CHAPTER I
INTRODUCTION
1. Introduction
1.1. Antioxidant
Billions of dollars are spent each year on antioxidant supplements (nearly
$2 billion, in fact, just on beta carotene and vitamins C and E), believing they will
dramatically lower the risk of cancer, heart disease, and memory loss. Most experts, however,
are convinced that taking antioxidants is not a shortcut to good health or the answer to staying
young [1].
Oxidation is a chemical reaction that transfers electrons or hydrogen from a substance
to an oxidizing agent. Oxidation reactions can produce free radicals. In turn, these radicals
can start chain reactions. When the chain reaction occurs in a system, it can cause damage to
the system. Antioxidants terminate these chain reactions by removing free radical
intermediates, and inhibit other oxidation reactions. They do this by being oxidized
themselves, so antioxidants are often reducing agents such as thiols, ascorbic acid, or
polyphenols. Free radicals are believed to play a role in preventing the development of such
chronic diseases as cancer, heart disease, stroke, Alzheimer's disease, rheumatoid arthritis,
and cataracts. Generation of free radicals not only affects the biological system, it also affects
the lipids [2].
Electron transfer is one of the fundamental processes in chemistry. The passage of an
electron or a pair of electrons from a donor (reducing species) to an acceptor (oxidizing
species) results in a change in properties of both partners in the reaction [3].
A typical schematic of oxidation reaction is given in Figure 1.1. Oxidation was once defined
as the incorporation of oxygen into a substance, but now can be more precisely defined as the
conversion
of
a
chemical
substance
into
another
having
fewer
electrons.
The propensity of chemical compounds to undergo reduction or oxidation has been studied for
nearly 300 years, probably beginning with the Becher-Stahl theory of combustion, popularly
known as the "phlogiston theory," formulated in the early 18th century [2, 4].
1
Figure.. 1.1 : Schematic diagram of redox reaction
Oxidative stress occurs when the production of free radicals is beyond the protective
capability of the antioxidant defenses. Free radicals are chemically active atoms or molecular
fragments that have a charge due to an excess or deficient number of electrons. Examples of
free radicals are the superoxide anion, hydroxyl radical, transition metals such as iron and
copper, nitric acid, and ozone [2, 5].
Damage by free radicals is impossible to avoid.. Free radicals arise from sources both
inside (endogenous) and outside (exogenous) our bodies. Oxidants that develop from
processes within our bodies form as a result of normal aerobic respiration,
respiration metabolism, and
inflammation. Exogenous free radicals form from environmental factors such as pollution,
sunlight, strenuous exercise, X-rays,
X
smoking and alcohol (Figure 1.2) [6].
Figure
Figure.1.2
: Reasons for free radical formation
2
1.2. The Antioxidant’s Function
Antioxidants block the process of oxidation by neutralizing free radicals. In doing so,
the antioxidants themselves become oxidized. That is why there is a constant need for
antioxidant replenishment.
How they work can be classified in one of the following two ways:
•
Chain-breaking - When a free radical releases or accepts an electron, a second
radical is formed. This molecule then turns around and does the same thing to a third
molecule, further continuing the generation of unstable products.
•
Preventive - Antioxidant enzymes like superoxide dismutase, catalase and
glutathione peroxidase prevent oxidation by reducing the rate of chain initiation.
That is, by scavenging initiating radicals, such antioxidants can thwart an oxidation
chain from ever setting in motion [2, 4].
Some phytochemicals that are currently under study for their antioxidant activities are
listed in table 1.1.
Phytochemical
Source
Allyl sulfides
Onions, garlic, leeks, chives
Carotenoids (e.g., lycopene, lutein,
zeaxanthin)
Tomatoes, carrots, watermelon, kale, spinach
Curcumin
Turmeric
Flavonoids (e.g., anthocyanins, resveratrol,
quercitin, catechins)
Grapes, blueberries, strawberries, cherries,
apples, grapefruit, cranberries, raspberries,
blackberries
Glutathione
Green leafy vegetables
Indoles
Broccoli, cauliflower, cabbage, Brussels
sprouts, bok choy
Isoflavones (e.g., genistein, daidzeins)
Legumes (peas, soybeans)
Isothiocyanates (e.g., sulforaphane)
Broccoli, cauliflower, cabbage, Brussels
sprouts, bok choy
Phytochemical
Lignans
Source
Seeds (flax seeds, sunflower seeds)
3
Monoterpenes
Citrus fruit peels, cherries, nuts
Phenols, polyphenols, phenolic
compounds (e.g., ellagic acid, ferulic acid,
tannins)
Grapes, blueberries, strawberries, cherries,
grapefruit, cranberries, raspberries,
blackberries, tea
Saponins
Beans, legumes
Table 1.1: Sources of antioxidants from plants
1.3. CLASSIFICATION OF ANTIOXIDANTS
Antioxidants may be broadly grouped according to their mechanism of action:
primary or chain breaking antioxidants, and secondary or preventive antioxidants. According
to this classification, some antioxidants exhibit more than one mechanism of activity,
therefore, referred to as multiple-function antioxidants. Another commonly used
classification categorizes antioxidants into primary, oxygen scavenging, and secondary,
enzymatic and chelating/sequestering antioxidants. However, synergistic antioxidants are not
included in this classification.
During the past two decades, several naturally occurring compounds have been added
into the list of antioxidants that are effective against oxidation of unsaturated fats and oils and
most of them fall into the multifunctional category. Classification of antioxidants according to the
mode of activity as primary and secondary is preferred in this discussion.
1.3.1. Primary Antioxidants
Primary antioxidants are also referred to as type 1 or chain-breaking antioxidants.
Because of the chemical nature of these molecules, they can act as free radical
acceptors/scavengers and delay or inhibit the initiation step or interrupt the propagation step
of autoxidation. Figure 1.3 illustrates possible events that primary antioxidants may interfere
with along the lipid autoxidation pathway. Primary antioxidants cannot inhibit
photosensitized oxidation or scavenge singlet oxygen.
ROO* + AH
ROOH +A*
R* + AH
RH +A*
ROO* + A*
RO**OA
RO* + AH
ROH + A*
4
RO* + A*
RO*A
A* + A*
AA
Figure. 1.3: Mechanism of primary antioxidant activity
The first kinetic study of antioxidant activity was conducted by Boland and tenHave
[7]. The primary antioxidants (AH) react with lipid and peroxy radicals (ROO*) and convert
them to more stable, nonradical products as shown in Figure. 1.3. These antioxidants are
capable of donating a hydrogen atom to lipid radicals and produce lipid derivatives and
antioxidant radicals (A*) that are more stable and less readily available to participate in
propagation reactions. Primary antioxidants have higher affinities for peroxy radicals than
lipids and react predominantly with peroxy radicals. Many reasons have been listed for their
high affinity. Propagation is a slow step in lipid oxidation process; thus, peroxy radicals are
found in comparatively larger quantities than other radicals. In addition, peroxy radicals have
lower energies than alkoxy radicals; therefore, they react more readily with the low energy
hydrogen of primary antioxidants than with unsaturated fatty acids. As the free radical
scavengers are found in low concentration, they do not compete effectively with initiating
radicals (e.g., hydroxyl radicals) [8, 9]. Therefore, primary antioxidants inhibit lipid oxidation
more effectively by competing with other compounds for peroxy radicals, and are able to
scavenge peroxy- and alkoxy-free radicals formed during propagation and other reactions in
autoxidation. The antioxidant radical produced because of donation of a hydrogen atom
exhibits very low reactivity toward the unsaturated lipids or oxygen; therefore, the rate of
propagation is very slow. The antioxidant radicals are relatively stable and do not initiate a
chain or free radical propagating autoxidation reaction unless present in very large quantities.
These free radical interceptors react with peroxy radicals (ROO*) to stop chain propagation
thereby inhibiting the formation of peroxides. Also, the reaction with alkoxy radicals (RO*)
decreases the decomposition of hydroperoxides to harmful degradation products. Most of the
primary antioxidants that act as chain breakers or free radical interceptors are
mono- or polyhydroxy phenols with various ring substitutions. The antioxidant effectiveness
is influenced by the chemical properties of the compound including hydrogen bond energies,
resonance
delocalization,
and
susceptibility
to
autoxidation.
The ability of the primary antioxidant molecule to donate a hydrogen atom to the free radical
is the initial requirement. The ability of the free radical interceptor (scavenger) to donate a
hydrogen atom to a free radical can be predicted from standard one-electron potentials. Each
5
oxidizing species is capable of accepting an electron (or H atom) from any reduced species.
species
That means when the standard one-electron
one electron reduction potential is concerned, the free radical
scavengers that have reduction potential below peroxy radicals
radicals are capable of donating an H
atom to peroxy radical and form a peroxide. The resulting antioxidant radical should be of
low energy, ensuring the lesser possibility of catalyzing the oxidation of other molecules. The
formed antioxidant radical is stabilized
stabilized by delocalization of the unpaired electron around the
phenol ring to form a stable resonance hybrid (Figure
(
1.4)) and as a result attains
attain low-energy
levels [10].
Figure. 1.4: Stable resonance structure of phenoxy radical of phenolic antioxidant
Antioxidant
tioxidant radicals are capable of participating in termination reactions with peroxy,
alkoxy,, or antioxidant radicals removing reactive free radicals from the system. Dimers of
antioxidant molecules are usually found in fats and oils containing phenolic antioxidants.
an
This is a good indication that antioxidant radicals readily undergo termination reactions and
form dimers as proposed in Figure 1.3.. When considering all of these, the primary
antioxidants or free radical scavengers can inactivate at least two free radicals, the first one
during the interaction with peroxy radical and the second in the termination reaction with
another peroxy radical.
The compounds that exhibit primary antioxidant activity include polyhydroxy
phenolics as well as the hindered phenolics.
phenolics. There are several synthetic ring substituted
phenolics as well as naturally occurring phenolic compounds that may perform via the
primary antioxidant mechanism.
mechanism A common feature of all of these antioxidants is that they
are mono- or polyhydroxy phenols
enols with various ring substitutes (Figure
(
1.5
1.5).
6
Figure. 1.5 : Chemical structure of synthetic phenolic antioxidants commonly used in fats and oils.
Substitution with electron-donating
electron donating group/s ortho and/or para to the hydroxyl group of
phenol increases the antioxidant activity of the compound by an inductive effect (e.g., 2,6-di2,6
tert-butyl-4-methylphenol
methylphenol or BHA). Thus, the presence of a second hydroxyl group in the 22
(ortho) or the 4-position
position (para) of a phenol increases the antioxidant activity (e.g., TBHQ). In
the dihydroxybenzene derivatives, the semiquinoid radical produced initially can be further
oxidized to a quinone by reacting with another lipid radical as shown in Figure 1.6. This
semiquinoid radical may disproportionate into a quinone and a hydroquinone
hydroquinone molecule, and
the process of this conversion contributes to antioxidant activity as peroxy
peroxy radical scavenging
potential [11, 12]. Table 1.22 summarizes most commonly used primary antioxidants in fats
and oils and lipid containing foods. Substitution
Substitution with butyl or ethyl group/s para to the
hydroxy groups also enhances the antioxidant activity. Substitution of branched alkyl groups
at ortho positions enhance the ability of the molecule to form a stable resonance structure that
reduces the antioxidant
nt radical’s participation in propagation reactions [13,, 14].
7
Figure. 1.6 : Mechanism of antioxidant activity in dihydroxy benzene derivative
To be most effective, primary antioxidants should be added during the induction or
initiation stage of the autoxidation reaction cascade. Antioxidants can then scavenge the
formed free radicals, as the cyclical propagation steps have not occurred at this stage.
Addition of primary antioxidants to a lipid that already contains substantial amounts of lipid
peroxides may result in loss of antioxidant activity [15]. Table 1.2 provides examples of some
of these compounds that exhibit secondary antioxidant activity.
Natural
Synthetic
Carotenoids
Butylated hydroxyanisole (BHA)
Flavonoids
Butylated hydroxytoluene (BHT)
Phenolic acids
Ethoxyquin
Tocopherols and
Tocotrienols
Propyl gallate (PG),
Tertiary butylhydroquinone (TBHQ)
Table 1.2 : Commonly used primary antioxidants.
1.3.2. Secondary Antioxidants
Secondary antioxidants are also classified as preventive or class II antioxidants. They
offer their antioxidant activity through various mechanisms to slow the rate of oxidation
reactions. Unlike primary antioxidants, secondary antioxidants do not convert free radicals
into stable molecules. They act as chelators for pro-oxidant or catalyst metal ions, provide H+
to primary antioxidants, decompose hydroperoxide to non-radical species, deactivate singlet
8
oxygen, absorb ultraviolet radiation, or act as oxygen scavengers. They often enhance the
antioxidant activity of primary antioxidants. Table 1.3 provides examples of some of these
compounds that exhibit secondary antioxidant activity.
Activity
Compounds in use
Metal chelation
Citric, Malic, Succinic and Tartaric acids,
Ethylenediaminetetraacetic acid, phosphates.
Oxygen scavenging and reducing
agents
Ascorbic acid, Ascorbyl palmitate, Erythorbic acid,
Sodium erythorbate, Sulfites
Singlet oxygen quenching
Carotenoids (Lycopene and Lutein)
Table 1.3: Compounds that exhibit secondary antioxidant activity
1.3.2.1. Sequestering/Chelating Agents
Sequestering/chelating agents or metal deactivators are heavy metals with two or
more valence states with a suitable oxidation-reduction potential between them (e.g., Co, Cu,
Fe, Mn, etc.), shorten the induction period and increase the maximum rate of oxidation of
lipids. Trace amounts of these metal ions are present in lipid-containing foods coming from
naturally present compounds or included during processing operations. The effectiveness of
copper as a catalyst for hydroperoxide decomposition has been reported [15, 16]. Transition
metals such as iron exhibit low solubility at pH values near neutrality [17]. That means in
foods, transition metals may exist chelated to other compounds. Many compounds form
complexes with these metals and change their catalytic activity. Chelation can increase the
pro-oxidant activity of transition metals by making them more non-polar, and some can
increase oxidative reactions by increasing metal solubility or altering redox potential [18].
According to Graf and Eaton, chelators may exert antioxidant activity by prevention of metal
redox cycling, occupation of all metal coordination sites, and formation of insoluble metal
complexes and steric hindrance of interactions between metals and lipids or oxidation
intermediates (e.g., peroxides) [19]. Chelation of these metal ions or use of metal deactivators
reduces the pro-oxidant activity by raising the energy of activation for initiation reactions. The
most effective form of chelating agents are secondary antioxidants, which form s-bonds with
metal ions because they reduce the redox potential and stabilize the oxidized form of the metal
ion. Chelating agents such as heterocyclic bases that form p-complexes raise the redox potential
and may accelerate metal-catalyzed hydroperoxide decomposition and act as pro-oxidants.
9
Multiple carboxylic acid compounds such as citric acid, ethylene diamine tetraacetic
acid (EDTA), and phosphoric acid derivatives (polyphosphates and phytic acid) are
commonly used in extending the shelf life of lipid-containing foods because of their metal
chelating properties. Typically these chelators are water soluble, but citric acid exhibits
solubility in lipids, which allows it to inactivate metals in the lipid phase [33]. Chelator’s
activity
depends
on
pH
and
the
presence
of
other
chelatable
ions
(e.g., Ca). Most food grade chelators are unaffected by food-processing operations and
storage; however, polyphosphates may decrease their antioxidant activity because of possible
hydrolysis by endogeneous phosphatases in foods, especially in raw meat [20].
1.3.2.2. Oxygen Scavengers and Reducing Agents
As oxygen is essential and is one of the reactants in the autoxidation process,
scavenging of oxygen molecular species is one way of providing antioxidant activity.
Ascorbic acid acts as a reducing agent and as an oxygen scavenger. Singlet oxygen is the
excited state oxygen, and its inactivation is an effective way of preventing initiation of lipid
oxidation. Carotenoids are capable of inactivating photoactivated sensitizers by physically
absorbing their energy to form the excited state of the carotenoid. Later, the excited state
carotenoid returns to ground state by transferring energy to the surrounding solvent. Other
compounds found in food, including amino acids, peptides, proteins, phenolics, urates, and
ascorbates also can quench singlet oxygen [20]. Compounds such as superoxide anion and
peroxides do not directly interact with lipids to initiate oxidation; they interact with metals or
oxygen to form reactive species. Superoxide anion is produced by the addition of an electron
to the molecular oxygen. It participates in oxidative reactions because it can maintain
transition metals in their active reduced state, can promote the release of metals that are
bound to proteins, and can form the conjugated acid, perhydroxyl radical depending on pH,
which
is
a
catalyst
of
lipid
oxidation.
The enzyme superoxide dismutase that is found in tissues catalyzes the conversion of
superoxide anion to hydrogen peroxide. Catalase is capable of catalyzing the conversion of
hydrogen peroxides to water and oxygen [21]. Glucose oxidase coupled with catalase is well
used commercially to remove oxygen from foods, especially fruit juices, mayonnaise, and
salad dressings [19]. Glutathione peroxidase that is found in many biological tissues also
helps to control both lipid and hydrogen peroxides [22]. These enzymatic reactions help to
10
reduce
various
types
of
radicals
that
could
be
formed
in
lipid-containing biological systems.
1.4. Synthetic Antioxidants
Synthetic antioxidants are manmade and are used to stabilize fats, oils, and lipid
containing foods and are mostly phenolic-based. Many compounds are active as antioxidants,
but only a few are incorporated into food because of strict safety regulations. These phenolic
derivatives usually contain more than one hydroxyl or methoxy group. Ethoxyquin is the only
heterocyclic, N-containing compound that is allowed for use in animal feeds. Synthetic
phenolic antioxidants are p-substituted, whereas the natural phenolic compounds are mostly
o-substituted. The p-substituted phenolic antioxidants are preferred because of their lower
toxicity. The m-substituted compounds are inactive. Synthetic phenolic antioxidants are
always substituted with alkyl groups to improve their solubility in fats and oils and to reduce
their toxicity [23, 24]. The primary mechanism of activity of these antioxidants is similar to
those of primary antioxidants. An antioxidant molecule reacts with a peroxy radical produced
by the oxidizing lipid, thus forming a hydroperoxide molecule and an antioxidant free radical.
A similar path of reaction may occur with the alkoxy free radicals formed during the
decomposition of hydroperoxides. The antioxidant free radical so formed may be deactivated
by a lipid peroxy or an alkoxy radical or with another antioxidant radical. Dimers and even
trimers of antioxidant molecules are formed because of the reaction of antioxidant radicals,
and these may have a modest antioxidant activity of their own. With the help of synergists
such as ascorbic acid, some of these original antioxidant molecules may be regenerated.
Quinones are formed from phenolic antioxidants by reaction with peroxy radicals. When
antioxidants are present in excess, the reaction of antioxidant free radicals with oxygen may
become important; even their reaction with polyunsaturated fatty acids has some impact on
the course of oxidation [24].
1.4.1. Butylated Hydroxyanisole (BHA)
BHA exists as a mixture of two isomers, 3-tertiary-butyl-4-hydroxyanisole (90%) and
2-tertiary-butyl-4-hydroxyanisole (10%). The 3-isomer shows a higher antioxidant activity
than the 2-isomer. BHA is commercially available as white, waxy flakes and is lipid soluble.
BHA exhibits good antioxidant activity in animal fats as compared to vegetable oils. It has
good carry-through properties but is volatile at frying temperatures. When BHA is included
11
into packaging materials it easily migrates to the containing food and delays lipid oxidation
[25, 26].
1.4.2. Butylated Hydroxytoluene (BHT)
BHT is also a monohydroxyphenol and is widely used in foods. This fat-soluble
antioxidant is available as a white crystalline compound. BHA is less stable than BHT at high
temperatures and has lower carry-through properties. BHA and BHT act synergistically, and
several commercial antioxidant formulations contain both of these antioxidants. BHT is
effectively used in oxidation retardation of animal fats. It is postulated that BHA interacts
with peroxy radicals to produce a BHA phenoxy radical. This BHA phenoxy radical may
abstract a hydrogen atom from the hydroxyl group of BHT. BHA is regenerated by the H
radical provided by BHT. The BHT radicals so formed can react with a peroxy radical and
act as a chain terminator [27].
1.4.3. Tertiary-Butylhydroquinone (TBHQ)
TBHQ is a diphenolic antioxidant and is widely used in a variety of fats and oils.
TBHQ has excellent carry-through properties and is a very effective antioxidant for use in
frying oils. It is available as a beige color powder that is used alone or in combination with
BHA or BHT. TBHQ can be used in a variety of lipid-containing foods and fats and oils.
Chelating agents such as monoacyglycerols and citrates enhance the activity of TBHQ,
mainly in vegetable oils and shortenings. TBHQ reacts with peroxy radicals to form a
semiquinone resonance hybrid. The semiquinone radical intermediate may undergo different
reactions to form more stable products; they can react with one another to form dimers,
dismutate, and regenerate as semiquinones; and they can react with another peroxy radical
[28].
2. Biodiesel
Relentless efforts in pursuing human comfort and luxury have led to the deterioration
of our environment paving the way for global warming. Automobiles that consume fossil
fuels lead to production of tones of toxic gases like carbon monoxide, carbon dioxide, oxides
of nitrogen, oxide of sulphur, hydro-carbons etc., that not only cause pollution but also
reasons for critical changes in climate. Depletion of fossil fuels and a steady increase in their
price necessitate the search of alternative renewable sources of fuels that will meet the
increasing energy demand. One possible alternative to fossil fuel is the use of oils of plant
12
origin like vegetable oils and tree borne oil seeds. These alternative diesel fuels can be termed
as biodiesel. Such fuel is biodegradable, free of sulphur and aromatic compounds, non-toxic,
renewable and has low emission profiles as compared to petroleum diesel. The fuel must be
technically feasible, economically competitive, environmentally acceptable and readily
available. Chemically, the oils/fats consist of triglyceride molecules of three long chain fatty
acids that are ester bonded to a single glycerol molecule. These fatty acids differ by the
length of carbon chains, the number, orientation and position of double bonds in these chains.
Thus biodiesel refers to lower alkyl esters of long chain fatty acids, which are synthesized
either by transesterification with lower alcohols or by esterification of fatty acids [26].
2.1. GLOBAL STATUS OF BIODIESEL
With the crude fossil fuel prices near all-time high, biodiesel has emerged as one of
the fastest growing industries worldwide. Several countries, especially United States and
members of European Union are actively supporting the production of biodiesel from the
agriculture sector. It is interesting to note that 75% of the total biodiesel production comes
from European countries. This is mainly due to substantial support from the European
government such as consumption incentives (fuel tax reduction) and production incentive
(tax incentives and loan guarantees) which is expected to catalyze the growth of biodiesel
market in the next ten years. The United States has spent around US$ 5.5 billion to 7.3 billion
a year to accelerate biofuel production during the last few years. As a result, around 450
million gallons of biodiesel was produced in the United States in the year 2007 compared to
only 25 million gallons in 2004 [25]. This 1700% rise was indeed a shocking increase in the
entire history of biodiesel production. However, by the year 2020, it is predicted that
biodiesel production from Brazil, China, India and some South East Asian countries such as
Malaysia and Indonesia could contribute to as much as 20% of the total global production.
The driving forces for development of biodiesel in these countries are economic, energy and
environmental security, improving trade balances and expansion of agriculture sector. In
addition, Brazil, China and India each have set targets to replace 5 to 20% of total diesel with
biodiesel by the year 2010 with emphasis on second generation non-edible feedstock. Figure
1.7 illustrates the world biodiesel production and capacity till 2008 [29].
13
Figure. 1.7: Biodiesel production and capacity till 2008
14
2.2. COMPARISION OF PETRODIESEL Vs BIODIESEL
Diesel exhaust consists of hundreds of gas-phase, semi-volatile, and particle-phase
organic compounds that are produced through the combustion of fossil fuel [30]. Increasing
energy demand, climate change and carbon dioxide (CO2) emission from fossil fuels make it
a high priority to search for low-carbon energy resources. Biodiesels represent a key target
for the future energy market that can play an important role in maintaining energy security. It
is primarily considered as a potentially cheap, low-carbon energy source. Most life-cycle
studies of biodiesel have found that bio-ethanol made from corn or sugarcane generally
reduces greenhouse gases, replacing gasoline (petrol). Biodiesel have been increasingly
explored as a possible alternative source to gasoline with respect mainly to transport. To
summarise, interest in biodiesel is increasing for a number of reasons:
1. Reduced reliance on fossil fuels
2. Reduction in greenhouse gas emission
3. National independent security of fuel supply
4. Employment and economic benefits through the development of a new fuel
production.
2.3. TRANSESTERIFICATION
Vegetable oils cannot be used directly due to high viscosity, polyunsaturated characters
and low volatility [31]. To solve these limitations, vegetable oil or animal fats are reacted
with simple alcohol to produce fatty acid methyl esters (FAME). Transesterification is an
important method of converting vegetable oils to biodiesel. Transesterification (also called
alcoholysis) is the reaction of a fat or oil with an alcohol to form esters and glycerol. A
catalyst is usually used to improve the reaction rate and yield. Excess alcohol is used to shift
the equilibrium toward the product because of reversible nature of reaction. For this purpose
primary and secondary monohybrid aliphatic alcohols having 1-8 carbon atoms are used [32].
15
2.3.1. CHEMISTRY OF TRANSESTERIFICATION
Transesterification also called alcoholysis which is the displacement of alcohol from
an ester by another alcohol in a process similar to hydrolysis except that an alcohol is used
instead of water. This has been widely used to reduce the viscosity of the triglycerides [33].
Figure 1.8 represents a typical transesterification reaction
Figure. 1.8: Basic transesterification reaction
2.3.2. TYPES OF TRANSESTERIFICATION
2.3.2.1. Alkali Catalyzed Transesterification
The reaction mechanism for alkali catalyzed transesterification was formulated in
three steps [34]. The first step is an attack on the carbonyl carbon atom of the triglyceride
molecule by the anion of the alcohol (methoxide ion) to form a tetrahedral intermediate that
reacts with an alcohol to regenerate the anion of alcohol. The base-catalyzed process is
relatively fast but is affected by water content and free fatty acids of oils or fats. Free fatty
acids can react with base catalysts to form soaps and water. Soap not only lowers the yield of
alkyl esters but also increases the difficulty in the separation of biodiesel and glycerol, and
washing with water because of the formation of emulsions [34].
2.3.2.2. Acid Catalyst Transesterification
An alternative process is to use acid catalysts that some researchers have claimed are
more tolerant of free fatty acids [35]. The protonation of carbonyl group of the ester leads to
the formation of carbocation, which after a nucleophilic attack of the alcohol produces a
tetrahedral intermediate. This intermediate eliminates glycerol to form a new ester. Generally,
acid-catalyzed reactions are used to convert FFAs to esters, or soaps to esters as a
pretreatment step for high FFA feedstocks [36]. Acid catalyst process requires excess alcohol,
hence
the
transesterification
reactor
and
alkali
distillation
column
of
acid-catalyzed process are larger than alkali catalyzed process for same biodiesel production
16
capacity. High conversion efficiencies with acid-catalyzed transesterification can be achieved
by increasing the molar ratio of alcohol to oil, reaction temperature, concentration of acid
catalyst and the reaction time [37].
Figure 1.10: Mechanism of alkali catalyst transesterification.
Figure. 1.11: Mechanism of acid catalyst transesterification.
2.3.2.3. Lipase Catalysed Transesterification
This transesterification process was similar to alkali transesterification, except that the
ratio of catalyst and solvent stirring time was varied. Lipase was used as catalyst in this
transesterification [38]. The process is explained as follows:
Figure. 1.12: Steps in production of biodiesel using lipase.
Lipases are known to have a property to act on long-chain fatty alcohols better than
on short-chain ones. Thus, in general, the efficiency of the transesterification of triglycerides
with methanol (methanolysis) is likely to be very low compared to that with ethanol in
systems with or without a solvent. Various studies have been conducted with different
catalyst,
alcohol
and
methanol
molar
17
ratios
at
different
temperatures
Linko et. al. have demonstrated the production of a variety of biodegradable esters and
polyesters with lipase as the biocatalyst [39].
TYPE OF
CATALYST
ADVANTAGES
• Very fast reaction rate - 4000
times faster than acid-catalyzed
transesterification
Homogeneous
base catalyst
• Reaction can occur at mild
reaction condition and is less
energy intensive
• Catalysts such as NaOH and KOH
are relatively cheap and widely
available
• Relatively faster reaction rate
compared to acid-catalyzed
transesterification
• Reaction can occur at mild
reaction conditions and is less
energy intensive
Heterogeneous
base catalyst
• Easy separation of catalyst from
product
• Catalyst reuse and regeneration
highly possible
DISADVANTAGES
• Sensitive to FFA content in the
oil
• Soap formation occurs if the
FFA content in the oil is more
than 2 wt.%
• Too much soap formation
decreases biodiesel yield and
hinders product purification
especially generating huge
amount of wastewater
• Poisoning of the catalyst when
exposed to ambient air
• Sensitive to FFA content in the
oil due to its basicity
• Soap formation occurs if the
FFA content in the oil is more
than 2 wt.%
• Too much soap formation
decreases biodiesel yield and
hinders product purification
• Leaching of catalyst active sites
may result in product
contamination
18
•
•
TYPE OF T
Heterogeneous
acid catalyst
•
•
•
ADVANTAGES
Insensitive to FFA and water
content in the oil
Preferred method if low-grade oil
is used
Esterification and
transesterification occur
simultaneously
Easy separation of catalyst from
product
Catalyst reuse and regeneration
highly possible
•
•
•
•
DISADVANTAGES
Complicated catalyst synthesis
procedures lead to higher cost
Normally, high reaction
temperature, high alcohol to oil
molar ratio and long reaction
time are required
Energy intensive
Leaching of catalyst active sites
may result in product
contamination
Table 1.4 : Advantages and disadvantages of various types of catalysts
2.3.2.4. Supercritical Transesterification
The simple transesterification process is confronted with two problems, i.e. the
process is relatively time consuming and it needs separation of the catalyst and saponified
impurities from the biodiesel. The first problem is due to the phase separation of the vegetable
oil/methanol mixture, which may be dealt with by vigorous stirring. These problems were not faced
in the supercritical methanol method of transesterification. This is perhaps due to the fact that the
tendency of two-phase formation of vegetable oil/methanol mixture was not encountered and a
single phase was formed due to decrease in the dielectric constant of methanol in the supercritical
state. As a result, the reaction was found to be complete in a very short time of about 2–4 min.
Further, since no catalyst was used, the purification on biodiesel was much easier, trouble free and
environment friendly [40].
2.3.3. PROCESS VARIABLES
The most important variables that influence the transesterification reaction are:
1. Reaction temperature
2. Ratio of alcohol to oil
3. Catalyst
4. Mixing intensity
5. Purity of reactants
2.3.3.1. Reaction Temperature
19
Previous reports have revealed that the rate of reaction is strongly influenced by the
reaction temperature. However, the reaction is conducted close to the boiling point of
methanol (60–70°C) at atmospheric pressure for a given time. Such mild reaction conditions
require the removal of free fatty acids from the oil by refining or pre-esterification. Therefore,
degummed and deacidified oil is used as feedstock [41]. Pre-treatment is not required if the
reaction is carried out under high pressure (9000 kPa) and high temperature (240ºC), where
simultaneous esterification and transesterification take place with maximum yield obtained at
temperatures ranging from 60 to 80°C at a molar ratio of 6:1 [42].
2.3.3.2. Ratio of Alcohol to Oil
Another important variable is the molar ratio of alcohol to vegetable oil.
As indicated earlier, the transesterification reaction requires 3 mol of alcohol per mole of
triglyceride to give 3 mol of fatty esters and 1 mol of glycerol. In order to shift the reaction to
the right, it is necessary to either use excess alcohol or remove one of the products from the
reaction mixture. The second option is usually preferred for the reaction to proceed to
completion. The reaction rate was found to be highest when 100% excess methanol was used.
A molar ratio of 6:1 is normally used in industrial processes to obtain methyl ester yields
higher than 98% (w/w) [41].
2.3.3.3. Catalysts
Alkali metal alkoxides are found to be more effective transesterification catalysts
compared to acidic catalysts. Sodium alkoxides are the most efficient catalysts, although
KOH and NaOH can also be used. Transmethylation occurs in the presence of both alkaline
and acidic catalysts [43]. As they are less corrosive to industrial equipment, alkaline catalysts
are preferred in industrial processes. A concentration in the range of 0.5–1% (w/w) has been
found to yield 94–99% conversion to vegetable oil esters [44] and further increase in catalyst
concentration does not affect the conversion but adds to extra cost, as the catalyst needs to be
removed from the reaction mixture after completion of the reaction.
20
2.3.3.4. Mixing Intensity
It has been observed that during the transesterification reaction, the reactants initially
form a two-phase liquid system. The mixing effect has been found to play a significant role in
the slow rate of the reaction. As phase separation ceases, mixing becomes insignificant. The
effect of mixing on the kinetics of the transesterification process forms the basis for process
scale-up and design.
2.3.3.5. Purity of Reactants
Impurities in the oil affect the conversion level considerably. It is reported that about
65–84% conversion into esters using crude vegetable oils has been obtained as compared to 94–
97% yields refined oil under the same reaction conditions [41]. The free fatty acids in the crude
oils have been found to interfere with the catalyst. This problem can be solved if the reaction is
carried out under high temperature and pressure conditions.
2.3.4. METHODS TO ANALYZE BIODIESEL
Various analytical methods have been developed for analyzing mixtures containing
fatty acid esters and mono-, di-, and triglycerides obtained by transesterification. Analyses have
been performed by thin layer chromatography/flame ionization detector (TLC/FID), gas
chromatography (GC), high performance liquid chromatography (HPLC), gel permeation
chromatography
(GPC),
proton
nuclear
magnetic
resonance
1
( H-NMR) and near infra-red spectroscopy (NIR). Most reports on the use of GC for
biodiesel analysis employ flame-ionization detectors (FID), although the use of mass
spectrometric detector (MSD) was employed in some cases to eliminate any ambiguities
about the nature of the eluting materials since mass spectra unique to individual compounds
would be obtained [43].
2.3.5. Disadvantages of using Biodiesel
One of the major drawbacks in maintaining the quality of biodiesel and its widespread
commercialization is its oxidation stability. Unlike petroleum diesel fuel, the nature of
biodiesel makes it more susceptible to oxidation or auto oxidation during long term storage.
Storage conditions like exposure to water and exposure to oxygen, which are naturally
present in the ambient air, influence the rate of oxidation. The biodiesel stability generally
depends upon the fatty acid composition of the parent oil. Unsaturated fatty acids are
significantly more reactive to oxidation than saturated compounds. With respect to long-chain
21
FAMEs, polyunsaturated fatty esters are approximately twice as reactive to oxidation as
monounsaturated esters. This is attributed to the fact that these unsaturated fatty acid chains
contain the most reactive sites, which are particularly susceptible to free-radical attack. While
bis-allylic methylenes are much more susceptible to oxidation, biodiesel stability can also
depend upon the presence of allylic methylenes in the hydrocarbon chain [45].
The bis-allylic configurations, where the central methylene group is activated by the
two double bonds (i.e., -CH=CH-CH2-CH=CH-), react with oxygen via the auto oxidation
mechanism, with the radical chain reaction steps of initiation, propagation, chain branching,
and termination. During these reaction steps, several products can be formed, such as
peroxides and hydro-peroxides, low molecular weight organic acids, aldehydes and keto
compounds, alcohols, as well as high molecular-weight species (dimers, trimers, and cyclic
acids) via polymerization mechanisms. The use of antioxidant additives can help slow the
degradation process and improve fuel stability up to a point [46 - 50]. Fuel properties degrade
during
long-term
storage
as
follows:
(i) oxidation or autoxidation from contact with ambient air; (ii) thermal or thermal-oxidative
decomposition from excess heat; (iii) hydrolysis from contact with water or moisture in tanks
and fuel lines; or (iv) microbial contamination from migration of dust particles or water
droplets containing bacteria or fungi into the fuel [51].
Monitoring the effects of auto oxidation on biodiesel fuel quality during
long-term storage presents a significant concern for biodiesel producers, suppliers, and
consumers [52]. Engine and injection pump producers insist on the parameter for oxidation
stability which was finally fixed at a minimum limit of a 6-hour induction period at 110 °C
[53]. The method adopted for determination of the oxidation stability is the so called
Rancimat method which is commonly used in the vegetable oil sector. Especially high
contents of unsaturated fatty acids, which are very sensitive to oxidative degradation, lead to
very low values for the induction period. Thus, even the conditions of fuel storage directly
affect the quality of the product. Several studies showed that the quality of biodiesel over a
longer period of storage strongly depends on the tank material as well as on contact to air or
light. Increase in viscosities and acid values and decreases in induction periods were observed
during such storage [54, 55]. To improve the storage and oxidative stability of biodiesel, it
has become a necessacity to find new additives. Intensive research is underway in this area to
22
investigate storage stability of biodiesel as highlighted by literature presented in chapters IV
and V.
The objective of this study was to investigate the oxidative stability and storage
stability of Pongamia pinnata and Jatropha curcus oil methyl esters. The Rancimat
procedure for oxidation stability and the ASTM procedure for storage stability have been
used in this study. Using different antioxidants in different concentrations, the fuel properties
such as acid value (AV), kinematic viscosity (KV) and peroxide value (PV) of Pongamia
biodiesel (PBD) and Jatropha biodiesel (JBD) were determined at regular periods of time.
Chapter IV discusses the effect of commercially available antioxidants such as BHT, BHA,
GA, TBHQ and PY (Figure 1.5) on the stability of biodiesel.
3. Natural Antioxidants
Use of plant parts (barks, leaves, seeds, etc.) and their extracts to preserve food from
developing a rancid taste is a practice that has continued since prehistoric times. There is
evidence that even for the industrial materials plant-based components were used as antidrying agents to prevent oxidation and polymerization of polyunsaturated fatty acid-rich plant
oils [55, 56]. The high compatibility of biodiesel with petroleum diesel characterizes it as a
good alternative capable of supplying most of the existing diesel fleet without great
adaptations. It is also biodegradable and renewable, has a lubricant capacity in the pure form
and is competitive with diesel in terms of fuel properties [52]. However, unlike fossil fuels
that are relatively inert and maintain their essential characteristics with little alteration during
storage, biodiesel degrades with time and can be altered due to the action of air, light,
temperature and moisture. Contact with contaminants, both of inorganic and microbial nature,
can also tend to introduce variations in product quality. Biodiesel is prone to undergo
oxidative degradation when it is exposed to atmospheric air [49, 52]. In order to inhibit or
delay oxidation in oils, fats and fatty foods, phenolic chemical compounds known as
synthetic antioxidants and/or stabilizers are used [54]. Antioxidants occur naturally in
vegetable oils and the most common are the tocopherols. However, some plant oil production
processes include a distillation step to purify the triglycerides. The biodiesel obtained from
these oils normally has little or no natural antioxidants so they become less stable and
therefore antioxidants need to be applied to increase the biofuel stability and extend its
properties for a longer period [56].
23
Factors which influence the oxidative stability of biodiesel include fatty acid
composition, natural antioxidant content, the level of total glycerin, and the conditions of fuel
storage such as temperature, exposure to light and air, and tank material [58, 59, 60, 61].
Previous studies have found that antioxidants can be effective in increasing the stability of
biodiesel [58, 60, 62, 63]. However, these effects have not been fully elucidated and results
have been inconclusive or conflicting. Sendzikiene et al. found that butylated hydroxyanisole
(BHA) and butyl-4-hydroxytoluene (BHT) have nearly the same effect on the oxidative
stability of rapeseed oil, and tallow-based biodiesel, and the optimal level of synthetic
antioxidants was determined to be 400 ppm [60]. Mittelbach et al. reported that pyrogallol
(PY), propylgallate (PG), and t-butylhydroquinone (TBHQ) could significantly improve the
stability of biodiesel obtained from rapeseed oil, used frying oil, and beef tallow, whereas
BHT was not very effective [64]. Moreover, Domingos et al. found that BHT had the highest
effectiveness for refined soybean oil-based biodiesel, while BHA displayed little
effectiveness [59]. Natural antioxidants are constituents of many fruits and vegetables and
they have attracted great deal of public and scientific attention because of their
anticarcinogenic potential. Biodiesel is an environment friendly liquid fuel similar to
petrodiesel in combustion properties.
A new alternative to delay the biodiesel oxidative degradation process may be the use
of natural antioxidants present in spices, bearing in mind that they do not damage the
environment and are easily obtained [65]. According to some studies, rosemary (Rosmarinus
officinalis L.) and sage (Salvia officinalis L.) are the spices with the greatest antioxidant
potential [65, 66]. The antioxidant activity of carnosol and carnosic acid, found in rosemary,
was validated in an emulsion containing methyl linoleate [67]. According to Nakatani and
Inatani, the addition of natural antioxidants such as carnosol and carnosic acid, at a 0.01%
concentration, in a linoleic acid emulsion, have activity levels similar to those of the synthetic
antioxidants BHA (butyl hydroxy anisole) and BHT (butyl hydroxy toluene) added in the
same concentration [68]. Five different phenolic compounds were isolated from oregano
(Origanum vulgare L.); all presented antioxidant activity and one of them was identified as
rosmarinic acid [69]. Furthermore, the study carried out by Bragagnolo and Mariuti reported
several other phenolic compounds that were isolated from oregano, including luteolin, pcoumaric acid, carvacrol, thymol, p-cimen, and campherol [70]. These findings demonstrate a
great possibility of using these spices as good antioxidants and possible substitutes for the
synthetic antioxidants, especially in mixtures consisting of unsaturated carbon compounds as
24
substrate. Numerous strong antioxidant compounds have been identified in fruits, vegetables [71,
72] and in different beverages [73]. For example, high concentrations of antioxidants have been
found in berries [74], apples [75], and citrus fruits [76].
3.1. Application of Antioxidants
3.1.1. As food preservatives
Antioxidants are used as food additives to help guard against food deterioration.
Exposure to oxygen and sunlight are the two main factors in the oxidation of food, so food is
preserved by keeping in the dark and sealing it in containers or even coating it with wax, as
with cucumbers. However, as oxygen is also important for plant respiration, storing plant
materials in anaerobic conditions produces unpleasant flavors and unappealing colors [77].
Consequently, packaging of fresh fruits and vegetables contains about 8% oxygen
atmosphere. Antioxidants are an especially important class of preservatives as, unlike bacterial
or fungal spoilage, oxidation reactions still occur relatively rapidly in frozen or refrigerated food.
These preservatives include natural antioxidants such as ascorbic acid (AA, E300) and
tocopherols (E306), as well as synthetic antioxidants such as propyl gallate (PG, E310), tertiary
butylhydroquinone
(TBHQ),
butylated
hydroxyanisole
(BHA,
E320)
and
butylated
hydroxytoluene (BHT, E321) [78].
The most common molecules attacked by oxidation are unsaturated fats; oxidation
causes them to turn rancid. Since oxidized lipids are often discolored and usually have
unpleasant tastes such as metallic or sulfurous flavors, it is important to avoid oxidation in
fat-rich foods. Thus, these foods are rarely preserved by drying; instead, they are preserved
by smoking, salting or fermenting. Even less fatty foods such as fruits are sprayed with
sulfurous antioxidants prior to air drying. Oxidation is often catalyzed by metals, which is
why fats such as butter should never be wrapped in aluminium foil or kept in metal
containers. Some fatty foods such as olive oil are partially protected from oxidation by their
natural content of antioxidants, but remain sensitive to photooxidation [79]. Antioxidant
preservatives are also added to fat-based cosmetics such as lipstick and moisturizers to prevent
rancidity.
3.1.2. Industrial Application
Antioxidants are frequently added to industrial products. A common use is as
stabilizers in fuels and lubricants to prevent oxidation, and in gasoline to prevent the
polymerization that leads to the formation of engine-fouling residues [78, 79].
25
In 2007, the worldwide market for industrial antioxidants had a total volume of
around 0.88 million tons. This created revenue of about 3.7 billion US-dollars. They are
widely used to prevent the oxidative degradation of polymers such as rubbers, plastics and
adhesives that causes a loss of strength and flexibility in these materials [80]. Polymers
containing double bonds in their main chains, such as natural rubber and polybutadiene, are
especially susceptible to oxidation and ozonolysis, which can be protected by anti-ozonants. Solid
polymer products start to crack on exposed surfaces as the material degrades and the chains break.
The mode of cracking varies between oxygen and ozone attack, the former causing a "crazy
paving" effect, while ozone attack produces deeper cracks aligned at right angles to the tensile strain
in the product. Oxidation and UV degradation are also frequently linked, mainly because UV
radiation creates free radicals by bond breakage. The free radicals then react with oxygen to
produce peroxy radicals which cause further damage, often in a chain reaction. Other polymers
susceptible to oxidation include polypropylene and polyethylene. The former is more sensitive
owing to the presence of secondary carbon atoms present in every repeat unit. Attack occurs at this
point because the free radical formed is more stable than one formed on a primary carbon atom.
26
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