composites, i.e. in-situ or solvent method or usually by mixing melted polymer matrix.
One of the first nanofillers that achieved technological success was montmorillonite that
is layered aluminosilicate from smectite group. This layered nanofiller was used with Nylon-6 to
obtain a nanocomposite which has shown clear improvement of mechanical and thermal
properties. The second group of nanofillers are particles of linear structure in form of nanotubes
or nanofibers. Currently, carbon nanotubes (CNTs) are usually used which are composed of
graphene layers and have good mechanical and electrical properties. Moreover, it is possible to
control electric properties of nanocomposite containing CNTs by chemically modifying nanotube

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surfaces. CNTs have been used in combination with matrices such as polyethylene,
polycarbonate, polystyrene, poly(methyl methacrylate), polylactide, polyvinyl alcohol etc.
Polymer composites: general applications
At present, the world’s total output of composite materials is in megatons4. Polymer composites
holds a significant share of the total production. Composite materials find their applications in
variety of areas including aircraft and aerospace industries, automobiles and transports, marine
industries, civil and constructions, electronics, energy, medical and sports sector. Polymer
composites have wide application particularly in medical industries to prepare artificial implants,
sports industries to prepare different equipments, electronics and electrical industries to prepare
heat and shock resistant materials and many more. While polymer composites are now being
used for target specific and precision drug delivery, their application in agricultural field, in
specific pesticide delivery is at very pre-mature stage. Only recently, the scientific communities
have understood the potential of using polymer composites as carriers for pesticide delivery.
Though cost of preparation, availability of raw materials, infrastructures and other associated
problems had put some initial barriers in the progress of the path of this magnificent technology
to come to its full potential in the area of pesticide delivery, still there is lot of scope for the
technology to flourish in the area of targeted delivery, controlled delivery, sustained delivery and
a rational delivery of pesticides.
Polymer composites: application in pesticide delivery
In the area of pesticide science polymer composites are taking initial baby-steps towards
maturity. A majority of the applications are focusing at developing sensors for pesticides and
manufacturing composites for pesticide removal. However, a potential area of application for
customized polymer composites could be as carriers for pesticide delivery. The age-old problems
of pesticide applications including residue, resistance, environmental load etc. can be addressed
by developing custom-made formulations employing polymer composites with controlled and
targeted release properties. Significant amount of work is going on worldwide to develop such
formulations.
In recent time, polymer-clay composites/nanocomposites have been widely explored for
this purpose. In general, layered silicates have layer thickness on the order of 1 nm and a very
high aspect ratio (e.g. 10–1000). Thus a few weight percent of layered silicates when properly
dispersed throughout the polymer matrix can create much higher surface area for polymer/filler
interaction as compared to conventional composites. Depending on the strength of interfacial
interactions between the polymer matrix and layered silicate, three different types of
composites/nanoconposites are thermodynamically achievable: i) intercalated ii) flocculated or
phase separated and iii) exfoliated (Fig. 3).

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Fig 3. Different types of clay-polymer composites/nanocomposites

If the polymer is unable to intercalate into the galleries, a phase separated composite is
formed, whose properties are similar to that of traditional microcomposites; the poor interaction
between the organic and the inorganic component results in relatively poor mechanical
performance. On the other hand, an intercalated nanocomposite is obtained when extended
polymer macromolecules diffuse between unchanged clay sheets, leading to a well ordered
multilayer structure of alternating polymeric and inorganic layers with a repeating distance of
few nanometers between them5,6. The most significant changes in physical properties are
observed in exfoliated hybrids, where clay layers are separated and uniformly dispersed,
maximizing thus the polymer-clay interactions.
As normal smectites are generally highly hydrophilic species, they are naturally
incompatible with a wide range of polymer types. Thus, the use of modified organoclays, being
organophillic, play pivotal role in most of the clay-polymer composites. Delamination of the clay
depends on various factors including the exchange capacity of the clay, the polarity of the
reaction medium and the chemical nature of the interlayer cations (e.g. onium ions). By
modifying the surface polarity of the clay, onium ions allow thermodynamically favourable
penetration of polymer precursors into the interlayer region. The ability of the onium ion to assist
in delamination of the clay depends on its chemical nature such as its polarity7. The loading of
the onium ion on the clay is also crucial for success. The correct selection of modified clay is
essential to ensure effective penetration of the polymer or its precursor into the interlayer spacing
of the clay and result in the desired exfoliated or intercalated product. Polymer can be
incorporated either as the polymeric species itself or via the monomer, which is polymerized in
situ to give the corresponding polymer-clay nanocomposite. Polymers can be introduced either
by melt blending (extrusion) or by solution blending. Melt blending (compounding) depends on
shear to help delaminate the clay and can be less effective than in situ polymerization in
producing an exfoliated nanocomposite. The large variety of polymer systems are used in

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nanocomposites preparation with layered silicates e.g. vinyl polymers, condensation polymers,
polyolefins, heterocyclic polymers, biodegradable polymers etc.
One of the most considerable effects of clays in the polymer matrix properties is the
dramatic improvement of barrier properties of polymers. Clay sheets are naturally impermeable.
Clays increase the barriers properties of polymers by creating a maze or tortuous path that retards
the diffusion of gas molecules through the polymer matrix (Fig. 4). The degree of enhancement
in the barrier properties depends on the degree of tortuousity created by clay layers in the
diffusion way of molecules trough the polymer film. The tortuous factor is determined by the
ratio of actual distance which diffusive molecule is walked to the shortest distance to diffuse
(polymer film thickness). This factor is affected by the aspect ratio of clay dispersed in the
matrix. Increasing the lateral length of clay sheet as well as increasing of exfoliation or
dispersion degree cause to the more barrier enhancement in the polymer matrix. It has also been
observed that the addition of platelet fillers like as layered silicates, effectively improves the
anticorrosive barrier effect of polymer coatings by increasing the length of the diffusion
pathways for aggressive species. Clay materials due to their platelet structure and high aspect
ratio, in well dispersed state, decrease the permeability of polymer coating films by increasing
the diffusion pathways.

Fig 4. Mechanism of barrier improvement by the addition of clay platelets in polymer matrix

A significant number of recent literatures reported preparation of such clay-polymer


composites for controlled pesticide delivery. A novel composite gel composed of
carboxymethyl-chitosan and bentonite was used as the carrier for encapsulating atrazine and
imidacloprid to control their release in water and retard their leaching in soil8. According to the
results of release experiments in water, the composite carrier showed double advantages of both
encapsulation by the polymer and sorption by the bentonite. The time taken for 50% of active
ingredients to be released, was prolonged for both the pesticides. Controlled release formulations
of metribuzin were prepared by carboxymethyl cellulose-kaolinite composite9 and
polyacrylamide-bentonite composite10. Both the formulations showed prolonged release of
metribuzin. Similar results were obtained for acephate when it was encapsulated in
carboxymethyl cellulose-kaolinite composite11. A controlled release formulation of imazapyr
was prepared by binding the anionic herbicide to polydiallyldimethylammonium-chloride
(PDADMAC)-montmorillonite composites12. Imazapyr release from the controlled release
formulation applied on a thin layer of soil was found substantially slower than its release from
143 Winter School on "New generation smart agrochemical oriented approaches for crop
and human health management", 07-27 Dec, 2017
the commercial formulation and leaching was found significantly less. A modified kaolin-
alginate-chitosan composite bead had shown slow and sustained release of acetamiprid to
promote the efficient use of pesticide to reduce pollution effect on the environment13. In another
study, kaolin and bentonite were used as filler materials to control the release profile of thiram
from starch-alginate matrix14. Similar study reported controlled isoproturon release profile from
carboxymethyl starch-montmorillonite composite microparticles15. Biopolymeric clay hydrogels
composites were synthesized from crosslinking of carboxymethyl cellulose with citric acid in the
presence of bentonite, which were used to develop base triggered release formulations (TRFs) of
thiamethoxam through ex-situ encapsulation technique16. The study showed higher release rate of
thiamethoxam at alkaline pH than neutral condition. A number of similar studies are available
where clay polymer composites have been explored for controlled delivery of pesticides.
Though clay polymer composites have remained the primary focus for developing
pesticide formulations due to their relatively cost effectiveness and ease of preparation, polymer
composites with other types of fillers or reinforcements have also been explored. The mechanism
of controlled release for such composites is to provide barrier properties just like clay
composites. However, some fillers like activated carbons, carbon nanotubes provide extra
adsorption of loaded pesticide and thereby prolonged release17. Nano silver or copper themselves
provide fungicidal activity as well as controlled release properties18,19.
Conclusion
Polymer composites hold a huge potential to become the future of pesticide delivery strategy.
Much research still remains in order to understand the complex structure-property relationships
in various nanocomposites which can be used for pesticide delivery. Novel composites with
different biopolymers and cheap fillers can be explored to develop cost effective and efficient
delivery systems. With the current need of environment-friendly, biodegradable agrochemical
formulations, polymer composites are holding the key of future.
References
1. Jose JP, Malhotra SK, Thomas S, Joseph K, Goda K and Sreekala MS (2012) Advances
in polymer composites: macro- and microcomposites – state of the art, new challenges,
and opportunities. In: Polymer composites: volume 1, First edition, S Thomas, K Joseph,
SK Malhotra, K Goda and MS Sreekala (Eds), Wiley-VCH Verlag GmbH & Co., pp 3-
16.
2. Huang H and Talreja R (2006) Numerical simulation of matrix micro-cracking in short
fiber reinforced polymer composites: initiation and propagation. Compos Sci Technol,
66(15), 2743-2757.
3. Barton J, Niemczyk A, Czaja K, Korach L and Sachermajewska B (2014) Polymer
composites, biocomposites and nanocomposites: production, composition, properties and
application fields. CHEMIK 2014, 68(4), 280-287.
4. Wang RM, Zheng SR and Zheng Y (2011) Polymer matrix composites and technology.
First edition, Woodhead Publishing Limited, pp 568.
5. LeBaron PC, Wang Z and Pinnavaia TJ (1999) Polymer-layered silicate nanocomposites:
an overview. Appl Clay Sci, 15, 11–29.

144 Winter School on "New generation smart agrochemical oriented approaches for crop
and human health management", 07-27 Dec, 2017
6. Sinha Ray S and Okamoto M (2003) Polymer/layered silicate nanocomposites: a review
from preparation to processing. Prog Polym Sci, 28, 1539–1641.
7. Dutta A and Singh N (2015) Surfactant-modified bentonite clays: preparation,
characterization, and atrazine removal. Environ Sci Pollut Res, 22, 3876–3885.
8. Li J, Yao J, Li Y and Shao Y (2012) Controlled release and retarded leaching of
pesticides by encapsulating in carboxymethyl chitosan /bentonite composite gel. J
Environ Sci Health, Part B, 47(8), 795-803.
9. Kumar J, Nisar K, Shakil NA, Walia S and Prasad R (2010) Controlled release
formulations of metribuzin: Release kinetics in water and soil. J Environ Sci Health, Part
B, 45(4), 330-335.
10. Sahoo S, Manjaiah KM, Datta SC, Shabeer TPA and Kumar J (2014) Kinetics of
metribuzin release from bentonite-polymer composites in water. J Environ Sci Health,
Part B, 49(8), 591-600.
11. Nisar K, Kumar J, Shakil NA, Pankaj, Walia S and Parmar BS (2009) Controlled release
formulations of acephate: water and soil release kinetics. J Environ Sci Health, Part B,
44(6), 533-537.
12. Radian A and Mishael YG (2008) Characterizing and designing polycation-clay
nanocomposites as a basis for imazapyr controlled release formulations. Environ Sci
Technol, 42(5), 1511-1516.
13. Yan H, Chen X, Wu T, Feng Y, Wang C, Li J and Lin Q (2014) Mechanochemical
modification of kaolin surfaces for immobilization and delivery of pesticides in alginate-
chitosan composite beads. Polym Bull, 71(11), 2923-2944.
14. Singh B, Sharma DK, Kumar R and Gupta A (2009) Controlled release of the fungicide
thiram from starch–alginate–clay based formulation. Appl Clay Sci, 45, 76-82.
15. Wilpiszewska K, Spychaj T and Pazdzioch W (2016) Carboxymethyl
starch/montmorillonite composite microparticles: properties and controlled release of
isoproturon. Carbohydr Polym, 136, 101-106.
16. Sarkar DJ and Singh A (2017) Base triggered release of insecticide from bentonite
reinforced citric acid crosslinked carboxymethyl cellulose hydrogel composites.
Carbohydr Polym, 156, 303-311.
17. Fernández-Pérez M, Villafranca-Sánchez M, Flores-Céspedes F, Garrido-Herrera FJ and
Pérez-García S (2005) Use of bentonite and activated carbon in controlled release
formulations of carbofuran. J Agric Food Chem. 53(17), 6697-6703.
18. Sambhy V, MacBride MM, Peterson BR and Sen A (2006) Silver bromide
nanoparticle/polymer composites:  dual action tunable antimicrobial materials. J Am
Chem Soc, 128(30), 9798-9808.
19. Cioffi N, Torsi L, Ditaranto N, Tantillo G, Ghibelli L, Sabbatini L, Blevezacheo T,
D’Alessio M, Zambonin PG and Traversa E (2005) Copper nanoparticle/polymer
composites with antifungal and bacteriostatic properties. Chem Mater, 17(21), 5255-
5262.

145 Winter School on "New generation smart agrochemical oriented approaches for crop
and human health management", 07-27 Dec, 2017
Innovative Controlled Nutrient Delivery Devices
Suman Manna and Anupama Singh

Division of Agricultural Chemicals


ICAR-Indian Agricultural Research Institute, New Delhi

Introduction
Green revolution has revolutionized the agricultural productivity by adopting semi-dwarf
varieties of cereals which enables global agriculture to feed world’s current population.
According to the Food and Agriculture Organization, fertilizer globally contributes 40-60% of
the yield increases (FAO, 2012). But the excessive use of fertilizer also called for negative
consequences like green house gas emission, increased nutrient leaching, nutrient run-off,
eutrophication, etc.
Nutrient use efficiency (NUE) is a critically important concept in the evaluation of crop
production systems. It can be greatly impacted by fertilizer management as well as by soil- and
plant-water management. The objective of nutrient use is to increase the overall performance of
cropping systems by providing economically optimum nourishment to the crop while minimizing
nutrient losses from the field. NUE addresses some but not all aspects of that performance.
Therefore, system optimization goals necessarily include overall productivity as well as NUE.
Thus nutrient use efficiency is decreased due to losses of nutrients in the environment. Rakshit et
al (2015) reported that nutrient use efficiency (NUE) of different nutrients as 30-50% nitrogen,
~18% phosphorous, ≥80% potassium, ~11% sulphur and ≤5% micronutrients require for
agricultural production. Thus poor nutrient use efficiency leads to decreased agricultural
production. Thus to increase the nutrient use efficiency the fertilizer nutrient should be
formulated such a way so that the loss of the nutrients become minimum. In this context slow or
controlled release fertilizers/nutrients come into the picture which have low water solubility
thereby release the nutrients over an extended period of time. Different the slow release crop
nutrient formulations are discussed below.
Classification of controlled release fertilizers (CRF)
CRFs may be classified in the following way.
1. Organic-N-Low-Solubility Compounds: These can be subdivided into biologically
decomposing compounds usually based on urea-aldehyde condensation products, such as
urea-formaldehyde (UF), urea-triazone (UT), crotonylidene diurea (CDU), and
chemically decomposing compounds, such as isobutylidene-diurea (IBDU). Succinctly,
UF is prepared by reacting excess urea under controlled conditions of pH, temperature,
U-F ratio, and reaction time. UT solution is based on the reaction of urea-ammonia-
formaldehyde. CDU is prepared by reacting urea with acetaldehyde under the catalysis of
an acid. IBDU is prepared by reacting liquid isobutyraldehyde with solid urea.

2. Fertilizers in Which a Physical Barrier Controls the Release: These can be


subdivided into granules coated by hydrophobic polymers or as matrices in which the
soluble active material is dispersed in a continuum that restricts the dissolution of the
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fertilizer.The coated fertilizers can further be divided into fertilizers with organic polymer
coatings that are either thermoplastic or resins and fertilizers coated with inorganic
materials such as sulphur or mineral based coatings. The materials used for preparation of
matrices can also be subdivided into hydrophobicmaterials such as polyolefins and rubber
and gel-forming polymers (hydrogels) which are hydrophilic in nature.

3. Inorganic Low-Solubility Compounds. This type of CRFs includes fertilizers such as


metal ammonium phosphate (e.g., MgNH4PO4) and partially acidulated phosphate rocks
(PAPR). Besides, the biologically and microbially decomposed N products, such as UF,
are commonly referred to in the trade as slow-release fertilizers and coated or
encapsulated/ occluded products as controlled-release fertilizers.

Different types of slow release fertilizers:


Slow release potash fertilizer
Slow release fertilizers serves as a modern and advanced way of supplying nutrients to
crops. Different approaches have been made to develop slow release potash fertilizer which
minimizes the nutrient loss. Slow release can be achieved by using coatings of different materials
like waxes, plaster of paris and resins. Pellet formulation of potash was prepared by mixing
potash with wet clay. To ensure same dimension to all the pellets, casting is done in an unique
cylindrical mold. The dried pellet is coated with toothpaste to ensure proper attachment of
polyacrylamide to the surface of pellet. The toothpaste also supplies Ca as secondary nutrient.
The pellet is then dipped in polyacrylamide solution to get the polymer coated pellet. The
purpose of choosing polyacrylamide is it prevents soil erosion.
The developed pellets were dipped in 100 mL and 200 mL water. The release study of the
developed pellets showed in fig. 1. It can be concluded that lower the amount of potash, lower
the dissolution rate and slow release of fertilizer (Subbarao et al., 2013).

Fig.1. Dissolution rate of potash with and without coating in 100 mL of water
Effect of water on dissolution of potash was studied by fixing the amount of potash to
0.25 g in 100 mL and 200 mL water. From the figure it is evident that in the absence of coating

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only 0.18 gm out of 0.25 gms of potash could be leached in one hour where as in presence of
coating, 0.11 gms of potash could be leached there by justifying the purpose of coating.

Fig. 2. Release of potash from pellet with or without coating at 100 mL of water
Slow release formulation of nitrogenous fertilizers
Sulphur coated urea (SCU)
Sulfur-coated urea was one of the first CRF and nitrogen release is controlled by the thickness of
the sulfur coating (Goertz 1993). SCU was developed at the Tennessee Valley Authority in 1961.
It is prepared by spraying molten sulphur over granular urea to yield a product containing
between 31 to 38% N. A wax sealant is then sprayed to seal the cracks in the coating and thereby
reduce the leakage and microbial degradation of the S coating. This is followed by a layer of a
conditioner such as attapulgite or talc. The wax sealant may be replaced by an organic polymer
layer to produce a polymer-coated SCU called PSCU. One example of a polymer coating over
SCU is produced by fi rst coating an isocyanate-reactive component that includes a polyether
(polyol) onto fertilizer particles and then applying an isocyanate component. Polymer-cum-
sulphur-based coatings of different fertilizers and SCU form the majority of coated products now
in practice.
Polymer-coated multi-nutrient fertilizers
Polymer coated controlled release fertilizer (PCRF) of N, P, and K has been developed and the
coated fertilizer particle is known as ‘prill’. Nutrient release has been controlled by chemical
composition and thickness of the coating material. This technology was applied to for other
secondary and micronutrient also. The release rate of nutrients can be as short as 3 moths or as
long as 18 months.

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Fig. 3. Polymer coated controlled release fertilizers or ‘prills’ (A and B). Dissolution of nutrients
inside prills, moving towards the soil or growing medium along an osmotic gradient.
The release of nutrients from prills occurred in two way. When exposed to soil or growing
medium, the moisture entered into prills, condensing the soluble fertilizer nutrients which
increase the osmotic pressure of the prills. Secondly, the increased pressure within the prills
causes fertilizer ion to diffuse into the surrounding medium (fig. 3C).
Types of polymer coated controlled release fertilizers
Several brand names are available in market for PCRF for agricultural use and can be
categorized based on nutrient content, release pattern and longevity (Jacobs et al., 2003).
Osmocote®: Osmocote is a coated NPK fertilizer that releases nitrogen, phosphate, potassium,
trace elements over a pre-chosen period of time. It is one the oldest PCRF and its coating is
classified as polymeric resin. Osmocote fertilizers can release nutrients for a period of 3-4
months to 14-16 months. A wide variety of Osmocote PCRF is available for different crops and
production cycles including a “miniprill” formulation (Figure 3B). The nutrients encapsulated in
the organic resin coating are protected from leaching and releases only a little every day.
Throughout the period the level of release of nutrients is related to the temperature only, no other
external stimuli influence the release. The above release period is based on the average
temperature of 21 oC, at warmer climate zone or in green house, if used, its release is accelerated
and subsequently release period will be shorter. At lower temperate climate the release will slow
down and longevity will be extended.

Fig. 4. Release mechanism of nutrients from osmocote®

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Every granule of osmocote® is covered with an organic, semi-permeable coating of
biodegradable resin made of vegetable oils. After application of Osmocote®, water penetrates
through the semi-permeable coating and starts to dissolve the nutrients present in the granule.
The release of nutrients starts once they have been partially dissolved. A pump-like action is
initiated due to differences in osmotic pressure. The plant is able to take up the released
nutrients. After releasing all the nutrients, the membrane starts to degrade.
Different types of osmocote are available in market viz.Osmocote Pro, Osmocote Start 6W,
Osmocote Exact Mini 3-4M, Osmocote Bloom 2-3M etc.
Apex®: It is N, P, K formulation which utilizes two reactive monomers over fertilizer core in a
continuous coating drum, resulting in an ultrathin polyurethane membrane coating. The resultant
PCRF delivers nutrient through a solute concentration gradient permeation process that is
unaffected by soil moisture, microbial activity, or pH levels. A variety of Apex formulations are
available in market. Apex Native, is specially formulated for plants that are sensitive to high
rates of P, and therefore aids in the colonization of mycorhizal fungi (Simplot, 2008).
Multicote® and Nutricote®: They use thermoplastic resin coatings blended with special release-
controlling agents to determine the nutrient release rate and longevity. 2 brands of PCRF—
Multicote® are available in the U.S. and Canada, and Nutricote®, which is only available in the
western U.S. (Sun Gro Horticulture 2008). Multicote® is available in a wide variety of nutrient
formulations with release rates from 4 to 16 months (Table 1).
Diffusion®: These polymeric fertlizers are customized for different temperature zones, and come
in many nutrient formulations with longevities from 3 to 9 months (Green Valley Agricultural
2008).
Table 1. Macro nutrient composition (N-P-K) and longevity of polymer-coated controlled release
fertilizers

Longevity at 21 Osmocote Apex Multicote Diffusion


o
C classic
3 to 4 months 14-14-14 - 15-7-15 17-6-17, 22-2-3
19-6-12 18-6-18
5 to 6 months - - 15-7-15 17-6-17, 22-4-9
18-5-18
8 to 10 months 13-13-13 13-13-13 15-7-15 17-6-17, 22-4-8
19-6-12 16-8-16, 18-6-12 17-7-14 18-4-18
12 to 14 months 19-6-12 17-6-12 14-7-14, 20-5-12 -
17-6-14
14 to 16 months 19-6-12 16-5-11 14-7-14, 20-5-10 -
17-5-14

Slow release urea fertilizers based on acrylamide copolymer


Most of the soil applied urea fertilizers (40-60%) is lost due to leaching, volatilization and other
processes. Thus several efforts were taken for the development of controlled release formulations

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based on polymers which acts as soil conditioners. Urea granules were coated with the
copolymer of acrylamide and divinylbenzene (DVB)/N,N’-methylenebisacrylamide
(NNMBA)/tetraethyleneglycol diacrylate (TTEGDA) or pentaerythritol triacrylate (PETA) and
sealant materials (wax and polystyrene). Among the crosslinking agents, DVB is rigid and
hydrophobic in nature. TTEGDA is very flexible and hydrophilic and NNMBA is in between
DVB and TTEGDA in these properties. PETA is a trifunctional hydrophilic monomer and was
included in the study to determine the suitability of the peculiar three-dimentional net structured
copolymer that it forms with acrylamide as a coating material. Urea content (%)of the developed
coated fertilizer were as follows: DVB-urea (84.1), NNMBA-urea (88.1), TTEGDA-urea (81.6)
and PETA-urea (82.7).The release behavior (in term of leaching loss) of the polymer coated urea
fertilizers (PCUF) in soil column showed urea coated with copolymer of acrylamide-
tetraethyleneglycol diacrylate was found to be having a better slow-release property among the
nnother polymer coated fertilizers (Table 2). More than 80% of applied N was leached out from
the uncoated urea by 2nd day only (Abraham and Pillai, 1996).
Table 2. Total N (%) leached from PCUF and controlled systems

Time (days) DVB-urea NNMBA-urea TTEGDA-urea PETA-urea Control-urea


1 22.9 24.0 20.0 23.3 42.4
2 23.1 24.4 20.8 20.3 41.6
7 22.2 23.3 20.9 25.4 5.5
14 8.4 8.7 7.0 13.3 1.3
21 4.0 3.1 5.9 4.9 0.8
28 1.5 1.5 3.9 1.6 0.4
45 0.3 0.8 2.8 0.3 0.2

Other controlled release fertilizer formulation:


Nanoclay polymer composite: Nanoclay–polymer composite (NCPC) superabsorbent nutrient
carriers were prepared. These NCPCs were based on the reactions of different types of nanoclays
(10 wt %) with partially neutralized acrylic acid and acryl amide by a free-radical aqueous
solution copolymerization reaction with N,N’-methylene bisacrylamide as a crosslinker and
ammonium persulfate as an initiator (Sarkar et al., 2014). Kaolinite, mica and
montmorillonitewere tried for the NCPC’S. The water absorbency decreased in the NCPCs
compared to that of the pure polymeric hydrogel. In case of the pure polymer, the entire amount
of nutrient loading released within 15 h of incubation; this was higher than that of the NCPCs. In
the initial stage (up to 15 h), no significant differences in nutrient release were observed among
the different polymer/clay composites, but there were differences in later stages. Among the
different NCPCs, the percentage release of nutrients at 48 h ranged from around 70% in the
polymer/clay montmorillonite composite to 90% in the polymer/clay kaolinite composite.
Microcapsule fertilizer from ethylene vinyl acetate polymer: Slow release microcapsules
containing iron fertilizer was made and its release pattern evaluated (Abedi-Koupai et al., 2012).
Three polymers ethyl cellulose (EC), glycerol mono stearate (GMS) and Compritol 888 ATO,
were mixed with avicel and lactose and the microcaopsules were made by

151 Winter School on "New generation smart agrochemical oriented approaches for crop
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extrusion/spheronization method. The dissolution test carried out to determine the Fe release rate
versus time. The results showed that, microcaopsules containing Compritol 888 ATO, released
iron ions slower than that of EC and GMS. The release rate reached 85% after 7 days. In order to
reduce the release rate, these microcaopsules were coated with ethylene vinyl acetate (EVA)
polymer by dip coating method. It is concluded that the combination of matrix type formulation
and the coating with EVA polymer caused a significant decrease in the release rate of iron.
Advantages of Using Polymer-Coated Controlled-Release Fertilizers
Polymeric coated controlled release fertilizers offers following advantages to crop plants over
conventional fertilizers.
(1) Easy to adjust fertilization type and rate for different crops: Growers can easily customize
their fertilization programs due to wide variety of NPK formulations and nutrient release
timings
(2) Better fertilizer use efficiency: As the PCRF are placed directly in the root zone of crop,
its use efficiency is sufficiently high compared to other conventional and liquid
fertilizers. There is also least chance of loss of fertilizer nutrients from PCRF.
(3) Less fertilizer pollution in wastewater: The loss of fertilizer in terms of runoff and
leaching from PCRF is less thereby decreasing the possibility of fertilizer pollution.
(4) No rinsing required after fertilization: In fertigation method the concentrated fertilizer
solution need to be rinsed off from plant foliage to prevent burning. But in PCRF, there is
no need of rinsing as the fertilizer is placing in the root zone and the release from
formulation is controlled.
(5) Nutrients present at root initiation: Incorporation of PCRF in soil helps in root
development of crops. The nutrients are available as soon as root forms.
References
1. FAO (Food and Agriculture Organization of the United Nations), 2012. World
Agriculture towards 2030/2050, the 2012 Revision. Retrieved from. http://www.
fao.org/docrep/016/ap106e/ap106e.pdf.
2. Rakshit A, Kumari S, Pal S, Singh A and Singh HB (2015) Bio-priming mediated
nutrient use efficiency of crop species. Nutrient Use Effic. Basics Adv. 181-191.
3. Subbarao CV, Karthik G and Sirisha D (2013) Slow release of potash fertilizer through
Polymer coating. International Journal of Applied Science and Engineering 11(1): 25-30.
4. Jacobs DF, Rose R and Hasse DL (2003) Incorporating controlled release fertilizer
technology into outplanting. In: National Proceedings: forest and conservation nursery
association-2002, Riley LE, Dumroese RK, Landis TD, technical coordinators. Fort
Collins (C)O): USDA Forest Service, Rocky Mountain Research Station. Proceedings
RMRS-P-28. p 37-42.
5. Simplot (2008) Apex nursery fertilizer: a higher standard in plant nutrition. URL:
http://www.simplot.com/turf/apex/index.cfm (accessed 27th July, 2017).
6. Sungrow Horticulture (2008) Nutricote controlled release fertilizer. URL:
http://www.sungro.com/products_displayProBrand.php?brand_id=6 (accessed 27th July,
2017).

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7. Green Valley Agricultural (2008) Diffusion controlled release fertilizers. URL:
http://www.diffusionfertilizer.com/7128.html (accessed 27th July, 2017).
8. Abraham J and Pillai VNR (1996) Membrane-encapsulated controlled-release urea
fertilizers based on acrylamide copolymers. Journal of Applied Polymer Science, 60:
2347-2351.
9. Sarkar S, Dutta SC and Biswas DR (2014) Synthesis and characterization of nanoclay–
polymer composites from soil clay with respect to their water-holding capacities and
nutrient-release behavior. Journal of Applied Polymer Science, 131: 39951-39958.
10. Abedi-Koupai J, Varshosaz J, Mesforoosh M and Khoshgoftarmanesh AH (2012)
Controlled release of fertilizer microcapsules using ethylene vinyl acetate polymer to
enhance micronutrient and water use efficiency. Journal of Plant Nutrition, 35: 1130–
1138.

153 Winter School on "New generation smart agrochemical oriented approaches for crop
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Advances in the Development, Regulation and Use of Biopesticides
against Insects
Opender Koul
Insect Biopesticide Research Centre
30 Parkash Nagar, Jalandhar-144003, India

It has been estimated that global population will touch 10.12 billion by 21001, therefore, demand
for food will grow that would need increased agricultural production. In order to increase the
productivity, the appropriate pest and disease management is required along with the suitable
yield enhancement from crops2. Chemical pesticides do take care of such pests to a larger extent,
but at the same time, they are hazardous tothe environment and human health. Thus best
approach during last few decades has been the research and development of biopesticides;
suggested also to reduce the overall costs of pesticide applications3. Another important
component is the biological control by natural enemies, though only 15% of them have been
identified. In all successful biocontrol programs; most important parasitoids are Hymenoptera
and predators (Neuroptera, Hemiptera, and Coleoptera). Globally more than 125 species of
natural enemies are commercially available for biological control programs such as
Trichogramma spp.; Encarsiaformosa, and Phytoseiuluspersimilis4.Biopesticides which are
generally termed as environmentally friendly also help in reducing the chemical pesticide load5.
As of today, several biopesticides have been developed frommicroorganisms (bacteria, fungi,
viruses, nematodes, and protozoans), plant products (botanical biopesticides), biochemicals
(semiochemicals) and genetically modified organisms. Globally, there are 175 registered
biopesticide active-ingredients and 700 products available in the market. The global market for
biopesticides was valued at the US $1.3 billion in 2011, and it is expected to reach the US $3.2
billion by 2017 end. The increasing demand for residue-free crop production is one of the key
drivers of the biopesticide market. The growing organic food market and easier registration than
chemical pesticides are other important driving factors for the growing biopesticide market6,7.
However, it will be beyond the scope of this compilation to deal with all aspects of
biopesticides against a variety of pests, therefore, the objective here will be to concentrate on the
advances, regulation, and use of biopesticides against insects and also to demonstrate the
commercial potential of such products.

Microbial biopesticides
Out of all the biopesticides used today, microbial biopesticides constitute the largest group of
broad-spectrum biopesticides, which are pest specific (i.e., do not target non-pest species and are
environmentally benign). There are at least 1500 naturally occurring insect-specific
microorganisms, 100 of which are insecticidal8. Over 200 microbial biopesticides are available in
30 countries affiliated to the Organization for Economic Co-operation and Development
(OECD)9. There are 53 microbial biopesticides registered in the USA, 22 in Canada and 21 in the
European Union (EU) 10, 11 although reports of the products registered for use in Asia are
variable. Overall, microbial biopesticide registrations are increasing globally, the expansion of
various technologies has increased the scope for more products and the change in the trend to

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develop microbial products is definitely on the rise 12, 13. A comprehensive list of commercial
microbial biopesticides developed has been documented earlier14.

Bacteria
The bacterial microbial control agents include gram-positive, spore-forming Bacillus
thuringiensis (Bt) and Lysinibacillus sphaericus (Ls), the gram-negative, non-spore-forming
Serritia entomophila, and most recently Chromobacterium substsugae. Out of these various
subspecies of Bt are commonly used microbial agents globally and have over 50% of all
microbial product sales15. The majority of Bt products are largely used against lepidopterans and
some are also potentially effective against forest pests, mosquitoes and black flies16. Insecticidal
activity of Bt and Ls is due to protein toxins occurring in parasporal inclusions, which become
toxic after ingestion by the pest. The toxins become solubilized in the high pH of the midgut
where the gut proteolytic enzymes cleave them to toxic moieties. These toxins recognize
receptors and perforate enterocytes that ultimately causes cell rupture, compromising the
integrity of the gut epithelium. Various examples of target pests on various crops are shown in
Table 1. Details of host range factors affecting larvicidal activity, safety and production of
entomopathogenic bacteria have been comprehensively discussed recently16.

Viruses
A wide range of viruses have been identified from a variety of insects and arthropods and about
16 families of viruses have been recorded17. The major ones include baculoviruses,
polyhedroviruses, entomopoxviruses, and nudiviruses. Baculoviruses are the major class of
viruses that host about 600 species of insects 18, 19. Most commonly used strategy for their use is
inundation augmentative application. Out of 4 genera of baculoviridae the alpha baculovirus
(nucleopolyhedrovirus; NPVs) and betabaculovirus (granuloviruses; GVs) are widely used
microbes20. The differences between the two are mainly the host range and proteinaceous
occlusion bodies (OBs). The morphology of NPVs has been comprehensively discussed recently
21
and they may have wide host range19 like AucaMNPVs, which are active against 95 species in
15 families of Lepidoptera or some may have the specific host as in Lymantria
dispar22.Generally, baculovirus OBs act via ingestion mode enter the midgut epithelium and
multiply and subsequently invade other tissues which lead to replication and occlusion.
Baculoviruses are the only ones that have been mass-produced, commercialized and applied
widely. All baculovirus products produced today are from insects under laboratory conditions 23.
Usually, the insects are harvested from the fields and inoculated in production facilities to obtain
the virus in bulk. Field production of baculoviruses is also economical for developing countries
where farmers can easily afford to have low-cost products of several baculoviruses24.

Fungi
There are reports to demonstrate that about 1000 species of fungi cause fungal diseases in
arthropods25, 26, thus potential microbes to control insects and mites. These fungi mostly belong
to Hypocreales and Entomophthorales. The common species of such entomopathogens are
Beauveria bassiana, Metarhizium species, Isaria fumosorosea, and Lecanicillium species (Table
1). These fungi attack the cuticle of insects after spores germinate on the surface of the host and
germ tube penetrating the cuticle via lytic enzymatic activity. Following the growth of hyphae
and blastospores within the host, sporulation of the fungus occurs on the host cuticle. Faria and
Wraight (2007) have recorded 110 commercial products based on entomopathogenic fungi and

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40% of these are B. bassiana based products and 39% are based on M. anisopliae. Other
products include B. brongniarii, Isarea fumosorosea, I. farinosus, Lecanicillium longisporum
and L. muscarium. Several products have been introduced in Asian and Latin American markets
and even several multinational companies like BASF, Monsanto, DuPont, etc. are now producing
entomopathogen based products 28. A large number of infectious propagules like aerial conidia or
blastospores are important components for inundative treatment with foliar applications.
Quantitatively fungal spores at the rate of 2 × 1012 to 5 × 1013/ha are usually required to control
pests under field conditions29. Therefore, the commercial success of the products is directly
related to efficient and cost- effective mass production. Mass production of entomopathogenic
fungi has been comprehensively reviewed30.

Table 1. Some examples of microbial entomopathogens

Microbe group Specific microbe Target Crops


VIRUSES
NPV HearSNPV, HezeSNPV Helicoverpa and Corn, cotton,
(Alphaviruses) Heliothis spp. soyabean,
SemNPV tobacco
Spodopteraexigua Vegetables and
ornamentals
GV CpGV Cydiapomonella Pome fruit and
(Betaviruses) walnut
AdorGV Adoxophyesorana Apple
BACTERIA
Bacillus thuringiensis subsp. Lepidoptera Vegetables and
kurstaki and aizawai fruits, forest
and stored
products
Bacillus thuringiensis sub sp. Diptera: Culicidae,
israelensis Simuliidae Aquatic habitats

Lysinibacillus spharicus Diptera: Culicidae Lentic aquatic


habitats

Serratia entomophila Costelytrazea landica Pasture

Chromobacterium subtsugae Leptinotarsa Diverse crops


decemlineata and some
Hemiptera and Acarina

FUNGI
Hypocreales Beauveria bassiana Broad host range Diverse crops

Metarhizium anisopliae Very broad host range Diverse crops

Metarhizium acridum Grasshoppers, locusts Greenhouse


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crops
Isaria fumosorosea Hemiptera Diverse crops

Lecanicillium sp. Hemiptera Diverse crop


Entomophthorale Neozygitestanajoae Mononychellus tanajoa Cassava
s
Entomophaga maimaiga Lymantria dispar Deciduous trees
NEMATODES
Steinernema carpocapsae Broad host range Field crops,
orchards
Steinernema feltiae Coleoptera, Lepidoptera Vegetables,
ornamentals
Steinernema scapterisci Orthoptera Lawn and turf

Heterorhabditis Coleoptera, Lepidoptera Vegetables,


bacteriophora ornamentals,
turf mushrooms,
orchard
Heterorhabditis megidis Coleoptera Lawn and turf
Modified from the Source: Lacey (2016)

Nematodes
Entomopathogenic nematodes (EPNs) as insect control agents have been studied for decades and
number of them have been reported from insects 31, 32, 33. Amongst these nematodes from two
families, Steinernematidae and Heterorhabditidae are major microbial agents of this class and
they have mutual and obligate association with Xenorhabdis and Photorhabdis bacteria carried
inside the gut of infective juveniles (IJs). It is these IJs that actively seek a host insect. They gain
entry into the host insect via spiracles or via inter-segmental membranes. Inside the host the
mutualistic bacteria are liberated that kill the host and then digest host tissue. Subsequently, IJs
moult and start feeding; there could be just one generation or in some cases 2 to 3 generations as
well depending upon the nutrients available. Once nutrients exhaust, the IJs leave the host and
then search for the new host. Production and application technology is critical for the success of
EPNs in biological control. Production approaches include in vivo, and in vitro methods (solid or
liquid fermentation). For laboratory use and small scale field experiments, in vivo production of
EPNs appears to be the appropriate method. In vivo production is also appropriate for niche
markets and small growers where a lack of capital, scientific expertise or infrastructure cannot
justify large investments into in vitro culture technology. In vitro technology is used when large-
scale production is needed at reasonable quality and cost. Infective juveniles of
entomopathogenic nematodes are usually applied using various spray equipment and standard
irrigation systems. Enhanced efficacy in EPN applications can be facilitated through improved
delivery mechanisms (e.g., cadaver application) or optimization of spray equipment. Substantial
progress has been made in recent years in developing EPN formulations, particularly for above
ground applications, e.g., mixing EPNs with surfactants or polymers or with sprayable gels. Bait
formulations and insect host cadavers can enhance EPN persistence and reduce the number of
nematodes required per unit area. This subject has been comprehensively reviewed recently 34.

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Protozoans
Although ptotozoans infect a wide range of pests naturally and induce chronic and debilitating
effects that reduce the target pest populations, the use of protozoan pathogens as biopesticide
agents has not been very successful. Protozoa are taxonomically subdivided into several phyla,
some of which contain entomogenous species. Microsporan protozoans have been investigated
extensively as possible components of integrated pest management programmes. Microsporidia
are ubiquitous, obligatory intracellular parasites that are disease agents for several insect species.
Two genera, Nosema and Vairimorpha, have some potential as they attack lepidopteran and
orthopteran insects and seem to kill hoppers more than any other insect35. A study of Nosema
pyrausta, a microsporidium infecting the European corn borer, Ostrinia nubilalis, suggests that
in a horizontal transmission, a spore is eaten by a European corn borer larva, which germinates in
the midgut, extrudes a polar filament and injects sporaplasm into a midgut cell. The sporaplasm
reproduces and then forms more spores, which can infect other tissues. Spores in infected midgut
cells are sloughed into the gut lumen and are eliminated along with feces to the maize plant.
These spores remain viable and are consumed during larval feeding so that the infection cycle is
repeated in midgutcells of the new host. If a female larva is infected, Nosema is passed to the
filial generation by vertical transmission. As the infected larva develops through to an adult the
ovarial tissue and developing oocytes become infected with N. pyrausta. The embryo is infected
within the yolk and when larvae hatch, they are infected with N. pyrausta. Both horizontal and
vertical transmissions maintain N. pyrausta in natural populations of European corn borer. N.
pyrausta suppresses populations of European corn borer by reducing oviposition, percentage
hatch and survival of infected neonate larvae 36. The only protozoan registered for use as a
biopesticideis the microsporidian, Nosema locustae, which infects grasshoppers. This organism
is most effective when ingested by nymphal stages of grasshoppers and kills them within three to
6 weeks post-infection 36. However, not all infected grasshoppers are killed by this protozoan
infection.

Future Scenario
Some recent studies reveal that pattern and impact of microbial toxins vary from species to
species, depending on the ecosystem, the route of exposure and the non-Bt control against which
effects are quantified 37. As such, the effect of microbial biopesticides on microbial communities
must be carefully monitored38.In fact, there is a need for well-defined selection criteria and a
complete process description for the development of a microbial pest control product. For a
commercial microbial product, three specific criteria for selection are required, i.e. toxicity,
production efficiency and safety of the product. That means while screening process toxicity of
the product will be relative to dose rate, mode-of-action, the speed of kill, host range, sensitivity
to abiotic factors and persistence. Secondly, mass production will be critical criteria and should
be a high-yield-oriented process. Thirdly, the safety of product will be essential in relation to
registration requirements and the costs involved. An important question, however, is when
microbial biopesticides are appropriate? Generally, scientists and biocontrol companies seem to
develop their products without a well-developed plan, though the approach should be to develop
a product to solve a problem or to grasp an opportunity. It is essential to make a detailed
characterization like which pest, crop, region, time of the problem, solutions available,
acceptable costs and market potential. If these aspects are considered and details are provided, a
potential microbial product could be obtained. Therefore, recommended steps to obtain a good
microbial pest control product would be: (i) collection of isolates and identification of perfect

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isolate, (ii) laboratory screening for efficacy, (iii) assessment of production efficiency, (iv)
mode-of-action and toxicological properties, (v) glasshouse trials and (vi) evaluation of efficacy
under commercial conditions. If all these factors are considered, success is inevitable.
The roadmap to successful development and commercialization of a microbial pest
control product is amply illustrated in new flow diagrams recently, which provide the details of
various phases’ involved and output information leading to consecutive steps for decision
making and ultimately the market potential28. Commercialization is the final and most difficult
step in the development of a microbial product. The most critical factors are developmental cost
and time to market. Costs amount to US$14 – 21 million for a new entrepreneur and the time to
market including registration is no less than 5–7 years. Therefore, to examine all these critical
factors in the successful commercialization of microbial pest control products is essential in the
developmental process of a product and these critical factors have been comprehensively
discussed earlier28.

Botanical biopesticides

Commercial use of phytochemical biopesticides began in the nineteenth century with the
introduction of nicotine from Nicotiana tabacum(L.), rotenone from Lonchocarpus sp., derris
dust from Derris elliptica (Wallich) Benth and pyrethrum from Tanacetum
cinerariifolium(Trevir) (previously Chrysanthemum cinerariifolium). The use of these
compounds, their efficacy, and commercial potential has been comprehensively discussed39. This
successful use of traditional botanicals has aroused further interest in exploring plant
biodiversity40 for new bioactive phytochemicals and extractives as a possible source of pest
control agents 41, 42. In fact, several species of a wide diversity of plants are known to have anti-
insect properties. During last decade there has been specific emphasis on essential oils and the
components there in. In fact, Pesticides based on plant essential oils or their constituents have
demonstrated efficacy against a range of stored product pests, domestic pests, blood feeding
pests and certain soft-bodied agricultural pests, as well as against some plant pathogenic fungi
responsible for pre- and post-harvest diseases. They may be applied as fumigants, granular
formulations or direct sprays with a range of effects from lethal toxicity to repellence and/or
oviposition deterrence in insects. These features indicate that pesticides based on plant essential
oils could be used in a variety of ways to control a large number of pests 43. Various compounds
isolated with insect control potential have been comprehensively dealt with in various studies
mentioned above. However, there are some recent leads too to demonstrate that there is ample
scope for finding newer molecules in future. For example, phytochemical investigation of the
chloroform extract of Tinospora cordifolia yielded a new clerodane diterpenoid tincordin along
with tinosporide, 8-hydroxytinosporide, columbin, 8-hydroxycolumbinand 10-hydroxycolumbin.
The structure of the new compound was elucidated comprehensively using 1D and 2D NMR
methods. All major clerodane diterpenoids isolated were tested for their efficacy as insect
antifeedants against Earias vitella, Plutellaxy lostella and Spodoptera litura 44. The activity of
these compounds ranged between 65.0 to 80.0% at a concentration of 10.0 µg/cm2 for all the 6
compounds. Similarly, Pieris formosa is a poisonous plant to livestock and is used as an
insecticide in rural areas of China. Two novel polyesterified 3,4-seco-grayanane diterpenoids,
pierisoids A and B, were isolated from its flowers and were identified by spectroscopic analysis
and X-ray diffraction. Both compounds showed obvious antifeedant activity against cotton

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bollworm, indicating their toxic properties, suggesting a defensive role of polyesterified 3,4-
seco-grayanane diterpenoids for P. formosa against herbivores45.

TincordinTinopsoride 8-Hydroxytinosporide

Columbin 8-Hydroxycolumbin 10-Hydroxycolumbin

PierisoidsA : R = COCH2CH3
PierisoidsB : R = COCH3
Another potential example is of four prenylated flavonoids, isoglabratephrin, (+)-
glabratephrin, tephroapollin-F and lanceolatin-A, that were isolated from Tephrosia apollinea L.
and tested against three stored grain insects. A nutritional bioassay, using a flour disc and test
concentrations of 0.65, 1.3 and 2.6 mg/g, revealed a significant reduction in the relative growth
rate, relative consumption rate and efficiency of conversion of ingested food by all insects. The
studies show that these compounds could be potential antifeedants and activity significantly
varies in relation to structures among the tested flavonoids46.

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Isoglabratephrin (+)-Glabratephrin Tephroapollin-FLanceolatin-A

Therefore, it is obvious that botanicals have greater scope if more potential molecules are
isolated and various technologies developed that can extend the efficacy of botanical insecticides
over longer periods of time. A better understanding of the behavior and bioactivity of individual
components of botanical insecticides coupled with more advanced methods of
compartmentalization and formulation will allow greater degrees of control over the availability
and activity of individual components of complex botanical mixtures and, consequently, should
enhance the efficacy of botanicalinsecticides47.
Future Scenario
The practice of using plant biodiversity as a bioresource allows us to develop and exploit
naturally occurring plant defense mechanisms, thereby reducing the use of conventional
pesticides. Biodiversity-rich countries like Brazil, Columbia, China, and India should quickly
survey their traditionally used flora to document pesticidal plants. Appropriate protection of
species and ecological communities needs to be put in place. Such efforts will help protect the
biodiversity resource from threats. A sound knowledge of the biodiversity
resource is key to reduce biopiracy (the commercial development of naturally occurring
biological materials, such as plant substances or genetic cell lines, by a technologically advanced
country or organization without fair compensation to the peoples or nations in whose territory the
materials were originally discovered) and establish each country’s sovereign right to any
botanical pesticides developed from such plants. The Nagoya protocol48 emphasizes the fair and
equitable sharing of the benefits arising from the utilization of genetic resources. This could be
achieved by appropriate access to genetic resources and by appropriate transfer of relevant
technologies, taking into account all rights over those resources and to technologies, and by
appropriate funding, thereby contributing to the conservation of biological diversity and the
sustainable use of its components.
The literature survey as of today suggests49 that greater efforts are required to investigate
the utility of plant extracts for crop protection with trials in farmer’s fields. In terms of the future
research in order to make botanical insecticides as potential commercial products, few aspects
need to be considered. Advanced extraction procedures need to be followed that will preserve the
integrity of phytochemical mixtures. Formulation strategies need to be modified and
nanotechnology can definitely help in preventing unwanted reactions, however, complexity and
cost-effectiveness could be the constraints 47. The delivery of the products under field condition
requires specific technologies that will provide quantitative and qualitative release of botanical
products for potential control of pests. Use of botanicals as biorational mixtures50, 51, 52, 53 will

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also help in preventing resistance development and several such studies are progressively taking
shape and these could be useful for future development of potential products

Semiochemicals
A semiochemical is a chemical signal produced by one organism that causes a behavioural
change in an individual of the same or a different species. Insect sex pheromones are a major
class of such chemicals used in crop protection. Many of them have been synthesized and used in
crop protection via mass trapping technology for monitoring of insect pestsand subsequently,
lure-and-kill systems and mating disruption is induced to control pest populations. Worldwide,
mating disruption is used on over 660 000 ha and has been particularly useful in orchard crops 54.
Generally, semiochemicals are utilized for the management of insect pests through the
detection of invasive species, monitoring the populations of endemic species to synchronize the
timing of insecticide treatments and increasing the effectiveness of biological control by
increasing the predation/parasitism rates of predators and parasitoids. Kairomones could also be
applied to plants to increase the rate of parasitization through increasing the search rate of
parasitoids 55. Attract and kill approach is also a useful technique wherean attractant or
semiochemicalis usedto lure an insect to a point source that contains a killing agent. The
technique leads to the reduction of the insect population by killing the target insect or reducing
its fitness and fecundity or disabling it by causing disease55.
Another strategy for semiochemical-based control is the push-pull strategy that involves the
behavioural manipulations of insect pests and their natural enemies by the use of behaviour
modifying stimuli which makes the main crop comparatively unattractive and unpalatable to the
pests while diverting them to the more attractive sources from where they are removed. The
push-pull effect is established by the use of exploiting semiochemicals which deter the pests
from the main crop (“push”) and attract them into trap crops (“pull”). Intercropping or
companion cropping is done for semiochemical delivery which masks host stimuli and insects
are repelled56. Farming systems for pest control, based on the stimulo-deterrent diversionary
strategy or push–pull system, have become an important tool in sub-Saharan Africa using a
combination of companion plants delivering semiochemicals, as plant secondary metabolites, for
smallholder farming cereal production, initially against lepidopteran stem borers. The same
strategy is being used for other regions as well for mainstream arable farming systems. A good
push and pull example where secondary metabolites like (E)-4,8-dimethyl-1,3,7-nonatriene
repelling pests and attracting beneficial insects is well known57.

Future Scenario
For improvement of semiochemicals, understanding the mechanisms of communication systems
of insects, behaviour and mating systems among target insects andnon-target organisms is
important. At the same time theeffects of different meteorological and
physiochemicalcharacteristics of insects and plants should be understood. Delivery systems for
controlled released strategies and stability of products are a constraint that needs to be looked
into. Future research on semiochemicals for insect pest management also should focus on
innovative formulation for field deployment as well as on optimization of controlled-release
technologies and trapping efficiency. More research on the chemical ecology of target insect pest
is of paramount importance for the development of semio-chemically-based insect pest
management programs.

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Commercialization
As per the latest report on the global biopesticides market, it is estimated to be valued at USD
3.36 Billion in 2016 and projected to reach USD 8.82 Billion by 2022, at a CAGR of 17.4% from
2016. The global biopesticides market has been segmented on the basis of type, crop type, origin,
formulation, and mode of application. It has been further segmented on the basis of a region into
North America, Europe, Asia-Pacific, and the Rest of the World. The main objectives of the
report are to define, segment, and project the size of the global market for biopesticides, with
respect to the above-mentioned segmentation and to provide a detailed study of key factors
influencing the growth of the market, along with profiling the key players in the market and their
core competencies.
Worldwide there are about 1400 biopesticide products being sold58. At present, there are
68 biopesticide active substances registered in the EU and 202 in the USA. The EU biopesticides
consist of 34 microbials, 11 biochemicals and 23 semiochemicals59, while the USA portfolio
comprises 102 microbials, 52 biochemicals and 48 semiochemicals 60. To put this into context,
these biopesticide products represent just 2.5 percent of the total pesticide market. However, a
number offeatures of the agricultural economy make it difficultfor companies to invest in
developing new biopesticide products and, at the same time, make it hard forfarmers to decide
about adopting the new technology. Main reasons for this are lack of profit from niche market
products, fixed costs, Farmers risk aversion and IPM portfolio economies 54.

Regulation barriers
There are a variety of organisms and chemicals that come under biopesticide umbrella, therefore,
there are vast differences in their characteristics, modes-of-action and behaviour. They also come
under global regulatory regimes in order to protect the life and environment.
Thus only authorized biopesticide products can be used legally for crop protection. The guidance
of the Organization of Economic Corporation Development (OECD) is that biopesticides should
only be authorized if they pose minimal or zero risks. For example, the OECD guidance for
microbial biopesticides is that: ‘the micro-organism and its metabolites pose no concerns of
pathogenicity or toxicity to mammals and other non-target organisms which will likely be
exposed to the microbial product; the microorganism does not produce a known genotoxin; all
additives in the microbial manufacturing product and in end-use formulations are of low toxicity
and suggest little potential for human health or environmental hazard’61. The constraints for
biopesticide registration data are lots of data generation and involved costs. In fact, information
about the mode of action, toxicological and eco-toxicological evaluations, host range testing, etc.
is required.Until very recently, it is true to say that government regulators—with the probable
exception of the USA—were unfamiliar with biologically based pest management and were
therefore slow to appreciate the need to make the regulatory process appropriate for biopesticides
rather than treat them in the same way as synthetic chemical pesticides. Some regulatory
authorities, the UK, for example, have acknowledged that basing the regulatory system for
biopesticides on a chemical pesticides model has been a barrier to biopesticide
commercialization. Actually, regulatory innovation is possible but pieces of evidence are
required to demonstrate that candidate biopesticides presents minimal risk and the regulators can
modify the data requirements. For example, the OECD regards semiochemicals used for
arthropod control as presenting the minimal hazard, with straight chain lepidopteran pheromones
that form the majority of semiochemical-based biopesticides being thought sufficiently safe as to
justify ‘substantial reductions in health and environmental data requirements’61.Similarly, in case

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of botanical biopesticides the actions were taken in Canada by the Pest Management
RegulatoryAgency, which approved an experimental use permit allowing the aerial application
of neem for control of forest - defoliating sawflies based on HPLC analysis of the neem
concentrate in which the major ten limonoids, accounting for 90% of the UV - visible material,
were identified and quantified62. In the United States, the regulatory changes have led to
streamlining regulatory processes to favour products that are ‘generally regarded as safe’
(GRAS) and allow botanicals as a category different from conventional pesticides 63. In India, to
achieve this goal, provisional registrations have been given to manufacturers and the products are
being sold in the market. However, it is imperative for producers to fulfil the requirements within
the period stipulated by the regulatory authorities. Western countries should adopt this policy if
biological pesticides are to make any impact in the near future in the conventional insecticide
market. Neem has already provided a modern paradigm for the development of biopesticides and
others have to follow the direction.

Future prospects
It is quite obvious that for more than five decades synthetic chemical pesticides have played a
significant role in crop protection, however, their use is definitely declining due to new
legislation and their hazardous environmental impacts globally. Therefore, strategic alternatives
have been researched for a long time now and biopesticides are making an impact. As discussed
above, biopesticides include the variety of products based on micro-organisms, boatnicals,
semiochemicals and even the natural enemies have been included under biopesticide umbrella.
While all these have potential for management of pests, the regulatory barriers have hindered
their inclusion because most of the regulations are similar to the ones used for chemical
pesticides that impose heavy costs on the biopesticide industry. Technical barriers for production
of biopesticides are also impediments for their development. Accordingly, innovative agriculture
policy for their regulation is necessary. There are also new opportunities for developing
biopesticides in integrated pest management (IPM) by combining ecological science with post-
genomics technologies. The new biopesticide products that will result from this research will
bring with them new regulatory and economic challenges that must be addressed through joint
working between social and natural scientists, policy makers and industry.
In fact, biopesticides are attracting global attention as safer strategy to manage pest
populations such as weeds, plant pathogens and insects while posing less risk to human being
and the environment, however, regulatory constraints need to be managed, though it is important
to maintain the quality and availability of the biopesticides at affordable cost, and public-private
sector approach to the development, manufacturing and sale of environmentally friendly
alternatives to chemical pesticides is required. Research in production, formulation and delivery
may greatly assist in the commercialization of biopesticides. The interest in organic farming and
pesticide residue-free agricultural produce would certainly warrant increased adoption of
biopesticides by the farmers. Awareness campaign among the farmers, manufacturers,
government agencies, policymakers and the common men to switch-over to biopesticides for
pest management requirements is necessary. It is also believed that biological pesticides may be
less vulnerable to genetic variations in plant populations that cause problems related to pesticide
resistance. If deployed appropriately, biopesticides have potential to bring sustainability to global
agriculture for food and feed security.

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52. Pavela R (2015) Acute toxicity and synergistic and antagonistic effects of the aromatic
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http://www.epa.gov/oppbppd1/biopesticides/

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Major Characterization Techniques for Nanoformulations
Pradip Roy*, Nabanita Mukherjee, Satakshi Basu, Samarendra Barik and Arunava
Goswami
Agricultural and Ecological Research Unit, Indian Statistical Institute,
203 B. T. Road, Kolkata 700108.

Abstract
Nanotechnology research in India has started growing with moderate amount of public funding
in the last five years. Many researchers have opined that food, agriculture and medicine will be
immensely benefitted by this new technology. However, there are a large number of cross-roads
where necessary infrastructure has to be installed. One of them is installation of regulatory
agency for approval of indigenously produced nano-formulations (with nanoparticles) as
fertilizers, pesticide, food, food supplements and drugs (both allelopathic and ayurvedic) etc.
When submitted to regulatory agency for approval, the producer has to supply active ingredient
(nanoparticle) in pure form and other ingredients like surfactants, co-surfactants and stabilizers
etc. separately as well as in full formulation form. This review focuses on the types of
formulations which can be prepared with naked, quantum, solid, encapsulated nanoparticles
(within the definition of nano-formulations) and their typical characterization techniques.

Different types of conventional formulations and nano-formulations


Formulation in traditional sense applies to homogeneous and stable mixture of active and other
ingredients (stabilizers, stickers etc.) which make the final product simpler to use in agricultural
field (e.g. nano-agro-chemicals1), as drug (e.g., veterinary animals2, human body3) & food
additives etc. Even though, while preparing nano-formulations, usually chemically suitable
surfactants, co-surfactants, stabilizers, stickers are used, one has to make sure that these sets of
chemicals do not alter the chemistry of nanoparticles. Some of the important parameters are
thermodynamics of mixing, phase equilibrium, solutions, surface chemistry, colloids, emulsions
and suspensions4. Now-a-days, sophisticated commercial liquid and solid nano-formulations
often contain active ingredient(s) along with surfactants5, dispersants6, wetting agents7, solvents8,
emulsifiers9, defoamer10, stabilizer11, anti-microbials12, anti-freeze13, pigments/colorants14,15,
buffers16, etc. Nano-formulations are defined as formulations which contains nanoparticles alone
or in combinations with other ingredients as stated above. Quantum and naked nanoparticles
used for making formulations remain in non-equilibrium state and the property of each
nanoparticle (even made from the same metal) depends on the size, shape, structure etc.17. Nano-
formulations of these kinds have spontaneous tendency of going back to constituent phases.
Some nano-formulations possess relatively very high kinetic energy and remain stable for several
years18. The reason for this kind of behaviour is not well understood. Majority of the
formulations made using naked and quantum nanoparticles are covered by patents and very little
information is available in public domain. In fact, it is generally assumed that formulations
techniques so far used for other larger nanoparticles, chemicals or protein or drugs will remain
applicable for aforesaid special nano-formulations. Till date, all nano-formulations prepared
using solid and encapsulated nanoparticles have been made based on the conventional techniques
and they have worked well19.

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Different kinds of nano formulations and examples
Some of the advanced applications of nanoformulations to agriculture, food and medicine are
given below: (a) fluorescent biological labels for pathogen detection in agro-processed foods20,
(b) pesticide, drug and gene delivery21-23, (c) detection of pathogens24, (d) detection of foreign
proteins in the food and vaccines25, (e) probing of viral as well as other pathogen DNA and RNA
in food and medicine26, (f) tissue engineering or making scaffolds27, (g) tumour destruction via
heating (hyperthermia)28, (h) MRI contrast enhancement29, (i) pharmacokinetic studies30 etc. A
few examples which are connected to agriculture, food and medicine industry is given below.
For precision farming, carbon nanotubes, dendrimers and nano-cantilevers of many dimensions
are now used by many farmers in USA, Japan, China and EU31-33.They are effective enough to
indirectly measure carbon footprints as well34. These nanoparticles are mixed with several
formulation components to bring stability for use in open field conditions. Nano-encapsulation
and controlled release method have revolutionized the domain of pesticide and herbicide35,36.
The nano-formulations can be dissolved more effectively in water and can be mixed with gels,
creams and inorganic / organic liquids so that the resultant product would have multiple
applications for preventive measures, treatment or preservation of the harvested product 37-42.
Syngenta’s prime growth regulating product, Primo MAXX (nano-emulsion), which if applied
prior to the onset of stress such as heat, drought, disease or traffic can strengthen the physical
structure of turf grass, and allow it to withstand ongoing stresses throughout the growing
season43. Nanoparticle farming has shown that alfalfa plants grown in gold rich soil absorb gold
nanoparticles through their roots and accumulate these in their tissues. The gold nanoparticles
attached to alfalfa proteins and lipids can be mechanically separated from the plant tissue
following harvest44. Nanocheck (Altairnano Inc.) contains lanthanum nanoparticles that absorb
phosphates from ponds and prevents growth of algae. This nanoformulation with some
modification can be geared to benefit commercial fish farmers which spend huge amounts of
money to remove algae from their ponds per year45. For building smart packaging, on demand
preservatives, and interactive foods, all food giants like Nestle, Kraft, Heinz, and Unilever are
screening thousands of nanocapsules containing flavour or colour enhancers, or added nutritional
elements (such as vitamins) and some of the products are already and more will be in the nano-
food market in the next decade46. A large number of examples of applications of nano-
formulations can be obtained from the internet and study resources47. A particular area of
application is highlighted below, which is important for Indian agro-industry (tea industry) and
have not been highlighted in any other review. After seasonal pruning of the highly valuable
commercial trees, shrubs and during clone preparation, non-toxic and ecologically safe paints
should be used48. A large number of conventional paints have been developed over years, but
none of them could combine all the requirements in a single paint formulation. Nano-composite
coatings are produced by ball milling of paint materials to nanometer level, which can be later
transformed into a polymeric viscous fluid product and can be used as paint48. Effective choice
of nanoparticles and successful choice of formulation technique could give many advantages of
multi-purpose coatings with a little cost difference49. Among notable differences is development
of scratch resistant coatings to self cleaning surface coatings as well as corrosion resistant
coatings to weathering resistance coatings49-54. Unique physical and chemical property displayed
by nanoparticles in the size range below 30 nm can be harnessed, for example, when nano-TiO2,
nano-SiO2 and nano-ZnO are used55-57. They are non-toxic in nature. Tea industry would be one
of the notable targets for spray after seasonal pruning. Micron size based fillers used for making
nano-formulations which generate matt or semi-matt appearance and cause notable amount

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scattering of visible light58. Nanoparticle based fillers (30 to 70 nm size) reduces scattering
significantly. Nano-SiO2 embedded in UV curable lacquers (less than 30 nm) shows improved
abrasion resistance and lead to low pigment loading per gram of coatings and due to transparent
appearance, the coatings will not hamper photosynthesis and transpiration rate of plants59. Nano-
formulations can be classified as follows- (1) Dry-Sprayable- (WP: Wettable powders and WG
or WDG: Water dispersible granule)60; (2) Liquid Sprayable- (SL: Soluble Concentrate; SC:
Suspension Concentrate; EC: Emulsifiable Concentrate; ME: Microemulsion; OD: Oil
Dispersion; CS: Microencapsulated Particles61; (3) Dry-Spreadable Granule (GR: Soil applied
Granule on inert carrier)62. Properties of some of the heavily used aforesaid formulations are- (i)
WP (Wettable Powder): Solid final product where nanoparticles has been added in powder form
and typically applied as suspended particles after dispersion in water. Basic solid active
ingredients (a.i.: 5%-75%) will be nano-powder (nanotized to 30 nm-70 nm) along with
negatively charged anionic dispersant and anionic / non-ionic wetting agent. Preparation is
simple and easy to mix with water, but dusty and the characterisation of the a.i. is difficult as the
preparation settles down when dispersed in water and therefore needs sophisticated instruments
like FE-SEM (Field Emission Scanning Electron Microscope)63; (ii) Suspension concentrate (a.i.
5%-70%): A stable suspension of solid nanoparticle (less than 30 nm) in a fluid usually intended
for final use with water. Ideally the suspension should be stable and should not settle out. Soild
nanoparticles are mixed with anionic / non-ionic dispersant and polymeric viscosity stabilizers
are added. One of major properties is that the a.i. nanoparticles must be water insoluble with
friable crystals64; (iii) Emulsifiable concentrate (EC): A long term stable solution of liquid
nanoparticles (a.i. 4%-40%) with emulsifying agents in a water insoluble solvent is EC65. Apart
from the aforesaid three, another one area which has advanced quite well is the emulsification
techniques of nanoparticles. They are loosely classified as `nano-emulsions’. Nano-emulsions
fall in the category of EC, but they exhibit novel thermodynamic properties due to presence of
nanoparticles as a.i66. Three major components of nano-emulsions are oil, surfactant /co-
surfactant and aqueous phase. Following both high energy and low energy and combined
methods are used- (a) High energy: Most commonly used methods are ball mill, high energy
centrifugation followed by stirring, ultrasonic emulsification and homogenization and all these
might include micro-fluidics and membrane emulsification67; (b) Low energy: Most commonly
used methods are emulsion inversion point method, phase inversion point methods and
spontaneous emulsification68; (c) Combined method: Using both high energy and low energy
emulsification methods, as stated above, reverse nanoemulsions are prepared in highly viscous
systems69.

Different characterization techniques


In nano-formulations, nanoparticles prepared by both `top-down’ and `bottom-up’ methods are
used. Nanoparticles prepared by top-down are often heterogeneous in molecular size, distance
and stoichiometry whereas nanomaterials prepared by `bottom-up’ methods are relatively more
homogenous in nature70. Direct quantification of smaller size (<10 nm) nanoparticles are very
difficult due to scale and difficulties in handling of nanomaterials. Special techniques like ultra-
centrifugation, light scattering, surface plasmon resonance, mass spectrometry, atomic force
microscopy, electron microscopy, NMR and X-ray crystallography are used71. Indirect methods
like mathematical modelling for determination of stoichiometry, structure, size and distance are

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often used by industries72. A number of other studies (thermodynamic stability studies, droplet
size analysis, refractive index, active ingredient content analysis etc.) are performed on nano-
formulations for better characterization of the formulations. (1) Measurement of refractive
index and active ingredient: For solid including liposomal nano-formulations, the refractive
index, n, of a medium is defined as the ratio of the speed, c, of a wave such as light or sound in a
reference medium to the phase speed, vp, of the wave in the medium represented by following
equation- n=c/vp. The value is determined using an Abbes type refractrometer73. Active
ingredient content is determined by gas chromatography (GC) and reverse phase HPLC method
using different columns of appropriate porosity74. (2) Thermodynamic stability studies:
Nanoformulations are subjected to various storage conditions of temperature and humidity to
assess their stability as per ICH [International Council for Harmonisation of Technical
Requirements for Registration of Pharmaceuticals for Human Use (ICH)] guidelines Q1A (R2).
Physical and chemical stability of nanoemulsion are evaluated for six months by storing the
formulation at 300C ± 200C / 65 ± 5% RH and 400C ± 200C / 75 ± 5% RH. To estimate
metastable nano-formulations (as declared by the party), thermodynamic stability tests are
performed. Among of these tests, simplest one is where formulations are centrifuged at 3500 rpm
for 30 minutes and checked whether those formulations show any phase separations take for the
heating and cooling cycle. Similarly, in another case, formulations are taken through six cycles
of low (4ºC) and high temperature (45ºC) over a period of 48 hours and stability is judged by
HPLC. The formulations which show stability in the aforesaid temperature cycles are subjected
to harsh conditions in the form of freeze-thaw cycle test. Three freeze-thaw cycles with
temperature range –21ºC and +25ºC are applied. Those formulations that survive thermodynamic
stability tests are selected for further studies75. (3) Droplet size analysis and light scattering:
The droplet size of the nano-formulations (where a.i. is dispersed well) is determined by photon
correlation spectroscopy76. One of the most popular methods is dynamic light scaterring. In
short, the formulation (0.1 mL) is dispersed in 50 mL of water in a volumetric flask and gently
mixed by inverting the flask. Scattering is measured by using a Zetasizer and light scattering
monitor at 25ºC at 90ºor 180º angle77. Laser light incident on a sample of nano-meter sized
(preferentially spherical in shape) nanoparticles in water scatter into a random pattern of spots of
varying size, shape and intensity. The pattern of spots is the result of interference of light waves
emanating from nanoparticles. If the nanoparticles are well dispersed (i.e. poly-dispersity index
is low) then nanomaterials would be in constant Brownian motion inside the cuvette of the DLS
machine. As the incident light intensity changes, the pattern also would also change continuously
and the intensity of the light at particular scattering angle is measured. Noise or random
fluctuations can be effectively deducted from the effective value using signal-computed
correlation (statistical or small modelling) function or power-spectrum (or wavelength
representation). The verification of Brownian motion and use of aforesaid measurement methods
would lead to the measurement of value of the diameter of nanomaterials78. This method works
quite well in the size range of 30-50 nm, as very small nanoparticles generate rapid fluctuations
in scattered light intensity and very big nanoparticles show higher diffusion coefficient. An
upscale method for determining molecular weight of nanomaterials present in nano-formulations
is the use of size exclusion chromatography with online light scattering and UV and UV
absorbance detection (SEC-LS). This method offers almost absolute determination of weight-
average solution molecular weight (Mw). The size exclusion column chromatography is used
solely for the separation of molecular species. Molecular weights of the separated species are
calculated from the column and matched with combination of results obtained from light

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scattering and UV spectrophotometry. In this way negative aspects of both column
chromatography (i.e. interaction with column matrix) and light scattering (round shape) can be
avoided to a large extent79.

Figure 1. Light scattering studies using silica nano-formulations in dilute human serum (Rahman,
Brahmachary and Goswami, 2017, unpublished data).
(4) Surface plasmon resonance: Metal nanoparticles used as active ingredient in the nano-
formulations are characterized by surface plasmon resonance or SPR. If the poly-dispersity index
is low, then thin metal films (plasmon) reflect incident light a narrow band80. A small fraction of
the incident light at a defined angle interacts with the delocalized electrons in the metal and
reduces the reflected light intensity. If binding takes place to the sample immobilized onto the
plasmon then local refractive index changes. This leads to a significant change in SPR angle.
SPR can estimate real time molecular interactions and it is possible to immobilize molecules of
interest and monitor interactions with their binding partners in solution under different divalent
metal ion concentration, pH etc. Following such approaches, both association and dissociation
data are obtained and thus overall affinity for complex formation as well as stoichiometric data
for interacting components is determined81. (5) Mass spectroscopy: Mass spectroscopy and
electrospray (including MALDI spectroscopy) are used for determining the mass of the
nanoparticles in nano-formulations. Mass spectroscopy can measure mass with an accuracy
factor of 0.01% of the total molecular weight of the sample (i.e. within 0.3 da for a sample of 1
Kda). This measurement is sufficient to detect batch to batch variation of nano-formulations
containing nano-encapsulated pesticide molecules. Several mass spectrometers, when placed in
tandem can give detail structural information by fragmenting the sample and analysing the
products generated82. On the other hand, electrospray or MALDI spectrometers rely on different
methods. In this method, nanomaterials in the nano-formulations are ionized and molecular
weights are measured. Ions formed from nano-materials are sorted by a number of mass
analysers (quadruples, time-of-flight analysers, magnetic sectors and both Fourier transform and
quadruple ion traps) and all these mass analysers work according to their mass-to-charge (m/z)
ratio. Both these methods have advantages and disadvantages. For example, mass spectrometry
is more accurate than ionization methods when samples are measured in water, but might give
noise when nanoformulations are made in buffers and pure salt solutions. On the other side,
nanoformulations from any materials can be used for mass spectrometry whereas electrospray
works well with metal nanoparticles and not well with semi-conductor nanoparticles83. (6) AFM
and Cryo-AFM: Atomic force microscopy (AFM) allows us to gather the topographical data of

173 Winter School on "New generation smart agrochemical oriented approaches for crop
and human health management", 07-27 Dec, 2017
nanomaterials present in the nano-formulations on the order of tens of thousands or more. A
significant advantage of this method is that one can use directly very dilute solution of nano-
formulations and collect data. In this method, the surface contours of the nanomaterials are
obtained with a surface force probe. The probe goes very near to the surface of the nanomaterials
and depending on the minute attractions or repulsion experienced by the probe, detail picture of
the nanomaterials are obtained. Since the distance between the probe and the sample is very
small, the probe gets a lot of freedom to move and scan over a very small carpet area of the
surface of the nanomaterials. The probe in AFM also is used sometimes to pull-apart multi-
domain nanoparticles and characterize individual domains84. For many nanomaterials, due to
softness of the sample, AFM does not work well as the molecular vibration is high at room
temperature. When taken to crystalline phase, these samples give lower noise. For these kinds of
nano-formulations, Cryo-AFM works effectively near or below 77 K. At extremely low
temperature, the microscope reduces the possible thermal motion of the nanomaterials with
multiple units85. (7) Conventional and high resolution transmission electron microscopy:
Microscopes are always ranked on the basis of their spatial resolution power86. Conventional
transmission electron microscopes (CTEM) usually have following essential components and
have many of the same optical principles of the light microscope- electron beam production
system and condenser, image-producing system, image-recording system, pumps and gauges,
valves and power supplies and a vacuum system87. Since electrons are tiny and easily can get
deflected by hydrocarbons or gas molecules, so a series of high level pumps are used to produce
high vacuum in the electron travel path. Rotary or roughing pump are used in the first step so
that electrons travel in the 10-3 mm of Hg range. In addition to rotary, a series of pumps
(diffusion pump, turbo, ion and cryo etc.) are used in tandem by various manufacturers, when
greater vacuum is required. (a) Electron gun- Gun generates electron beam and the condenser
focuses the electron beams on the nanomaterials present in very low concentration inside the
nanoformulations. The gun is like a light bulb and its filament acts the source of electrons.
Typically, in CTEM, the filament is hairpin shaped tungsten wire. Fixed amount of negative high
voltage is applied to the surrounding cathode cap (Wenelt cylinder). A small amount of emission
current is then applied to the filament for releasing electrons. When the gun gives steady flow of
electrons (saturation point), the observation on nano-formulation sample is started. In order to
create steady flow, following conditions has to be met as well. Cathode cap should be slightly
more negative than the filament. A resister (bias) creates the difference in negative voltage
between the filament and the cathode cap. This, in turn allows the assembly of electrons inside
the cathode cap and finally electron cloud is produced. The anode located below the gun
assembly is kept electrically at ground and thus creates a positive attraction for the negatively
charged electrons. This is how the microscope overcomes the negative repulsion of the cathode
cap and accelerates electrons through the small hole in the anode. (b) image producing system
and condesors- Image producing system consists an array of objective lenses, intermediate and
projector lenses as well as movable specimen stage. This assemblage allows the microscope to
produce highly magnified image from the electrons passing through the nano-formulations. As
glass lenses obstruct electrons, therefore, CTEM lenses are electromagnetic converging lenses.
The real advantage of the aforesaid lenses is that a tightly wrapped copper wire makes up the
magnetic field. A metal shroud around the copper coils shrugs off the charge when the
microscope is in off condition. The electrons move through the center hole in the solenoid with a
brass lining, called pole piece. Condensor in CTEM like light microscope gather electrons of the
first cross-over image and focus them on the nanoparticle samples. Condenser and objective

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aperture is used to reduce spherical aberration and enhance specimen (nanoparticle) image.
Objective lens and specimen stage inserted within objective lens for primary magnification and
imaging purposes respectively. Cold finger or anticontaminator consists of a thin copper rod
(kept in liquid nitrogen) which attracts contaminants and thereby reducing drift to a large extent.
(c) image-recording system- Image recording system with a fluorescent screen is used for
viewing and captures the image for permanent records. Image recording needs intermediate
lenses which magnify the image and finally sends to projector lenses. The final image comes
onto a phosphorescent screen which gives off photons when irradiated by the electron beam. A
film camera is located beneath the phosphorescent screen. In earlier days, a special photographic
film with a thicker emulsion layer than light film was used. Now-a-days improved CTEM use
digital capture with a computer digitizing and archiving (CCD) camera. Metal and metal oxide
nanoparticles used in nano-emulsions give good contrast and high resolution, but, for example,
oxide nanoparticles do not give good resolution, as the nanomaterials are not electron dense.
Another problem encountered often is that the nanoparticles less than 10 nm size does not good
results. In order to overcome these difficulties, high-resolution transmission electron
microscopes (HR-TEM) are used88.

Figure 2. HR-TEM pictures of different sized gold nanoformulations (Das S, Debnath N,


Pradhan S, Goswami A, 2017, Gold Bulletin, pp 1–11. DOI: 10.1007/s13404-017- 0214-z. Epub
7 August 2017).

HR-TEM images are typically interference patterns of the electron wave functions with
themselves after they are diffracted from the specimen. Interferences are highly dependent on
phases of waves. The most important advantage of HR-TEM is that the machine can calculate
very accurately the phase difference between electron wave front and to that of the waves altered
by small nanoparticles (less than 10 nm). HR-TEM technology has been heavily used as ultimate
technology for nullifying the defects in imaging of various nanoparticles. In favourable
preparation of specimen it is capable of giving 2-D projection of the crystals and their defects. In
HR-TEM, a very thin slice of the crystal from the nano-formulations is kept in tilted position so
that a low-index direction is near exact perpendicular to the incoming electron beam. All lattice
planes in nanoparticles about parallel to the electron beam will be close enough the Bragg
position (X-ray crystallography) and the primary electron beams will get diffracted from the
sample. The diffraction pattern will be the fourier transform of the periodic potential for the
electrons in 2-D. In the objective lens, all diffracted beams and the primary are brought together

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and human health management", 07-27 Dec, 2017
again and their interference is calculated. The interference provides a back-transformation and
leads to huge magnification (x106) on the screen following electron-optical system. Both TEM
and HR-TEM provide the measurement of the size of nanoparticles and EDX machine
attachment gives an estimate about the chemical composition of the nanoparticles which can be
further substantiated by techniques like atomic absorption spectroscopy, ICP-OES, ICP-MS
etc89. Negative stain TEM is used to heighten the contrast between samples (e.g. semi-conductor
nanoparticles) and the background. Heavy metals of high atomic numbers are used as stain. As a
result, the background becomes dark and the contours of the nanoparticles become very clear and
easily measurable. This method is easy and it takes only few minutes to prepare the grids.
However, background staining needs careful monitoring. Otherwise, there will be huge artefacts
and heterogeneous background staining. (8) Scanning transmission electron microscopy
(STEM): STEM measures the image and mass of unstained freeze-dried nanoparticles present in
nano-formulations. For nanocomposites of mass between 30kDa-10GDa the accuracy of
measurement is ~0.5-10%. In this kind of microscopy, when operated in dark field, scattered
electrons are sequentially measured for each irradiated sample unit. The number of elastic
scattered electrons is proportional to mass at the corresponding specimen unit90. (9) X-ray
diffraction: X-ray diffraction peaks are produced by constructive interference of a
monochromatic beam of x-ray at a given angle from each set of lattice planes in a sample91. The
peak intensities are determined by distribution of atoms within the lattice. Therefore, the X-ray
diffraction pattern is the fingerprint of the periodic atomic arrangements in a given material. A
search of the ICDD (International Centre for Diffraction Data) database of X-ray diffraction
patterns enables the phase identification of a large variety of crystalline samples. This method is
very quick and essential for many reasons. Sometime, crystalline nature of nanoparticles leads to
the toxicity of nano-formulations. By performing XRD, one can find out the nature of the
material easily and this method is one of the pillars of regulatory set up. Main applications of
XRD analysis are- (a) Identification/quantification of crystalline phase, (b) Measurement of
average crystallite size, strain, or micro-strain effects in bulk materials and thin-film, (c)
Quantification of preferred orientation (texture) in thin films, multi-layer stacks, and
manufactured parts, (d) Determination of the ratio of crystalline to amorphous material in bulk
materials and thin-films92. (10) Modelling: Various modelling tools like (numerical
computational modelling, statistical modelling, smart data mining etc.) are available for fine
tuning the measurement data obtained from the aforesaid nine methods93. For example, (a)
Numerical computer modelling tools have been successfully used for elucidation of the structure
of nano-composites present in nano-formulations94; (b) Traditional numerical modelling
techniques have been used for enhancing the resolution of cryo-TEM95; (c) the use of smart
modelling for complex nanoparticles present in the nano-formulations (e.g. bifurcation theory,
graph invariants and topology, pattern recognition, fuzzy logic, neural networks, genetic
algorithms etc.) is now gaining popularity96-99. Structural data obtained from all the
characterization techniques are used inputs. Typically in smart modelling, networks train the
input dataset and try to match with specific output. In case of complex nanoparticles, data output
from characterization techniques are used as inputs and dataset from individual component of the
complex nanoparticles are used as target. When the match happens with the target, one can
calculate the stoichiometry of the components of nanoformulations.
In this review, we have deliberately focused on a small number of characterization techniques
applicable to less than 100 nm sized nano-materials- when they would be used in making nano-

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and human health management", 07-27 Dec, 2017
formulations. Already started with promising results, nano-formulations (materials, devices and
systems) are making inroads into the agriculture, food and medicine market. In conclusion, a
decade ago nanoparticles were studied by mainly material scientists for their size dependent
unique and novel physical and chemical properties. Now, the products have entered the
commercial exploration period which has been welcomed by industries around the globe.
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89. Pradhan S, Patra P, Mitra S, Dey KK, Jain S, Sarkar S, Roy S, Palit P, Goswami A (2014)
Manganese nanoparticles: impact on non-nodulated plant as a potent enhancer in nitrogen
metabolism and toxicity study both in vivo and in vitro. J Agric Food Chem 62: 8777–8785.
90. Ponce A, Mejia-Rosales S, Jose-Yacaman M (2012) Scanning transmission electron
microscopy methods for the analysis of nanoparticles. Methods Mol Biol 906: 453-471.
91. Govarthanan M, Selvankumar T, Manoharan K, Rathika R, Shanthi K, Lee K, Cho M,
Kamala-Kannan S, Oh B (2014) Biosynthesis and characterization of silver nanoparticles
using panchakavya, an Indian traditional farming formulating agent. Int J Nanomedicine 9:
1593–1599.
92. Klaus T, Joerger R, Olsson E, Granqvist C (1999) Silver-based crystalline nanoparticles,
microbially fabricated. Proc Natl Acad Sci USA 96: 13611–13614.
93. Jones DE, Ghandehari H, Facelli JC (2016) A review of the applications of data mining and
machine learning for the prediction of biomedical properties of nanoparticles. Comput
Methods Programs Biomed 132: 93-103.
94. Zenga QH, Yua AB, Lub GQ (2008) Multiscale modeling and simulation of polymer
nanocomposites. Prog Polym Sci 33: 191–269.
95. Leung AKK, Tam YYC, Chen S, Hafez IM, Cullis PR (2015) Microfluidic mixing: a general
method for encapsulating macromolecules in lipid nanoparticle systems. J Phys Chem B 119:
8698–8706.
96. Johnston RL (2013) Evolving better nanoparticles: Genetic algorithms for optimising cluster
geometries. Dalton Trans, 1: 4193–4207.
97. Rossi G, Ferrando R (2009) Searching for low-energy structures of nanoparticles: a
comparison of different methods and algorithms. J Phys Condens Matter 21: 084208.
98. Bonifazi G, Serranti S (2014) 2D-fractal based algorithms for nanoparticles characterization.
Proceedings Volume 9019, Image Processing: Algorithms and Systems XII; 90190K; doi:
10.1117/12.2038392.
99. Rondina GG, Da-Silva JLF (2013) Revised basin-hopping monte carlo algorithm for structure
optimization of clusters and nanoparticles. J Chem Inf Model 53: 2282–2298.
100. Tan M, Li Y, Jiao H (2013) Design of sewage treatment using iron nanoparticles based on
fuzzy logic control. In: Sun Z., Deng Z. (eds) Proceedings of 2013 Chinese Intelligent
Automation Conference. Lecture Notes in Electrical Engineering, vol 254. Springer, Berlin,
Heidelberg.

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Nanosizing of Molecules and their aApplications
Rajesh Kumar and Indu Chopra
Division of Agricultural Chemicals
ICAR-Indian Agricultural Research Institute, New Delhi 110 012
Nanotechnology is the application of scientific knowledge to manipulate and control
matter in the nanoscale. This technology provides a unique approach to create specific properties
of a material by preparing it in the form of nanoparticles or with a suitable nanostructure. These
specific nanoeffects have been used for the development of a large number of products in the
fields of agriculture, advanced materials and coatings, catalysis, electronics, energy and water
management, sensors, drug delivery and other medical applications.
Nanoparticles of noble metals such as gold, silver and palladium are important due to
their applications in various fields of industry. Silver nanoparticles have been known as effective
antibacterial agents. Over the past few decades, many synthetic methods of silver nanoparticles
have been reported. Chemical method for synthesizing silver nanoparticles is one of the most
common methods.
Procedure
Synthesis of silver nanoparticles using tri sodium citrate (TSC) as a reducing agent
Prepare the solutions of silver nitrate (0.001 M) and trisodium citrate (1%) in distilled water.
Heat 50 ml of 0.001 M silver nitrate solution to boil. To this solution, add 5 mL of 1 % trisodium
citrate drop by drop. During the process, mix the solutions vigorously and heat until change of
color was evident (pale yellow). Then remove it from the heating device and stirr until the
reaction mixture attains room temperature.
The reaction could be expressed as follows:
4Ag+ + C6H5O7Na3 + 2H2O → 4AgO + C6H5O7H3 + 3Na+ + H+ + O2↑
Characterize the colloidal solution of silver nanoparticles by using UV-Visible spectroscopy and
SEM.
Synthesis of silver nanoparticles using sodium borohydride as a reducing agent
Sodium borohydride is needed in excess to reduce the ionic silver and to stabilize the resulting
silver nanoparticles. Add different volumes of 0.001M silver nitrate drop wise (about 1 drop per
second) to 30 mL of cold 0.002 M sodium borohydride solution (chilled in an ice bath). Stirr the
reaction mixture vigorously on a magnetic stirrer. The solution will turn to light yellow after the
addition of 2 mL of silver nitrate and to brighter yellow after addition of all of the silver nitrate.
AgNO3 + NaBH4 → Ag + H2+ B2H6 + NaNO3
The entire addition process will take about 3 minutes, after which stop the stirring and remove
the stir bar.

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Precautions

• Reaction conditions including stirring time and relative quantities of reagents must be
carefully controlled to obtain the stable yellow colloidal silver.

• If stirring is continued after addition of total required silver nitrate solution, aggregation
will begin as the yellow solution first turn to darker yellow then violet and eventually
grayish after which the colloid will break down and particle will settle out.
References
Beyene HD, Werkneh AA, Bezabh HK and Ambaye TG (2017) Synthesis paradigm and
applications of silver nanoparticles (AgNPs), a review. Sustainable Materials and
Technologies 13: 18–23.
Natsuki J, Natsuki T and Hashimoto Y (2015) A review of silver nanoparticles: Synthesis
methods, properties and applications. International Journal of Materials Science and
Applications. 4(5): 325-332.

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and human health management", 07-27 Dec, 2017
Overview of Chromatographic Techniques
Tirthankar Banerjee and Mukta Chakrabarty

Division of Agricultural Chemicals,


ICAR-Indian Agricultural Research Institute, New Delhi-110012
The term "chromatography" is derived from the original use of this method for separating yellow
and green plant pigments. The term chromatography means writing in colour (in Greek:
Khromatos-colour, and graphos- written). It was discovered by Mikhail Tswett in 1906. Russian
scientist Tswett in 1906 used a glass columns packed with finely divided CaCO3 to separate
plant pigments extracted by hexane. The pigments after separation appeared as colour bands that
can come out of the column one by one.
Chromatography has been developed into a new method of separation of mixture of
substances mainly when they are available in small amounts. This method is very useful when
the components of a mixture have almost the same physical and chemical properties and hence
can’t be separated by other usual methods of separations. Chromatographic methods have high
"resolving power", i.e. they are capable of sharp separations of closely related compounds,
particularly when very small samples are used.

Principles of chromatography

Chromatography is based on selective adsorption of compounds on a solid (or liquid) with high
surface area (the stationary phase). As the solute mixture passes over the solid, the components
are adsorbed and then released from the surface at differing rates with a mobile phase. This
means that the solutes are continuously partitioned between the adsorbent and the mobile phase,
either a gas or a solvent mixture. The stronger the interaction of the solute with stationary phase,
the slower the solute will progress. The motion of the solute and solvent through the stationary
phase in is called elution. The process is analogous to fractional distillation or extraction, in
which different compounds are partitioned between liquid and vapour, or between two
immiscible liquids, respectively.
Differential affinities (strength of adhesion) of the various components of the analyte
towards the stationary and mobile phase results in the differential separation of the components.
Affinity, in turn, is dictated by two properties of the molecule: ‘Adsorption’ and ‘Solubility’.
Adsorption can be defined as the property of how well a component of the mixture sticks to the
stationary phase, while solubility is the property how well a component of the mixture dissolves
in the mobile phase.
• Higher the adsorption to the stationary phase, the slower the molecule will move through the
column.
• Higher the solubility in the mobile phase, the faster the molecule will move through the column.
So, the interplay between the above two factors determines the differential rates at which the
different components of the analyte will move through the column. Adsorption and solubility of
a molecule can be manipulated by choosing the appropriate stationary phase and mobile phase.
Now, the question arises why do different compounds possess different affinities towards the
stationary and mobile phases? “Polarity” of the compounds dictates their affinities towards the
stationary and mobile phases.

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Examples of the application of chromatographic methods are (i) the purification of reaction
mixtures in chemical synthesis, (ii) the purification of bio-molecules such as proteins for
pharmaceutical research, (iii) the analysis of complex sample mixtures such as those obtained in
forensics (body fluids, paints etc) and (iv)the analysis of environmental samples/natural
products.

Key terms:
Adsorption: Interaction of solute molecules (or atoms or ions) with the surface of the stationary
phase (that it is different from absorption where the molecules fill the pores of a solid).
Stationary phase: The part of the chromatography system that is fixed in place. Most commonly
a solid e.g. the packing in column chromatography or gas chromatography or the coating on a
chromatographic plate.
Mobile phase: The part of the chromatography system that is mobile. Commonly a solvent
mixture (as in column chromatography or thin layer chromatography or a gas (as in gas
chromatography).
Eluent: The mobile phase (usually for solvents or carrier gases)
Elution: Motion of the mobile phase through the stationary phase
Elution time: The time taken for a solute to pass through the system. A solute with a short
elution time travels through the stationary phase rapidly, i.e. it elutes fast.
Eluate: fluid exiting the column (that is collected in flasks)
Normal phase: “Unmodified” stationary phase where POLAR solutes interact strongly and run
slowly
Reverse phase: “Modified” stationary phase where POLAR solutes run fast i.e. reverse order
Resolution: Degree of separation of different solutes. In principle, resolution can be improved
by using a longer stationary phase, finer stationary phase (e.g. column packing or TLC plate
coating) or slower elution.
Analyte : mixture whose individual components have to be separated and analyzed
Types of chromatography

• Column chromatography
• Paper chromatography
• Thin-layer chromatography
• Ion-exchange chromatography
• Gel-permeation (molecular sieve) chromatography
• Affinity chromatography
• Gas chromatography
• Dye-ligand chromatography
• Hydrophobic interaction chromatography
• Pseudoaffinity chromatography
• High-pressure liquid chromatography (HPLC)
• Supercritical fluid chromatography (SFC)
• For special applications, scientists sometimes employ reverse-phase chromatographic
techniques where the scenario is reversed i.e. the stationary phase is non-polar while the
mobile phase is polar.

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Column chromatography
Since organic molecules have difference in polarity, stationary phase used, and binding capacity,
each one of these characteristic components can be purified using chromatographic methods.
Among these methods, most frequently column chromatography is applied. This technique is
used for the purification of biomolecules. On a column (stationary phase) firstly the sample to be
separated, then wash mobile phase are applied. Their flow through inside column material placed
on a fiberglass support is ensured. The samples are accumulated at the bottom of the device in a
tme-, and volume-dependent manner.

Column chromatography

Illustration of a column chromatographic separation

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Ion- exchange chromatography
Ion- exchange chromatography is based on electrostatic interactions between charged protein
groups, and solid support material (matrix). Matrix has an ion load opposite to that of the protein
to be separated, and the affinity of the protein to the column is achieved with ionic ties. Proteins
are separated from the column either by changing pH, concentration of ion salts or ionic strength
of the buffer solution. Positively charged ion- exchange matrices are called anion-exchange
matrices, and adsorb negatively charged proteins. While matrices bound with negatively charged
groups are known as cation-exchange matrices, and adsorb positively charged proteins.

Gel- permeation (molecular sieve) chromatography


The basic principle of this method is to use dextran containing materials to separate
macromolecules based on their differences in molecular sizes. This procedure is basically used to
determine molecular weights of proteins, and to decrease salt concentrations of protein solutions.
In a gel- permeation column stationary phase consists of inert molecules with small pores. The
solution containing molecules of different dimensions are passed continuously with a constant
flow rate through the column. Molecules larger than pores can not permeate into gel particles,
and they are retained between particles within a restricted area. Larger molecules pass through
spaces between porous particles, and move rapidly through inside the column. Molecules smaller
than the pores are diffused into pores, and as molecules get smaller, they leave the column with
proportionally longer retention times . Sephadeks G type is the most frequently used column
material. Besides, dextran, agorose, polyacrylamide are also used as column materials.

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Gel-permeation (molecular sieve) chromatography

Affinity chromatography
This chromatography technique is used for the purification of enzymes, hormones, antibodies,
nucleic acids, and specific proteins [13]. A ligand which can
make a complex with specific protein (dextran,
polyacrylamide, cellulose etc) binds the filling material of the
column. The specific protein which makes a complex with
the ligand is attached to the solid support (matrix), and
retained in the column, while free proteins leave the column.
Then the bound protein leaves the column by means of
changing its ionic strength through alteration of pH or
addition of a salt solution.

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Affinity chromatography

Paper chromatography
In paper chromatography support material consists of a layer of
cellulose highly saturated with water. In this method a thick
filter paper comprised the support, and water drops settled in its
pores made up the stationary “liquid phase.” Mobile phase
consists of an appropriate fluid placed in a developing tank.
Paper chromatography is a “liquid-liquid” chromatography.

Paper chromatography
Thin-layer chromatography
Thin-layer chromatography is a “solid-liquid adsorption” chromatography. In this method
stationary phase is a solid adsorbent substance coated on glass plates. As adsorbent material all
solid substances used. in column chromatography (alumina, silica gel, cellulose) can be utilized.
In this method, the mobile phase travels upward through the stationary phase The solvent travels
up the thin plate soaked with the solvent by means of capillary action. During this procedure, it
also drives the mixture priorly dropped on the lower parts of the plate with a pipette upwards
with different flow rates. Thus the separation of analytes is achieved. This upward travelling rate
depends on the polarity of the material, solid phase, and of the solvent.
In cases where molecules of the sample are colorless, florescence, radioactivity or a specific
chemical substance can be used to produce a visible coloured reactive product so as to identify
their positions on the chromatogram. Formation of a visible colour can be observed under room
light or UV light. The position of each molecule in the mixture can be measured by calculating
the ratio between the the distances travelled by the molecule and the solvent. This measurement
value is called relative mobility, and expressed with a symbol Rf. Rf. value is used for qualitative
description of the molecules.

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Thin-layer chromatography

Gas chromatography
In this method stationary phase
is a column which is placed in
the device, and contains a liquid
stationary phase which is
adsorbed onto the surface of an
inert solid. Gas chromatography
is a “gas-liquid”
chromatography. Its carrier
phase consists of gases as He or
N2. Mobile phase which is an
inert gas is passed through a
column under high pressure. The
sample to be analyzed is
vaporized, and enters into a
gaseous mobile phase phase. The
components contained in the sample are dispersed between mobile phase, and stationary phase
on the solid support. Gas chromatography is a simple, multifaceted, highly sensitive, and rapidly
applied technique for the extremely excellent separation of very minute molecules. It is used in
the separation of very little amounts of analytes. This gas stream can be introduced straight into a
mass spectrometer (MS) to give the very important technique of GCMS which facilitates the
separation and identification of samples within a mixture.

Dye- ligand chromatography


Development of this technique was based on the demonstration of the ability of many enzymes
to bind purine nucleotides for Cibacron Blue F3GA dye. The planar ring structure with
negatively charged groups is analogous to the structure of NAD. This analogy has been
evidenced by demonstration of the binding of Cibacron Blue F3GA dye to adenine, ribose
binding sites of NAD. The dye behaves as an analogue of ADP-ribose. The binding capacity of
this type adsorbents is 10–20-fold stronger rhat that of the affinity of other adsorbents. Under

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appropriate pH conditions, elution with high-ionic strength solutions, and using ion-exchange
property of adsorbent, the adsorbed proteins are separated from the column.
Hydrophobic interaction chromatography (HIC)
In this method the adsorbents prepared as column material for the ligand binding in affinity
chromatography are used. HIC technique is based on hydrophobic interactions between side
chains bound to chromatography matrix.
Pseudoaffinity chromatography
Some compounds as anthraquinone dyes, and azo-dyes can be used as ligands because of their
affinity especially for dehydrogenases, kinases, transferases, and reductases The mostly known
type of this kind of chromatography is immobilized metal affinity chromatography (IMAC).
High-prssure liquid chromatography (HPLC)
Using this chromatography technique it is possible to perform structural, and functional analysis,
and purification of many molecules within a short time, This technique yields perfect results in
the separation, and identification of amino acids, carbohydrates, lipids, nucleic acids, proteins,
steroids, and other biologically active molecules, In HPLC, mobile phase passes throuıgh
columns under 10–400 atmospheric pressure, and with a high (0.1–5 cm//sec) flow rate. In this
technique, use of small particles, and application of high presure on the rate of solvent flow
increases separation power, of HPLC and the analysis is completed within a short time.
Essential components of a HPLC device are solvent depot, high- pressure pump, commercially
prepared column, detector, and recorder. Duration of separation is controlled with the aid of a
computerized system, and material is accrued.

Supercritical fluid chromatography (SFC)


It is a form of normal phase chromatography that uses a supercritical fluid such as carbon
dioxide as the mobile phase.[1][2] It is used for the analysis and purification of low to
moderate molecular weight, thermally labile molecules and can also be used for the separation
of chiral compounds. Principles are similar to those of high performance liquid
chromatography (HPLC), however SFC typically utilizes carbon dioxide as the mobile phase;
therefore the entire chromatographic flow path must be pressurized. Because the supercritical
phase represents a state in which liquid and gas properties converge, supercritical fluid
chromatography is sometimes called convergence chromatography.
Different types of chromatographic techniques described above can be summarized
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Technique Stationary Mobile Basis of Notes
phase phase separation
*Paper polarity of compound spotted directly on a
chromatography solid (cellulose) liquid molecules cellulose paper
*Thin layer glass is coated with thin layer of
chromatography solid (silica or polarity of silica on which is spotted the
(TLC) alumina) liquid molecules compound
*Liquid column solid (silica or polarity of glass column is packed with
chromatography alumina) liquid molecules slurry of silica
Size exclusion solid liquid size of small molecules get trapped in
chromatography (microporous molecules the pores of the stationary phase,
beads of silica) while large molecules flow
through the gaps between the
beads and have very small
retention times. So larger
molecules come out first. In this
type of chromatography there
isn’t any interaction, physical or
chemical, between the analyte
and the stationary phase.
Ion-exchange solid (cationic liquid ionic charge molecules possessing the
chromatography or anionic resin) of the opposite charge as the resin will
molecules bind tightly to the resin, and
molecules having the same
charge as the resin will flow
through the column and elute
out first.
Affinity solid (agarose liquid binding if the molecule is a substrate for
chromatography or porous glass affinity of the enzyme, it will bind tightly
beads on to the analyte to the enzyme and the unbound
which are molecule to analytes will pass through in the
immobilized the mobile phase, and elute out of
molecules like molecule the column, leaving the
enzymes and immobilized substrate bound to the enzyme,
antibodies) on the which can then be detached
stationary from the stationary phase and
phase eluted out of the column with an
appropriate solvent.
Gas liquid or solid gas boiling samples are volatilized and the
chromatography support (inert point of the molecule with lowest boiling
gas like molecules point comes out of the column
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argon or first. The molecule with the
helium) highest boiling point comes out
of the column last.

Application areas of chromatography in medicine

Chromatography technique is a valuable tool for biochemists, besides it can be applied easily
during studies performed in clinical laboratories For instance, paper chromatography is used to
determine some types of sugar, and amino acids in bodily fluids which are associated with
hereditary metabolic disorders. Gas chromatography is used in laboratories to measure steroids,
barbiturates, and lipids. Chromatographic technique is also used in the separation of vitamins,
and proteins.

Life Science Applications of Chromatography


Chromatography is widely used in various life science applications. Some important applications
of chromatography in the food, molecular biology, and forensic sectors are discussed below.

Food industry
Spoilage detection
Chromatography can be used in flavor studies and to detect spoilage in foods. Determining the
amount of organic acids in foods provides key information about the quality of foods. Column
chromatography is used to detect and quantify spoilage indicators such as pyruvic acid in milk.
Pyruvic acid content is a measure of psychrotrophic bacteria present in milk.
The same separation method is used to assess total organic acid profile of milk and to measure
lactose, which indicates the level of sweetness. Chromatography enables rapid analysis when
compared with techniques such as bacterial plating, which may take several days to yield results.
Rapid analysis is crucial in the food industry to prevent outbreak of spoilage and to minimize
possible health risks.

Additive detection
Additives are added to foods to enhance their flavors or to give them a visual appeal. For
example, the presence of added malic acid in apple juice is more difficult to detect because apple
juice naturally contains malic acid. However, synthetic malic acid contains fumaric acid as a
contaminant and hence its level in an apple juice sample is an indicator of the commercial malic
acid. Chromatography has been successfully used to detect and quantify fumaric acid in apple
juice.

Determining nutritional quality


Vitamin C depletion in foods can be an indicator of depletion of other nutrients and so the
vitamin C content of foods and beverages is closely monitored during all stages of food
processing using column chromatography. This analysis can be carried out rapidly using modern
acid analysis columns coupled with electrochemical detection even in complex samples. This
technique is used to quantitate vitamin C in juices, powdered drinks, and both fresh and frozen
vegetables and fruits.

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Forensics
Crime scene testing
Gas chromatography is used to test evidence such as blood or hair from a crime scene. This
allows investigators to understand the crime better and to develop theories on what exactly
happened and where the victim has been earlier, based on the material found.
Forensic pathology
Gas chromatography (GC) has been widely used in forensic pathology to identify the type of
compounds and fluids present in the human body, post death. This testing can help detect the
presence of alcohol or drugs or poisonous substances in the body at the time of death, thus
assisting in determining the possible motive and cause of death.

Arson investigation
GC is a low cost technique used to identify ignitable / flammable liquids from fire debris. On
comparison with a list of flammable liquids publically available, the exact kind of liquid used
can be concluded. Mass spectrometry (MS) characterization of the separated components yields
better and more precise results.

Molecular biology studies


Hybrid techniques that combine electrochemistry (EC) and MS with chromatography are
powerful tools in the study of redox reactions involving various bioorganic molecules. ESI-MS is
coupled with liquid chromatography (LC) separation for the characterization of the reaction
mixture. EC–LC–MS is applied in the study of biomolecules such as proteins, peptides, and
nucleic acids.

Metabolomics and proteomics


EC–LC–MS is essential in mimicking biotransformation reactions, such as phase I oxidative
reactions in drug metabolism studies. The technology has been applied in the study of
pharmaceutical compounds such as; acetaminophen, diclofenac, lidocaine, clozapine,
haloperidol, flunitrazepam, chlorpromazine, alprenolol, albendazole and verapamil.
In proteomics, this technique is used to analyze oxidation of proteins and peptides and in
selective labeling of these substances. Chromatography techniques are also widely used in
purification of plasma proteins, hormones, monoclonal antibodies, and vaccines as part of their
development.

Nucleic acids research


Electrochemistry coupled with LC, MS, or gas chromatography (GC) has been successfully used
to identify the oxidation products of nucleobases, nucleotides, and nucleosides. This has
accelerated the identification of these compounds compared to long drawn-out isolation steps.

Conclusion
Initially chromatographic techniques were used to separate substances based on their color as
was the case with herbal pigments. With time its application area has been extended
considerably. Nowadays, chromatography is accepted as an extremely sensitive, and effective
separation method.

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ca/applications/bioprocessing-applications/
30. http://www.chromatographytoday.com/news/gc-mdgc/32/breaking news/how is gas
chromatography used in forensics/30185/sthash.CThm4qHP.dpuf

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Recent Trends in IR Spectroscopy
Suman Gupta, Neethu Narayanan, Tirthankar Banerjee and Anupama
Division of Agricultural Chemicals,
ICAR-Indian Agricultural Research Institute, New Delhi-110012

Spectroscopy is the study of matter and its interaction with electromagnetic radiation.
Electromagnetic radiation is a form of energy that is produced by oscillating electric and
magnetic disturbance, or by the movement of electrically charged particles traveling through a
vacuum or matter. The electric and magnetic fields come at right angles to each other and
combined wave moves perpendicular to both magnetic and electric oscillating fields thus the
disturbance. These electric and magnetic waves travel perpendicular to each other and have
certain characteristics, including amplitude, wavelength, and frequency.

Infrared (IR) spectroscopy is the study of interaction of infrared light with matter, which can be
used to identify unknown materials, examine the quality of a sample or determine the amount of
components in a mixture. Infrared light refers to electromagnetic radiation with wavenumber
ranging from 13000 – 10 cm-1 (corresponding wavelength from 0.78 – 1000 μm). The Infra red
regions of interest in the electromagnetic spectrum are:

Infrared Wavelength Wave Source of radiation Information gathered


region number
Near 0.7 μm to 2.5 14000- Tungsten-halogen lamps Study of overtones and
Infrared μm 4000 cm-1 and metallic conductors harmonic or
(NIRS) coated with ceramic combination vibrations
Middle 2.5 μm to 25 4000-400 Globar (silicon carbide), Study of fundamental
Infrared μm cm-1 Nernst glower (oxides of vibrations and the
(MIRS) zirconium, yttrium and rotation-vibration
erbium) and metallic helices structure of small

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(chromium-nickel alloy or molecules
tungsten)
Far 25 μm to 400-10 mercury high-pressure lamp Study of inorganic and
Infrared 1000 μm cm-1 metal-organic species
(FIRS)

Middle infrared region is most widely investigated region as most of the molecules absorb
radiations in this region to induce the vibrational excitation of functional groups. All matter
contains molecules; these molecules have bonds that are continually vibrating and moving
around. These bonds can vibrate with stretch motions or bend motions. Simple diatomic
molecules have only one bond, which may stretch. In polyatomic molecules, each atom is having
three degrees of freedom in three directions which are perpendicular to each other. Thus, a
molecule of n atoms has 3n degrees of freedom. For a linear molecule, two degrees of freedom
describe rotation and three degrees describe translation, so the remaining 3n -5 are number of
fundamental vibrations. While for a non-linear molecule, three degrees of freedom describe
rotation and three degrees describe translation, so the remaining 3n -6 are number of
fundamental vibrations. More complex molecules have more than one bond and different types
of vibrations may occur. Vibrations fall into the two main categories of stretching and bending.
Stretching can either be symmetric or asymmetric. In symmetric stretching, bond length increase
or decrease symmetrically whereas in asymmetric stretching, length of one bond increases and
the other one decreases.

In bending vibrations, a change in bond angle occurs between bonds with a common atom, or
there is a movement of a group of atoms with respect to the remainder of the molecule without
movement of the atoms in the group with respect to one another. The bending vibrations are also
called as deformation vibrations. Deformation vibrations are of two types: In-plane bending
vibrations and Out of plane bending vibrations. In in-plane bending type of vibrations, there is a
change in bond angle. This type of bending takes place within the same plane. In plane bending
are of two types: Scissoring and Rocking. In Scissoring, the bond angle decreases whereas in
rocking, the bond angle is maintained but both bonds moves within the same plane. The out of
plane bending takes place outside of the plane of molecule. It is of two types: Wagging and
twisting. Wagging vibrations are those in which both atoms move to one side of the plane while
twisting is the one in which one atom is above the plane and the other is below the plane.
In order for a bond to be promoted to the excited state, it must be exposed to radiation of the
exact same frequency as the energy difference between ground and excited states (ΔE).
Determining these frequencies and representing them allows us to determine the bonds that exist
in a molecule. Infrared Spectrometer passes infrared radiation through a sample of an unknown

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compound and uses a detector to plot percent transmission of the radiation through the molecule
versus the wavenumber of the radiation. A downward peak on the plot represents absorption at a
specific wavenumber. In sum, IR spectroscopy is useful in determining chemical structure
because energy that corresponds to specific values allows us to identify various functional
groups within a molecule. An IR spectrum usually extends from radiation around 4000 cm-1 to
600 cm-1 and can be split into the functional group region (4000-1450 cm-1) and the fingerprint
region (1450-600 cm-1). Two different molecules may have similar functional group regions
because they have similar functional groups, but they will always have a different fingerprint
region. Due to the high information content of its spectrum, infrared spectroscopy has been a
very common and useful tool for structure elucidation and substance identification. Vibrational
frequency range of some of the most common functional groups is shown below:

Instrumentation
Most commonly used instruments in infrared spectroscopy are dispersive infrared spectrometer
and Fourier transform infrared spectrometer.
Dispersive infrared spectrometer:

Dispersive infrared
instruments
are sometimes
called grating or scanning spectrometers. A dispersive infrared instrument has a source and
mirrors, but the similarities to an FT-IR end there. The source energy is sent through both a
sample and a reference path, through a chopper to moderate the energy reaching the detector, and
directed to a diffraction grating. This grating is similar to a prism. It separates the wavelengths of
light in the spectral range and directs each wavelength individually through a slit to the detector.
Each wavelength is measured one at a time, with the slit monitoring the spectral bandwidth and
the grating moving to select the wavelength being measured. The detector produces an electrical
signal and results in a recorder response. Two types of detectors are employed in dispersive
infrared spectrometer, namely, thermal detectors and photon detectors.

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Fourier transform infrared spectrometer
An FT-IR instrument uses a system called an interferometer to collect a spectrum. The
interferometer consists of a source, beam splitter, two mirrors, a laser and a detector. The energy
goes from the source to the beam splitter which splits the beam into two parts. One part is
transmitted to a moving mirror; one part is
reflected to a fixed mirror. The moving mirror
moves back and forth at a constant velocity.
This velocity is timed according to the very
precise laser wavelength in the system which
also acts as an internal wavelength calibration.
The two beams are reflected from the mirrors
and recombined at the beam splitter. The beam
from the moving mirror has traveled a different
distance than the beam from the fixed mirror.
When the beams are combined, an interference
pattern is created, since some of the
wavelengths recombine constructively and
some destructively. This interference pattern is
called an interferogram.
This interferogram then goes from the
beam splitter to the sample, where some energy is absorbed and some is transmitted. The
transmitted portion reaches the detector. The detector reads information about every wavelength
in the infrared range simultaneously. To obtain the infrared spectrum, the detector signal is sent
to the computer, and an algorithm called a Fourier transform is performed on the interferogram
to convert it into a single beam spectrum. A reference or “background” single beam is also
collected without a sample and the sample single beam is ratioed to the background single beam
to produce a transmittance or “%T” spectrum.
One of the important features of FTIR spectroscopy is the ability to signal average a large
number of scans in a relatively short amount of time. Thus spectra on very small quantities of
material, or on highly absorbing materials, in which the signal to noise ratio of individual scans
is very poor, can be achieved. In practice, the interferograms are signal averaged first, and a
single Fourier transform then done, as the Fourier transform can take up quite significant
amounts of computing time. The second major advantage of FTIR over conventional dispersive
instruments is the high throughput of infrared radiation, since narrow slits are no longer
necessary to achieve resolution. The FTIR spectrometer is also capable of very high resolution of
absorption bands. Higher resolution is achieved by moving the mirror further while maintaining
the same starting point. FTIR spectroscopy, therefore, has two main advantages over
conventional IR spectroscopy, improved sensitivity and improved computational ability.

Modern trends in IR Spectroscopy


An infrared spectrum is commonly obtained by passing infrared radiation through a sample and
determining what fraction of the incident radiation is absorbed at a particular energy. Aside from
the conventional IR spectroscopy of measuring light transmitted from the sample, the reflection
IR spectroscopy was developed using combination of IR spectroscopy with reflection theories. In
the reflection spectroscopy techniques, the absorption properties of a sample can be extracted

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from the reflected light. Reflectance techniques may be used for samples that are difficult to
analyze by the conventional transmittance method. In all, reflectance techniques can be divided
into two categories: internal reflection and external reflection. In internal reflection method,
interaction of the electromagnetic radiation on the interface between the sample and a medium
with a higher refraction index is studied, while external reflectance techniques arise from the
radiation reflected from the sample surface. External reflection covers two different types of
reflection: specular (regular) reflection and diffuse reflection. The former usually associated with
reflection from smooth, polished surfaces like mirror, and the latter associated with the reflection
from rough surfaces (Khoshhesab, 2012; Frost and Roberts 2003).

A) Internal reflectance spectroscopy (IRS) or Attenuated total reflectance


spectroscopy: The technique of Attenuated Total Reflectance (ATR) has in recent years
revolutionized solid and liquid sample analyses because it combats the most challenging aspects
of infrared analyses, namely sample preparation and spectral reproducibility. It may be used to
study i) the surface of solid samples, provided good contact can be achieved between the sample
and the ATR element. Depth profiling is also possible by varying the angle of incidence provided
it does not exceed the
critical angle. This
technique is ideal for solid
samples that can make good
contact with the ATR
element, in this class fall
rubber, polymer films
fabrics, coated and painted
surfaces.

ii) ATR has been used with excellent results to investigate the solid water interface. The
application of FTIR-ATR Spectroscopy to aqueous systems has opened up a whole new area of
study, from the clotting mechanism of blood on foreign surfaces to the mode of action of
flotation collectors on mineral surfaces.
An infrared beam is directed onto an optically dense crystal (Zinc Selenide (ZnSe) or
Germanium or diamond) with a high refractive index at a certain angle. This internal reflectance
creates an evanescent wave that extends beyond the surface of the crystal into the sample held in
contact with the crystal. It can be easier to think of this evanescent wave as a bubble of infrared
that sits on the surface of the crystal. This evanescent wave protrudes only a few microns (0.5 µ -
5 µ) beyond the crystal surface and into the sample. Consequently, there must be good contact
between the sample and the crystal surface. In regions of the infrared spectrum where the sample
absorbs energy, the evanescent wave will be attenuated or altered. The attenuated energy from
each evanescent wave is passed back to the IR beam, which then exits the opposite end of the
crystal and is passed to the detector in the IR spectrometer. The system then generates an
infrared spectrum.
For the technique to be successful, the following two requirements must be met:
1. The sample must be in direct contact with the ATR crystal, because the evanescent wave or
bubble only extends beyond the crystal 0.5 µ - 5 µ.
2. The refractive index of the crystal must be significantly greater than that of the sample or else
internal reflectance will not occur – the light will be transmitted rather than internally reflected in

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the crystal. Typically, ATR crystals have refractive index values between 2.38 and 4.01 at 2000
cm-1. It is safe to assume that the majority of solids and liquids have much lower refractive
indices.
ATR is an IR sampling technique that provides excellent quality data in conjunction with the
best possible reproducibility of any IR sampling technique. It has revolutionized IR solid and

liquid sampling through (1) Faster sampling (2) Improving sample-to-sample reproducibility (3)
Minimizing user to user spectral variation.

The ATR-FTIR technique finds its application in different fields such as biology, medicine,
forensics, process analytical chemistry and organic chemistry. Bach and Miller (2001)
investigated mixtures of cholesterol with dimyristoyl phosphatidyl serine or
deuterated dimyristoyl phosphatidylserine were by using polarized and non polarized attenuated
total reflection (ATR) Fourier transform infrared (FTIR) Spectroscopy. Attenuated total
reflection (ATR) Fourier transform-infrared (FT-IR) spectroscopy was also used to analyze
synthetic fibers and natural hairs of human, cat, and dog origin by Manheim et al (2016). In that
study, they have used chemometric analysis to differentiate hair spectra from the three different
species, and to predict unknown hairs to their proper species class, with a high degree of
certainty. From a forensic perspective, this technique would be complementary to microscopic
hair examination, and in no way replace it. More importantly, this approach is non-destructive,
rapid, can provide reliable results, and requires no sample preparation, making it of ample
importance to the field of forensic science. Attenuated total reflection Fourier transform infrared
(ATR-FTIR) spectroscopy is also an excellent vibrational spectroscopic technique for the
analysis of serum due to its rapidity and ease of translation into the clinical environment. FTIR
spectroscopy combined with appropriate data handling frameworks over the years has proved a
useful tool in biomedical research, particularly in the identification and diagnosis of cancer and
other diseases, through the discovery of diagnostic biomarkers from the complexity of the
biological background (Dorling and Baker, 2013).

B) Specular reflectance spectroscopy Specular reflectance techniques basically involve a


mirror-like reflection from the sample surface that occurs when the reflection angle equals the
angle of incident radiation. It is used for samples that are reflective (smooth surface) or attached
to a reflective backing. Thus, specular techniques provide a reflectance measurement for

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reflective materials, and a reflection–absorption (transflectance) measurement for the surface
films deposited on, or pressed against reflective surfaces.
In absorption-reflection measurement, one fraction of the radiation is reflected on the upper
interface and contributes towards the spectrum via specular reflection. Another part of the

radiation penetrates the surface film and is reflected by the reflective surface, thus, the light
passes through the surface layer twice-to and from the reflective surface, leading to increase the
intensity of the reflectance spectrum as compared to the normal transmission. The most common
applications of this technique are evaluation of surfaces such as: coating, thin films,
contaminated metal surface.

C) Diffuse Reflectance Spectroscopy (DRS) In diffuse reflectance spectroscopy, the


electromagnetic radiation reflected by roughened surfaces is collected and analyzed. When this
technique is applied in (FT) IR region, it is termed as diffuse reflectance infrared Fourier
transform spectroscopy (DRIFTS). Light incident onto a solid sample may be partly reflected
regularly (specular reflection) by the sample surface, partly scattered diffusely, and partly
penetrates into the sample. The latter part may be absorbed within the particles or be diffracted at
grain boundaries, giving rise to diffusely scattered light in all directions. Diffuse reflectance
spectroscopy associated with the reflected lights which are produced by diffuse scattering. Since
regular reflection distorts the DRS spectra, thus, the regular reflection component should be
eliminated in diffuse reflectance measurement. The DRIFTS accessory is designed to eliminate
the specularly reflected radiation. In diffuse reflectance spectroscopy, there is no linear relation
between the reflected light intensity (band intensity) and concentration, in contrast to traditional
transmission spectroscopy in which the
band intensity is directly proportional to
concentration. Therefore, quantitative
analyzes by DRIFTS are rather
complicated.
Diffuse reflectance technique is used for
powders and solid samples having rough
surface such as paper, cloth. In diffuse
reflectance technique, particles size,
homogeneity, and packing density of powdered samples play important role on the quality of
spectrum. A sample with smaller particle size having narrow size distribution is preferred. Thus,
in order to obtain a qualified spectrum, the sample should be ground into smaller size.
Diffuse reflectance measurement in near-IR is more common than in mid-IR. Because
nonabsorbing scattering substrates are rare in mid-IR, and also more efficient scattering occurs at
shorter wavelengths (near-IR). Additionally, due to lower efficiency of the scattering in mid-IR,

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diffuse reflectance is very weak in this region, as a consequent, in mid-IR, diffuse reflectance
could only be measured by FT-IR spectrometer (DRIFTS).
In diffuse reflectance spectroscopy, diffusely scattered light can be directly, collected from
material in a sampling cup or, alternatively, collected by using an abrasive sampling pad. In mid-
IR, the diffusely reflected light from sample is generally collected by large ellipsoidal mirrors,
which cover as much area above the sample as possible. In near-IR, diffuse reflectance spectra
are usually measured by an integrating sphere, described by Ulbrich. The inner surface of
"Ulbrich sphere" is coated by strongly scattering, nonabsorbing powder. After repeated
reflection, all radiations reach the detector. Thus, with Ulbrich sphere, the entire radiation
reflected by the sample is integrated. In mid-IR region, the inner surface of diffuse scattering
sphere is treated with gold vapor to guarantee a high degree of reflection, while in near-IR
region, Ulbrich sphere consists of spectralon (a thermostatic resin) which is applied as white
standard due to its high degree of reflection. DRIFTS is a versatile technique for analyzing
nontransparent samples, powders, roughened surfaces and coating. It offers the advantages of
easy sample preparation and applicability to analyze samples at elevated temperature and
pressure.

D) Photoacoustic Spectroscopy It has the great advantage of requiring very little sample
preparation and in contrast to the ATR method the sample does not have to be in contact with an
element. Photoacoustic infrared spectroscopy differs from traditional infrared spectroscopy in
one important way: in its most common implementation, a microphone is used to detect acoustic
waves that result from absorption of infrared radiation by a sample. Higher surface/volume ratios
of the sample increase the signal intensity. Thus powdered samples and rough surface
morphologies are more favorable in PAS. The technique has been used with great success in the
depth profiling of solid surfaces. Previously variable angle ATR was the only method for depth
profiling and the maximum depth was of the order of a micron. PAS allows maximum depths of
many microns by using low mirror velocities. As with the ATR method the sampling depth is
wavelength dependent, making quantification of surface concentrations difficult. PAS is best
suited to intractable solid samples.

E) Fourier Transform Infrared Emission


Spectroscopy. The technique of measurement of
discrete vibrational frequencies emitted by thermally
excited molecules is known as Fourier Transform
Infrared Emission Spectroscopy (FTIR ES or simply
IES). IES has the great advantage that no sample
preparation is involved apart from micronising the
sample to < l m size. In the IES technique, the
heated sample acts as a source of the infrared
radiation and so no IR source is required. The
technique is particularly useful for the study of, for
example, thin films, polymers, catalyst and mineral
surfaces. The major advantage of IES is that the
samples are measured in situ at elevated
temperatures. Hence the technique removes the uncertainties introduced by heating the sample to

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elevated temperatures and quenching before measurement, as IES measures the surface
phenomena as it is actually taking place. Thus phase changes, dehydration, de hydroxylation,
surface adsorption and desorption may be readily studied. The technique when combined with
self-absorption is useful for the study of chemical changes on catalyst surfaces.

F) Surface Enhanced Infrared Absorption Spectroscopy (SEIRAS) Recent development in


the area of Surface Enhanced Infrared Absorption Spectroscopy (SEIRAS) overcomes some of
the limitations of sensitivity of detection presented by using traditional ATR FTIR spectroscopy
to study proteins. Complimentary to surface enhanced Raman spectroscopy, this technique
utilises the surface plasmon effect from the interaction of light with metallic nanoparticles. These
nanoparticles, mainly silver or gold are deposited onto the surface forming metal island films
which provide localised enhancement of IR absorption. It is important to note that unlike SERS,
the effect of enhancement in SEIRAS is not limited to gold or silver but has also been shown for
other metals. It is thought that the main contributing factor to the SEIRAS effect is the
polarization of the metal island films by the incident IR radiation. This induced dipole generates
a local electromagnetic field and the coupling of the local electromagnetic field with that of the
incident IR radiation produces one that is stronger than the incident photon field, in the vicinity
of the metal islands. The dipolar moment of molecules absorbed onto the metal islands is
enlarged because of this, enhancing the infrared absorption of the molecules. One of the major
drawbacks of SEIRAS can be the loss of spectral intensity due to the metal film deposited on the
IRE surface if, for example, the film is too thick. Therefore, preparation of the metal island film
is very important. The signal enhancement of adsorbed protein achievable from SEIRAS is in the
range of 2 orders of magnitude greater than without enhancement. The high sensitivity of the
technique allows it to provide information from monolayer level and this is useful for examining
the structure, function and assembly of molecules at the ATR interface. As with traditional ATR
FTIR spectroscopy, measurements are carried out in-situ meaning that dynamic processes on a
molecular level can be studied using SEIRAS. It is worth mentioning that the signal
enhancement drops off rapidly within 10 nm from the surface and as such it is necessary to
ensure that the biological material being studied is attached to the metal island film, often
achieved through chemical modification of the film itself.
SEIRAS has been widely used to address the biochemical problems. Use of SEIRA results in
high surface sensitivity by enhancing the signal of the adsorbed molecule by approximately two
orders of magnitude and has the potential to enable new studies, from fundamental aspects to
applied sciences. It is reported to use in the studies of DNA and nucleic acid adsorption to gold
surfaces, development of immunoassays, electron transfer between metal electrodes and proteins,
and protein–protein interactions. Another area in which SEIRAS has been exploited is in the
cancer research where it is used as a diagnostic criterion. This technique is crucial for
determining the properties of nucleic acids of tumor tissues and their interaction with anticancer
drugs.
G) Polarization modulated infrared reflection spectroscopy (PM-IRRAS) Polarization
modulated infrared reflection spectroscopy is also often used to study biological monolayers,
particularly lipid monolayers. Whilst this method can provide information on orientation and
structure of protein monolayers, the technique lacks the sensitivity to detect minute structural
changes that could occur during functional studies, which is one of the main advantages of
SEIRAS. PM-IRRAS is known to improve the infrared signal sensitivity of spread molecules at

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the interface, using a polarization modulation that reduces the strong anisotropic water
interference in the amide I and II region of the infrared spectra. This technique has been utilized
in the study of state of photosystem II core complex (PS II CC) in order to get molecular
information about the structure of this protein complex. This technique has a powerful capacity
to provide direct structural information on membrane proteins when spread in monolayers at the
air-water interface. With this technique, it is possible to prepare monolayers of membrane
proteins where its native secondary structure is maintained (Gallant et al, 1998). PM-IRRAS is a
highly surface specific FT-IR method that is capable of detecting chemical compositions from
interfacial films down to one molecule thick films. The PM-IRRAS technique allows enhanced
detection on substrates and measurements from the air-water interface. Changes in the PM-
IRRAS signal intensity and position can be used to infer molecular absorption/desorption
behavior and kinetics, molecular packing, phase transitions, hydration, hydrogen bonding and
different surface reactions in a thin film. This technique is also useful in studying interaction of
biomolecules using cell membrane models to understand reactions related to drug delivery and
the membrane behavior itself. These kinds of model systems are employed in several application
areas, such as drug development, food technology, and biological and biochemical research.
H) Nanoscale IR Spectroscopy: Atomic Force Microscopy (AFM) and infrared (IR)
spectroscopy (AFM-IR) The combination of atomic force microscopy (AFM) and infrared (IR)
spectroscopy in the technique of AFM-IR is one of the most important recent developments in
the field of IR micro spectroscopy and chemical imaging. However, the fundamental physical
limit imposed by diffraction has prevented the use of this technique in applications requiring
high spatial resolution, which is the case for many applications in polymers and the life sciences.
AFM-IR uses an AFM probe as the IR absorbance sensor and hence breaks through the
diffraction limit to attain spatial resolution improvements of up to two orders of magnitude over
traditional IR micro spectroscopy. A spatial resolution breakthrough has been achieved with a
novel technique that uses a nanoscale probe from an atomic force microscope acting as the IR
absorbance detector. The nature of the IR absorbance detection results in simultaneous
measurements of nanoscale mechanical properties and nanoscale morphology, along with the
chemical composition. The technique also integrates nanoscale thermal property mapping,
resulting in a multifunctional tool that provides nanoscale structure, chemical, mechanical, and
thermal properties. In the life sciences, AFM-IR has the ability to perform chemical spectroscopy
at the sub-cellular level. Specifically, the AFM–IR technique provides a label-free method for
mapping IR-absorbing species in biological materials. On the polymer side, AFM–IR was used
to map the IR absorption properties of polymer blends, multilayer films, thin films for active
devices such as organic photovoltaics, microdomains in a semicrystalline polyhydroxanoate
copolymer, as well as model pharmaceutical blend systems. By taking advantage of the ability to
arbitrarily control the polarization direction of the IR excitation laser, it is possible to obtain
important information regarding molecular orientation in electrospun nanofibers.

I) Planar-Array IR (PAIR) Spectroscopy PAIR Technologies makes instruments combining


dispersive elements like prisms and gratings with a standard globar and a two-dimensional (2D)
IR focal plane array to characterize dynamics in materials in real time. It is a real double-beam
instrument that takes sample and reference simultaneously using a very fast IR camera. One can
get a spectrum in 100 μs. The x-axis is the frequency domain and the y-axis provides spatial

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information on samples placed in the beam, either in transmission or using a specially developed
hemispherical attenuated total reflectance accessory.
J) Synchrotron radiation based Fourier-transform infrared (SR-FTIR) microspectroscopy
This is an emerging technique, which is increasingly employed in analytical sciences. This
technique combines FTIR spectroscopy (namely specific identification of molecular groups
within a variety of environments: organic/inorganic, crystallized/amorphous, solid/liquid/gas)
with high brightness, and therefore small spot size and faster acquisition of high-quality spectral
imaging data from a synchrotron light source. IR emission from a synchrotron radiation (SR)
source has potentially higher brilliance compared to a thermal source. The synchrotron source
offers brightness (or brilliance or spectral radiance) 2–3 orders of magnitude higher than a
thermal (laboratory-based) IR source, a high degree of polarization, as well as light pulses in the
2–10 ps time scale. They are thoroughly exploited in the far-IR (FIR)/THz, and mid-IR (MIR)
regions. In the long-wavelength domain, both flux and brightness exceed those of the thermal
source. The most rapidly expanding application of the synchrotron IR source is micro
spectroscopy on individual sample spots, as well as for chemical imaging. The principal
advantages of the method are enhanced lateral resolution typically at or very close to the
diffraction limit combined with superior signal-to-noise ratio obtained without resorting to
prohibitively long acquisition times. By using the SR-FTIR technique, intensities and the
distribution of the biological components (such as lignin, protein, lipid, structural and non-
structural carbohydrates and their ratios) in the microstructure of plant tissue within cellular
dimensions could be imaged. From this we can chemically define the intrinsic feed structure and
compare feed tissues according to spectroscopic characteristics, functional groups, spatial
distribution and chemical intensity. The SR-FTIR technique finds a good place in feed science
and animal nutrition research.
Near Infrared Spectroscopy
Near-infrared (NIR) spectroscopy is based on the absorption of electromagnetic radiation at
wavelengths in the range 780–2500 nm. NIR spectra of foods comprise broad bands arising from
overlapping absorptions corresponding mainly to overtones and combinations of vibrational
modes involving C-H, O-H and N-H chemical bonds. The concentrations of constituents such as
water, protein, fat and carbohydrate can in principle be determined using classical absorption
spectroscopy. However, for most food samples, this chemical information is obscured by
changes in the spectra caused by physical properties such as the particle size of powders. This
means that NIR spectroscopy becomes a secondary method requiring calibration against a
reference method for the constituent of interest. As a consequence of the physics of diffuse
transmittance and reflectance and the complexity of the spectra, calibration is normally carried
out using multivariate mathematics (chemometrics). NIR spectroscopy is used routinely for the
compositional, functional and sensory analysis of food ingredients, process intermediates and
final products. The major advantage of NIR is that usually no sample preparation is necessary,
hence the analysis is very simple and very fast (between 15 and 90 s) and can be carried out on-
line. One of the strengths of NIR technology is that it allows several constituents to be measured
concurrently. In addition, for each fundamental vibration there exists a corresponding series of
overtone and combination bands with each successive overtone band approximately an order of
magnitude less intense than the preceding one. This provides a built-in dilution series which
allows several choices of absorptions of different intensity containing the same chemical

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information. Finally, the relatively weak absorption due to water enables high-moisture foods to
be analyzed. The major limitation of NIR spectroscopy in food analysis is its dependence on
less-precise reference methods. Both NIR and MIR provide very useful qualitative and
quantitative information of different classes of antioxidants due to its simplicity and low cost.
Near infrared (NIR) and mid infrared (MIR) spectroscopy have been used to quantify several
compounds having a diverse antioxidant capacity such as carotenoids, polyphenols, fatty acids
and glucosinolates in a wide range of food commodities, for example, wine, dairy products, tea,
fruit, vegetables, herbs, spices and cereals. IR spectroscopy combined with chemometric
techniques such as Principal Components Analysis (PCA) is an extremely useful technique for
food analysis. Near- and Mid-IR are both non-destructive techniques requiring no sample
preparation. However, the longer effective pathlength and better spatial averaging available with
Near-IR diffuse reflectance is beneficial for adulterant detection, and using glass sample
containers allows for rapid analysis with no accessory cleaning required. Larger sampling areas
and the ability to spin the material during Near-IR analysis results in more reproducible and
representative sampling of food products with an inhomogeneous nature. Thus Near-IR
spectroscopy is the ideal solution for herb and spice authenticity and adulteration detection
analysis. There are also reports of using near-infrared (NIR) and mid-infrared (MIR)
spectroscopic techniques for process monitoring, quality control, and authenticity determination
in cheese processing.

References
1. Ataka, K., Stripp, S.T. and Heberle, J. (2013) Surface-enhanced infrared absorption
spectroscopy (SEIRAS) to probe monolayers of membrane proteins, Biochimica et
Biophysica Acta (BBA) - Biomembranes, 1828 (10): 2283-2293.
2. Bach, D. and Miller, I.R. (2001) Attenuated total reflection (ATR) Fourier transform
infrared spectroscopy of dimyristoyl phosphatidyl serine–cholesterol mixtures,
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1514 (2): 318–326.
3. Cozzolino, D (2015) Infrared spectroscopy as a versatile analytical tool for the
quantitative determination of antioxidants in agricultural products, foods and plants,
Antioxidants, 4: 482-497; doi:10.3390/antiox4030482
4. Dorling, K. M. and Baker, M. J. (2013) Highlighting attenuated total reflection Fourier
transform infrared spectroscopy for rapid serum analysis, Trends in Biotechnology, 31
(6): 327-328.
5. Frost, R.L. and Roberts, N.K. (2003) Vibrational Spectroscopy of Surfaces. In Surface Analysis
Methods in Materials Science, D.J. O'Connor, B.A. Sexton, R.St.C. Smart (Eds.) Chapter 8, pp
203-227.
6. Gallant, J., Desbat, B., Vaknin, D. and Salesse, C. (1998) Polarization-modulated infrared
spectroscopy and x-ray reflectivity of photosystem II core complex at the gas-water
interface, Biophysics Journal, 75 (6): 2888-2899.
7. Khoshhesab MZ (2012) Reflectance IR Spectroscopy. In Infrared Spectroscopy – Materials
Science, Engineering and Technology, Theophile Theophanides (Editor), Chapter 11, pp 233-244.
8. Manheim, J., Doty, K. C., McLaughlin, G. and Lednev, I. K. (2016) Forensic hair differentiation
using attenuated total reflection fourier transform infrared (ATR FT-IR) spectroscopy, Applied
Spectroscopy, 70 (7): 1109-1117.

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Application of NMR for Molecule Identification
Irani Mukherjee, V.S. Rana and Mukta Chakraborty
Division of Agricultural Chemiclas
ICAR- Indian Agricultural Research Institute, New Delhi

Since the early 1960s, NMR spectroscopy has become an indispensable tool for the
characterization of organic molecules. Although early spectrometers were relatively insensitive
and performed simple experiments at low-field strengths, they gave reliable measurements of
chemical shifts and coupling constants. Later, spectrometers were much more sensitive and
attained much higher field strengths, enabling the use of powerful 2D NMR experiments. In
particular, the pairing of COSY (correlation spectroscopy) with directly detected heteronuclear
correlation experiments (which link carbon atoms and nearby protons) proved particularly
efficacious for determining structural connectivity.

Nuclear Magnetic Resonance spectroscopy is a powerful and theoretically complex analytical


tool. The chemical environment of specific nuclei is deduced from information obtained about
the nuclei.

Nuclear spin and the splitting of energy levels in a magnetic field

Subatomic particles (electrons, protons and neutrons) can be imagined as spinning on their axes.
In many atoms (such as 12C) these spins are paired against each other, such that the nucleus of
the atom has no overall spin. However, in some atoms (such as 1H and 13C) the nucleus does
possess an overall spin. The rules for determining the net spin of a nucleus are as follows;

1. If the number of neutrons and the number of protons are both even, then the nucleus
has NO spin.
2. If the number of neutrons plus the number of protons is odd, then the nucleus has a half-
integer spin (i.e. 1/2, 3/2, 5/2)
3. If the number of neutrons and the number of protons are both odd, then the nucleus has
an integer spin (i.e. 1, 2, 3)

The overall spin, I, is important. Quantum mechanics tells us that a nucleus of spin I will have
2I + 1 possible orientations. A nucleus with spin 1/2 will have 2 possible orientations. In the
absence of an external magnetic field, these orientations are of equal energy. If a magnetic field
is applied, then the energy levels split. Each level is given a magnetic quantum number, m.

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When the nucleus is in a magnetic field, the initial populations of the energy levels are
determined by thermodynamics, as described by the Boltzmann distribution. This is very
important, and it means that the lower energy level will contain slightly more nuclei than the
higher level. It is possible to excite these nuclei into the higher level with electromagnetic
radiation. The frequency of radiation needed is determined by the difference in energy between
the energy levels.

Calculating transition energy

The nucleus has a positive charge and is spinning. This generates a small magnetic field. The
nucleus therefore possesses a magnetic moment, , which is proportional to its spin,I.

The constant, , is called the magnetogyric ratioand is a fundamental nuclear constant which has
a different value for every nucleus. h is Plancks constant.

The energy of a particular energy level is given by;

Where B is the strength of the magnetic field at the nucleus.

The difference in energy between levels (the transition energy) can be found from

This means that if the magnetic field, B, is increased, so is E. It also means that if a nucleus has
a relatively large magnetogyric ratio, then E is correspondingly large.

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If you had trouble understanding this section, try reading the next bit (The absorption of radiation
by a nucleus in a magnetic field) and then come back.

The absorption of radiation by a nucleus in a magnetic field

In this discussion, we will be taking a "classical" view of the


behaviour of the nucleus - that is, the behaviour of a charged particle
in a magnetic field.

Imagine a nucleus (of spin 1/2) in a magnetic field. This nucleus is in


the lower energy level (i.e. its magnetic moment does not oppose the
applied field). The nucleus is spinning on its axis. In the presence of a
magnetic field, this axis of rotation will precess around the magnetic
field;

The frequency of precession is termed the Larmor frequency, which


is identical to the transition frequency.

The potential energy of the precessing nucleus is given by;

E=- B cos

where is the angle between the direction of the applied


field and the axis of nuclear rotation.

If energy is absorbed by the nucleus, then the angle of


precession, , will change. For a nucleus of spin 1/2,
absorption of radiation "flips" the magnetic moment so that
it opposes the applied field (the higher energy state).

It is important to realise that only a small proportion of


"target" nuclei are in the lower energy state (and can absorb
radiation). There is the possibility that by exciting these
nuclei, the populations of the higher and lower energy levels
will become equal. If this occurs, then there will be no further absorption of radiation. The spin
system is saturated. The possibility of saturation means that we must be aware of the relaxation
processes which return nuclei to the lower energy state.

Relaxation processes

How do nuclei in the higher energy state return to the lower state? Emission of radiation is
insignificant because the probability of re-emission of photons varies with the cube of the
frequency. At radio frequencies, re-emission is negligible. We must focus on non-radiative
relaxation processes (thermodynamics!).

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Ideally, the NMR spectroscopist would like relaxation rates to be fast - but not too fast. If the
relaxation rate is fast, then saturation is reduced. If the relaxation rate is too fast, line-broadening
in the resultant NMR spectrum is observed.

There are two major relaxation processes;

• Spin - lattice (longitudinal) relaxation


• Spin - spin (transverse) relaxation

Spin - lattice relaxation

Nuclei in an NMR experiment are in a sample. The sample in which the nuclei are held is called
the lattice. Nuclei in the lattice are in vibrational and rotational motion, which creates a complex
magnetic field. The magnetic field caused by motion of nuclei within the lattice is called
the lattice field. This lattice field has many components. Some of these components will be equal
in frequency and phase to the Larmor frequency of the nuclei of interest. These components of
the lattice field can interact with nuclei in the higher energy state, and cause them to lose energy
(returning to the lower state). The energy that a nucleus loses increases the amount of vibration
and rotation within the lattice (resulting in a tiny rise in the temperature of the sample).

The relaxation time, T1 (the average lifetime of nuclei in the higher energy state) is dependant on
the magnetogyric ratio of the nucleus and the mobility of the lattice. As mobility increases, the
vibrational and rotational frequencies increase, making it more likely for a component of the
lattice field to be able to interact with excited nuclei. However, at extremely high mobilities, the
probability of a component of the lattice field being able to interact with excited nuclei
decreases.

Spin - spin relaxation

Spin - spin relaxation describes the interaction between neighbouring nuclei with identical
precessional frequencies but differing magnetic quantum states. In this situation, the nuclei can
exchange quantum states; a nucleus in the lower energy level will be excited, while the excited
nucleus relaxes to the lower energy state. There is no net change in the populations of the energy
states, but the average lifetime of a nucleus in the excited state will decrease. This can result in
line-broadening.

Chemical shift

The magnetic field at the nucleus is not equal to the applied magnetic field; electrons around the
nucleus shield it from the applied field. The difference between the applied magnetic field and
the field at the nucleus is termed the nuclear shielding.

Consider the s-electrons in a molecule. They have spherical symmetry and circulate in the
applied field, producing a magnetic field which opposes the applied field. This means that the
applied field strength must be increased for the nucleus to absorb at its transition frequency.
This upfield shift is also termed diamagnetic shift.

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Electrons in p-orbitals have no spherical symmetry. They produce comparatively large magnetic
fields at the nucleus, which give a low field shift. This "deshielding" is termed paramagnetic
shift.

In proton (1H) NMR, p-orbitals play no part (there aren't any!), which is why only a small range
of chemical shift (10 ppm) is observed. We can easily see the effect of s-electrons on the
chemical shift by looking at substituted methanes, CH3X. As X becomes increasingly
electronegative, so the electron density around the protons decreases, and they resonate at lower
field strengths (increasing H values).

Chemical shift is defined as nuclear shielding / applied magnetic field. Chemical shift is a
function of the nucleus and its environment. It is measured relative to a reference compound.
For 1H NMR, the reference is usually tetramethylsilane, Si (CH3)4.

Spin - spin coupling

Consider the structure of ethanol;

The 1H NMR spectrum of ethanol (below) shows the methyl peak has been split into three peaks
(a triplet) and the methylene peak has been split into four peaks (a quartet). This occurs because
there is a small interaction (coupling) between the two groups of protons. The spacings between
the peaks of the methyl triplet are equal to the spacings between the peaks of the methylene
quartet. This spacing is measured in Hertz and is called thecoupling constant, J.

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To see why the methyl peak is split into a triplet, let's look at the methylene protons. There are
two of them, and each can have one of two possible orientations (aligned with or opposed against
the applied field). This gives a total of four possible states;

In the first possible combination, spins are paired and opposed to the field. This has the effect of
reducing the field experienced by the methyl protons; therefore a slightly higher field is needed
to bring them to resonance, resulting in an upfield shift. Neither combination of spins opposed to
each other has an effect on the methyl peak. The spins paired in the direction of the field produce
a downfield shift. Hence, the methyl peak is split into three, with the ratio of areas 1:2:1.

Similarly, the effect of the methyl protons on the methylene protons is such that there are eight
possible spin combinations for the three methyl protons;

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Out of these eight groups, there are two groups of three magnetically equivalent combinations.
The methylene peak is split into a quartet. The areas of the peaks in the quartet have the ration
1:3:3:1.

In a first-order spectrum (where the chemical shift between interacting groups is much larger
than their coupling constant), interpretation of splitting patterns is quite straightforward;

• The multiplicity of a multiplet is given by the number of


equivalent protons in neighbouring atoms plus one, i.e. the n + 1 rule
• Equivalent nuclei do not interact with each other. The three methyl protons in ethanol
cause splitting of the neighbouring methylene protons; they do not cause splitting among
themselves
• The coupling constant is not dependant on the applied field. Multiplets can be easily
distinguished from closely spaced chemical shift peaks.

Nuclear Magnetic Resonance Spectrometers:


The basic elements of a typical n.m.r. spectrometer consist of the main parts;

(1) A magnet with strong, stable homogeneous field. The field must be constant over the area of
the sample.
(2) A radio frequency oscillator (transmitter) connected to coil which transmits energy to the
sample in a direction perpendicular to the magnetic field.
(3) A sample container, usually a glass tube spun by an air driven turbine to average the
magnetic field over the sample dimensions.
(4) A radio frequency receiver connected to a coil encircling the sample. The two coils are
perpendicular to each other and to the magnetic field.
(5) A read out system. The other supporting parts are— consisting of an amplifier, recorder and
additional components for increasing sensitivity, accuracy or convenience.
(6) A sweep generator which supplies a variable d-c current to a secondary magnet so that the
total applied magnetic field can be varied (swept) over a limited range.

Two ways have been employed in NMR experiments for getting the desired particular
combinations:

Fig 1 Schematic diagram of Simple NMR instrumentatiom

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(i) In one way, the magnetic field remains constant and radio frequency is varied.

(ii) In second, the radio frequency remains unchanged and magnetic field is varied till resonance
conditions are obtained and there is detectable absorption by the nucleus.

Instrument:
In block diagram, the blocks labelled N and S represent the poles of the large HO magnet, which
is generally an electromagnet operated through a stabilized power supply. A field of up-to 1400
gauss and a pole of about 1.75 — 1.8 inch is necessary for high resolution spectra. The frequency
and field strength are related to each other by Larmor condition.

v = үH0/2π
[This equation represents the condition of resonance.]

where HO = magnetic field,

v = is the frequency of radiation associate with transition from one state to another. It is generally
known as Larmor frequency,

ү = proportionality constant or gyromagnetic ratio.

The most important molecular parameter determined by NMR is the chemical shift. The
chemical shift is defined as a measure of the resonance frequency of the nuclei in a given
chemical environment.The magnitude of the chemical shift is proportional to the strength of
applied field and is caused by the circulations of surrounding electrons about the protons.

Applications of NMR

Today, all but the most complex organic molecules are amenable to routine analysis, even with
submilligram sample quantities. Recent work has uncovered the structural details of not only
natural products of every class but also synthetic creations. Well-characterized examples include

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platensimycin (1),(Singh etal., 2006) a broad spectrum nonmevalonate terpenoid antibiotic,
maoecrystal V (2), (Li et al., 2004 ) an antitumor diterpenoid, chlorofusin, a peptide-based fungal
metabolite with anticancer properties,(Duncan 2001) daphlongeranine B (3),(Li , 2007) an
unusual polycyclic alkaloid, and cytosporic acid (4),( Jayasuriya , 2007) a polyketide- derived
HIV-1 integrase inhibitor, as well as β,β_-disilyl- substituted vinyl cations 5 (Muller, 2005 ) and
cyanoresorc (Li etal ., 2004) larene 6. D’Acquarica, 2006).

NMR-based compound identification from mixtures: a paradigm shift

Until recently identification of new biogenic small molecules – usually obtained as part of a
complex metabolite mixture - largely relied on the investigator’s ability to isolate the compound
of interest chromatographically. Chromatographic fractionation, often guided by assays for
specific biological activities, followed by NMR spectroscopic characterization of pure, isolated
compounds constitutes the traditional approach of structure determination in natural products
chemistry and chemical biology. Usually, achieving a high degree of purity was deemed
essential for the success of subsequent NMR-spectroscopic analyses . Moreover, recent examples
show that activity-guided isolation and characterization of metabolites from complex mixtures
may overlook biologically important compounds.

Identification of unstable alkaloids from unfractionated biofluids


The first examples for using NMR spectroscopy to identify new compounds from complex
mixtures originated from arthropod natural products studies [16,17]. Myrmicaria ants produce
copious amounts of a highly toxic poison gland secretion, however, mass spectrometric analyses
of some secretion samples revealed only non-toxic monoterpene hydrocarbons. It was then
observed that the secretion rapidly lost its toxic properties after collection, likely due to
decomposition upon exposure to air. Consequently, fresh, unfractionated secretion was subjected
to NMR-spectroscopic analyses, which lead to the identification of myrmicarin 430A (1,Figure

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2) and related structures, representing a new family of heptacyclic alkaloids. Instrumental for the
identification of myrmicarin 430A from the unfractionated ant secretion, which also contained
large quantities of monoterpene hydrocarbons and other alkaloids, was the use of high-resolution
dqfCOSY spectra [17]. Because dqfCOSY crosspeaks contain detailed coupling constant
information and due to their high fidelity (excellent peak shape), structural assignments were
possible even in cases were signals overlapped or signals of very low intensity had to be
discerned.

Single insect NMR

Dossey et al. extended the scope of 2D NMR-spectroscopic analyses of unfractionated biofluids


by demonstrating the utility of reduced-volume probes to increase mass sensitivity. Using a 1-
mm HTS cryogenic probe that afforded 25-fold greater sensitivity than conventional probes at
that time [18], unfractionted defensive secretion from individual male walking sticks
(Anisomorpha buprestoides) was analyzed via COSY, TOCSY, ROESY, and natural abundance
(1H,13C)-HMQC and HMBC [19]. Based on these spectra, the walking stick secretion was
shown to consist of glucose and a mixture of monoterpene dialdehydes that are stereoisomers of
the known dolichodial (2, Figure 2). For the case of A. buprestoides, the authors further
demonstrated the use of a computational deconvolution procedure for the interpretation of
TOCSY spectra of mixtures (“DemixC”) which will be discussed in a following section. In
addition to known compounds, Dossey et al. discovered novel metabolites using 2D NMR of
unfractionated extracts. Using the same low-volume HTS cryogenic probe, the defensive spray
of the walking stick Parectatosoma mocquerysi was found to contain the novel monoterpene
parectadial [20]. After partitioning the defensive spray between D2O and benzene-d6, COSY,
(1H,13C)-HMQC, (1H,13C)-HMBC, and NOESY acquired.

Figure 2. Structures of new natural products identified via NMR-spectroscopic analyses of


complex mixtures. Myrmicarin 430A (1) and bacillaene (6) represent members of a
small but growing class of metabolites that have never been isolated in pure form.

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New fungal metabolites from NMR-spectroscopic whole-metabolome analyses

Differential analyses of 2D NMR spectra (DANS), a method for graphic comparison of 2D NMR
spectra representing different biological states, was first applied to a small library of metabolite
extracts derived from cultures of the filamentous fungus, Tolypocladium cylindrosporum. This
study aimed to detect and identify products of PKS/NRPS pathways induced by specific
environmental conditions, for example nutritional stress, and used DANS to compare dqfCOSY
spectra obtained for metabolite extracts derived from seven different culturing protocols [13].
The dqfCOSY spectra representing different conditions were overlayed and processed using a
simple algorithm that highlighted spectroscopic signals that are strongly differential between
spectra, i.e. signals that represent compounds whose biosynthesis is strongly induced under
specific culturing conditions. This approach led to detection and identification of two new indole
alkaloids, TC-705A (5, Figure 2) and TC-705B. These structures were proposed based on NMR-
spectroscopic analyses of the unfractionated extracts and subsequently were confirmed through
additional spectroscopic analyses of chromatographically enriched samples of TC-705A and TC-
705B. In addition to highlighting those extracts that contained structurally interesting new
metabolites, the DANS analyses of the unfractionated fungal metabolite extracts continued to be
of use, as (1) the fractionation could be guided by NMR-spectroscopic signals recognized as
representing differentially expressed compounds, and (2) the original DANS spectroscopic data
provided positive evidence that the enriched metabolites were not artifacts of the isolation
process.
Nuclear magnetic resonance (NMR) spectroscopy can be used to both identify and quantify
chemicals from complex mixtures. This can be done semi-automatically by comparing the
mixture of interest to a library of reference spectra derived from pure compounds of known
concentrations. This particular approach is now being exploited to characterize the metabolomes
of many different biological samples in what is called quantitative metabolomics or targeted
metabolic profiling.
References
1. Acquaric I. Da, L. Nevola, G. D. Monache, E. Gács-Baitz, C.Massera, F. Ugozzoli, G.
Zappia, B. Botta, Eur. J. Org. Chem.2006, 3652–3660.
2. Brey WW, Edison AS, Nast RE, Rocca JR, Saha S, Withers RS. Design, construction,
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3. Validation of a 1-mm triple-resonance high-temperature-superconducting probe for
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4. Caenorhabditis elegans. Proc Natl Acad Sci U S A 2009;106: 7708–7713. [PubMed:
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5. Dossey AT, Walse SS, Rocca JR, Edison AS. Single insect NMR: A new tool to probe
chemical biodiversity. ACS Chem Biol 2006; 1: 511–514. [PubMed: 17168538]
6. Dossey AT, Walse SS, Conle OV, Edison AS. Parectadial, a monoterpenoid from the
defensive spray of Parectatosoma mocquerysi. J Nat Prod 2007;70: 1335–1338.
[PubMed: 17661519]
7. Duncan S. J., J. Am. Chem. Soc. 2001, 123, 554–560.
8. Forseth Ry R. and Frank C. Schroeder (2011) NMR-spectroscopic analysis of mixtures:
from structure to function Curr Opin Chem Biol. 2011 15(1): 38–47

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9. Jayasuriya H., Z. Guan, J. D. Polishook, A. W. Dombrowski, P. J. Felock, D. J. Hazuda,
S. B. Singh, J. Nat. Prod. 2003, 66,551–553.
10. Koehn FE, Carter GT. The evolving role of natural products in drug discovery. Nat Rev
Drug
11. Discov 2005; 4:2 06–220. [PubMed: 15729362]
12. Li S.-H., J. Wang, X.-M. Niu, Y.-H. Shen, H.-J. Zhang, H.-D. Sun, M.-L. Li, Q.-E Tian,
Y. Lu, P. Cao, Q.-T. Zheng, Org.Lett. 2004, 6, 4327–4330.
13. Li C.-S., Y.-T. Di, H.-P. He, S. Gao, Y.-H. Wang, Y. Lu, J.-L. Zhong, X.-J. Hao, Org.
Lett. 2007, 9, 2509–2512.
14. MülleT. D. Margraf, Y. Syha, J. Am. Chem. Soc. 2005, 127, 10852–10860.
15. Pungaliya 1C, Srinivasan J, Fox BW, Malik RU, Ludewig AH, Sternberg PW, Schroeder
FC. A shortcut to identifying small molecule signals that regulate behavior and
development in
16. Schroder F, Franke S, Francke W, Baumann H, Kaib M, Pasteels JM, Daloze D. A new
family of tricyclic alkaloids from Myrmicaria ants. Tetrahedron 1996; 52 :13539–13546.
17. Schroder F, Sinnwell V, Baumann H, Kaib M. Myrmicarin 430A: A new heptacyclic
alkaloid from Myrmicaria ants. Chemical Communications 1996:2139–2140.
18. Singh S. B., H. Jayasuriya, J. G. Ondeyka, K. B. Herath, C.Zhang, D. L. Zink, N. N.
Tsou, R. G. Ball, A. Basilio, O. Gen yilloud, M. T. Diez, F. Vicente, F. Pelaez, K. Young,
J. Wang, J. Structural Elucidation with NMR Spectroscopy Am. Chem. Soc. 2006, 128,
11916–11920.

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Techniques for Pesticide Decontamination from Fruits and
Vegetables
Shashi Bala Singh, Neera Singh, Indu Chopra and Birendra Kumar
Division of Agricultural Chemicals
ICAR-Indian Agricultural Research Institute, New Delhi-110 012

Agricultural crops during their growth period are infested by many insect pests and diseases. To
combat these pest problems and assure high crop production pesticides are widely used plant
protection tools farmers can depend upon. They are utilized during cultivation and post-harvest
treatment of agricultural commodities (FAO/WHO, 2004). Use of agricultural pesticides has
enabled mass and stable production of fruits and vegetable in present day agriculture. However
use of un-recommended pesticides and dosages many a times lead to pesticide residues.
Additionally, sometimes due to lack of storage facilities or due to their perishable nature,
harvested produce are rushed to market, leaving large amounts of pesticides residues on fruits
and vegetables. The issue is particularly serious when these commodities are consumed fresh.
Although humans are exposed to small amounts of pesticide residues after pesticides are
metabolized by plants or decomposed by environmental agents, trace amounts of pesticide
residing in the human body for long periods have been linked with a wide range of human
wellbeing menace, extending from transient effects, for example, cerebral pains and nausea to
chronic effects like cancer, conceptive harm, and endocrine disturbance (Chen et al., 2011) and
can cause chronic diseases like cancer (Carrozza et al., 2009). As a result, the food commodities
are often found with unsafe levels of pesticides and produce unforeseen situation for consumers.
Pesticide removal techniques for fruits and vegetables

Use of agricultural pesticides has enabled mass and stable production of fruits and vegetables in
present day agriculture. However, pesticide residues in food produce pose adverse risks to
consumers, inducing effects such as immuno, reproductive and neuro toxicities. Half-life periods
of many pesticide residues in food commodities kept in typical storage conditions of 30ºC and
50% relative humidity ranged from 2 to 70 weeks. Unprocessed foods with high water content
like berries are kept in cold storage (0−5ºC), or under deep freeze conditions (-10 to -20ºC) for
longer storage. Under such storage conditions initial levels of residues showed much lower
decrease even less than 20%. Therefore, the development of effective techniques for post-harvest
removal and degradation of pesticides at consumer level become necessary. Detectable level of
pesticide residues in fruits and vegetables are reported from time to time by different countries.
Washing before consumption of fruits and vegetables is an age old practice. Even when no
pesticide was used in agriculture, the washing was used to get rid of surface microbial load as
well as other dirty particles. With modernization of agriculture and advancement of technologies
human brain has devised many techniques for overproduction including pesticides as unavoidable inputs
and the presence of residue in food commodities is also obvious. Therefore various techniques which are
being used from time to time to get rid of these notorious chemical for safe consumption are investigated.
To name a few are as follows-

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(i) Washing
(ii) Peeling
(iii) Cooking
(iv) UV radiation, sonication and pulsed electric field
(v) Washing with reagents and commercial washes
(vi) Chemical dips – hypochlorous solution and ozone
Washing
Washing procedures in households are generally carried out at moderate temperatures with
standing or running water. The effect depends on the properties of the pesticides, such as
volatility, water solubility, hydrolytic rate constant and octanol-water partition coefficient as well
as the location of the residues. Washing processes generally reduce the hydrophilic residues
located on the outer surface of the fruits and vegetables. The residue level is influenced by the
temperature of the water used for washing and the nature of washing. Washing with hot water
and the use of detergent are more effective than that of cold water washing. Washing along with
gentle rubbing of surface under tap water for one minute significantly dislodges the pesticide
residues (Barooah and Yein, 1996). However, lipophilic and systemic pesticide residues are not
significantly removed by washing. For example, Geisman (1975) reported that washing of field
tomatoes could reduce the residues of almost all the pesticides except that of methamidaphos
which is highly polar and systemic in nature. Also, there are reports that suggest that the removal
of pesticide residues from crop produce surfaces by washing decreases over the time. This may
be due to the movement of residues to deeper layers or cuticular waxes. Residues of fenitrothion
or methidathion on cauliflower when subjected to washing or blanching were found to be
inversely related to the number of days passed after spraying (Street, 1969). Reduction of DDT
residues after washing with water varies for different vegetables, from 20% in potatoes up to
91% in tomatoes. It has been reported that residue levels of pesticides such as lindane and
hexachlorobenzene could be removed with water only (Holland et al., 1994; Soliman et al.,
2001). However, these methods remove pesticide residues by transferring them to the washing
solution. Therefore, such treatment contributes to increase in pesticide active ingredient levels in
soil or water mediums. The efficiency of removal by washing is dependent on many factors like
location of pesticide residues within the fruit, time passed after exposing to active ingredient and
solubility of residue in water. Washing type, washing time and temperature also impact this
process. Considerable improvement in efficiency may be achieved by increasing the solution
temperature and addition of additional agents such as detergents.
Peeling
Pesticide residues are often present in greater amount on the outer leaves of vegetable crops
because of the pesticide application during the growing season. Therefore, trimming or peeling
methods are useful in reducing the pesticide residues in leafy vegetables. Peeling of bulb, tuber
and root vegetables using a knife is common practice in households. Peeling may remove more
than 50% of the pesticide residues present in the commodity. Sugibayashi et al. (1996) reported

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that among household methods, peeling was most effective in removing the pesticide from the
vegetables followed by frying. The non-systemic types of surface residues are generally
concentrated in the peels of fruits and vegetables. In the case of systemic pesticides, the peeling
method may not prove as effective.
Cooking
Cooking of food for different conditions such as duration, temperatures, water content or food
additives level, and whether the system is open or closed may have an effect on the residue level.
Generally, pesticide residues are decreased during cooking by volatilization process or by the
process of hydrolysis. Also, adding cooking liquid dilutes the residues. Many studies have been
reported for the decontamination of pesticides residues in food by cooking. The degradation of
the organophosphorus pesticides, fenitrpothion, prothiophos, chlorfenvinphos, methidathion and
isoxathion during cooking was reported by Nagayama (1996) in tea green leaves, fruits and
spinach. Cooking process decreased these pesticides corresponding to the boiling time. Similarly,
Thanki et al. (2012) compared the raw, cooked and roasted brinjal samples for their residues of
p,p’ DDT, cypermethrin, monocrotophos, carbaryl, endosulphan, quinalphos, p,p’ DDD,
captafol, permethrin, phorate and pendimethalin. It was found that the pesticide residues in
eggplant were substantially decreased by processing. The study showed that the roasting was
better method for pesticide removal than cooking. In another study, Sengupta et al. (2010)
reported that the initial level of pesticides in chicken meat was considerably reduced after
cooking and baking. Further, the study concludes that cooking is better method than roasting in
decreasing the pesticide load in meats of different animal sources.
Ultra violet radiation, sonication and pulsed electric field
Ultra violet radiation has been used for photocatalytic degradation of pesticide residues.
Bhatkhande et al. (2004) reported the photocatalytic degradation in the case of chlorobenzene
using artificial UV and solar radiations. Photocatalytic activity of Mo6+, V5+ and Th4+ doped
polycrystalline TiO2 was studied for the degradation of residues of chlorpyrifos under ultra violet
and solar light (Devi et al., 2009). Gong et al. (2011) studied the effect of sonication along with
ozone on degradation of carbendazim, malathion, chlorothalonil and diphenylamine in apple
fruits. The effect of combination of ozone and ultrasound treatment was found to be better than
ultrasound alone and single ozone water process. Chen et al (2009) reported that pulsed electric
field (PEF) has significant effects on the degradation of methamidophos and chlorpyrifos. PEF
treatment was effective in degradation of chlorpyrifos and methamidophos residues in apple
juice. Of the two pesticides, chlorpyrifos was more labile to pulsed electric field than
methamidophos.
Washing with reagents and commercial washes
Home remedies such as use of vinegar solution and baking soda, can also be used for pesticide
removal. Soaking in 50% vinegar solution and then rinsing with water is effective. Also,
spraying with a combination of one table spoon lemon juice, two table spoons of baking soda,
and one cup of water can be done. Zohair (2001) reported that acidic reagents such as acetic acid,
ascorbic acid and citric acid were more useful in reducing the residual content of pesticides than
neutral or alkaline solution, as well as plain water. Washing potatoes with solutions of acetic acid
and sodium chloride at different concentrations enabled reduction of residues within ranges
224 Winter School on "New generation smart agrochemical oriented approaches for crop
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18.2−40.1% for DDT, 18.8−65.3% for lindane and 23.7−59.7% for HCB. However, removal of
organophosphorus insecticides residues such as dimethoate, primiphos-methyl and malathion
was more effective with alkaline solutions. Reduction of fungicides such as myclobutanil
(triazole), fenhexamid (hydroxyanilide) and boscalid (carboxamide) was possible within the
range of 17.3−65.4% for washing procedures where water, sodium carbonate (Na2CO3), sodium
hypochlorite (NaClO), glycerol and acetic acid were utilized.
Potassium permanganate solutions effectively remove a variety of chemical pesticide residues
from fruits and vegetables, making them safer for consumption. Dilute solutions of potassium
permanganate degrades the polyaromatic hydrocarbons, chlorinated solvents, organo-pesticides
phenolics and substituted aromatics. Leafy Chinese-Kale treated with potassium permanganate
solution (0.001%) effectively removed pesticide residues. Washing vegetables in potassium
permanganate removed more pesticide residue than washing in water alone (Klinhom et al.,
2008). Potassium permanganate can also reduce the number of pests present on the surface of
plants themselves. For example, potassium permanganate effectively kills bacteria and harmful
micro-organisms such as Fasciola, a flatworm parasite.
Sodium chloride solution is largely used to decontaminate the pesticide residues from different
fruits and vegetables. There are several studies to prove the efficacy of salt water washing to
dislodge the pesticides from crops. In this process, chopped vegetables and fruit samples are
placed in a beaker having 5% sodium chloride solution. After 15 minutes the plant samples are
gently rubbed by hand in salt solution and all water is decanted. Kumar et al. (2000) reported
that dipping green chillies in 2% sodium chloride solution for 10 min followed by washing with
water prove to be effective method, facilitating 32.56 and 84.21% removal of residues
respectively at 0 and 5 days after the spraying of 700g a.i. ha-1 triazophos while the acephate
residues were removed to an extent of 78.95% at zero day.
Veg n Fru Wash is a commercially available fruits and vegetables wash. It is based on patented
edible ingredients. The product is claimed to wash surface contaminants like pesticide residues,
microorganisms, wax, sand, grime dirt etc. in single wash. Kalra et al. (2015) reported the
removal of malathion residues up to 71% from eggplants using Veg n Fru wash.
Pusa reagent, a commercial wash developed by Division of Agricultural Chemicals, IARI,
contains 0.5% of edible alkali and 0.1% KMnO4. Washing with Pusa reagent could remove
malathion residues upto 91% from eggplant fruits (Kalra et al., 2015).
Chlorine based washes have been used for microbial sanitation purposes. However, these may
produce toxic compounds like trihalomethane in water or on food surfaces. Due to their possible
effect on health, these products have been restricted for use in many countries (Beltran, 2005).
Chemical dips
Hypochlorous solution generators are commercial equipments which produce electrolyzed
oxidizing and acidified chlorinated water solutions for the sanitation purposes. These generators
kill bacteria and microorganisms and thus find application in medical field (Park et al., 2001).
Water electrolyzer are available in market which produce electrolyzed oxidizing (EO) water for
reducing bacteria, pathogens and viruses (Gomez et al., 2012). The electrolyzer produces acid

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and alkaline solutions at the same time. Several reports are available on the efficacy of EO water
in removing various microbes and pathogens from surfaces of fruits like apple (Okull and
Laborde, 2004) strawberries and broccoli (Hung et al., 2010). The effect of these techques on the
quality of fruits has also been checked (Rico et al., 2007; Ji et al., 2012).
Ozone
Ozone (O3) is a natural gas at ambient and refrigerated temperatures. It is generated by the
passage of air or oxygen gas through a high voltage electrical discharge or by ultraviolet light
irradiation. Ozone has been used for deodorization, sterilization, virus inactivation, bleaching,
organic matter decomposition, mycotoxin degradation and others. It is Generally Recognised as
Safe (GRAS) for food contact application and it changes to oxygen by autolysis (USFDA, 1997).
Due to its quick decomposition nature, ozone overcomes the concerns of residues and therefore it
has been of particular interest for food safety uses. Ozone is effectively applied to drinking water
treatment and wastewater treatment for its powerful oxidization potential. Ozone has been
widely used as fumigant in the storage of fruits and vegetables as it helps in preventing them
from many fungal and microbial diseases. It has also been evaluated for use in the field for pest
control. Ozone has also been used for the disinfection of swimming pools, bottled water, waste
water treatment and for preventing fouling of cooling towers.
Recently, ozone water dips have been commercialized for removal of pesticides and microbes
from food surfaces. The equipment generally contains an output hose of ozone gas which is
immersed in water. Fruit, vegetables and meat products are treated by dipping in this ozonated
water. Concentration of 0.25 ppm ozone in water enabled reduction of over 50% of residue
levels of substances such as azinphos-methyl, captan (phthalimide fungicide) and formetanate
hydrochloride in fresh apples and their products. Aqueous solution containing 2 ppm of ozone
enabled 60% reduction of initial residue level of diazinon, parathion, methyl-parathion and
cypermethrin in vegetables (Balawejder, 2013). A standard chlorpyrifos solution (1 mg L-1)
when treated with 1 MHz ultrasonic irradiation, ozone and their combination for 0, 10, 20, 30,
40, 50 and 60 min, the combined treatment was found to have a synergistic effect in decreasing
chlorpyrifos concentrations, with the maximum reduction occurring within the first ten minutes.
Chlorpyrifos residues on bird chillies (Capsicum frutescens Linn.) after ultrasonic and ozone
washing were reduced 73.03% in 60 min (Pengphol, 2012).
Degradation percentage varies for different pesticides. In a study, aqueous solution of diazinon
(initial concentration of 10–30mg L-1) was completely degraded in 20 min, while only 80% of
either methyl parathion or parathion at an initial concentration of 20 mg L-1 was degraded in
30min by ozonation at 1.4 mg L-1. The possibilities of utilizing low level (1.4–2.0 mg L-1) of
dissolved ozone for reduction of the residues of four pesticides on vegetable (Brassica rapa)
surface were also tested. Ozone was mainly effective in the removal of cypermethrin (Wu et al.,
2007). Ozone enriched solution in water resulted in 59% reduction of residues of mancozeb in
blackcurrants. However, utilization of gaseous ozone permitted a 38% reduction of mancozeb
residues (Antos et al., 2013). Removal of 60% of chlorothalonil from table grapes (pulp and
skin) was observed regardless of ozone concentration. Ozone treatment of table grapes at a
concentration of 3 mg L−1 changed most of the quality parameters evaluated. Treatment at 2 mg
L−1 maintained the fruit quality for a longer storage period compared to the untreated control
table grapes (Heleno et al., 2015). Lychee fruits when treated with both ozone gas and ozone-

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containing water reduced chlorpyrifos residue but exposure to ozone gas for 60 min was found to
be most effective (Wangchai et al., 2011). Fungicides residues on berries at gaseous ozone
concentrations of 900 ± 12 ppmv (μL L–1) over 2 h were decreased for fenhexamid, cyprodinil,
and pyrimethanil 64%, 38% and 35% respectively (Walse and Karaca, 2011). Pesticides in
soybean sprouts by the treatment of soaking and watering with water for 5 days, those by 0.3
ppm ozone-water watering, and those by soaking and watering with 0.3 ppm ozone water were
destroyed to 85~99, 89~100 and 94~100%, respectively (Soon-Dong K, 2000). Tomato fruits
treated (in field) with imidaclorprid when subjected to ozonation wash of 20 mL of a 10 ppm
solution for a period of 10 and 30 minutes resulted in 21 and 43 per cent reduction in the
imidacloprid residue respectively. However, ozonation caused a significant reduction in the
ascorbic acid content (40.7 %) and had no effect on color of tomato samples (Ergen et al., 2015).
Tomato fruits having mancozeb residues when treated with ozone solutions for 20 min showed
43% and 60% reductions of the residue levels at 1 and 3 mg L–1 ozone concentration
respectively. However, dipping into the chlorine solution at 100 mg L–1 for 20 min resulted in
71% reduction (Cengiz and Certel 2014). In tomatoes, the concentration of fenitrothion after at
10 min after the start of ozone micro bubble treatments of the decompression type and the gas-
water circulation type was 84 and 95%, respectively, showing less pesticide removal (Ikeura et
al., 2011). Pesticides like chlorpyrifos, azoxystrobin, cypermethrin, chlorthalonil, hexaconazole
& methyl parathion were found to be reduced from 37-95% in tomato, capsicum, grape & apple
after ozonated water dip (Swami et al., 2015). Carotene, cynadine-3-glucoside, ascorbic acid in
tomato and apple were found to be decreased. Phenols found to be altered with different
duration of ozone treatment (Swami et al., 2016).
Conclusion
The literature review gives a number of methods used for removal of surface residues of
pesticides including washing and peeling as safe method. Systemic pesticides are affected by
cooking to some extent but sometimes formation of toxic products may be a point of concern.
Variable reports are available for other methods which detoxify the residues in washings too.
Few reports indicate that some methods are also affecting the nutritional quality of fruits or
vegetables to different extent depending upon the concentration and duration of removal
technique used.
References
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19. Kumar K, Jagdishwar Reddy D, Narasimha Reddy K, Ramesh Babu T, Narendranath VV
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22. Pignatello, J. J., & Sun, Y. (1995). Complete oxidation of metolachlor and methyl
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carrot. Nippon shokuhin Kagaku Gakkaishi, 2(2), 97-101.
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ozone treatment on the reduction of chlorpyrifos residues in fresh lychee fruits. Ozone-
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potatoes during soaking in different solutions. Food Chem Toxicol 39(7): 751-755.

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Agriwaste Adsorbent Chemistry to Decontaminate Environmental
Matrices
Neera Singh, Abhishek Mandal and S.B. Singh

Division of Agricultural Chemicals,


ICAR-Indian Agricultural Research Institute, New Delhi-110012

Use of pesticide in modern agriculture is inevitable as high yielding crop varieties are more
prone to pest attack. The role of plant protection chemicals as saviours of mammalian food, feed
and fibre has remained unchallenged, even though, these plant protection chemicals have serious
environmental concerns like soil and water pollution and deleterious effects on the non-target
flora, fauna and other organisms including man. Indiscriminate and non-judicious use of these
plant protection chemicals has further increased the problem. Green methods to decontaminate
environmental matrices, soil and water, are need of the hour. Agri-waste adsorbent, generally
termed as low cost adsorbents, hold the key for future environmental friendly green techniques
for remediation of contaminated soil and water.
Agricultural runoff and industrial discharge are the main cause of contamination of water
bodies (Bereswillet al., 2012).Further, conventional wastewater treatment plants (WWTPs) have
been singled out as one of the main point-sources of xenobiotic transfer into the aquatic
environment (Bendz et al. 2005;Carballa et al. 2004; Carballa et al. 2005; Lishman et al. 2006;
Vieno et al. 2006; Palmer et al. 2008; Spongberg and Witter 2008) and treated effluents released
by them still contain significant concentrations of pesticides. There are regular reports of effluent
concentrations over 0.1 μg L-1 for several pesticides (diuron, glyphosate,
aminomethylphosphonic acid) and pharmaceuticals (Ternes et al. 2004; Gabet-Giraud et al.
2010; Martin Ruel et al. 2010; Falås et al. 2012). Photocatalytic degradation, combined photo-
fenton, advanced oxidation, aerobic degradation, filtration, ozonation, coagulation, flocculation,
precipitation, and adsorption are the commonly adopted methods to treat waste water. But, due to
ease of operation, economy and efficiency, adsorption has superior advantages over other
methods for removing pollutants at low concentration (Yakout and Daifullah, 2013). The term
adsorption refers to a process wherein a material is concentrated at a solid surface from its liquid
or gaseous surroundings. The term adsorption is used to differentiate surface accumulation from
intermolecular penetration. Adsorption process is surface accumulation of material. The most
important property that a good adsorbent should possess is a porous structure resulting in high
surface area. In addition, the time taken for adsorption equilibrium to be established should be as
small as possible so that it can be used to remove contaminants in lesser time. Thus, for removal
of pollutants, one looks to adsorbents with high surface area and porosity and showing fast
adsorption kinetics. Activated carbon is the most commonly employed adsorbent for removal of
contaminant from wastewaters, but, expensive cleanup cost limits its use. The use of non-
conventional, low-cost biosorbents prepared from agricultural wastes and byproductscan not
only reduce a large quantity of solid waste but also be very attractivedue to low investment cost,
insensitivityto toxicants and better efficiency for very low concentrations of contaminant
(Abdeen and Mohammad, 2013). Ata et al. (2012) reported thatbiosorption could reduce 20%,
36% and 28% of capital, operationaland total cost in comparison with conventional systems.
This chapter on use of agri-waste to decontaminate environmental matrices is divided in
sections: (i) utilization of biosorbents for pesticide removal, (ii) modification of agriwaste for
230 Winter School on "New generation smart agrochemical oriented approaches for crop
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better adsorption, (iii) Pyrolyzed carbon product (biochars) from agrowaste/organic waste, (iv)
factors affecting pesticide removal, (v) mechanism of pesticide removal

Utilization of biosorbents for pesticide removal

Use of locally available low-cost adsorbents viz., sawdust, rice husk, date stones, watermelon
peels, rice bran, pine sawdust, oak sawdust, tea leaves, wood sawdust, chestnut shells, bamboo
canes, straw, mango kernel, peanut shells, and peach nut shells etc. for removal of pesticide from
water has been widely studied

Bras et al. (1999) studied removal of organochlorines pesticides from water using pine
bark fixed bed mini-columns. The removal of organochlorine pesticides from spiked (1 to 10 μg
L-1) water solutions was approximately 97% for heptachlor, aldrin, endrin, dieldrin, DDD, DDT,
and DDE. Lindane could not be efficiently adsorbed by this methodology (38% removal). When
compared with activated carbon, pine bark displays analogous response, suggesting that for
compounds with similar physicochemical characteristics the pine bark can be effectively
used.Ratolaet al. (2003a,b) used pine bark to remove lindane and heptachlor from aqueous
solutions. The average efficiency of lindane and heptachlor removal was 80.6% and 93.6%,
respectively, after 24h equilibration using 125–300 µm particle size fraction. Presence of metals
in wastewater did not affect the efficiency of lindane removal.

Akhtar et al. (2007, 2009a, b) studied the sorptive potential of rice bran (RB),
Moringaoleifera pods (MOP), rice husk (RH) and chickpea husk (CH) for the removal of methyl
parathion (MP) and triazophos (TAP) from aqueous solution. Langmuir isotherm best explained
the sorption process and sorption was exothermic in nature.The pesticide may be stripped by
sonication with methanol, making the regeneration and reutilization of sorbents promising.These
sorbent have potential for application in water decontamination, treatments of industrial and
agricultural waste waters.
Memonet al. (2007, 2008, 2009) evaluated chest nut and peach nut for the removal of
carbofuran, methyl parathion and endosulfan. Ninety nine percent removals was achieved for
methyl parathion at 0.38–3.80 ×10−4mol dm−3 and carbofuran at 0.45–4.5 ×10−4mol dm−3
concentrations, using 0.4 g chest nut per 100 mL of solution after 30 min agitation.Peach nut
shells was able to remove 95% endosulfan at 0.24 × 10−4 mol dm−3 concentration using 0.1 g of
adsorbent in 20 mL of solution after 60 min was 95 ± 1%. The developed procedure was
validated using real waste water samples from agricultural fields and 95-99% pesticide removal
was achieved.
Sharma et al. (2008) evaluated saw dust and coconut fiber to remove atrazine from water.
The atrazine removal efficiency of sawdust and coconut shell was 78.5–80.5% and 92.4%–
95.2%, respectively.
Boussahelet al. (2009) investigated the removal of a chlorinated pesticide (4,4-DDT)
from aqueous solutions using wood sawdust and cork waste. The influence of the adsorbent
particle size and the organic matter content of water (humic acids) on the removal process were
also studied. The adsorption capacity (qm) calculated using the Langmuir isotherm were
69.44 mg g−1and, 19.08 mg g−1 for saw dust and cork waste, respectively. The DDT removal
process was highly dependent on the adsorbent particle size and humic acid content in water
negatively influenced the adsorption process.

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Islam et al. (2009) studied multiple response optimizations for the removal of quinalphos
from aqueous solution onto used tea leaves. The optimum conditions for maximum quinalphos
(96.31%) removal were: pH 8.83, concentration 7 mg L−1 and dose 0.40 g L-1. Quinalphos
adsorption capacity of tea leaves was 196.07 μg g−1.
El Bakouriet al. (2009) evaluated ten natural biosorbents (bamboo canes, peanut shells,
olive stones, avocado stones, date stones, leaves of Eucalyptus gomphocephala, Raphanus
raphanistrum, Nerium oleander, Origanum compactum and Cistus ladaniferus) for the removal
of alachlor, aldrin, atrazine, chlorpyrifos, chlorfenvinphos, dieldrin, alpha-endosulfan, endrin,
hexachlorobenzene, beta-HCH, gamma-HCH (lindane), simazine and trifluralin. The date stones
were identified as the best adsorbents for the removal of selected pesticides and endosulfan
sulfate was the best removed pesticide with KF value of 13.54 μg g–1.
Nanseu-Njiki et al. (2010) studied biosorption of paraquat on Ayous
(Triplochitonschleroxylon) sawdust by batch method. Results suggested that paraquat adsorption
was mainly attributed to cation exchange as indicated by the adsorption energy determined by
the Dubinin–Radushkevich model (E = 12.0736 kJ mol−1). Some other interactions resulting
from the affinity through organophilic interactions between paraquat and sawdust too contributed
towards herbicide adsorption.
Silva et al. (2013) evaluated banana peel for the removal of atrazine and ametryne from
river and treated waters. The optimum conditions for herbicides removal were: sample volume =
50 mL, banana mass = 3.0 g, stirring time = 40 min. Removal efficiencies attained for atrazine
and ametryne were 59.8 and 75.3%, respectively. The Freundlich adsorption constant (Kf) values
for atrazine and ametryne were 35.8 and 54.1, respectively.
Al-Zaben and Mekhamer (2013) investigated the use of coffee waste (CW) to remove the
4-chloro-2-methyl phenoxy acetic acid (MCPA) from aqueous solutions. The adsorption capacity
of adsorbent for MCPA was found to be 0.34 g g-1.
Rojas et al. (2015) used sunflower seed shells and rice husk as potential adsorbents for
the removal of atrazine, alachlor, endosulfan sulfate and trifluralin from aqueous solution. The
study revealed that the maximum removal efficiency (73.9%) was reached using 1 g of rice husk
and 50 mL of pesticide solution (200 μg L-1). A pseudo first order model was found to be more
suitable for the sorption of atrazine and alachlor while the pseudo second order model best
described the endosulfan sulfate and trifluralin adsorption.

Modification or byproducts from agri-waste

Modification and activation of adsorbents to increase specific surface area and pore volume, pore
size distribution, and surface functional groups significantly influence the adsorption capacity of
adsorbents. Adsorption capacity increases with increase in specific surface area due to the
availability of a number of adsorption sites, while pore size and micropore distribution are
closely related to the composition of the adsorbents and is affected by the nature of feedstock
used. Modification of agriwaste can include: Chemical (acid or alkali) treatment, thermal
treatment and ash obtained after complete burning of agrowaste

Gupta et al. (2002) used bagasse fly ash, obtained from the local sugar industry, as an
inexpensive adsorbent for the removal of lindane and malathion from wastewater. Up to 97–98%
removal of the two pesticides was achieved under optimum conditions; therefore, bagasse fly ash
was very useful and economic for the removal of these insecticides. Similarly, Akhtar et al.

232 Winter School on "New generation smart agrochemical oriented approaches for crop
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(2007) used bagasse fly ash for the removal of methyl parathion (MP) and results suggested that
maximum sorption capacities calculated using Freundlich and Langmuir isotherm were 5.3±1.4
and 0.39±0.005 mmol g−1, respectively. Sharma et al. (2008) evaluated fly ash and baggasse
charcoal to remove atrazine from water. The fly ash effectively removed about 84.1%–88.5%
atrazine from water at 0.05 and 0.1 ppm levels while baggasse charcoal showed 76.5–84.6%
atrazine removal. Recently, Deokar et al. (2016) used rice husk ash (RHA) for removal of 4-
chloro-2-methyl phenoxy acid (MCPA). Results suggested that MCPA adsorption capacity of
RHA determined by packed bed method was3019 mg/L for 90% saturation of bed. Similar
results were reported by Trivedi et al. (2016) for mustard plant ash for 2,4-
dichlorophenoxyacetic acid (2,4-D) removal.
Memonet al. (2007, 2008, 2009) evaluated chemically and thermally treated watermelon
peel for the removal of carbofuran, methyl parathion and endosulfan. Treated watermelon peels
showed 99 ± 1% methyl parathion removal at 0.38–3.80 × 10−4 mol dm−3 concentration using
0.1 g adsorbent in 20 mL of solution after 60 min agitation time at pH 6.
Boudesocqueet al. (2008) tested lignocellulosic waste (sequentially acid-base treated rice
bran) for the removal of terbumeton, desethylterbumeton, dimetomorph and isoproturon from
wastewaters.Results suggested that at 2×10−7 - 3×10−4mol L−1 concentrations the amounts of
adsorbed pesticide varied from 1 to 8 g kg−1 of adsorbent suggesting that the waste can provide a
simple, effective and cheap method for removing pesticides from contaminated waters.
Rodrıguez-Cruz et al. (2008) studied the effect of different wood pre-treatments on
linuron and metalaxyl sorption. Water, NaOH, HCl and octadecyltrimethylammonium bromide
(ODTMA) treated pine (softwood) and oak (hardwood) woods and untreated woods were used as
sorbents. The Freundlich sorption constant (KF) values in untreated pine and oak wood were 96.2
and 74.4 (linuron) and 8.28 and 4.95 (metalaxyl), respectively. Wood pre-treatment resulted in
1.04−2.35-fold (linuron) and 1.33−2.17-fold (metalaxyl) increase in pesticide sorption.
Hsu and Pan (2007) and Hsu et al (2009) used several modified biosorbents for paraquat
removal from water.Results suggested that methacrylic acid treated rice husk
hadmaximumadsorption capacity for paraquat (317.70 mg/g). Abdeen and Mohammad (2013)
prepared activated carbon by phosphoric acid treatment of apricot stone and used it for
ethoprophos removal. The maximum monolayer adsorption capacity of activated charcoal for
ethoprofos was 20.04 mg/g. Similarly, Ahmed and Mohammad (2014) suggested that apricote
stone activated carbon showed removed 147.05 mg/g of oxamyl from water.
Activated carbons prepared by pyrolysis and physical activation from corn cob, olive
kernel, soya stalks and rapeseed stalks showed bromopropylate adsorption little less than or
equal to commercial activated carbon (Ioannidou et al., 2010). The maximum adsorption
capacities of bromopropylate were 15.6x10-2 mg/g (Filtrasorb400), 21.17 x10-2 mg/g (NORIT®
GL50) and 18.9 x10-2, 12.3 x10-2, 11.6 x10-2, 7.9 x10-2 mg/g for activated carbons from corncob,
olive kernel, soya stalks, rapeseed stalks,respectively.
El Bakouriet al. (2009) described the potential applicability of chemically and thermally
treated date stones for removing drin pesticides (aldrin, dieldrin and endrin) from aqueous
solutions. Maximum removal efficiency (93%) was reached using 0.1 g of acid-treated date
stones (ATDS) (63–100 μm) and 100 mL of aldrin (0.5 mg L−1) solution. The removal efficiency
of drin pesticides decreased in the order: aldrin>dieldrin>endrin. Removal efficiency decreased
with increase in temperature suggesting exothermic nature of sorption. Experimental data were
modelled using the Langmuir, the Freundlich and the Dubinin–Radushkevich (D–R) isotherms.

233 Winter School on "New generation smart agrochemical oriented approaches for crop
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The Freundlich isotherm model (R2 = 0.98–0.99) fitted the equilibrium data better than the
Langmuir and theD–R models.
Further, authors (El Bakouriet al., 2010) studied the fate of endosulfan metabolites, in
small-scale biopurification systems, using treated and untreated date, olive and avocado stones.
Maximum removal efficiency (94.8%) for endosulfan sulfate (0.1 mg L−1) was achieved using
acid-treated date stones (<125 μm fraction, solid/liquid ratio: 1 g L−1). Date stones showed
higher removal efficiency followed by olive and avocado stones. Thermo-chemical treatment
notably improved the pesticides adsorption efficiency of adsorbents and adsorption decreased
with increase in pesticide aqueous solubility.
Mandal and Singh (2017) used eucalyptus bark (EB), corn cob (CC), bamboo chips (BC),
rice straw(RS) and rice husk (RH) to study atrazine and imidacloprid sorption. Studies suggested
that biosorbents greatly varied in their pesticide sorption behaviour. The EB was the best
biosorbent to sorb both atrazine and imidacloprid with KF values of 169.9 and 85.71,
respectively.
Cara et al. (2017) studied the adsorption potential of alkaline treated straw (wheat and
corn) in mixture with soil, for the removal of sulfonylurea molecules from aqueous solutions. An
increase (337.22 mg g−1) of adsorption capacity of sulfonylurea molecules was obtained for all
studied straws.

Pyrolyzed carbon product (biochars) from agrowaste/organic waste

Agriwaste and other organic waste can be converted in to biochars via


pyrolysis.Pyrolysis produces biochar, liquids and gases from biomass by heating the biomass in a
low/no oxygen environment. The absence of oxygen prevents combustion. Temperatures of 400–
500°C produce more char, while temperatures above 700°C favor the yield of liquid and gas fuel
components. Feedstock and pyrolysis conditions and temperature (highest treatment temperature
HTT) are the most important factors controlling the physical and chemical properties of the
resulting biochar. Due to high surface area and porosity biochars have high potential to adsorb
pesticides.
Zheng et al. (2010) reported that atrazine and simazine sorption ability of biochars
increased with decreasing solid/solution (w/v) ratio (451-158 mg kg-1at 1:50 and 243-1066 mg
kg-1. Low pH favoured the sorption of both herbicides while co-existence of two herbicides
decreasedsorption (Kf) for atrazine from 435 to 286 and 514 to 212 for simazine.Similarly,
Uchimiyaet al. (2010) reported that sorption of deisopropylatrazineon broilerlitter-derived
biocharsincreased with increasing pyrolysis temperature and steam-activation. Zhao et al. (2013)
compared the atrazine sorption potential of corn straw biochars, untreated (CS450) and
NH4H2PO4/ADP treated (ADPCS450). Results suggested that atrazine sorption capacity of
ADPCS450 was much higher than the sorption capacity of CS450.
Sun et al. (2011) investigated the sorption behaviour of norflurazon and fluridone on
wood and grass derived biochars produced (HTT 200-600°C). Amorphous biochars
(HTT=400°C) exhibited the highest sorption of both the herbicides, emphasizing the importance
of amorphous structural arrangement of aromatic moieties in these chars. Further, authors (Sun et
al., 2012) compared the sorption of fluridone and norflurazon on thermal (poultry litter and
wheat straw biochar at 400°C HTT) and hydrothermal (poultry litter and swine solids at 250°C
HTT) biochars. The hydrothermal biochar showed relatively high herbicide sorption efficiency
compared to the thermal biochar.

234 Winter School on "New generation smart agrochemical oriented approaches for crop
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Tsai and Chen (2013) compared the paraquat adsorption potential of swine waste derived
biochar and activated carbon. The adsorption capacity of paraquat onto the activated carbon was
slightly lower than that on swine-manure-derived biochar (4.7 mg g-1). Results suggested that
swine waste derived biochars can be used as a low-cost adsorbent for the removal of cationic
organic pollutants from the aquatic bodies.
Klassonet al. (2013) studied adsorption of dibromochloropropane (DBCP) on almond
shell derived activated biochars prepared at 800°C for 45 min to give activated biochar with
specific surface area of 344 m2 g-1. The maximum DBCP adsorption capacity of activated
biochar was 102 mg g-1.
Zhang et al. (2013) studied the adsorption and catalytichydrolysis of carbaryl and atrazine
on original and deashed pig manure biochars. Both pesticides were substantially adsorbed on
biochars and organic carbon normalized sorption coefficient (KOC) values of 102.65-103.66 L kg-1
for carbaryl and 101.90-103.57 L kg-1 for atrazine at Ce of 0.5 mg L-1 were observed. The
pesticides got hydrolysed in the presence of biochars and in the presence of biochar prepared at
700°C, 71.8% and 27.9% of carbaryl and atrazine, respectively, were hydrolyzed in 12 h. It was
found that hydrophobic effect alone could not explain the sorption, and several other processes
including pore-filling and π-π electron donor-acceptor interactions were involved in the pesticide
adsorption. Adsorption increased greatly on the deashed biochar, indicating that some organic
sorption sites in the original biochars were blocked or difficult to access due to their interactions
with inorganic moiety.
Cabrera et al. (2014) reported that nature of feedstock affected pesticide adsorption in
biochars. Compared to wood chip pellet biochar the macadamia nut shell biochar exhibited
lower pesticide sorption. This result was attributed to the higher dissolved organic carbon content
of macadamia nut shell biochar that competed with pesticides for mineral soil surfaces and thus
decrease pesticide sorption.
Taha et al. (2014) studied the adsorption of a mixture of 15 different pesticides from
water by untreated and phosphoric acid treated rice straw and corn stover biochars and charcoal.
Biochars exhibited better removal of pesticides than the charcoal. Further, treated
biochars/charcoals were better than the untreated biochars/charcoals. Treated rice straw biochar
took 2 h (adsorption contact time) to reduce individual pesticide concentration in water (pH 7) to
≤0.005 μg L-1 with the exception of oxamyl, which was reduced to ≤0.068 μg L-1 after 24 h.
Xiao and Pignatello (2015) examined the adsorption of a series of triazine herbicides on
hardwood biochars prepared at different temperatures. Results suggested that adsorption of
triazine herbicides was primarily affected by the mesoporosity and microporosity of biochars.
Further, π–π electron donor-acceptor and steric effects also played important roles in adsorption.
Wang et al. (2015) investigated the adsorption mechanism of chlorpyrifos on wheat
straw-derived biochars (HTT250, 350, 450, 550, 650 and 750°C). Results suggested that WS750
was the best to adsorb chlorpyrifos (16 mg g-1) and π-π stack between the aromatic ring of
chlorpyrifos and aromatic areas on WS750 surface were the driving force for adsorption.
Sun et al. (2015) studied propiconazole sorption on plant-residue (PLABs) and animal
waste-derived biochars (ANIBs) obtained at three HTT (300, 450 and 600°C. Results indicated
that pore-filling in nanopores within aromatic C dominated as propiconazole sorption mechanism
whereas HTTs or C contents did not necessarily regulate the sorption. Removal of minerals from
BCs450 elevated propiconazole sorption as minerals may exert certain influence on sorption via
impacting spatial arrangement of polar groups and/or organic matter (OM)–mineral interactions.

235 Winter School on "New generation smart agrochemical oriented approaches for crop
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Liu et al. (2015) reported that physicochemical properties of biochars produced from
different feedstock (soybeanSBB, corn stalk CSB, rice stalk RSB, poultry manure PMB, cattle
manure CMB, and pig manure PgMB) greatly varied in their physicochemical characteristics and
atrazine adsorption. The adsorption capacity decreased in the order:
SBB > RSB > CMB > CSB > PMB > PgMB and total pore volume and biochar pH played
important roles in determining the adsorption capacity. Similar results were reported by Mandal
et al. (2017) for atrazine and imidacloprid sorption.
White et al. (2015) used sugarcane bagasse (BC350, BC700 prepared at 350 and 700°C)
and pine wood (WC400 prepared at 400°C) biochars to adsorb metribuzin. Results indicated that
BC700 showed highest metribuzin sorption and the Freundlich sorption coefficient (Kf) for the
BC700 (47.2) was 4.8X and 14X greater than the BC350 or WC400 values. Highest metribuzin
sorption in BC700 was attributed to its surface area, which for the BC700 was 31X and
170Xgreater than the BC350 or WC400, respectively.
Mayakaduwaet al. (2016) reported that glyphosate adsorption on woody biochar was best
explained by Temkin and Freundlich models and maximum adsorption capacity determined by
the Temkin isotherm was 44 mg g-1.
Mandal et al. (2017) compared the atrazine and imidacloprid adsorption potential of
biochars prepared from corncob, eucalyptus bark, wheat straw, rice straw and rice husk. Results
suggested that rice straw biochar and acid activated rice straw biochars were the best to remove
both pesticides. Higher efficiency of these biochars was attributed to their aromaticity, porosity,
polarity and pH.
Yanget al. (2017) used N-doped porous carbon sheets (NPCS) resulting from wheat
straws were fabricated using molten salts via the carbonization functionalization progress, which
show unique hierarchical structure, large pore volume and high surface area with affluent
micropores. Results indicated that there existed many hierarchical pores consisting of the single
carbon sheet with ultrathin nature, owing to the template role of molten salt mixtures at high
temperature. Such superior structure can bring about desired performance of adsorption capacity
of 82.8 mg/g and quick adsorption rate of 1.43 L/h with an initial concentration of 35 mg/L at
25 °C.
Mandal et al. (2017) investigated the mechanisms of 2,4-Dichlorophynoxy acetic acid
(2,4-D) sorption on biochars of various green wastes (tea, burcucumber, and hardwood) at two
pyrolytic temperatures (400 and 700 °C) and steam activated tea waste (700 °C) biochar in
aqueous solutions. The steam activated biochar produced from tea waste showed the highest
(58.8 mg g−1) 2,4-D sorption capacity, which was attributed to the high specific surface area
(576 m2 g−1). The sorption of 2,4-D was found to be strongly dependent on the biochar properties
such as specific surface area, surface functional groups, and microporosity.
Matos et al. (2017) reported the removal of pesticides from water using activated and
magnetized biochars produced from exhausted husk, and dry tannin from barks of black wattle
(Acacia mearnsii). At the longest time examined, the amounts of thiacloprid and thiamethoxam
adsorbed per gram of activated biochar adsorbent were 1.02 and 0.97 mg, respectively, while
values of 0.73 mg (thiacloprid) and 0.40 mg (thiamethoxam) were obtained using magnetized
biochar.

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Factors affecting adsorption

Removal of pesticide on agrowaste biosorbents was affected by the nature of pesticide, physic-
chemical characteristics of adsorbent, temperature, solution pH, adsorbent dose and pesticide
concentration. Important factors include:

pH: Solution pH has a pronounced effect on pesticide sorption on biosorbents as pH greatly


affect the solubility of pesticide, charge distribution on pesticide molecule and surface functional
groups of adsorbents. Opposite charge on pesticide and adsorbent favoured adsorption. El
Bakouri et al., (2009) reporting that increasing solution pH from 2 to 10 decreased biosorption
of endosulfan sulphate on bamboo canes, date stones, peanut shells, and avocado stones. Acidic
pH favoured positive charge on adsorbents and thereby the interaction between sorbents and
polar pesticide increases.

Temperature: Temperature is another important factor affecting pesticide adsorption.


Generally, adsorption of pesticide is an exothermic process, therefore increasing temperature will
decrease adsorption.

Concentration: Concentration of contaminant in solution greatly affects the percent removal of


contaminant. Generally, pesticides are sorbed more at low initial pollutant concentration and
adsorption decreases at higher concentration. Total contaminant adsorption increases with
increase in adsorbent quantity.

Adsorbent properties: Agri-waste adsorbents are lignocellulosic in nature and their important
properties which affect pesticide adsorption are adsorbent nature (chemical composition,
functional groups), specific surface area, pore size distribution and presence of surface functional
groups are of major importance towards pesticide molecules uptake from aqueous solutions.
Adsorbent dosage is also an important parameter as it determines the adsorption capacity of an
adsorbent for a given initial concentration. Memon et al., (2007) and Mandal and Singh (2015)
reported a rapid increase of sorption percent of pesticide with increasing the amount of sorbent
dosage.

Mechanism of pesticide adsorption

Adsorption of pesticide on agriwaste biosorbents is affected by the properties of both adsorbent


and adsorbate. Sorption of pesticide molecules, which are mainly non-ionic compounds, is
mainly governed by van der Waal forces, hydrogen bonding, dipole dipoleinteraction or other
electrostatic interactions. However, pesticides, which are ionic in nature may get sorbed by
chemical bonding. Not much details are available for pesticide adsorption on agriwaste
biosorbents.
El Bakouri et al. (2009) reported that Van der Waals and hydrogen bond were dominant
mechanisms for drin pesticides adsorption onto acid treated olive stone. Similar sorption
mechanism was reported for 4-chloro-2-methyl phenoxy acetic acid sorption on coffee wastes
(Al-Zaben and Mekhamer, 2013). Paraquat, which is a cationic pesticide, was sorbed by cation
exchange mechanism on Ayous sawdust (Nanseu-Njikiet al., 2010) as adsorption energy for
herbicide adsorption was 12.0247 kJ/mol.

237 Winter School on "New generation smart agrochemical oriented approaches for crop
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Biochars, which are pyrolyzed product ofagri- or organic-waste, are composed of both
carbonized and non-carbonized domains, which have different affinity for pesticide molecules
with different sorption mechanisms. Generally, the sorption of organic compounds on carbonized
domains of biochar is nonlinear in nature and is dominated by pore-filling mechanism; however,
sorption on the non-carbonized domains is better described by a partitioning mechanism and is
linear and non-competitive. Thus, mechanism of pesticide adsorption on biochars is pesticide
specific. Oxygenated groups, such as carboxyl and phenolic surface active groups, on the biochar
surface which have proven to be binding sites for pesticides and other organic contaminants
(Uchimiya et al., 2010). The π-π electron donor-acceptor interaction between π-electron rich
graphene surface of biochar and π-electron deficient positively charged organics have been
observed (Qiuet al., 2009; Teixidoet al., 2011; Sun et al., 2012). Even electrostatic interactions
have been reported as the principal mechanisms for the sorption of ionic and ionizable organic
compounds (Zheng et al., 2013; Inyang and Dickenson, 2015). Mandal et al (2017) suggested
that atrazine and imidacloprid can bind to biochars both specifically (H-bondingand charge-
transfer interactions) as well as non-specifically (hydrophobic-like interactions) with surface
active groups of the adsorbent.

Conclusion

Cost of lignocellulose based biosorbents and efficiency of pesticide removal suggested that they
can be exploited as low-cost biosorbents for removing pesticidesfrom water. However, till now,
most of the studies have focused on single contaminants removal in laboratory. Therefore,
research should focus on multi-contaminant system at large/pilot scale with focus on minimizing
the use of adsorbent and standardizing parameters for maximum removal.

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Nutraceuticals: Role in Human Health Improvement

Suresh Walia and Supradip Saha

Division of Agricultural Chemicals


Indian Agricultural Research Institute, New Delhi-110 012

Nutraceuticals derived from food and non-food crops provide health promoting and medicinal
properties. Such products are increasingly used as natural antioxidants and food colorants in
functional foods and dietary supplements. Vegetable like red and black carrot, beet root, tomato,
capsicum, red cabbage, and fruits like pomegranate, watermelon grapes and jamun, cereal crop
like rice and non-food plant like marigold and gulmohar are rich source of natural antioxidants
and food colorants. Some biomolecules extracted from such plants like carotenoids, phytosterols,
isoflvones, polyphenols, tocopherols and tocotrienols, anthocyanins etc. exhibit outstanding
antioxidant activity and thus protect the body against the damaging effects of free radicals. As
intense pigments, β-carotene, lycopene, capsanthin, and anthocyanins are used as natural
colorant in food, beverage and pharmaceutical applications. Greater emphasis is being laid to
develop customized nutraceutical products fortified with medicinal herbs and/or vigor promoting
ingredients. Nano-technological and other interventions have led to new innovative products
with increased bioavailability, stability and health benefitting effects. .

INTRODUCTION
High value food and non-food crops are rich source of multitude of nutraceuticals and other
bioactive natural products. Such products enhance ability of the body to fight, cure and prevent
diseases through anti-oxidant, anti-carcinogenic, anti-inflammatory, anti-bacterial, anti-
proliferative and other properties. Plant-derived antioxidants are particularly important because
they are believed to protect the body against free radicals, the harmful molecules that can cause
heart disease, premature aging, Alzheimer’s disease, blindness and a variety of cancers. They
have been shown to scavenge superoxide and hydroxyl radicals to prevent lipid peroxidation and
undue oxidation of other biological substrates. Horticultural crops are an excellent source of
nutraceuticals to provide health benefits, and phyto-pharmaceuticals to alleviate human
sufferings. Such bioactive compounds (flavonoides, carotenoides, anthocyanins, polyphenolics,
sterols, vitamins etc.) provide health benefits of disease prevention through their antioxidant
activity and nutritional traits of preventing disease risks. Some vegetables (carrots, tomato,
capsicum, red cabbage, beetroot), fruits (pomegranate, jamun, watermelon, grapes), cereal crops
(black rice), and non-food flowering plants like marigold (Tagetes spp.) and gulmohar (Dalonix
regia) are potential source of natural antioxidants, food colorants and other phytoceuticals with
health benefitting and disease prevention effects (Walia 2009, 2012, 2013a). These are believed
to be responsible for many reported health benefits including preventing Alzheimer’s disease,
age-relating macular degeneration and anti-ageing by enhancing existing neuronal connections
and improving neuronal regeneration. Some such food and non-food crops have been processed
to develop natural antioxidants and food colorants and converted into value added functional
foods with improved shelf life and nutrition value (Walia, 2013b). Potential of some of the
potential food and non-food crops is highlighted below

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Food Crops
Carrots (Daucus carota): Carrot (Daucus carota, Fam. Apiaceae or Umbelliferae) is available in
red, yellow, purple/black colour. The variety of pigments contained in them performs a range of
protective functions in the human body. Red carrotsare thought to have originated in India and
China in the eighteenthcentury. Red carrots derive their color mainly from lycopene, a type of
carotene believed to guard against heart disease and cancers. These are thought to have
originated in India and China in the eighteenthcentury. Red strains of carrot contain abundant
lycopene in addition to theprovitamin A, and carotenoids such as - and -carotene. Yellow
carrots accumulate xanthophylls, pigments similar to β-carotene that support good eye health.
Purple or black carrots possess anthocyanins which act as powerful antioxidants. Tissue specific
accumulation of carotenoids in carrot roots has been reported in carrots and related vegetables
(Baranska et al 2006). Red strains of carrot contain abundant lycopene in addition to carotenoids
such as - and -carotene (Saha et al., 2015a). It qualifies as a functional food because, in
addition toproviding essential vitamin A, it may offer protection againstchronic disease through
the putative benefits of lycopene.Studies have revealed that lycopene in the red carrot is about
44% as bioavailable as that from tomato paste. Red carrots, therefore, provide an alternative to
tomato paste as a good dietary source of lycopene and also provide bioavailability of β-carotene
(Horvitz et al 2004).

Black or purple carrot has been known for centuries in several countries namely Turkey,
Afghanistan, Egypt, Pakistan, India and in the Far East. Black carrots possess an entirely
different class of pigmentsanthocyanins, which act as powerful antioxidants. It is a rich source of
more stable acylated anthocyanins showing highest antioxidant activity. The anthocyanins are
acylated with p-coumaric, ferulic, p-hydroxybenzoic and sinapic acids. They have high
anthocyanin content (1750 mg/kg) (Kirca et al., 2007). The use of anthocyanins from black
carrot for coloring food is advantageous for health reasons. Purple carrot extract provides an
excellent bright purple shade at acidic pH and is an excellent choice for coloring other fruit
juices, nectars, beverages, candy, preserves, jellies confectionary and bakery items. The
conventional method of anthocyanin extraction from finely cut, sliced or homogenized black
carrot involves extraction with dilute methanol or ethanol containing a small amount of acid. The
identification and quantification of anthocyanins has been the subject of several studies (Dougall
et al 1998, Elham et al 2006, Kammerer et al 2004). Enzyme -assisted extraction methods are
gaining more attention for high extraction efficiency (Landbo et al 2004). Versatility of
pressurized acid water extraction has also been explored for the extraction of anthocyanins from
black carrot (Gizir et al 2008).

The black carrot extract concentrate is readily soluble in water, stable in pH < 4, temperature
resistant and can be used to color jams, jellies, ice creams, pastries, fruit juices, drinks, and other
beverages. The colour of canned fruits can be significantly stabilized using commercial black
carrot extract as the natural colorant. The colour of less stable strawberries and beverages based
on them can be stabilized using black carrot concentrates due to stable acylated anthocyanins.
(Kammerer et al 2006). Microencapsulation of black carrot anthocyanins using spray drier has
led to products with increased stability and half-life (Seda and Unai 2007). Among the various
Indian carrot varieties, the red carrot (IPC-13) and Pusa Rudhira (IPC-122) are the richest source
of carotenoid pigments. WhileIPC-13 reportedly contains 4887 µg/100 g of total carotenoid,
IPC-122 contained 3354 µg/ 100g of the carrot.

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Solanum lycopersicum (Tomato): The tomato (Solanum lycopersicum, syn. Lycopersicon
lycopersicum; Solanaceae) has received significant attention as its primary constituent lycopene
has a role in cancer risk reduction (Gerster 1997). China, the largest producer of tomato
accounts for about one-fourth of the global output (31 million tons). India (7.6 MT) accounted
for 6.1 % of the total world production.It has a variety of cultivars which vary in size and shape
from the tiny and sweet cherry style tomatoes to big juicy tomatoes. The lycopene content of
tomatoes varies from species to species and increases as the fruit ripens. These are consumed
either cooked or raw and are low in calories and an excellent source of nutraceuticals such as
lycopene, vitamins A, C and E.

Lycopene is one of the most powerful natural antioxidants which help prevent prostate cancer
and benefit the heart.Tomatoes and tomato based sauces, juices, and ketchup account for more
than 85% of the dietary intake of lycopene for most people. Processed tomato products such as
pasteurized tomato juice, soup, sauce, and ketchup contain the highest concentrations of
bioavailable lycopene. They account for more than 85% of the dietary intake of lycopene. Unlike
other fruits and vegetables, where nutritional content such as vitamin C is diminished upon
cooking, processing of tomatoes increases the concentration of bioavailable lycopene. Lycopene
in tomato paste is four times more bioavailable than in fresh tomatoes. It is an efficient scavenger
of singlet oxygen and thus, have anti-cancer effect (Clinton 1996, Giovannucci 1995, 2002, Kaur
et al 2013).

Capsicum annum L. (Red Chilli): Chilies (Capsicum sp, Fam. Solanaceae) are the dried red
fruits of the genus Capsicum. The two well known species are Capsicum annum L. and C.
frutenscens L., the former being widely grown and economically important of the domesticated
chilli peppers. India is the largest producer and exporter of chillies. Pungency and color are the
two main quality attributes in chillies. In the spice extraction industry, chilli oleoresin is
produced by extraction of chilli powder with solvents such as acetone, ethylene dichloride and
hexane followed by de-solventization to get deep colored oleoresin. Selective enzyme mediated
extraction using alcohol as the solvent has been employed for the efficient extraction of
capsaicinoids and carotenoids (Santamaria et al 2000, Williams et al 2004). A microwave-
assisted method afforded optimum extraction of pigments from paprika powders (Csiktusnadi et
al 2000). The red color of chillies is due to the presence of capsanthin (major, 35%), zeaxanthin,
violaxanthin, cryptoxanthin, β-carotene etc. These pigments are present in chillies mainly in the
esterified form and to a small extent in non-esterified forms. The hot flavour of chillies is due to
the presence of a group of closely related compounds namely capsaicin (8-methyl-N-vanillyl-6-
nonenamide) and dihydrocapsaicin that account for approximately 90% of the pungency. These
two compounds are about twice as potent as the minor capsaicinoids - nordihydrocapsaicin,
homodihydrocapsaicin, and homocapsaicin. Despite popular belief, the seeds do not produce any
capsaicins, instead, it is present in large quantities in the placental tissue which holds the seeds,
the internal membranes and to a lesser extent in the fleshy parts of the capsicum fruits. Pure
capsaicin is a hydrophobic, colorless, odorless crystalline to waxy compound having
irritant/pungent properties. Besides capsaicinoids, hot peppers are a good source of dietary
antioxidants such as flavonoids, phenolic acids, carotenoids, vitamin A, ascorbic acid and
tocopherols (Rosa et al 2002; Saha et al 2015b).

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Chilli oleoresin is used in many Indian processed foods like sausages, meat products to impart
desired taint and pungency. Pungency-free food colorants from chilli are therefore, desired to
obtain high value commercial chilli oleoresin. Capsaicin is currently used in tropical ointments in
concentrations between 0.025-0.075% to relieve minor aches, muscle pains, joint pains,
backache, peripheral neuropathic pains, osteoarthritis pain as well as strains and sprains.
Capsaicin is also being explored as a possible cure for diabetes, prostate cancer and as a pesticide
to deter pests. Burning and painful sensations associated with capsaicin results from its chemical
interaction with sensory neurons and depletion of neurotransmitters.

Purple or red cabbage (Brassica oleracea)


Purple cabbage is a colorful variety from the brassica family. Due to the anthocyanin pigment
content, cabbage grows more reddish in acidic soils, more purple in neutral soils, and greenish-
yellow in alkaline soils. Its leaves have a relatively high content of natural colorant, nutrients and
vitamins. The extraction process involves various steps of washing, slicing, extraction, filtration,
purification, vacuum concentration, and spray-drying. It contains several anthocyanins having
different substituents and functional groups. Cabbage anthocyanins concentrate has been
partially purified (Coutinho et al 2004)(8) to obtain food grade colorant. HPLC-MS/MS of the
extract concentrate has led to the identification of twenty derivatives of cyanidin glucosides
(Wiczkowski et al 2013)(27), wherein the glucoside residues were non-acylated, monoacylated
and/or diacylated with sinapic, ferulic, caffeic and/or p-coumaric acids. The predominant
anthocyanins include non-acylated cyanidin-3-diglucoside-5-glucoside, cyanidin-3-(sinapoyl)
(sinapoyl)-diglucoside-5-glucoside, cyanidin-3-(p-coumaroyl)-diglucoside-5-glucoside,
cyanidin-3,5-diglucoside, and cyanidin-3-monoglucoside. Among these, cyanidin-3-diglucoside-
5-glucoside diacylated with sinapic acid exhibited the highest radical-scavenging activity.
Good stability of red cabbage anthocyanins is attributed to the presence of acyl groups in the
glycone moiety. The water soluble food colorant is noted for its excellent heat stability and light
fastness. It has greater stability at pH 3.8 or lower and at higher pH, the colorant becomes less
stable. It generates a reddish-magenta solution at pH 3; a pinkish solution at pH 4-6; a purple
solution in a pH of 7; a bluish-green solution in pH 8-9; and yellowish solution in pH > 9. As a
result of the typical changes at different pH, red cabbage powder is used as a pH indicator.
Purple colour of red cabbage pigment can be changed to rarely occurring sapphire blue by
addition of baking soda. Red Cabbage can be used in wine, beverages, fruit sauce, candy, cake,
sauerkraut etc. Since red cabbage anthocyanins are more stable than those from other fruits and
vegetables, it can have many applications in functional foods, beverages, food supplements, and
pharmaceutical industry.
Beet root (Beta vulgaris)
Beetroot (Beta vulgaris) is a rich source of betalain group of natural colorants. The name
"betalain" comes from the Latin name of the common beetroot (Beta vulgaris), from which
betalains were first extracted. There are two categories of betalains - betacyanins which appear
reddish to violet, and betaxanthins which appear yellow to orange in colour. They mainly
comprise of red–violet-colored betacyanins (betanin,isobetanin, probetanin and neobetanin) and
yellow–orange-colored betaxanthins. Fresh beets, beet powder or extracted pigments are often
added to improve the red colour of tomato pastes, sauces, soups, desserts, jams, jellies, ice

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creams, sweets and breakfast cereals etc. They also contribute to consumers’ health and
wellbeing because betalains are known to have antioxidant properties (Singh and Hathan 2014).
Beet root colorant is comparatively unstable and prone to degradation when exposed to light,
heat, and air. It also degrades during cooking if cooking time is increased. Due to its poor heat
stability it is ideally used in products which are designed for a short shelf life e.g. frozen foods.
Beet extract concentrate is pH dependent and generates a bluish-red shade at pH 4-5, a brighter
red as the pH rises, and yellowish-brown if the pH becomes too alkaline. The bluish-red color is
attributed to betanin which is stable at a higher pH. In the dry powder state, it is more stable than
in the liquid state. It finds wide use in soup, ice cream, yogurts powdered beverages,
confectionary products, and frozen foods of various types. Beet root extract is also a good
source of nitrates. Recent studies have suggested that proper intake of nitrates in the food may be
beneficial to human body. Thus intake of beetroot extract containing desired nitrate content is
more beneficial as it can regulate vasodilation, improve blood flow, and boost human stamina,
vigour and athletic performance

Fruits
Punica granatum (Pomegranate): The pomegranate (P. Granatum, Fam. Lythraceae) fruit is a
storehouse of several disease-fighting antioxidants and nutraceuticals. The polyphenol rich
pomegranate juice protects LDL against oxidation, which is responsible for hardening of the
arteries. Much of the work on pomegranate over the past years has focused on antioxidant
activity of various components (Singh et al 2002, Ricci et al 2006). The fruit is a good source of
sugars (14-16%), minerals (0.7-1.0%) and a fair source of iron (0.3-0.7 mg/100 g.). The extract
obtained from pomegranate solids (pericarp, inner membrane and seeds) also exhibit excellent
antioxidant potential. Punicalagin is a powerful antioxidant, protecting cardiovascular function
and accurate cellular replication. It is responsible, in part, for the high antioxidant activity of the
extract. The presence of other phytochemical compounds in the extract, or the synergistic effect
of these phytochemicals, may also be responsible for the anti-oxidant and other beneficial health
effects (Longtin, 2003; Seeram et al 2006). Seeram et al (2005) has reported in vitro
antiproliferative, apoptotic and antioxidant activities of punicalagin, ellagic acid and tannin
extract. In addition to punicalagin, other high molecular weight polyphenols include ellagitannin
and other hydrolysable tannins, such as punicacortein A, punicalin, pedunculagin, and gallotanin
dimmers and trimers. A large number of anthocyanins have also been characterized in the extract
of the pomegranate arils and solids. These include pelargonidin 3-glucoside, cyanidin 3-
glucoside, delphinidin 3-glucoside, pelargonidin 3,5-diglucoside, cyanidin 3,5-diglucoside, and
delphinidin 3,5-diglucoside. The pomegranate peel has also been found to be a rich source of
phenolics, flavonoids and proanythocyanidins than the pulp. As compared to pulp that yielded 24
milligrams per gram (mg/g) of phenolics, the peel yielded significantly higher (250 mg/g)
phenoilc content. Flavonoid content was also significantly greater in the peel than the pulp (59
versus 17 mg/g), as was proanythocyanidins (11 versus 5 mg/g).
Pomegranate peel performed significantly better than the pulp in all of the tests of antioxidant
activity. The phytochemical, pharmacological and clinical investigations on various
components of pomegranate has suggested a wide range of applications for the treatment and
prevention of cardiovascular diseases (Aviram and Dornfeld 2003), cancer, as well as other
diseases (Adams et al 2006, Jeune et al 2005, Lansky and Newman 2007) where chronic
inflammation is believed to play a significant role. Studies have shown that pomegranate juice

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has more polyphenol antioxidants than red wine, green tea, blueberry juice, cranberry juice and
orange juice.

Syzygium cumini (Jamun): Jamun (Syzygium cumini L., Syn: Eugenia jambolana, Syzygium
jambolana, Fam. Myrtaceae), is a tropical and sub-tropical evergreen tree that yields purple
ovoid fleshy fruit and has astringent taste. This underutilized tropical fruit is valued for its
medicinal and therapeutic properties. In traditional Indian medicine, it has been extensively used
against diabetes and apart from that, it is also administered against various ailments like
carminative, febrifuge, antibacterial, diuretic, and diarrhea. The antioxidant activity of jamun
fruit has been attributed to its total phenolic compounds including anthocyanins (Shahnawaz et al
2010). Ethanolic extract of jamun kernel exhibited antioxidant activity superior to catechin and
other commercial synthetic antioxidants (Benherlal and Arumughan 2007). Activity has been
attributed to the presence of anthocyanin content in the fruits. Among 30 volatile constituents in
the fruit, three esters namely dihydrocarvyl acetate, geranyl butyrate and terpinyl valerate were
probably responsible for the characteristic flavour of the jamun fruits (Vijayanand et al 2001).

Jamun fruit is valued for its diverse chemical constituents as well as medicinal and therapeutic
properties (Benherlal and Arumughan 2007; Sagrawat et al 2006). Previous studies have
identified the major anthocyanins in S. cuminii. fruit pulp/skin as diglucosides of delphinidin,
petunidin and malvidin (Veigas et al 2007; De Brito et al 2007; Li et al 2009). Some of the
bioactives in the fruits have been identified as delphinidin-3-gentiobioside, jambocine, malvidin-
3-laminaribiside and petunidin-3-gentiobioside. These anthocyanins are responsible for
imparting the ripened fruit its bright purple color. Fruits also contained citric, malic and gallic
acids. Seeds contain β-sitosterol. Diluted juice of fruits is diuretic and prevents enlargement of
spleen. Seed powder of jamun is used as good diabetic medicine. Jamun fruit extract has been
shown to exhibit anticancer activity against early stage human HCT-116 colon cancer cells and
ciolon cancer stem cells. The activity has been attributed to the synergistic presence of various
anthocyanin constituents in the fruits. (Charepalli et al 2015).

Citrullus lanatus (Watermelon): Watermelon (Citrullus lanatus, Fam. Cucurbitaceae) is an


emerging source of lycopene, known as natural antioxidant and food colorant. It contains large
amount of lycopene than the raw tomatoes. Enzyme (cellulase or pectinase) mediated extraction
generally enhances the juice yield. This pigment gives watermelon its color. Being a strong
antioxidant, it reduces the risk of cancer and other diseases. Watermelon also contains moderate
amounts of potassium, a mineral that is considered essential for proper functioning of body cells,
organs and tissues. Besides lycopene, β -carotene and α- trocopherols have also been detected in
significant concentration (Charoensiri et al 2009).

Watermelon is also a rich source of a non-essential amino acid-citrulline which is a potential


antioxidant and vasodilator. Red flesh watermelons had slightly less citrulline than the yellow or
orange flesh watermelons. Rind contained more citrulline than flesh (Rimando and Perkins-
Veazie 2005). Citrulline has also been reported from other cucurbitaceous fruits like bittermelon,
cucumber, muskmelon, pumpkin, bottle gourd, and wax gourd. Citrulline is an efficient hydroxyl
radical scavenger, a strong antioxidant and plays an important role in drought tolerance
also.Citrulline in the form of citrulline malate is sold as a performance-enhancing athletic diet
supplement, which reduce muscle fatigue (Bendahan et al 2002). Since citrulline is a precursor
248 Winter School on "New generation smart agrochemical oriented approaches for crop
a