CHAPTER-1

INTRODUCTION

1.1 Cyclodextrins

1.1.1 History of Cyclodextrins

1.1.1.1 Discovery Period

The first paper was published in 1891 by Villiers that reported the formation of some

unidentified crystalline substance during fermentation of starch [1]. Villiers, the French

author, assumed that this substance is some kind of cellulose and named it “cellulosine.” An

Australian Microbiologist, Franz Schardinger about 15 years later, whilst studying those

microorganisms which play a role in the deterioration of foods, isolated a microorganism

(named Bacillus macerans) which produced reproducibly two distinct crystalline substances

when cultivated on starch medium [2]. Because most of their properties were similar to the

already known partial degradation products of starch, he named them as α-dextrin and β-

dextrin Freudenberg and his co-workers elucidated the cyclic structure of these two dextrins

in the mid 1930s. This period from 1891-1936 is called the discovery stage in the history of

CDs.

1.1.1.2 The exploratory period from 1936-1970

After 1930s, Freudenberg and his co-workers came to the conclusion that the crystalline

Schardinger dextrins are built from maltose units and contain only α-(1, 4) glycosidic

linkages [3]. In 1950, γ-CD was discovered and its structure elucidated.

Freudenberg, Cramer and Plieninger were granted a patent in 1953 [4]. D. French published

the first fundamental review on cyclodextrins 1957 [5].

1.1.1.3 The utilization period: from 1970-onward

The first international symposium on CDs was organized in 1981 [6]. An international CD

symposium has been organised every 2nd year from 1984 onwards. By the end of 2003, the

total numbers of CD related papers/publications were over 26000. P. R. Sundrarajan and V.

S. R. Rao demonstrated by conformation energy map calculations that CDs with less than six

members cannot be formed due to steric considerations [7]. F. V. Lichtenthaler and S. Immel

coined the term “cyclodextrin” as a generic nomenclature for all the Cyclo-oligosaccharides

depending upon the numbers of glucose units present in a ring [8].

The spectacular development of CD technology relies on a series of reasons such as:

They are natural products, produced form a renewable natural material (starch) by a

relatively simple enzymatic conversion;

They are produced in 1000 tons/yr amounts by environmental technologies;

Their initial high price have dropped to levels where they become acceptable for most

of the industrial purposes;

Through their inclusion complex forming ability, important properties of the

complexed substances can be modified significantly. This unprecedented “molecular

encapsulation” is utilized in many industrial products, technologies and analytical

methods;

Any of their toxic effects is of secondary character and can be eliminated by selecting

the appropriate CD type, derivative, or mode of application;

CDs can consequently be consumed by humans as ingredients of drugs, foods and

cosmetics.

1.1.2 Structural features of Cyclodextrins (CDs)

Cyclodextrins comprises a family of three well known industrially produced major, several

rare and minor cyclic oligosaccharides. The three major CDs are crystalline, homogeneous,

nonhygroscopic substances, which are torus like macro rings built up from gluocopyranose

units. The α-cyclodextrin (Schardinger’s α-dextrin, cyclomaltohexose, cyclohexaglucan,

cyclohexaamylose, α-CD, ACD, C6A) consisting six gluocopyranose units, β-cyclodextrin

(Schardinger’s β-dextrin, cyclomaltoheptaose, cycloheptaglucan, cycloheptaamylose, β-CD,

BCD, C7B) consisting of seven gluocopyranose units and γ-cyclodextrin (Schardinger’s γ-

dextrin, cyclomaltooctaose, cyclooctaglucan, cyclooctaamylose, γ-CD, GCD, C8A)

consisting of eight gluocopyranose units.

1.1.3 Shapes and size of Cyclodextrins (CDs)

Cyclodextrins (CDs) have been described as “seductive molecules, appealing to investigators

both in pure research and applied technologies. Cyclodextrins have truncated cone or bucket

shaped structure (Fig. 1.1.1). CDs are water soluble, cyclic, non-reducing oligosaccharides

consisting of D-(+)-gluocopyranose units linked through α-(1, 4) glycosidic linkages. The

major and most common three cyclodextrins, α-cyclodextrin, β-cyclodextrin and γ-

cyclodextrin consist of six, seven and eight D-(+)-gluocopyranose units, respectively. Native

CDs are crystalline, non-hygroscopic, homogeneous substances that are torus (bucket)

shaped. In Cyclodextrins, the glucose units adopt 4C1 chair conformation and orient

themselves so that the molecule forms a toroidal truncated cone shaped structure. As a

consequence of the 4C1 conformation of the gluocopyranose units, all secondary hydroxyl

groups of gluocopyranose units are situated on one edge of the ring, where as all primary

hydroxyl groups are placed on the other edge. These two edges of the ring are called

secondary and primary face respectively. The ring of gluocopyranose units in CDs is a

conical cylinder which is frequently characterized as a doughnut or wreath shaped truncated

cone. The CD cavity is lined by the hydrogen atoms and glycosidic oxygen bridges. The non-

bonding (lone pair of electrons) electron pairs of glycosidic oxygen bridges are directed

towards the inside of the cavity, producing a high electron density and giving it Lewis base

characters [9]. The C-2-hydroxyl group of one gluocopyranose unit can form a hydrogen

bond with C-3-hydroxyl group of the adjacent gluocopyranose units. In the β-CD molecule, a

complete secondary belt is formed by these H-bonds; therefore, β-CD is a rather rigid

structure. This intramoleculer H-bond formation is cause for the lowest water solubility of β-

CD of all CDs. The H-bond belt is incomplete in α-CD molecule, because one

gluocopyranose unit of six is in a distorted position. As a result, instead of the six possible H-

bonds, only four can be formed at a time. The γ-CD is a non-planer, more flexible structure,

that is why, it is more soluble of three CDs. Important physical properties of three major and

most common cyclodextrins have been intensively studied (Table 1.1.1). Cyclodextrins are

not hygroscopic, but form stable hydrates. A quasielectron neutron scattering study on α-

CD.6H2O, β-CD.7H2O and γ-CD.8H2O showed that 2, 6 and 8.8 water molecules are

included in the α-CD, β-CD and γ-CD cavity, respectively.

Fig. 1.1.1 Schematic Structure of native α-, β- and γ-cyclodextrins

Table 1.1.1 Some chemical and physical properties of three major cyclodextrins

Cyclodextrin Type α-CD β-CD γ-CD

Number of glucose

units

6 7 8

Mw(Da) 972 1135 1297

Solubility in water

(g/100 mL, 298K)

14.5 1.85 23.2

[α]D at 298 K 150 ± 0.5 162.5 ± 0.5 177.4 ± 0.5

Cavity diameter (pm) 470-530 600-650 750-830

Height of torus (pm) 790 ± 10 790 ± 10 790 ± 10

Diameter of outer

periphery (pm)

1460 ± 40 1540 ± 40 1750 ± 40

Melting point (0C) 255-260 255-265 240-245

Crystal water (wt. %) 10.2 13.2-14.5 8.13-17.7

Water molecules in

cavity

6 11 17

Crystal Form Hexagonal

plates

Monoclinic

parallelograms

Quadratic

prisms

C1’-O4-C4 angle(o) 119 117.7 112.6

O4-O4’ distance Ao 4.23 4.39 4.48

O2-O3 distance Ao 3.00 2.86 2.81

Da: Dalton; A0: Angstrom; pm: picometer.

1.1.4 Inclusion complexes of Cyclodextrins (CDs)

The most important, unique and exceptional property of CDs is the formation of inclusion

complexes in solution as well as in crystalline solid form with wide varieties of molecules

depending upon the size of guest molecules. This phenomenon is called “molecular

encapsulation.” Inclusion complexes are entities comprising two or more molecules; the

‘host’ includes a ‘guest’ molecule, totally or in part, by only physical forces, that is, without

covalent bonding. CDs are typical host molecules and may include a great variety of

molecules having the size of one or two benzene rings, or even compounds, which have a

side chain of comparable size, to form crystalline inclusion complexes.

1.1.4.1 Mechanism of Inclusion complex formation

In an aqueous solution, the slightly apolar cyclodextrin cavity is occupied by water molecules

that are energetically unflavored (polar-apolar interaction). Therefore, water molecules can be

readily substituted by appropriate “guest” molecules (Fig. 1.1.2) and (Fig. 1.1.3). The

cyclodextrin is the “host” molecule and part of the “driving force” of the complex formation

is the substitution of the high enthalpy water molecules by an appropriate “guest” molecule.

One, two or three CD molecules can trap one or more “guest” molecules. Most frequently,

the host guest ratio is 1:1. This is the simplest and most frequent case. However, 2:1, 1:2 and

2:2, or even more complicated associations, and higher-order equilibrium exist. The inclusion

complexes formed can be isolated as stable amorphous or microcrystalline substances. The

association and dissociation of CD-guest complex is governed by a thermodynamic

equilibrium (Eq. 1.1.1), which is characterized by the stability constant, K (Eq. 1.1.2).

The ‘driving force’ for inclusion complexation is not fully understood. The following factors

have been considered for the formation of an inclusion complex.

Substitution of the energetically unfavored-apolar interactions between the included

water molecules and CD cavity on one hand, and between water molecules and the

guest on the other hand by the more favored apolar-apolar interactions between the

guest and CD cavity and polar-polar interaction between bulk water and the released

cavity-water molecules;

CD-ring strain release on complexation;

Vander walls interactions and

In the case of some guest, hydrogen bonding between host and guest.

Inclusion complex of Cyclodextrins with different substrates are shown below (Fig. 1.1.4)

and (Fig. 1.1.5).

Fig. 1.1.2 Schematic representation of the formation and dissociation of the

“host” (cyclodextrin) and “guest” (resorcinol) inclusion complex. The formed

“guest-host” inclusion complex can be isolated as a microcrystalline powder.

Fig. 1.1.3 Formation of Host-guest inclusion complex of Cyclodextrin and PAN

also showing primary and secondary faces of Cyclodextrin

Fig. 1.1.4 Inclusion complex of Cyclodextrins with various substrates

Fig. 1.1.5 Inclusion complex of Cyclodextrins with various substrates showing

(1:1) stoichiometry

1.1.5 Synthesis of Cyclodextrins (CDs)

The synthesis of cyclodextrins involves treatment of ordinary starch with easily available

enzymes. In nature, the enzymatic digestion of starch by cyclodextrin glycosyltransferase

(CGTase) produces a mixture of α-, β- and γ-cyclodextrins (CDs). Cyclodextrins are obtained

in a large scale by enzymatic degradation induced by bacterial strains such as Bacillus

macerans (Fig. 1.1.6).

Fig. 1.1.6 Schematic representation of synthesis of Cyclodextrins (CDs) based

on enzymatic degradation of starch by CGTase enzyme

1.1.6 Cyclodextrin polymers

Cyclodextrin polymers reserve a special position amongst the derivatives of the cyclodextrins

(CDs). β-Cyclodextrin polymers (β-CDP) help in biding with the substrates having two or

more guest parts more efficiently [10]. This increased inclusion capacity is observed due to

the co-operativity effect caused by adjacent β-CD moieties in polymeric back bone [11-12].

When two or more cyclodextrins are covalently linked they are known as polymers. The

cyclodextrins fixed into polymeric structures behave differently from their monomeric

derivatives. Cyclodextrin polymers can be classified as one of two types; those that are

soluble in water and those that are water insoluble. By varying the reaction conditions, either

water soluble or insoluble polymers can often be obtained. A considerable amount of work

has been done using cyclodextrins to synthesize polyesters, polyurethanes and various other

types of polymeric materials. Much of this work has been completed in the last 10-20 years

with the majority of the work dealing with both native cyclodextrins and their derivatives. A

variety of polymers obtained by more exotic routes than simple polycondensation, radical or

even suspension reactions of functionalities attached to cyclodextrin are known. Girek et al.

[13] suggested two ways in which cyclodextrins can be polymerized, where one is to attach a

cyclodextrin molecule as a pendant group on another polymer chain. Polymers with

cyclodextrin pendants are usually prepared by radical polymerization of the functional

cyclodextrin monomers such as acryloyl cyclodextrin. These monomers are further

copolymerized with other compatible monomers such as acryl amide. The second type of

polymerization is to react cyclodextrin molecules with bifunctional agents. The commonly

used agent is epichlorohydrin, which result in both water soluble and water insoluble

polymers. Other agents such as dihalogenated hydrocarbons and dicarboxylic derivatives are

also used. These nucleophilic substitution reactions with bifunctional agents usually occur

under strong alkaline conditions in order to deprotonate the hydroxyl groups of the

cyclodextrin.

1.1.6.1 Water soluble cyclodextrin polymers

Water soluble polymers have been the subject of many investigations. These polymers have a

wide variety of potential applications such as controlling the release of a soluble substance

across a membrane and partitioning of organic compounds in an aqueous two-phase system.

As with other cyclodextrin-based compounds, the usefulness of these polymers lies mainly in

their ability to form inclusion complexes with lipophilic guests. The polymeric form can

increase the stability constant of the complexes. Renard et al. have investigated the

relationship between preparation conditions and properties of the water soluble cyclodextrin

polymers [14]. The polymers obtained were characterized by physicochemical measurements.

The preparation was achieved by reacting β-cyclodextrin with epichlorohydrin in an alkaline

medium by a two-step procedure. Several reaction parameters were considered to develop the

conditions of a reproducible synthesis. For example, the size of the reaction vessel, the speed

of the stirrer and the volume of the reaction mixture were kept constant. These parameters

have a strong influence on the preparation of water soluble β-cyclodextrin-epichlorohydrin

polymers. Again, the use of lower mole ratio of epichlorohydrin/β-cyclodextrin gave water

soluble polymers even for longer reaction times. It has been observed that at

epichlorohydrin/β-cyclodextrin ratios greater than 7, insoluble polymers are usually formed.

Szejtli also gives conditions by which water soluble polymers are obtained namely short

reaction times and lower concentrations of cyclodextrin [15].

1.1.6.2 Water insoluble cyclodextrin polymers

Although water-insoluble polymers have not been extensively investigated, some examples

are known. Insoluble products can often be obtained by increasing the ratio of

cyclodextrin/crosslinking agent in the polymerization process. These types of polymers

usually have a high water sorption capability that tends to make them swell in an aqueous

medium. Polymers that are insoluble can either be crosslinked or linear. One example of this

class of polymer is the polyurethanes. Polyurethane resins containing cyclodextrin units as

specific sorption sites were prepared by crosslinking β-cyclodextrin with diisocyanates.

1.1.6.3 Why Cyclodextrins Polymers (CD-Polymers)

CD polymers have several added advantages over other solid supports (Fig 1.1.7). These are

enlisted as:

Environmentally Benign

Thermally stable

Facile one pot synthesis

Stable over a wide range of pH

Applicability in different areas of research

Re-usable

1.1.7 Applications of Cyclodextrins polymers (CDPs)

Polymers of cyclodextrin have very diverse applications, some of which have been

highlighted in the previous sections.

(i) Sebille et al. reported on the properties of chemically modified cationic β-

cyclodextrin-epichlorohydrin polymer as a chiral support. The polymer was used

in liquid chromatography for enatioselective separations. In food, the

epichlorohydrin polymer of cyclodextrin plays a major role [16].

(ii) Shaw et al. Used β-cyclodextrin-epichlorohydrin polymer to reduce the bitterness

components in grape fruit and navel orange juices in a fluidized bed process.

These polymers were compared with neutral XAD resins used under similar

fluidized bed conditions. It was found that cyclodextrin polymers and XAD resins

show effective debittering potential to acceptable levels [17].

(iii) Baille et al prepared poly-(β-cyclodextrin) (PCD) resins by Crosslinking β-

cyclodextrin with different amounts of epichlorohydrin in which some

cyclodextrin hydroxyl groups were functionalized with alkyl quaternary

ammonium groups. These polymers were used to bind several bile salts from

phosphate buffer solutions. Aminated PCD resins have a higher binding capacity

for bile salts [18].

(iv) Liu et. al., (2005) Synthesized a novel reagent by reaction of β-Cyclodextrin

polymer (β-CDP) with 2-DHPH(2,4-dihydroxyacetophenone phenylhydrazone)

and exploited its use in the determination of Cd(II) in different water samples

[19].

(v) Gao and Zhao synthesized a polymer by the supramolecular reaction of β-

cyclodextrin crosslinking polymer with 1-(2-pyridylazo)-2-naphthol (PAN). The

polymer was used to prepare electrorheological materials of an inclusive complex,

named β-cyclodextrin-PAN polymer [20].

(vi) A porous tubular ceramic membrane impregnated with β-cyclodextrin polymer

used to obtain a chiral-selective membrane, was prepared by Krieg et al [21]. This

membrane was evaluated in terms of the effectiveness of impregnation as well as

the ability to separate the enantiomers of the racemic pharmaceutical

chlorthalidone.

1.1.8 Applications of CDs

Cyclodextrins (CDs) have wide range of applicability in different fields. Some of which are

discussed below [22-28].

(a) CDs in Drug Delivery

The following examples illustrate the utility of CD complexation in drugs:

(i) Garlic oil smells very bad and loses its active ingredients contents rapidly.

Cyclodextrin complexation is the ideal solution of the problem.

(ii) Cetrizine is a bitter antiallergic drug. Its bitter taste can be masked using β-

cyclodextrin cetirizine complex.

(iii) Ibuprofen in CD complexed form is appropriate for production of a table

formulation, which is devoid of bitter taste.

(iv) The dextrametamorphan-bromide is a very bitter taste drug used as a peadiatric

drug. It’s very bad taste can be reduced by complexation with β-CD.

(v) Solubility of Itraconazole can be increased using hydroxypropyl-β-CD complex.

(vi) Effectiveness of herbicides, fungicides and insecticides can be enhanced by

complexation with CDs.

(vii) The pernitro γ-CD and its complex with nitramine is an excellent missile

propellent.

(b) In textile industry

The CDs play a significant role in the textile industry. They can be used to remove surfactant

from the washed textile material, textile finishing, bonding the chemical on the fibres in order

to increase wettabiity, include fragrance and removing the insects etc.

(i) Monochlorotriazinyl group (MCT) is used for textile finishing. This anchor group

reacts with the cellulose hydroxyl group, forming permanent covalent bonds.

(ii) For inflammatory treatment: the intelligent textiles offer favourable solutions that

remove some of the inconveniencies of the traditional treatments, such as:

sensitivity of the certain patients to the medicine; administration by the mouth;

omission in the case when the children and old people forget to take medicine. A

bandage is able to gradually release the necessary medicine at the inflamed place

avoids the above mentioned difficulties. The structure of such bandage consists of

an absorbing matrix such as cotton cloth, which contains an anti-inflammatory

medicine for example, sodium diclofenac included in the cyclodextrin linked to

the textile material by means of a reticulating agent, viz., dimethyldihydroxy

ethylene urea (DMDHEU). The bandage is executed in two stages: medicine

complexing with cyclodextrin, followed by attaching the complex to cotton using

the reticulating agent. If the textile is impregnated in a vat containing ethanol, the

rate of medicine release increases at the beginning, when the inflammation is

severe. But if the bandage is not treated with ethanol, the initial release of

medicine is slow. At the same time, the increase of medicine release rate is not

proportional to the initial concentration.

(c) Masking unpleasant odour

Complexation ability of β-CD can be used to mask unpleasant flavors.

(i) Chamomile extracts have an antiphlogistic, bacteriostatic and wound healing

effect. These formulations often have an intense and unpleasant odor. This odor is

reduced by complexation with cyclodextrins without affecting the anti-

inflammatory activity of chamomile.

(ii) Mercapto compounds are used in waving lotions. Due to mercapto compounds, an

extremely unpleasant odor is generated during application. This odor can be

eliminated by complexation with cyclodextrins.

(iii) Glutathione shows various physiological activities. For example, it inhibits the

melanin pigment formation. Therefore, it is used for skin whitening and skin

improving effects. But glutathione generates an offensive odor upon use in the

cosmetic formulations. However, glutathione complex with cyclodextrin is free of

odor but has the same effect on skin as glutathione.

(iv) Dihdroxyacetone (DHA) is used as tanning agent. It is not stable in aqueous

solution. It also has an unpleasant odor which is difficult to be masked by using

perfumes. This odor vanishes using CD-complex. The slow release of

dihdroxyacetone (DHA) from the complex results in the more uniform tanning of

the skin.

(d) Solubilization of the guest molecules.

A large number of cosmetic components are nearly insoluble in water. All these chemical

substances are able to form inclusion complexes with CDs. As a result, these complexes

are more soluble compared to pure compounds. The following examples illustrate this

effect.

(i) Menthol acts as a cooling agent in different cosmetic products. But menthol is

only slightly soluble in water. However, menthol-CD complex is freely soluble in

water.

(ii) Retinol is used in topical anti-ageing formulations. It reduces wrinkles and

supports the restoration of UV damaged tissues. But, UV-light and atmospheric

oxygen induces the chemical oxidation of retinol. Thus, during oxidation some

peroxidic toxic intermediates are formed. Low solubility of retinol in aqueous

media is also a problem. Retinol forms complex with cyclodextrins that are stable

in the presence of light and oxygen without any effect in its activity.

(iii) Cyclodextrins are also used to increase the solubility of substances secreted by the

skin. Thus, CDs find their applicability in products for skin cleaning. They are

able to complex and dissolve skin fat. The resulting fat-CD complex can be easily

removed from the skin.

(iv) Salicylic acid is used for cleaning of the skin. Its action is mainly antibacterial and

keratolytic. The solubility of the acid or its derivatives in aqueous solution is low.

The complex with cyclodextrin is much more soluble and the irritating occurring

with the free acid are prevented due to better homogeneity. Moreover, as a

complex the disinfectant, keratolytic and bacteriostatic activities are also

enhanced.

(v) Triclosan is used as a topical antiseptic and disinfectant in many personal care and

cosmetic products. But it is insoluble in aqueous solution. However, cyclodextrin-

triclosan complex is soluble in water giving a clear solution.

(e) Protection of the guest molecules

(i) Hydroquinone (HQ) is used in skin whitening formulations. In aqueous solution

hydroquinone is stable in limited pH range. Therefore stabilizers are used. But as

a cyclodextrin-hydroquinone complex, the oxidation of hydroquinone is prevented

and has a greater stability. Moreover, the depigmentation property is also

enhanced.

(ii) Kojic acid (KA) is another substance used as a whitening agent in cosmetic

creams. Its use is limited because it is labile when exposed to light or heat. Due to

decomposition, it turns yellowish brown. But as a CD complex, it has an improved

stability. Also, the skin whitening properties of Kojic acid are enhanced as a

cyclodextrin complex.

(iii) Peroxyacetic acid (PAA) forms solid complexes with α- and β-cyclodextrin. These

stable powders are easy to handle and can be used in cosmetic formulations. They

also act as mild oxidants with significant disinfectant properties.

As illustrated by the above examples, a guest complexed with CD can be shielded from the

attack by the reactive species. But if the guest is large size molecule then only a portion of

that molecule will be included with in the CD cavity. Only the portion of the molecule that is

included in the CD cavity would be shielded from chemical attack. This concept can be

utilized in many ways for highly selective synthesis procedures. CDs can also be used as

phase transfer catalyst to transfer reactive components between two immiscible liquids.

Fig. 1.1.7 Schematic diagramme showing applications of CDs in different fields

1.2. Introduction to metal ions

Metals are inorganic substances that occur naturally in geological formations. They are

integral part of our environment. Some metals are essential for our life and are naturally

available in our food and water. Concentrations of metals that are too low may lead to health

problems on the other hand an excess is toxic to plants, animals and human alike.

Metals may be classified into four sub types:

1. Macrominerals are those which are needed in large amounts such as Ca, Mg, Na, K,

Cu, Zn, Fe and S etc.

2. Required Trace minerals include Mn, Cr, Se, B and Mo.

3. Possibly required minerals include Ru, Sn, Au and Ag.

4. Toxic metals are Hg, Pb, As, Sb and U along others.

1.2.1 Exposure to Toxic Metals

Toxic metals are trace elements that form poisonous soluble compounds when considered in

abnormally high toxic doses. Today mankind is exposed to the highest levels in recorded

history of Pb, Hg, Al, Cu, Ni, Sn, Sb, Bi and V. Levels are up to several thousand times

higher than in primitive man. Trace amounts of metals enter our water bodies supplies

naturally as rain percolates through rock, dissolving minute quantities into the water. This

water enters larger water bodies which we use as resource of drinking water and then they

enter our bodies via food, drinking water and air.

1.2.1.1 Modern Diets and Toxic Metals

The danger of toxic metal is greatly aggravated today by the low mineral content of most of

our food supply. An abundance of vital minerals protects our body against toxic metals.

Essentials metals protect body and compete against the toxic metals for absorption and

utilization in enzymes and other tissue structures. However, when food is low in essential

minerals, the body absorbs and makes use of more toxic metals. Causes for low mineral

content of almost all agricultural products are primarily:

Hybrid crops are grown for production or disease resistance, rather than superior nutrition.

Superphosphate fertilizers produce higher yields by stimulating growth, but do not provide all

the trace elements.

Monoculture, the growing of just one crop over and over again on the same land, eventually

depletes the soil. Toxic sprays damage soil microorganisms needed to help the plants to

absorb minerals from soil.

1.2.1.2 Food Sources

Food grown near highways or downwind of the industrial plants may contain Pb [29] and

other toxic amounts of metals. Even organic home gardens may be contaminated if, for

example, old house paint containing Pb leaches Pb into the soil. Sprays and insecticides still

often contain Pb, Hg and other toxic metals. Pesticides used on fruits, vegetables and many

other foods may contain As, Pb, Cu, Hg and other toxic metals.

1.2.1.3 Drinking water

This is the most common source of toxic metals for most people. Al, Cu and many other toxic

chemicals are added to many municipal water supplies. Al allows dirt to settle out of the

water, while Cu kills algae that grows in reservoirs. Wells and even municipal water may also

contain some Pb, As and other undesirable metals. Galvanized and black plastic pipes can be

an important source of Zn and Cd. Lead Soldered pipes and copper pipes may increase these

metals in the drinking water if the water is soft. It is an uncommon problem in hard water

areas. Hydrofluorosilicic acid, the chemical often used to fluoridate drinking water, is a

smokestack waste that contain Pb. Hg, Cd, As, Al, benzene (C6H6) and radioactive waste

material.

1.2.1.4 Airborne Sources of Toxic Metals

Most toxic metals are effectively absorbed by inhalation. Auto and particularly aircraft

exhaust, industrial smoke and products form incinerators are among the airborne sources of

toxic metals and other chemicals. Hg and coal-fired power plants burnt high in the

atmosphere and aircraft fuel deposits everywhere and affects everyone on earth. Burning coal

can release Hg, Pb and Cd among other metals. Iranian and Venezuelan oil are high in V.

Tetraethyl lead [(C2H5)4Pb] was added to gasoline for many years. Residues are present on

pavement and may settle on buildings, cropland and elsewhere. Today, Mn is added to

gasoline. Incineration of electronic parts, plastics treated fabrics and batteries release all the

toxic metals into the air. The use of scrubbers and newer methods of very high temperature

incineration are much better. Cigarette and tobacco smoke are high in Cd, found in cigarette

paper [30, 31].

1.2.1.5 Medications

Many patented prescriptions and over-the-counter drugs contain toxic metals. Thimerisol, a

mercury containing preservative, is used in some vaccines, including all flu shots. Thiazide

diuretics such as Maxzide, Diazide and many others contain Hg. Antacids such as Gelusil,

Digusil and many others are very high in Al content.

1.2.1.6 Direct Skin Content

Almost all anti-perspirants and many cosmetics contain Al. Dental amalgams contain Hg, Cu

and other metals. Dental bridges and other appliances often contain Ni. Soaps, body lotions

and creams often contain toxic compounds. A few hair dyes contain Pb. Selsun Blue

shampoo contains Se that is quite toxic in high doses. Household lawn and garden chemicals

may contain Pb, As and other compounds. Mercury treated seeds and As treated wood are

other common sources of toxic metals.

1.2.2 Toxic Metals Dangers

Toxicity is described as ‘acute’, ‘sub-acute’ or chronic, according to the level and duration of

exposure. ‘Acute toxicity’, which is a measure of the hazard to man and animal following a

single exposure to toxicant, is usually expressed as the dose in milligrams of the chemical per

kilograms of body weight of the test animal. This is referred to as the lethal dose; thus, the

LD50 is the dose which will kill 50% of a given population. Metals are systemic toxins with

specific neurotoxic, nephrotoxic, foetotoxic and teratogenic effects. Breathing heavy metal

particles, even at levels well below those considered non toxic, can have serious health

effects. All aspects of animals and human immune system function are compromised by the

inhalation of metal pollutants. Toxic metals can also increase the acidity of the blood. The

biological half-lives for heavy metals are variably long; the half life for Cd in kidneys is

decades. Most toxic metals are readily transferred across the placenta, found in breast milk,

and are well known to have serious detrimental effects on behaviour, intellect and the

developing nervous systems in children. Heavy metals alter pro-oxidant/antioxidant balance

and bind to free sulfhydryl groups, resulting in inhibition of glutathione metabolism,

numerous enzymes and hormone function. Nutritionally, toxic metals are directly

antagonistic to essential trace elements and compete with nutrient elements for binding sites

on transport and storage proteins, metalloenzymes and receptors. Pb and Hg are well known

for their direct, destructive effects on neuronal functions, while Cd has direct adverse effect

on cells in the arterial wall. Chronic, low level Hg exposure is a problem that goes well

beyond the controversial issue of the dental amalgams. Two primary mechanisms for the

toxic effects of Hg are:

1. Hg is a pro-oxidant which catalyses the production of peroxides and enhances the

subsequent formation of hydroxyl radicals and lipid peroxides.

2. Hg interferes with the body’s capacity to quench highly reactive oxygen species.

Table 1.1.2 Sources, uses and harmful effects of some toxic metals

Sr. No.

Metal Sources Uses Accumulation site

Harmful effects on the

body1. Antimony

(Sb)Semiconductor industry in production of diodes, plumbing, soldering, antifriction alloys and antiprotozoan drugs

Antimony compounds are used in flame proofing compounds, batteries and as an alloy for the lead’s hardness

Bone marrow Excess of Sb leads to pneumonitis, fibrosis and carcinomas

2. Cadmium (Cd)

By-product of Zn, Pb and Cu extraction, battery manufacturing, volcanoes and inhaled tobacco smoke

Ni-Cd batteries, pigments, stabilizers for plastics, electroplated onto steel, silver solder

Liver, bones, testes, kidney

and cardiovascular

system

Causes pneumonitis, cerebral and cerebellar damage, lung cancer and renal toxicityDisease: Itai-Itai

3. Cobalt (Co) As a by-product of Ni and Cu mining and refining, central component of the vitamin cobalamin or vitamin B12

Used in making jet engines, permanent magnets, electroplating industries, purification of histidine-tagged fusion proteins

Lungs Dilated cardiomyopathy and pneumoconiosis

4. Copper (Cu) Natural resources like forest fires and volcanoes. Human activities like mining and phosphate fertilizer production

In electrical wiring, electrical equipment, alloys, in insecticides, fungicides and herbicides

Stomach and small intestine

and liver

Causes hepatic and renal damage. Cu poisoning also leads to haemolytic anaemia and centrilobular hepatic necrosis.Disease: Wilson’s Disease

5. Iron (Fe) Ores such as Cargo ships, Vomiting,

hematite, magnetite and mining

food containers, steel vehicles

gastrointestinal haemorrhage,metabolic acidosis, hepatic cirrhosis

6. Zinc (Zn) Industrial activities such as mining, smelting and steel processing

In galvanizing Fe, alloys, pigment in paints, printing inks.

Causes vomiting, diarrhoea, anaemia, neurological degeneration and osteoporosis.

7. Mercury (Hg) Breakdown of minerals, fossil fuel combustion, mining, smelting, solid waste combustion and fertilizers

In barometers, manometers, thermometers, liquid electrode, insecticides, rat poison, disinfectant, skin ointment.

Brain, pituitary gland, thyroid and adrenals

Causes irreversible brain damage including cerebral palsy, mental retardation, causes chromosomal segregation and inhibits cell division in foetus.Disease: Pink Disease

8. Manganese (Mn)

Mining and mineral processing, municipal waste water discharge, sewage sludge, fossil fuel combustion and volcanoes.

In low cost stainless steel, disinfectant, fertilizers and ceramics.

Muscles and brain

Causes multiple neurological problems with symptoms similar to those of Parkinson’s disease, facial muscle spasms and even hallucinations.Disease: Manganism

9. Nickel (Ni) Mining and refining, stainless steel, Ni-Cd batteries, electroplating, electro refining and welding.

Coins, rechargeable batteries, stainless steel, electroplating, pigments for paints or ceramics.

Kidney, liver and lungs

Causes allergic reactions, encephalopathy, pulmonary fibrosis and reduced sperm amount. Ni compounds are considered as human

carcinogen.10. Lead (Pb) In ores of Zn,

Ag, Cu, gasoline, car engines and car exhaust

In Pb-acid batteries, ceramic glazes, solders, Pb crystal glassware, ammunition and bearings.

Soft tissues, bone and teeth

Causes hallucinations and excitement of central nervous system, renal failure and chromosomal aberrations. Pb is also gametotoxic and embryotoxic.

11. Vanadium (V)

Minerals such as patronite, vanadinite, carnotite & bauxite. Vanadium also occurs in carbon containing deposits such as crude oil & tar sands etc.

Used as a steel additive, in jet engines and high speed air frames, in axles, gears. Vandium oxide (V2O5) is used as a catalyst in manufacturing sulphuric acid & maleic anhydride

Acute effects: headache, nausea, vomiting, insomnia & irritability.

Chronic effects: Lung & nasal cancers

12. Arsenic(As) In bronzing, hardening and improving the sphericity of shot, wood preservation, as pesticides, pyrotechnics, semiconductor devices, LED, lasers and ICs

Blood and hair Hypertension, hyper pigmentation, hypo-pigmentation,hyperkeratosis, periorbital swelling

1.3 Introduction to Dithiocarbamate Pesticides

The dithiocarbamates form a very important group among fungicides. Most of these are

foliage fungicides, while some are used for soil and seed treatments. Tisdale [32] first

demonstrated the fungicidal possibilities, of the carbonates in 1931 in the laboratories of E.I.

Dupont Company, USA, but the commercial production started about a decade later. The

dithiocarbamates were discovered as a class of chemical compounds early in the history of

organosulphur chemistry e.g., Debus [33] gave the synthesis of dithiocarbamic acids in 1850.

Delepine [34] was one of the pioneers in the field to recognize the metal binding properties of

dithiocarbamates. This property is due to the insolubility of the metal salts, with the exception

of Na and other alkali and alkaline earth metals and to the capacity of the molecules to form

the chelate complexes. It is due to this characteristic that various dithiocarbamates e.g.,

sodium diethyl dithiocarbamate, have been found practical outlet in the field of inorganic

analysis and antioxidants.

Carbamates are the half amides of carbonic acid. Their sulphur analogs, the

dithiocarbamates are the half amides of dithiocarbonic acid. The synthesis and the general

formula of dithiocarbamates are given below:

2NH3 + CO2 → H2N―COONH4

The ammonium salt of carbamic acid

2NH3 + CS2 → H2N―CSSNH4

The ammonium salt of dithiocarbamic acid

The world wide consumption of dithiocarbamates is between 25,000 to 35,000 metric

tonnes per year. Dithiocarbamates are mainly used in agriculture as insecticides, herbicides

and fungicides. In industry, they are used as slimicides in water-cooling systems, in sugar,

pulp, and paper manufacturing, and as vulcanization accelerators and antioxidants in rubber

[35]. Because of their chelating properties, they are also used as scavengers in waste-water

treatment. Dithiocarbamates have been employed extensively as inhibitors in research on

enzymes. Their usefulness is due to their metal binding capacity and their ability to interact

with sulfhydryl-containing compounds. Because of their biological activity they have also

found a more practical application in the field of medicine. They were first used in medicine

in experiments on control of various dermatophytes.

Dithiocarbamates with hydrophilic groups form water-soluble, heavy-metal complexes, while

some of the dithiocarbamate metal complexes used as fungicides are insoluble in water and

soluble in non-polar solvents. Alkylene bisdithiocarbamates (containing two donor CS2

groups), which form polymeric chelates, are insoluble in both polar and non-polar solvents.

Dithiocarbamates may decompose under certain circumstances into a number of compounds

as ethylenethiourea (ETU), and ethylenediamine (EDA). ETU is fairly stable, has high water

solubility, and is of particular importance because of its specific toxicity.

1.3.1 Toxicity of pesticides

Dithiocarbamates do not belong to systemic fungicides but are protectant fungicide applied

prior to fungus infection. Therefore, they act upon damaging fungi chiefly by surface deposits

[36]. Dithiocarbamates can be absorbed by the organism via the skin, mucous membranes,

respiratory and the gastrointestinal tracts. Whereas dithiocarbamates are absorbed rapidly

from the gastrointestinal tract, metal complexed Alkylene bisdithiocarbamates are absorbed

poorly both from the gastrointestinal tract and through the skin. Dithiocarbamates are known

to have toxicological and mutational effects [37].

1.4 Introduction to spectrophotometry

The spectrophotometry is based on the variation of colour of the system with change in

concentration of coloured species in the solution. The absorbing group in a molecule is called

chromophore. A molecule containing chromophore is called a chromogen. An auxochrome

does not itself absorb radiation, but if present in a molecule, it can enhance the absorption by

a chromophore or shift the wavelength of absorption when attached to a chromophore. The

UV-Vis spectrophotometry is an important tool for identification and estimation of inorganic,

organic and biochemical compounds. The merit of this analytical method is that very small

quantities of substance can be detected and estimated with reasonable accuracy.

1.4.1 Lambert Beer’s Law

The amount of monochromatic light radiation absorbed by a medium is described by Lambert

Beer’s Law, according to which, “when a beam of monochromatic light radiation is allowed

to pass through a solution, the intensity of the emitted light radiation decreases exponentially

with increase in the thickness of absorbing medium.” It could be expressed as follows

-dI/dt = kI

I is the intensity of the incident light radiation of wavelength, t is the thickness of the medium

and k is the constant of proportionality. Upon integrating this equation and putting I = I0

when t = 0, it becomes

In I0/I = kt

It = I0 e-kt

Beer studied the effect of concentration while Lambert studied the effect of the thickness of

the absorbing medium over the transmission or absorption of light. Thus, Lambert Beer Law

could be represented as:

It = I0. log10. εct

log I0/ It = εct

A = εct

ε = A/ct

where ‘A’ is the absorbance, ‘c’ is the molar concentration i.e., in mol L-1, ‘t’ is the thickness

of the medium in centimetres and ‘ε’ is the molar extinction coefficient or molar absorptivity

in L mol-1 cm-1.

If there is only one absorbing species in the solution, then the quantitative estimation of an

unknown solution involves plotting the absorbance values verses concentration for a set of

standard solutions, keeping the wavelength fixed at a value for which the maximum

absorbance is obtained. After plotting the curve, the concentration of the unknown solution is

determined by measuring the absorbance and referring it to the calibration curve.

1.4.2 Sensitivity of Spectrophotometric methods

Sensitivity refers to the slope of a calibration curve, but is frequently used to mean the least

determinable concentration or amount of the species of interest. The objective numerical

expression of sensitivity of Spectrophotometric methods is the molar absorptivity (ε) at the

wavelength of the maximum absorbance of the coloured species.

ε = A/ct

(is expressed in the litre mol-1 cm-1).

Savvin suggested the following criteria for describing the sensitivity.

Low sensitivity: ε < 2 × 104

Moderate sensitivity: ε < 2-6 × 104

High Sensitivity: ε > 6 × 104

The sensitivity of the spectrophotometric methods is often expressed in terms of the

expression given by Sandal which represents the number of micrograms of the species

determined per millilitre of a solution having an absorbance of 0.001 for a path length of

1cm. Then sensitivity ‘S’ according to Sandal is expressed in µg/cm2.

1.5 Introduction to reagents

1.5.1 1-(2-pyridylazo)-2-naphthol (PAN)

The most widely used azo dye is [1-(2-pyridylazo)-2-naphthol] (PAN, Fig. ) a heterocyclic

azo dyeforms coloured complexes with most metals. PAN since been developed as a

spectrophotometric reagent with or without extraction. These are red with the alkaline earths,

Al(III), Sc(III), Y(III), Ti(IV), Zn(II), Cd(II), Hg(II), Ga(III), In(III), Tl(III), Pb(II), Bi(III),

Ni(II), Mn(II) and U(IV) etc. With Cu(II), V(IV) and V(V), Fe(II) and Fe(III) and Ru(III),

they are of varying shades from red to violet, whereas with Co(II), Pd(II) and Pt(II) they are

green. The alkali metals, Ge(IV), As Se and Te do not recact. The pH of the solution is of

great importance, and minimum pH for chelation varies from metal to metal. Correct pH

control is of absolute importance in all analytical work with PAN [38]. Many of these

chelates are insoluble in water but can be extracted into various organic solvents as

investigated in detail by Shibita [39], Berger et al. [40] PAN shows both hypsochromic and

bathochromic shifts on protonation and ionization respectively. It is insluble in water, dilute

acids and alkslis, but is soluble in strong acid (pH < 2) to give a yellow-green cation, and in

strong alkalis (pH > 12) to give a red anion as shown below:

PAN acts as tridendate ligand complexing with most metals through the ortho-hydroxyl

group, the azo nitrogen nearest to the phenolic ring and the heterocyclic nitrogen atom, giving

two stable, 5-membered chelate rings [41]. The most common metal-to-ligand ratios

encountered are 1:1 and 1:2 and the structure for these two types of complexes are as shown

below:

Most PAN complexes have absorption maxima lying between 530 and 570 nm, but some

complexes absorb at longer wavelengths, for instance, V(V) (615 nm), Pd(II) (620 and 675

nm) and Rh(III) (598 nm). The spectrophtotmetric application with a wide range of metals

make PAN a useful sensitive and selective reagent. Shibita [42] has reviewed its use for

spectrophtotmetric determination of a number of metals in various samples.