CHAPTER 1

INTRODUCTION

1.1 Rational and/or hypothesis

Aflatoxins are naturally occurring mycotoxins that are produced by many

species of Aspergillus, mostly A. flavus, A. niger and A. parasiticus. Aflatoxins are

found worldwide in air, soil, dead plants and animals. They contaminate a wide

variety of important agricultural products such as peanuts, maize, rice and cottonseed.

Many countries in tropical and subtropical regions with weather of relatively high

temperature and humidity are suitable for growth of the molds and for production of

the toxin [1-3]. The main biological effects of aflatoxins are highly toxic, mutagenic,

teratogenic and carcinogenic compounds that have been implicated as causative

diseases in humans and animals. Human exposure to aflatoxins can result directly

from ingestion of contaminated foods, or indirectly from consumption of foods from

animals previously exposed to aflatoxins in feeds. The contamination in livestock feed

frequently results in poor growth and feed conversion efficiency, increased mortality

rates and a greater susceptibility to diseases [4]. The problem of food and feed

contamination with aflatoxins is of current concern and has received a great deal of

attention during the last three decades. Removal or inactivation of aflatoxin in food

and feedstuffs is a major global concern. Aflatoxins can be detoxified or removed

from contaminated foods and feeds by physical, chemical and biological methods.

The physical methods include solvent extraction, adsorption, heat treatment and

irradiation [5]. The approach to reduce this problem has been the dietary inclusion of

high affinity adsorbents that can bind aflatoxin e.g. commercial toxin binder and

bentonite [6-7]. Clay minerals, are hydrous aluminium phyllosilicates, sometimes with

variable amounts of iron, magnesium, alkali metals, alkaline earth and other cations

have been employed as well [8]. The binding of clay particles to mycotoxins is a very

complex process. Using clay products having a high cation exchange capacity can

have undesirable nutritional consequences to the animal by binding to mineral

components in the diet. Various types of clays including montmorillonite,

clinoptilolite and kaolinite have been used to adsorb Aflatoxin B1 (AFB1) in aqueous

solution. These clays showed more effective than other adsorbents. The objective of

this study was to investigate the adsorption behaviors of AFB1 on different natural

adsorbents. One of the most encouraging approaches in solving the aflatoxin problem

is the adsorption process, which it is operative in most natural physical, biological and

chemical systems and is widely used in industrial applications [9]. The exact nature of

the bonding depends on the details of the species involved, but the adsorbed material

is generally classified as exhibiting physisorption or chemisorption [10].

1.2 Theory and literature review

1.2.1 Aflatoxin B1

Production of AFBl by A. flavus and A. parasiticus is higher in rice than in

peanut. Pure AFB1 is pale-white to yellow crystalline, odorless solid. Aflatoxins are

soluble in methanol, chloroform, acetone and acetonitrile. A. flavus typically produces

AFB1 and AFB2, wherever A. parasiticus produces AFG1 and AFG2 as well as AFB1

and AFB2. Four other AFM1, AFM2, AFB2A and AFG2A are produced in minor

2

3

amounts, were subsequently isolated from cultures of A. flavus and A. parasiticus. A

number of closely related compounds namely AFGM1, parasiticol and aflatoxicol are

also produced by A. flavus. AFM1 and AFM2 are major metabolites of AFB1 and

AFB2 respectively, found in milk of animals that have consumed feed contaminated

with aflatoxins [11]. Chemical and physical properties of aflatoxins are given in Table

1.1, and aflatoxins are normally refers to the group of difurano-coumarins and

classified in two broad groups according to their chemical structures; the

difurocoumarocyclopentenone series (AFB1, AFB2, AFB2A, AFM1 and AFM2) and the

difurocoumarolactone series (AFG1, AFG2 and AFG2A) are shown in Figure 1.1 The

IUPAC name for AFB1 as 2,3,6aα,9aα-tetrahydro-4-methoxycyclopenta[c]furo[3’, 2’:

4,5][1] benzopyran1,11-dione [12-13].

Table 1.1 Chemical and physical properties of aflatoxins [12]

Aflatoxin Molecular formula Molecular weight Melting point

AFB1 C17 H12O6 312 268-269

AFB2 C17 H14O6 314 286-289

AFG1 C17 H12O7 328 244-246

AFG2 C17 H14O7 330 237-240

AFM1 C17 H12O7 328 299

AFM2 C17 H14O7 330 293

AFB2A C17 H14O7 330 240

AFG2A C17 H14O8 346 190

4

O O OCH3

O

OO

O O OCH3

O

OO

O O OCH3

O

O

O

O

O O OCH3

O

O

O

O

O O OCH3

O

OO

OH

O O OCH3

O

OO

OH

O O OCH3

O

OO

HO O O OCH3

O

O

O

O

HO

Figure 1.1 Chemical structures of aflatoxins [13]

AFB1 AFB2

AFG1 AFG2

AFM1 AFM2

AFB2A AFG2A

5

1.2.1.1 Occurrence

Many agricultural commodities are liable to infestation by aflatoxigenic molds

producing aflatoxins. The growth of the molds and production of aflatoxins in natural

substrates are influenced by a number of factors including types of substrates, fungal

species, moisture content of the substrate, presence of minerals, and relative humidity

of the surroundings, temperature and physical damage of kernels.

(i) Raw agricultural products

Aflatoxins often occur in crops in the field prior to harvest. After harvest,

contamination can occur if crop drying is delayed and the crop storage is too moist.

Insect or rodent infestations facilitate mold invasion of some stored commodities.

Aflatoxins can be detected in milk, cheese, corn, peanuts, cottonseed, nuts, almonds,

figs, spices and a variety of other foods and feeds. Milk, eggs and meat products are

sometimes contaminated because of the animal consumption of aflatoxin-

contaminated feed. However, the commodities with the highest risk of aflatoxin

contamination are corn, peanuts, and cottonseed.

(ii) Processed foods

Corn is probably the commodity of greatest worldwide concern. It is

grown in climates that are likely to have perennial contamination with aflatoxins, and

corn is the staple food of many countries. However, procedures used in the processing

of corn help to reduce contamination the resulting food product. This is because

although aflatoxins are stable to moderately stable in most food processes, they are

unstable in processes such as those used in making tortillas that employ alkaline

6

conditions or oxidizing steps. Aflatoxin-contaminated corn and cottonseed meal in

dairy rations have resulted in AFM1 contaminated milk and milk products, including

non-fat dry milk, cheese and yogurt [14].

1.2.1.2 Toxicity

Humans are exposed to aflatoxins by consuming foods contaminated with

products of fungal growth. Such exposure is difficult to avoid because fungal growth

in foods is not easy to prevent. Even though heavily contaminated food supplies are

not permitted in the market place in developed countries, concern still remains for the

possible adverse effects resulting from long-term exposure to low levels of aflatoxins

in the food supply. Evidence of acute aflatoxicosis in humans has been reported from

many parts of the world, namely the Third World Countries, like Taiwan, Ouganda,

India, and many others. The syndrome is characterized by vomiting, abdominal pain,

pulmonary edema, convulsions, coma and death with cerebral edema and fatty

involvement of the liver, kidneys and heart.

Aflatoxins are toxic and carcinogenic to animals, including humans. After

entering the body, aflatoxins are metabolized by the liver to an intermediate reactive,

AFM1, an epoxide. The aflatoxins display potency of toxicity, carcinogenicity,

mutagenicity in the order of AFB1 > AFG1 > AFB2 > AFG2. Structurally the

dihydrofuran moiety, containing double bond, and the constituents liked to the

coumarone moiety are of importance in producing biological effects. Moreover,

aflatoxins have been associated with incidence of the disease kwashiorkor, a

consequence of protein energy malnutrition in children. Ingestion of aflatoxins leads

to substantial loss of productivity and degradation of meat quality in farm animals

7

consuming contaminated feeds. The discovery of aflatoxins dates back to the year

1960, more than 100,000 young turkeys on poultry farms in England died in the

course of a few months from an apparently new disease that was termed "Turkey X

disease". It was soon found that the difficulty was not limited to turkeys. Ducklings

and young pheasants were also affected and heavy mortality was experienced. A

careful survey of the early outbreaks showed that they were all associated with feeds,

namely Brazilian peanut meal. An intensive investigation of the suspect peanut meal

was undertaken and it was quickly found that this peanut meal was highly toxic to

poultry and ducklings with symptoms typical of Turkey X disease [13-15].

1.2.1.3 Detoxification

The reactions of aflatoxins to various physical, chemical conditions and

reagents have been studied extensively because of the possible application of such

reactions to the detoxification of aflatoxins contaminated material.

(i) Heat

Aflatoxins have high decomposition temperatures ranging from 237 °C to

306 °C. Solid AFB1 is relatively stable to dry heating at temperatures below its

thermal decomposition temperature of 267 °C. The use of heat to inactivate aflatoxin

in contaminated food has been attempted. Normal home cooking conditions such as

boiling and frying (~150 °C) failed to destroy AFB1 and AFG1 in the solid state.

Temperatures above 150 °C were necessary to attain partial destruction of the toxin.

The extent of the destruction achieved was very dependent on the initial level of

contamination, heating temperature and time. Moreover, the type of food and

8

aflatoxin also influenced the degree of inactivation achieved. Degradation of aflatoxin

by heat is also governed by the moisture content, pH and ionic strength of the food [1].

(ii) Oxidizing agents

Many oxidizing agents, such as sodium hypochlorite, potassium

permanganate, chlorine, hydrogen peroxide, ozone and sodium perborate react with

aflatoxin and change the aflatoxin molecule in some way as indicated by the loss of

fluorescence. The mechanisms of these reactions are uncertain and the reaction

products remain unidentified in most cases [16-17].

(iii) Extraction

Extraction with solvents has been used to remove aflatoxins from the

oilseeds, peanut, and cottonseed. Materials treated in this way may only be suitable

for animal feeding. The solvents used include 95% ethanol, 90% aqueous acetone,

80% isopropanol, hexane-methanol, methanol- water, acetonitrile-water, hexane-

ethanol-water and acetone-hexane-water. The solvent: sample ratio was found to be

crucial for recovery of the toxin. Solvent extraction can remove all traces of aflatoxin

from oilseed meals with no formation of toxic byproducts or reduction in protein

content and quality. However, large-scale application of this technique is limited by

high cost and a problem related to disposal of the toxic extracts [1, 16-17].

(iv) Irradiation

Radiation is classified into two categories: ionizing and non-ionizing. In

ionizing radiation (e.g. X-rays, gamma rays and ultraviolet rays) potential changes

9

may occur in molecules of the irradiated object with little or virtually no temperature

rise. These molecular changes might be quite harmful to living organisms exposed to

large doses of ionizing radiation. On the other hand, non-ionizing radiation (e.g. radio

waves, microwaves, infrared waves, visible light) in sufficient intensity leads to a rise

in temperature, usually accompanied by molecular changes that are of no hazardous

nature to man. The use of ionizing radiation to free foods from pathogenic

microorganisms is among the methods applicable in food preservation. Despite the

debate on safety of irradiated foods in connection with human health, however, food

irradiation is becoming a technique of potential application on a commercial scale to

render food products sterile [1].

(v) Adsorption

Several adsorbents can bind and thus remove aflatoxins from aqueous

solution. For example: the in vitro and in vivo applications of hydrated sodium

calcium aluminosilicate (HSCAS) to adsorb aflatoxins and other mycotoxins were

reported. The effect on animals due to dietary addition of HSCAS to feedstuffs

contaminated with mycotoxins was discussed in a variety of farm animals. HSCAS, a

sorbent compound obtained from natural zeolite, has demonstrated an ability to bind

mycotoxins with a high affinity. Addition of this compound to feedstuffs

contaminated with aflatoxins has shown a protective effect against the development of

aflatoxicosis in farm animals [18-19]. Many other materials have been reported for

AFB1 adsorption such as montmorillonite silicate, a commonly the main constituent

of the clays known as bentonites. NovaSil Plus is a naturally-occurring and heat

processed calcium montmorillonites that is commonly used as an anticaking additive

10

in animal feed [20-21], Novasil clay provided significant protection from the adverse

effects of aflatoxins in multiple animal species by decreasing bioavailability from the

gastrointestinal tract [22].

Kaolinite essentially has no spacing between the platelets, while smectites

such as naturally acidic montmorillonite clay have a considerable amount of spacing,

depending on the interlamellar cation. The direct result of an interlamellar space is

that the surface area of smectite clays can be considerable [23]. Sodium bentonite

from southern Argentina had a high ability to sorbs AFB1 from aqueous solution.

Adsorption of zearalenone, ochratoxin, and AFB1 on natural zeolite,

clinoptilolite, modified with different amounts of octadecyldimethyl benzyl

ammonium ions was investigated [24]. Natural zeolite and clinoptilolite modified

with different levels of octadecyldimethylbenzyl ammonium were effective in

adsorbing the ionizable mycotoxins zearalenone and ochratoxin A. However, the

adsorption of AFB1 was greatly reduced when compared to the unmodified zeolitic

tuff, which had a high affinity for AFB1 [25]. Obviously, there exists a need for a

systematic approach to test potential sorbent/mycotoxin combinations in vitro to rank

them for testing in vivo. These include single-concentration sorption, isotherms and

chemisorption index. There were compared as methods for predicting the adsorption

of AFB1 from solution by four adsorbents: HSCAS, charcoal, clinoptilolite and sand

[26]. Moreover, the adsorption of AFB1 by cation-exchanged clinoptilolite zeolitic

tuff and montmorillonite was investigated at 37 ºC and pH 3.8 from an aqueous

electrolyte having a composition similar to that of gastric juices of animals. The

impact of the mineral adsorbents on the reduction of essential nutrients present in

animal feed (Cu, Zn, Mn and Co) showed that the Ca-rich montmorillonite had a

11

higher capability for the reduction of the microelements than the Ca-rich clinoptilolite

[27].

Clay minerals are hydrous aluminium phyllosilicates, sometimes with

variable amounts of iron, magnesium, alkali metals, alkaline earth and other cations.

The phyllosilicates that contain large percentages of water trapped between the

silicate sheets. The silica form tetrahedral sheets, and the alumina forms octahedral

sheets. Some of clay particles have the ability to absorb moisture and will expand

while others do not. The difference is due to clay chemistry and the elements (cations)

that are components of the layers. There among the most important minerals are used

by manufacturing and environmental industries. The characteristics common to all

clay minerals derive from their chemical composition, layered structure, and size.

Clay minerals all have a great affinity for water. Some swell easily and may double in

thickness when wet. Most have the ability to soak up ions (electrically charged atoms

and molecules) from a solution and release the ions later when conditions change [28-

29]. The summary of clay mineral properties are given in Table 1.2

Another important property of clay minerals, the ability to exchange ions,

relates to the charged surface of clay minerals. Ions can be attracted to the surface of a

clay particle or taken up within the structure of these minerals. The property of clay

minerals that causes ions in solution to be fixed on clay surfaces or within internal

sites applies to all types of ions, including organic molecules like pesticides. Clays

can be an important vehicle for transporting and widely dispersing contaminants from

one area to another [30-34]. All clay minerals show different expansions, whereas

kaolinite, hydrous mica, and chlorite are non-expanding minerals and the others are

expanding minerals. In kaolinite the bonding is strong, montmorillonite and

12

vermiculite show very weak to weak bonding due to various cations between the

sheets, therefore they show a great expansion, especially in wet conditions. In chlorite,

the bonding is moderate to strong because of the positively charged octahedral layer

[35].

Table 1.2 Summary of clay mineral properties [34]

Secondary

mineral

Interlayer condition

/ Bonding

CEC

[cmol/kg]

Swelling

potential

Specific

surface

area

[m2/g]

Basal

spacing

[nm]

Kaolinite

lack of interlayer

surface, strong

bonding

3 - 15 almost

none 5 - 20 0.72

Montmorillonite very weak bonding,

great expansion 80 - 150 high 700 - 800 0.98 - 1.8 +

Vermiculite weak bonding, great

expansion 100 -150 high 500 - 700 1.0 - 1.5 +

Hydrous Mica partial loss of K,

strong bonding 10 - 40 low 50 - 200 1.0

Chlorite

moderate to strong

bonding, non-

expanding

10 - 40 none - 1.4

1.2.2 Adsorption study

Adsorption is a process that occurs when a liquid or gas (called adsorbate)

accumulates on the surface of a solid or liquid (adsorbent), forming a molecular or

atomic film (adsorbate). It is different from absorption, where a substance diffuses

into a liquid or solid to form a "solution". The term sorption encompasses both

13

processes, while desorption is the reverse process. Adsorption is operative in most

natural physical, biological and chemical systems, and is widely used in industrial

applications such as activated charcoal, synthetic resins and water purification. The

most common industrial adsorbents are activated carbon, silica gel and alumina,

because they present enormous surface areas per unit weight. Temperature effects on

adsorption are profound, and measurements are usually at a constant temperature.

Graphs of the data are called isotherms. Most steps using adsorbents have little

variation in temperature. Adsorption, ion exchange and chromatography are sorption

processes in which certain adsorptive are selectively transferred from the fluid phase

to the surface of insoluble, rigid particles suspended in a vessel or packed in a column.

Similar to surface tension, adsorption is a consequence of surface energy. In a bulk

material, all the bonding requirements (be they ionic, covalent or metallic) of the

constituent atoms of the material are filled. Nevertheless, atoms on the surface

experience a bond deficiency, because they are not wholly surrounded by other atoms.

Thus, it is energetically favorable for them to bond with whatever happens to be

available. The exact nature of the bonding depends on the details of the species

involved, but the adsorbed material is generally classified as exhibiting physisorption

or chemisorption [36-37].

There is also a greater attraction of surface atoms toward neighboring atom in

the liquid or solid. This results in stronger bonds between surface atom and closer

distances as compared with atoms underneath the surface. This tendency for the atoms

to compress gives rise to surface tension. The unfilled forces at the surface can be

satisfied by adsorption of atom or molecule of another species. This reduces the

attractions of the surface atoms or molecule of soil or liquid toward its neighbors of

14

the same kind and reduces the surface tension. Thus, adsorption is always

accompanied by a decrease in surface tension.

Table 1.3 The properties of physical and chemical adsorptions [38]

Physical adsorption Chemical adsorption

Low heat of adsorption

(< 2 or 3 times latent heat of evaporation)

High heat of adsorption

(> 2 or 3 times latent heat of evaporation)

Non specific Highly specific

Monolayer or multilayer

No dissociation of adsorbed

Only significant at relatively low

temperature

Monolayer only

May involve dissociation

Possible over a wide range of temperature

Rapid, non-activated, reversible

No electron transfer although polarization

of sorbate may occur

Activated, may be slow and irreversible

Electron transfer leading to bond

formation between sorbate and surface

1.2.2.1 Physical adsorption and chemisorption

All adsorption processes, whether physical or chemical in character, are

accompanied by decrease in a free surface-energy. Physical adsorption frequently

referred to as van der Waals adsorption, occurs where there are relatively weak

adhensional forces between adsorbate and adsorbent. The heat evolved when a gas is

physically adsorbed is usually similar to the heat of liquefaction of the gas. On the

other hand, chemical adsorption arises from the actual formation of a chemical bond

with the surface. The heat evolved is the same of the order as those liberated in

15

chemical reaction, from about 10 to 100 kcal per mole. Physical adsorption occurs

rapidly and is reversible. Chemisorption is irreversible or reversible with great

difficulty. Physical adsorption also differs from chemical adsorption in that the former

requires little of any activation energy. With chemisorption, the activation energy can

be very considerable [38]. The difference between physical and chemical adsorption

is illustrated in Table 1.3.

1.2.2.2 Classification of adsorption isotherm

Isotherms provide a significant amount of information about the adsorbent

used and the interaction with the adsorbate in the system. Adsorption takes place

because of the presence of an intrinsic surface energy. When a material is exposed to

a gas, an attractive force acts between the exposed surface of the solid and the gas

molecules. The result of these forces is characterized as physical (or Van der Waals)

adsorption, in contrast to the stronger chemical attractions associated with

chemisorption. The surface area of a solid includes both the external surface and the

internal surface of the pores. Due to the weak bonds involved between gas molecules

and the surface, adsorption is a reversible phenomenon. Gas physisorption is

considered non-selective, thus filling the surface step by step (or layer by layer)

depending on the available solid surface and the relative pressure. Filling the first

layer enables the measurement of the surface area of the material, because the amount

of gas adsorbed when the mono-layer is saturated is proportional to the entire surface

area of the sample. The complete adsorption/desorption analysis is called an

adsorption isotherm. The six IUPAC standard adsorption isotherms are shown below,

they differ because the systems demonstrate different gas/solid interactions.

16

Figure 1.2 Diagrammatic representation of isotherm classification [37]

The Figure 1.2 shows the possible shapes and information which may be

drawn from them is outlined below:

Type I Isotherm - these are typical of adsorbents with a predominantly

microporous structure, as the majority of micropore filling will occur at relative

pressures below 0.1. The adsorption process is usually complete at a partial pressure

of ~0.5. Examples include the adsorption of nitrogen on carbon at 77 K and ammonia

on charcoal.

Type II Isotherm - physical adsorption of gases by non-porous solids is

typified by this class of isotherm. Monolayer coverage is followed by multilayering at

high relative pressures. Carbons with mixed micro- and meso-porosity produce Type

II isotherms.

Type III Isotherm - the plot obtained is convex to the relative pressure axis.

This class of isotherm is characteristic of weak adsorbate-adsorbent interactions and is

most commonly associated with both non-porous and microporous adsorbents. The

weak interactions between the adsorbate and the adsorbent lead to low uptakes at low

relative pressures. However, once a molecule has become adsorbed at a primary

17

adsorption site, the adsorbate-adsorbate interaction, which is much stronger, becomes

the driving force of the adsorption process, resulting in accelerated uptakes at higher

relative pressure. This co-operative type of adsorption at high partial pressures is

known as cluster theory and examples include the adsorption of water molecules on

carbon where the primary adsorption sites are oxygen based.

Type IV Isotherm - A hysteresis loop, which is commonly associated with the

presence of mesoporosity, is a common feature of Type IV isotherms, the shape of

which is unique to each adsorption system. Capillary condensation gives rise to a

hysteresis loop and these isotherms also exhibit a limited uptake at high relative

pressures.

Type V Isotherm - these isotherms are convex to the relative pressure axis and

are characteristic of weak adsorbate-adsorbent interactions. These isotherms are

indicative of microporous or mesoporous solids. The reasons behind the shape of this

class of isotherm are the same as those for Type III and again water adsorption on

carbon may exhibit a Type V isotherm.

Type VI Isotherm - introduced primarily as a hypothetical isotherm, the shape

is due to the complete formation of monomolecular layers before progression to a

subsequent layer. The isotherms arise from adsorption on extremely homogeneous,

non-porous surfaces where the monolayer capacity corresponds to the step height.

One example known to exist is the adsorption of krypton on carbon black (graphitised

at 3000 K) at 90 K.

18

1.2.2.3 Isotherm equations

This category includes models where adsorption is described by a simple

mathematical relationship (the adsorption isotherm) between the concentration and

activity in the liquid and solid phase, at equilibrium and at constant temperature.

These adsorption isotherm are based only on empirical ground (Freundlich

isotherm), or derived from isotherm that were originally developed for different

system (Langmuir isotherm derived for gaseous adsorption on planar surfaces).

Empirical models have been widely applied since they are simple, give a good

description of experimental behavior in a large range of operating conditions, and are

characterized by a limited number of adjustable parameters, moreover, the application

of these models have been further extended to include effects such as competition

among different adsorbates for adsorption sites and heterogeneity of the sorption sites

at the solid surface, thus making their use more general.

(i) Langmuir adsorption isotherm

Langmuir developed the adsorption equation in 1916. Langmuir equation

was one of the first and most important equations based on theory. Langmuir

postulated that adsorption occurred as a monolayer film on over the surface of

adsorbent, and derived adsorption isotherm results from investigation of the

equilibrium that is set up between the gas phase and the partially formed monolayer

[39-40].

Many simplified assumptions were made in the derivation of this adsorption

isotherm. Thus, it was assumed that the heat of adsorption is independent of fraction

of the surface that is covered and that only elastic collision-monolayer-occurs on the

19

covered surface. While Langmuir’s equation fits experiment data in only a limited

number of cases, it is important in the further development of the theory. The basic

assumptions on which the model is based are:

1) Molecules are adsorbed at a fixed number of well-defined localized sites.

2) Each site can hold one adsorbate molecule.

3) All sites are energetically equivalent.

4) There is no interaction between molecules adsorbed on neighboring sites.

The linearized Langmuir isotherm expressed by the equation;

(1.1)

Where Ce is the concentration of unadsorbed aflatoxin at equilibrium (mol/L)

q is the amount of aflatoxin sorbed per unit of weight of adsorbent

(mol/L)

Q0 is the maximum capacity of the adsorbent

b is the affinity of the adsorbent

(ii) Fruendlich adsorption isotherm

Freundlich is the adsorption of substances onto animal bone. The

demonstrated that the ratio of the amount of solute adsorbed onto a given mass of

adsorbent to the concentration of the solute in the solution was not a constant at

different solution concentrations. The Freundlich expression is an empirical equation

based on the sorption on heterogeneous surface. The suggested that if the

concentration of solute in the solution at equilibrium, Ce, was raised to the power nf,

⎟⎟⎠

⎞⎜⎜⎝

⎛+⎟⎟⎠

⎞⎜⎜⎝

⎛=

bQQC

qC ee

00

1

20

where q is the amount of solute adsorbed, and then qCfn

e / was a constant at a given

temperature. Hence the empirical equation can be written as;

fn

ef CKq = (1.2)

Where Kf is the Frundlich’s constants related to adsorption capacity

nf is the affinity constant

A linear form of the Freundlich expression is;

ef

f CnKq logloglog += (1.3)

If the adsorption data followed the linearized Freundlich isotherm then

plotting log q versus log Ce would give a straight line from which Kf and nf values

could be calculated from the intercept and the slope, respectively. The parameters are

the indication of the adsorption capacity and affinity constant, respectively [41-45].

1.2.2.4 Determination of AFB1

Several methods for aflatoxin determination in various samples have been

developed and reported in the literature. Methods based on thin-layer chromatography

(TLC) and high performance liquid chromatography (HPLC), with UV-absorption,

fluorescence, mass spectrometry or amperometric detection, have been reported [46-

48]. Techniques to develop sensitive, reliable confirmatory procedures, based on

LC/ESI-MS/MS, for simultaneously analyzing AFB1, AFB2, AFG1 and AFG2.

Particular attention was paid to optimize both extraction and clean-up steps. Different

extraction techniques such as homogenization, matrix solid phase dispersion (MSPD),

and ultrasonic extraction have been tested and compared. SPE, tested for clean-up, are

less specific than immunosorbents, and this allows the developed methods to be used

21

for multi-analyte assays [49-53]. Determination of AFB1 and total aflatoxin

(AFB1+AFB2+AFG1+AFG2) by used to column chromatographic sample clean-up,

OPLC separation and fluorescence densitometric evaluation [54]. However, these

methods require well equipped laboratories, trained personnel, harmful solvents and

several hours to complete an assay. Novel methods for the detection of aflatoxins such

as the application of surface plasmon resonance biosensors, flow injection monitoring,

fibre optic sensors, capillary electrokinetics, electrochemical transduction, and

electrochemical immunosensor have been proposed. Electrochemical immunosensor

based on the indirect competitive enzyme linked immunosorbent,assay (ELISA), for

simple and fast measurement of AFB1 [54-57]. Levels of aflatoxins were rapidly

screened by ELISA, quantified by HPLC and confirmed by LC–MS [58-60].

(i) Thin-layer chromatography

TLC, also known as flat bed chromatography or planar chromatography is

one of the most widely used separation techniques in aflatoxin analysis. Since 1990,

it has been considered the AOAC official method and the method of choice to identify

and quantities aflatoxins at levels as low as 1 ng/g. The TLC method is also used to

verify findings by newer, more rapid techniques.

(ii) Liquid chromatography

LC is similar to TLC in many respects, including analyte application,

stationary phase, and mobile phase. Liquid chromatography and TLC complement

each other. For an analyst to use TLC for preliminary work to optimize LC separation

conditions is not unusual. Liquid chromatography methods for the determination of

22

aflatoxins in foods include normal-phase liquid chromatography (NPLC), reversed-

phase liquid chromatography (RPLC) with pre- or before-column derivatization

(BCD), RPLC followed by postcolumn derivatization (PCD), and RPLC with

electrochemical detection.

(iii) Immunochemical methods

TLC and LC methods for determining aflatoxins in food are laborious and

time consuming. Often, these techniques require knowledge and experience of

chromatographic techniques to solve separation and interference problems. Through

advances in biotechnology, highly specific antibody-based tests are now

commercially available that can identify and measure aflatoxins in food in less than

10 minutes. These tests are based on the affinities of the monoclonal or polyclonal

antibodies for aflatoxins. The three types of immunochemical methods are

radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) and

immunoaffinity column assay (ICA).

(iv) UV-VIS spectrophotometer

Ultraviolet-visible spectroscopy or ultraviolet-visible spectrophotometry

involves the spectroscopy of photons and spectrophotometry, the measurement of the

wavelength and intensity of absorption of near-ultraviolet and visible light by a

sample. It uses light in the visible and adjacent near ultraviolet and near infrared

ranges. In this region of energy space molecules undergo electronic transitions.

Ultraviolet and visible light are energetic enough to promote outer electrons to higher

energy levels. UV-VIS spectroscopy is usually applied to molecules and inorganic

23

ions or complexes in solution. The UV-VIS spectra have broad features that are of

limited use for sample identification but are very useful for quantitative measurements.

The concentration of an analyte in solution can be determined by measuring the

absorbance at some wavelength and applying the Beer-Lambert Law. The Beer-

Lambert law states that the absorbance of a solution is directly proportional to the

solution's concentration. Thus UV-VIS spectroscopy can be used to determine the

concentration of a solution. It is necessary to know how quickly the absorbance

changes with concentration. The method is most often used in a quantitative way to

determine concentrations of an absorbing species in solution, using the Beer-Lambert

law, is the linear relationship between absorbance and concentration of an absorbing

species. The Beer-Lambert law is written as;

(1.4)

Where ε is the wavelength-dependent molar absorptivity coefficient with

units of M-1 cm-1

A is the measured absorbance

b is the path length

c is the analyte concentration

Modern absorption instruments can usually display the data as transmit-

tance, %-transmittance, or absorbance. An unknown concentration of an analyte can

be determined by measuring the amount of light that a sample absorbs and applying

Beer's law. If the absorptivity coefficient is not known, the unknown concentration

can be determined using a working curve of absorbance versus concentration derived

from standards [61-63].

cbA ××= ε

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(v) High performance liquid chromatography (HPLC)

In the analytical procedures of aflatoxin analysis by HPLC, there are three

steps: extraction, purification or clean-up and quantitative determination. The most

common solvent system used for extraction is mixtures of chloroform and water,

methanol–water or acetonitrile–water. Whatever extraction method is used, the

resulted extract still contains, besides aflatoxins, various impurities (lipids, pigments)

requiring further clean-up steps. The most commonly used extraction technique is the

SPE, which replaced the traditional use of column chromatography and liquid–liquid

partition for clean-up. The most popular stationary phases of the SPE columns used

are the following: silica gel, C18 bondedphase and magnesium silicate commercialized

as Florisil. Multi-functional clean-up and antibody affinity SPE columns are also

widely used. Considering the complexity of the matrices, the use of silica gel and C18

bonded-phase are necessary for removing the above-mentioned compounds from

extracts, which interfere in the determination of target analytes. In this case, aflatoxins

were also partitioned into chloroform. The resulted solution was cleaned by silica gel

SPE, and the determination was carried out by HPLC. The increasing complexity of

samples frequently makes direct analysis difficult. A new opportunity of sample

clean-up is provided by applying overpressured layer chromatography (OPLC) as a

planar version of a special SPE system where the purification step is managed on

layer shaped sorbent bed. The layers make possible the application of uncleaned

samples as well. In spite of that, in many cases, samples must be cleaned before

analyzing. However, it should be mentioned that the plates are not reusable. In our

laboratory, OPLC methods have been developed for the determination of aflatoxins in

different food and feed matrices.

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1.3 Aims of research

1. To study adsorption behavior of AFB1 on different natural adsorbents at

different temperatures and pH, and solution composition

2. To evaluate the affinity and capacity constants of the adsorbent materials

for adsorption of AFB1 using various isotherms