CHAPTER-1

INTRODUCTION
1.1 Cyclodextrins
1.1.1 History of Cyclodextrins
[Link] 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 -dextrin and 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.

[Link] 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 -(1, 4) glycosidic
linkages [3]. In 1950, -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].

[Link] 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 -cyclodextrin (Schardingers -dextrin, cyclomaltohexose, cyclohexaglucan,
cyclohexaamylose, -CD, ACD, C6A) consisting six gluocopyranose units, -cyclodextrin
(Schardingers -dextrin, cyclomaltoheptaose, cycloheptaglucan, cycloheptaamylose, -CD,
BCD, C7B) consisting of seven gluocopyranose units and -cyclodextrin (Schardingers dextrin, cyclomaltooctaose, cyclooctaglucan, cyclooctaamylose, -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 -(1, 4) glycosidic linkages. The
major and most common three cyclodextrins, -cyclodextrin, -cyclodextrin and 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 nonbonding (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 -CD molecule, a
complete secondary belt is formed by these H-bonds; therefore, -CD is a rather rigid
structure. This intramoleculer H-bond formation is cause for the lowest water solubility of CD of all CDs. The H-bond belt is incomplete in -CD molecule, because one
gluocopyranose unit of six is in a distorted position. As a result, instead of the six possible Hbonds, only four can be formed at a time. The -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 CD.6H2O, -CD.7H2O and -CD.8H2O showed that 2, 6 and 8.8 water molecules are
included in the -CD, -CD and -CD cavity, respectively.

Fig. 1.1.1 Schematic Structure of native -, - and -cyclodextrins

Table 1.1.1 Some chemical and physical properties of three major cyclodextrins
Cyclodextrin Type
Number of glucose

-CD
6

-CD
7

-CD
8

units
Mw(Da)
Solubility in water

972
14.5

1135
1.85

1297
23.2

(g/100 mL, 298K)

[]D at 298 K
Cavity diameter (pm)
Height of torus (pm)
Diameter of outer

150 0.5
470-530
790 10
1460 40

162.5 0.5
600-650
790 10
1540 40

177.4 0.5
750-830
790 10
1750 40

periphery (pm)
Melting point (0C)
Crystal water (wt. %)
Water molecules in

255-260
10.2
6

255-265
13.2-14.5
11

240-245
8.13-17.7
17

Hexagonal

Monoclinic

Quadratic

plates
119
4.23
3.00

parallelograms
117.7
4.39
2.86

prisms
112.6
4.48
2.81

cavity
Crystal Form

C1 -O4-C4 angle( )
O4-O4 distance Ao
O2-O3 distance Ao

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.

[Link] 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 -, - and -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). -Cyclodextrin polymers (-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 -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.

[Link] 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 -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 -cyclodextrin-epichlorohydrin
polymers. Again, the use of lower mole ratio of epichlorohydrin/-cyclodextrin gave water
soluble polymers even for longer reaction times. It has been observed that at
epichlorohydrin/-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].

[Link] 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 -cyclodextrin with diisocyanates.

[Link] 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 cyclodextrin-epichlorohydrin polymer as a chiral support. The polymer was used
in liquid chromatography for enatioselective separations. In food, the

(ii)

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

Shaw et al. Used -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

(iii)

show effective debittering potential to acceptable levels [17].

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

(iv)

for bile salts [18].

Liu et. al., (2005) Synthesized a novel reagent by reaction of -Cyclodextrin
polymer (-CDP) with 2-DHPH(2,4-dihydroxyacetophenone phenylhydrazone)
and exploited its use in the determination of Cd(II) in different water samples

(v)

[19].
Gao and Zhao synthesized a polymer by the supramolecular reaction of cyclodextrin crosslinking polymer with 1-(2-pyridylazo)-2-naphthol (PAN). The
polymer was used to prepare electrorheological materials of an inclusive complex,

(vi)

named -cyclodextrin-PAN polymer [20].

A porous tubular ceramic membrane impregnated with -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.

(ii)

Cyclodextrin complexation is the ideal solution of the problem.

Cetrizine is a bitter antiallergic drug. Its bitter taste can be masked using -

(iii)

cyclodextrin cetirizine complex.

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

(iv)

formulation, which is devoid of bitter taste.

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

(v)
(vi)

drug. Its very bad taste can be reduced by complexation with -CD.
Solubility of Itraconazole can be increased using hydroxypropyl--CD complex.
Effectiveness of herbicides, fungicides and insecticides can be enhanced by

(vii)

complexation with CDs.

The pernitro -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

(ii)

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

(ii)

inflammatory activity of chamomile.

Mercapto compounds are used in waving lotions. Due to mercapto compounds, an
extremely unpleasant odor is generated during application. This odor can be

(iii)

eliminated by complexation with cyclodextrins.

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

(iv)

odor but has the same effect on skin as glutathione.

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

(ii)

water.
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
(iii)

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

(iv)

removed from the skin.

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

(v)

enhanced.
Triclosan is used as a topical antiseptic and disinfectant in many personal care and
cosmetic products. But it is insoluble in aqueous solution. However, cyclodextrintriclosan 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

(ii)

enhanced.
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

(iii)

cyclodextrin complex.
Peroxyacetic acid (PAA) forms solid complexes with - and -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.

[Link] 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.

[Link] 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.

[Link] 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 (C 6H6) and radioactive waste
material.

[Link] 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].

[Link] 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.

[Link] 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 bodys capacity to quench highly reactive oxygen species.

Table 1.1.2 Sources, uses and harmful effects of some toxic metals
Sr.
No.
1.

Metal

Sources

Uses

Antimony (Sb)

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

2.

Cadmium (Cd)

3.

Cobalt (Co)

Semiconductor
industry in
production of
diodes,
plumbing,
soldering,
antifriction
alloys and
antiprotozoan
drugs
By-product of
Zn, Pb and Cu
extraction,
battery
manufacturing,
volcanoes and
inhaled tobacco
smoke
As a by-product
of Ni and Cu
mining and
refining, central
component of
the vitamin
cobalamin or
vitamin B12

4.

Copper (Cu)

5.

Iron (Fe)

Natural
resources like
forest fires and
volcanoes.
Human activities
like mining and
phosphate
fertilizer
production

Ores such as
hematite,

Accumulation
site
Bone marrow

Harmful effects
on the body
Excess of Sb
leads to
pneumonitis,
fibrosis and
carcinomas

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

Liver, bones,
testes, kidney
and
cardiovascular
system

Used in making
jet engines,
permanent
magnets,
electroplating
industries,
purification of
histidine-tagged
fusion proteins
In electrical
wiring, electrical
equipment,
alloys, in
insecticides,
fungicides and
herbicides

Lungs

Causes
pneumonitis,
cerebral and
cerebellar
damage, lung
cancer and renal
toxicity
Disease: Itai-Itai
Dilated
cardiomyopathy
and
pneumoconiosis

Cargo ships,
food containers,

Stomach and
small intestine
and liver

Causes hepatic
and renal
damage. Cu
poisoning also
leads to
haemolytic
anaemia and
centrilobular
hepatic necrosis.
Disease: Wilsons
Disease
Vomiting,
gastrointestinal

magnetite and
mining

steel vehicles

6.

Zinc (Zn)

Industrial
activities such as
mining, smelting
and steel
processing

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

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.

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

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

Brain, pituitary
gland, thyroid
and adrenals

haemorrhage,
metabolic
acidosis, hepatic
cirrhosis
Causes vomiting,
diarrhoea,
anaemia,
neurological
degeneration and
osteoporosis.
Causes
irreversible brain
damage including
cerebral palsy,
mental
retardation,
causes
chromosomal
segregation and
inhibits cell
division in foetus.
Disease: Pink
Disease
Causes multiple
neurological
problems with
symptoms similar
to those of
Parkinsons
disease, facial
muscle spasms
and even
hallucinations.
Disease:
Manganism
Causes allergic
reactions,
encephalopathy,
pulmonary
fibrosis and
reduced sperm
amount. Ni
compounds are
considered as

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.

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
In bronzing,
hardening and
improving the
sphericity of
shot, wood
preservation, as
pesticides,
pyrotechnics,
semiconductor
devices, LED,
lasers and ICs

12.

Arsenic(As)

Soft tissues,
bone and teeth

human
carcinogen.
Causes
hallucinations
and excitement of
central nervous
system, renal
failure and
chromosomal
aberrations. Pb is
also gametotoxic
and embryotoxic.
Acute effects:
headache, nausea,
vomiting,
insomnia &
irritability.
Chronic effects:
Lung & nasal
cancers

Blood and hair

Hypertension,
hyper
pigmentation,
hypopigmentation,
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

H NCOONH
2

The ammonium salt of carbamic acid

2NH3 + CS2 H2NCSSNH4
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 CS 2
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 Beers Law

The amount of monochromatic light radiation absorbed by a medium is described by Lambert
Beers 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. ct
log I0/ It = ct
A = ct

 = 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 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 () at the
wavelength of the maximum absorbance of the coloured species.
= A/ct
(is expressed in the litre mol-1 cm-1).
Savvin suggested the following criteria for describing the sensitivity.
Low sensitivity: < 2 104
Moderate sensitivity: < 2-6 104
High Sensitivity: > 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 g/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.