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3. THEORETICAL ANALYSIS

3.1. EXTRACTION

Antioxidant compounds are usually present in rather low amounts in natural

materials. Therefore, large additions of antioxidant-containing material would be

required to obtain a significant improvement in stability against oxidation, which may

be accompanied by a negative effect on the flavour or functional properties of the

product. The easiest way to prepare more concentrated materials is to remove water

by a suitable drying procedure. The next most optimal procedure is extraction. The

choice of solvent is of crucial importance. Conventional methods to extract natural

antioxidants from plants are generally based on the employment of organic solvents.

The extraction of bioactive compounds from plant material has shown great potential.

Solvent extraction is more frequently used for isolation of antioxidants and both

extraction yield and AA of extracts are strongly dependent on solvent due to the

different antioxidant potential of compounds with different polarity1. This technique

may be a promising method for the selective and efficient extraction of antioxidant

constituents from plant materials.

Extraction of organic molecules of interest, involve breaking open the cells.

When choosing an extraction method, maintaining the activity of the extracted

compound(s) is the priority. Wet extractions involve solid material in direct contact

with a liquid solvent2. During the extraction, organic solvents diffuse into the solid

material and solubilize compounds with similar polarity. The nature of the solvent

used will determine the types of compounds likely extracted from the plant material.

Organic solvents for extractions include polar solvents such as water, methanol,

ethanol and acetone to non-polar solvents such as ethyl acetate, dichloromethane,

chloroform, carbon tetrachloride and hexane. Extracting the plant tissues with an

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array of solvents with regard to polarity allows for a range of bioactive compounds.

Organically soluble compounds are extracted with organic solvents. The extract

obtained is clarified by filtration and is then concentrated in vacuum in a rotary

evaporator and they should be stored under refrigerated conditions.

Properly dried plant materials can be used for extraction. The plant materials

are first ground and then thoroughly mixed with a solvent inside a tank. The choice of

solvent depends on several factors including the characteristics of the constituents

being extracted, cost and environmental issues. The solid-liquid extraction is a

heterogeneous, multi component operation involving the non steady transfer of

solutes from a solid to a fluid3. Once the solvent dissolves the phytochemicals of the

plant material, the mixture is called “miscella”. The miscella is then separated from

the plant material by filtration.

3.1.1. Purification and Concentration of phytochemicals

Miscella that has been separated from the plant material generally contains

unwanted substances such as tannins, pigments, microbial contaminants or residual

solvent. Methods such as decanting, filtration, sedimentation, centrifuging, heating,

absorption, precipitation and ion exchange are used to separate impurities from the

extract. The extract is sometimes concentrated in order to increase the proportion of

the desired substances. This is done through evaporation or vaporization. Solvent is

generally recovered and reused. The degree of concentration depends on the product.

Any method used to concentrate the extract must avoid excess heat because the active

compounds may be subjected to degradation.

94

The Rotary evaporator is a device used for gentle and efficient evaporation of

solvents from a mixture. It contains of a heated rotating vessel (usually a large flask)

which is maintained under a vacuum through a tube connecting it to a condenser. The

rotating flask is heated by partial immersion in a hot water bath. The flask’s rotation

provides improved heat transfer to the contained liquid; the rotation also strongly

reduces the occupancy of ‘bumps’ caused by superheating of the liquid. The solvent

vapors leave the flask by the connecting tube and condensed vapors drain into another

flask where they are collected. It is a very efficient way of rapidly removing large

quantities of solvent4.

3.2. TOTAL PHENOLIC ASSAY

Phenolic and polyphenolic compounds constitute the main class of natural

antioxidants present in plants, foods and beverages and are usually quantified

employing Folin’s reagent. The F-C method has been used for many years as a means

to determine total phenolics in natural products. The reaction that takes place is an

oxidation/reduction, so F-C assay can also be considered an antioxidant capacity

method. This assay has many variations.

The original F-C method developed in 1927 originated from chemical reagents

used for tyrosine analysis in which oxidation of phenols by molybdotungstate reagent

yields a colored product with λmax at 745-750 nm5

Na2WO4/Na2MoO4→ (phenol-MoW11O40)6 --------------------3.1

Mo (VI) (yellow) +e- Mo (V) (blue) --------------------3.2

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The method is simple, sensitive and precise. However, the reaction is slow at

acid pH and it lacks specificity. Singleton and Rossi7 1965 improved the method with

molybdotungstophosphoric heteropoly anion reagent that reduced phenols more

specifically; the λmax for the product is 765nm. Phenolic compounds react with FCR

only under basic conditions (adjusted by a sodium carbonate solution to pH 10).

Dissociation of a phenolic proton leads to a phenolate anion, which is capable of

reducing FCR. This supports the notion that the reaction occurs through electron

transfer mechanism. The blue compounds formed between phenolate and FCR are

independent of the structure of phenolic compounds, therefore, ruling out the

possibility of coordination complexes formed between the metal center and the

phenolic compounds. Despite the undefined chemical nature of FCR, the total

phenolic assay by FCR is convenient, simple, and reproducible. As a result, a large

body of data has been accumulated, and it has become a routine assay in studying

phenolic antioxidants.

The degree of the color change is proportional to the antioxidant

concentrations. The reaction end point is reached when color change stops. The

change of absorbance (¢A) is plotted against the antioxidant concentration to give a

linear curve. The slope of the curve reflects the antioxidant’s reducing capacity, which

is expressed as Trolox equivalence (TE) or Gallic acid equivalent (GAE).

3.3. TESTING ANTIOXIDANTS

There have been a number of methods developed to measure the efficiency of

antioxidants as pure compounds or in extracts. These methods focus on different

mechanisms of the antioxidant such as scavenging of oxygen and hydroxyl radicals,

reduction of lipid peroxyl radicals, chelation of metal ions or inhibition of lipid

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peroxidation. Some methods determine the ability of an antioxidant to scavenge free

radicals generated by the system such as the Oxygen Radical Absorbance Capacity8

(ORAC), Total Reducing Ability of Plasma9 (TRAP) and Trolox Equivalent

Antioxidant Capacity10 (TEAC).Methods such as DPPH●11 (2,2-diphenyl-1-

picrylhydrazyl) and ABTS●12 (2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)

measures the scavenging of a stable free radical by the antioxidant. Other methods

evaluate antioxidants by quantifying lipid peroxidation products such as

malenaldehyde by Thiobarbituric Acid Reactive Substances13 (TBARS) and volatile

organic acid decomposition products and OSI14.

3.3.1. 2, 2-Diphenyl-1-picrylhydrazyl Assay

DPPH is widely used to test the ability of compounds to act as free radical

scavengers or hydrogen donors and to evaluate AA in extracts. It has also been used

to quantify antioxidant in complex biological systems in recent years. The antioxidant

ability of a sample can also be estimated by determining the hydrogen donating

ability of the samples in the presence of 2, 2-diphenyl–1-picrylhydrazyl (DPPH)

radical at 517 nm on the basis of the method of Hatano et al14 (1988). The DPPH

method can be used for solid or liquid samples and is not specific to any particular

antioxidant component, but applies to the overall antioxidant capacity of the sample.

A measure of total antioxidant capacity will help us understand the functional

properties of a given sample.

Fig.3.3.1. 2, 2-diphenyl-1-picrylhydrazyl

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Representing the DPPH radical by Z• and the donor molecule by AH, the

primary reaction is

Z• + AH → ZH + A• ---------------3.3

Where ZH is the reduced form and A• is free radical produced.

The structure of DPPH and its reduction by an antioxidant are shown in

Fig.3.3.1. The odd electron in the DPPH free radical gives a strong absorption

maximum at 517 nm and is purple in colour. The color turns from purple to yellow as

the molar absorptivity of the DPPH radical at 517 nm reduces when the odd electron

of DPPH radical becomes paired with hydrogen from a free radical scavenging

antioxidant to form the reduced DPPH-H. The resulting decolorization is

stoichiometric with respect to number of electrons captured15. More recently, this

reaction has been measured by the decoloration assay where the decrease in

absorbance at 515-528 nm produced by the addition of the antioxidant to the DPPH●

in methanol or ethanol is measured16.

The DPPH radical is one of the few stable organic nitrogen radicals, which

bears a deep purple colour. It is commercially available and does not have to be

generated before assay like ABTS. This assay is based on the measurement of the

reducing ability of antioxidants toward DPPH•. A rapid simple and inexpensive

method to measure antioxidant capacity of the sample involves the use of the free

radical, 2, 2-Diphenyl-1-picrylhydrazyl. Using this reagent, the free radical

scavenging ability of the antioxidant can be determined by spectro photometric

methods.

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3.3.2. Reducing power (RP) determination

Heavy metals like Fe3+ and Cu2+ are known to catalyse oxidative process in

living organisms. Fe3+, for instance, is reduced to Fe2+ in the process. It follows that if

the 2+ state does not aid the oxidative process, then the process does occur. The ability

to reduce Fe3+to the 2+ state is known as reducing power and is an indication of its

antioxidant property17. The Fe3+ reducing power of the samples is determined based

on the chemical reaction of

Fe (III) → Fe (II) ----------------------3.4

The reducing capacity of a compound may serve as a significant indicator of

its potential AA. Oyaizu18 (1986) has described a dose-dependent method (which was

modified by Yildirim17 in 2001) for the determination of the reducing capacity of

samples. The sample’s ability to reduce the Fe (III) to Fe (II) is determined by

measuring the amount of the Fe (II) spectroscopically; the absorbance of the reaction

mixture is measured at 700 nm. Increased absorbance indicates increased reducing

power19.

3.3.3. Ferric Thiocyanate (FTC) Method 20

The FTC method measures the amount of peroxide formed at the primary

stage of linoleic acid oxidation, in which peroxide reacts with ferrous chloride and

form ferric ion. The ferric ion then combines with ammonium thiocyanate and

produce ferric thiocyanate, a reddish pigment. The thicker the colour, the higher the

absorbance. High absorbance is an indication of a high concentration of peroxides.

The concentration of peroxide decreases as the AA increases. Low absorbance values

measured via the FTC method indicate high antioxidant activity.

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3.3.4. β- Carotene linoleic acid model system

The mechanism of bleaching of β carotene is a free radical mediated

phenomenon resulting from the hydro peroxides formed from linoleic acid. β carotene

in this model system undergoes rapid discoloration in the absence of an antioxidant.

The linoleic acid free radical formed upon the abstraction of a hydrogen atom from

one of its diallylic methylene groups attacks the highly unsaturated β carotene

molecule and looses its chromophore and characteristic orange colour21. This method

is a preliminary and fast test to distinguish the AA of certain compounds.

3.4. ANALYTICAL INSTRUMENTATION

Analytical instrumentation plays an important role in the products and

evaluation of new products and in the protection of consumers and the environment.

Thus instrumentation provides the lower detection limits require to assure safe food,

drugs, water and air22. Instrumental methods of Chemical analysis have now become

the backbone of experimental chemistry23.

Points to be considered in the selection of a procedure include.

• Stability of the absorbance with respect to time, minor variations in pH, ionic

strength and temperature.

• Degree of selectivity of a complexing agent including the effect of other

species likely to be present and the effect of an excess reagent.

• Conformity of the Beer –Lamberts law and plot calibration data for the range

of concentration measured.

The classification of quantitative physiochemical methods generally depend

upon the character of the measured properties of the system. These methods are

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generally depending upon the character of the measured properties of the system.

These methods generally classified into two broad groups. Spectral and

Electrochemical methods24.

3.4.1. Spectroscopy

The spectral methods are based on the nature of absorption/ emission of

electromagnetic radiation by the system being analyzed. The various spectral methods

include UV spectroscopy, Visible spectroscopy, infra-red spectroscopy fluorescence

spectroscopy, phosphorescence Spectroscopy, Raman Spectroscopy, Atomic

Absorption spectroscopy, Turbidimetry, Nephlometry, etc., The electrochemical

methods are based on the inter dependence of electrochemical properties and the

composition of the system. The various electrochemical methods are

Electrogravimetry, Potentiometry, Conductometry, Polography etc.

• Ultraviolet-visible spectroscopy refers to absorption spectroscopy in the UV-

visible spectral region. This means it uses light in the visible and adjacent

(near-UV and near-infrared (NIR) ranges. The absorption in the visible range

directly affects the perceived color of the chemicals involved. In this region of

the electromagnetic spectrum, molecules undergo electronic. This technique is

complementary to fluorescence spectroscopy, in that fluorescence deals with

transitions from the excited state to the ground state, while absorption

measures transitions from the ground state to the excited state25

• A double beam UV-VIS spectrophotometer is routinely used in the

quantitative determination of solutions of transition metal ions and

highly conjugated organic compounds.

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• The instrument used in ultraviolet-visible spectroscopy is called a

UV/vis spectrophotometer. It measures the intensity of light passing through a

sample (I), and compares it to the intensity of light before it passes through the

sample (Io). The ratio I / Io is called the transmittance, and is usually expressed

as a percentage (%T). The absorbance, A, is based on the transmittance:

A = − log (%T / 100%) --------------------3.5

• In a double-beam instrument, the light is split into two beams before it reaches

the sample. One beam is used as the reference; the other beam passes through

the sample. Some double-beam instruments have two detectors (photodiodes),

and the sample and reference beam are measured at the same time. In other

instruments, the two beams pass through a beam chopper, which blocks one

beam at a time. The detector alternates between measuring the sample beam

and the reference beam.

• Samples are typically placed in a transparent cell, known as a cuvette.

Cuvettes are typically rectangular in shape, commonly with an internal width

of 1 cm. (This width becomes the path length, L, in the Beer-Lambert

law.) The type of sample container used must allow radiation to pass over the

spectral region of interest. The most widely applicable cuvettes are made of

high quality fused silica or quartz glass because these are transparent

throughout the UV, visible and near infrared regions.

• A complete spectrum of the absorption at all wavelengths of interest can often

be produced directly by a more sophisticated spectrophotometer. In simpler

instruments the absorption is determined at one wavelength at a time and then

compiled into a spectrum by the operator. A standardized spectrum is formed

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by removing the concentration dependence and determining the extinction

coefficient (ε) as a function of wavelength.

3.4.2. Chromatography

Chromatography is one of the most powerful techniques available to the

analyst for the separation of the mixtures. It is a group of techniques which work on

the principle of separation of components of a mixture, depending on their affinities

for the solutes, between two immiscible phases. One of the phases is a fixed bed of

large surface area, while the other is a fluid which moves through the surface of the

fixed phase. The fixed phase is called stationary phase, and the other is termed as the

mobile phase. The mobile phase may be either a gas or liquid or a supercritical fluid24.

The field of chromatography can be subdivided or organized in different ways,

according to the physicochemical principles involved in the separation or according to

various techniques applied. The modern instrumental techniques of GLC and HPLC

provide excellent separation and allow accurate assay of very low concentrations of

wide variety of substance in complex mixtures.

In paper chromatography, the stationary phase is a very uniform absorbent

paper. The mobile phase is a suitable liquid solvent or mixture of solvents. The paper

is suspended in a container with a shallow layer of a suitable solvent or mixture of

solvents in it. It is important that the solvent level is below the line with the spots on

it. Paper is made of cellulose fibres, cellulose which is a polymer of the simple sugar,

glucose. The chromatographic chamber is covered when the separation takes place.

The reason for covering the container is to make sure that the atmosphere in the

beaker is saturated with solvent vapour. Saturating the atmosphere in the beaker with

vapour stops the solvent from evaporating as it rises up the paper.

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TLC26 was first described in 1938, has largely replaced paper chromatography

because it is faster, more sensitive and more reproducible. (Both of these techniques

may be referred to as planar chromatography.) This technique is more or less similar

to paper chromatography as far as most of the operations are concerned. TLC is done

exactly as it says - using a thin, uniform layer of silica gel or alumina coated onto a

piece of glass, metal or rigid plastic. The silica gel (or the alumina) is the stationary

phase. The mobile phase is a suitable liquid solvent or mixture of solvents.

The resolution in TLC is greater than in paper chromatography because the

particles on the plate are smaller and more regular than paper fibers. One of the

greatest advantages of TLC is the speed at which the separation is achieved. Generally

10-30 minutes are sufficient. However, with certain compounds about 90 minutes

may be required. Compared to paper chromatography, TLC is more versatile, faster

and more reproducible.

As the solvent slowly travels up the plate, the different components of the

mixture travel at different rates and the mixture is separated into different coloured

spots. The solvent is allowed to rise until it almost reaches the top of the plate. That

will give the maximum separation of the components, for this particular combination

of solvent and stationary phase.

CC27 is a routinely carried out technique which is adaptable to all the major

types of chromatography. The columns are usually made up of glass or polyacrylate

plastic. It is the most useful method of separating compounds in a mixture.

Fractionation of solutes occurs as a result of differential migration through a closed

tube of stationary phase and analytes can be monitored while the separation is in

104

progress. The most common technique for wet-packing involves making a slurry of

the adsorbent with the solvent and pouring this into the column. As the sorbent settles,

excess solvent is drained off and additional slurry is added. This process is repeated

until the desired bed height is obtained.

The sample to be fractionated is dissolved in a minimum volume of mobile

phase and is applied at top of the column. The process of passing the mobile phase

through the column is called elution and the portion that emerges from the outlet end

of the column is sometimes called the elute (or effluent). Elution may be isocratic

(constant mobile-phase composition) or a gradient may be used. Gradient elution

refers to changing the mobile phase (eg., increasing solvent strength or pH) during

elution in order to enhance resolution and decrease analysis time.

Fresh solvent is to be added at the top of the column, without disturbing the

packed material. Then the tap is opened so that the solvent can flow down through the

column, and the eluant is collected in a beaker or flask at the bottom. As the solvent

runs through, fresh solvent is added to the top so that the column never dries out.

The commonly used glass columns have a sintered glass disc at the bottom to

support the stationary phase. It is necessary that the sample to be applied reaches the

surface of the column below the top layer of the solvent. The sample is allowed to just

run into the column. Solvent is then added to the column to a height of 5-10 cm. It is

necessary to apply the sample in as less a volume as possible. This gives an initial

tight band of material when the separation begins and results in a sharper final

separation. Continuous passage of a suitable eluant (mobile phase) through the packed

column separates the components of the sample applied to the column. This process is

105

known as column development. Column packing influences flow rate. Unevenly

packed column leads to distortion of the flow leading to unsatisfactory resolution.

More densely packed columns retard the flow of the mobile phase and decrease the

flow rate. It is therefore very important to pack the column optimally to obtain a good

flow rate. The effluent as it emerges from the column outlet is analysed. The

properties of a particular compound, viz., ultraviolet absorption, color or fluorescence

are exploited in its analysis27.

HPLC is basically a highly improved form of column chromatography. Instead

of a solvent being allowed to drip through a column under gravity, it is forced through

under high pressures of up to 400 atmospheres that makes it much faster. It also

allows us to use much smaller particle size for the column packing material which

gives a much greater surface area for interactions between the stationary phase and

the molecules flowing past it. This allows a much better separation of the components

of the mixture25.

• Since HPLC is basically separating technique, it is always used in conjunction

with another analytical tool for quantitative analysis.

• Injection of the sample-Injection of the sample is entirely automated.

• Retention time-The time taken for a particular compound to travel through the

column to the detector is known as its retention time. This time is measured

from the time at which the sample is injected to the point at which the display

shows a maximum peak height for that compound. Different compounds have

different retention times. The conditions have to be carefully controlled if

retention times are used as a way of identifying compounds.

106

• The detector -There are several ways of detecting when a substance has passed

through the column. A common method which is easy to explain uses ultra-

violet absorption or Photo Diode Array (PDA) detector.

• Interpreting the output from the detector-The output will be recorded as a

series of peaks - each one representing a compound in the mixture passing

through the detector and absorbing UV light.

• Identification of compounds by HPLC is a crucial part of any HPLC assay. In

order to identify any compound by HPLC a detector must first be selected.

Once the detector is selected and is set to optimal detection settings, a

separation assay must be developed. The parameters of this assay should be

such that a clean peak of the known sample is observed from the

chromatograph. The identifying peak should have a reasonable retention time

and should be well separated from extraneous peaks at the detection levels

which the assay will be performed. Identifying a compound by HPLC is

accomplished by researching the literature and by trial and error. A sample of

a known compound must be utilized in order to assure identification of the

unknown compound. Identification of compounds can be assured by

combining two or more detection methods.

• Quantification of compounds by HPLC is a process of determining the

unknown concentration of a compound in a known solution. It involves

injecting a series of known concentrations of the standard compound solution

onto the HPLC for detection. The chromatograph of these known

concentrations will give a series of peaks that correlate to the concentration of

the compound injected.

107

• The control module includes parameters for the HPLC pumps, column oven,

detectors (including PDA), auto sampler, etc. Advances in column technology,

are high pressure pumping systems and sensitive detectors which have

transformed liquid column chromatography into a high speed, high efficiency

method of separation.

• A dedicated PDA Extension enables the evaluation of data from PDA

systems. A PDA is a linear array of discrete photodiodes on an integrated

circuit (IC) chip. It can be thought of as an electronic version of photographic

film. Array detectors are especially useful for recording the full UV-Vis

absorption spectra of samples that are rapidly passing through a sample flow

cell, such as in an HPLC detector. PDAs work on the same principle as

simple photovoltaic detectors. If sample has three components, each

component has different wavelength maxima in that case it is not possible by

UV in single injection we can do it by PDA detector only. HPLC PDA

detector provides the most advanced level of sensitivity and stability.

LCMS25 is a powerful tool for the analysis of complex mixtures is obtained by

linking liquid chromatography with mass spectrometry. The former is capable of

separating mixtures on the basis of the time required for the individual components to

appear at the end of suitably packed columns. Mass spectrometry permits

identification of each component according to the mass of the fragments formed when

the compound is bombarded with a beam of electrons. LC/MS equipment may

produce as many as 100 spectra in less than an hour, with each spectrum being made

up of tens to hundreds of peaks. Conversion of these data to an interpretable form is

time consuming; thus, the data must be stored in digital form for subsequent printing.

108

Identification of a species from its mass spectrum involves a search of files of spectra

for pure compounds until a match is found; this process is also time consuming.

Programs have been described which permit the search of several thousands of spectra

in a minute or less. Such a search will frequently produce several possible

compounds. Further comparison of spectra usually makes identification possible.

3.5. ASSESSMENT OF QUALITY OF OILS

One of the most important indicators of the quality of oils is their oxidative

stability28, which is the resistance of oil to oxidation29. Methods used to determine the

extent of oxidation of oils are based on the measurement of the concentration of the

primary or secondary products of oxidation. Various methods such as OSI30, DSC,31

Rancimat method, the measurement of conjugated diene hydro peroxides, volatile

compounds such as aldehydes, AV32 and the measurement of PVs33 have been used

for assessing the extent of oxidation of edible oils. These methods employ accelerated

oxidation conditions such as exposing the oil at a determined constant temperature,

flow of air or the presence or absence of light and catalysts29 and assessing the extent

of oxidation. The measurement of PV and AV can be used to monitor the formation of

primary and secondary oxidation products respectively, as a means of determining the

quality of oils.

3.5.1. Peroxide value

Since hydro peroxides are the primary products of lipid oxidation, PV (a

measure of hydro peroxides concentration) is in most cases used as an indicator of the

initial stages of oxidation, based on an iodometric titration34 of I2, which is produced

when an excess of potassium iodide is added to the oil. The peroxides are reacted with

potassium iodide in the presence of acetic acid, liberating iodine (reactions 3.5 and

109

3.6), which is produced in equivalent amount to the peroxides present in fat or oil.

The iodine is titrated with standard sodium thiosulphate, (reactions 3.8).

2KI+2CH3COOH→ 2HI+2CH3COO●K+ --------------------3.6

ROOH+2HI→ROH+H2O+I2 --------------------3.7

I2+2Na2S2O3→ Na2S4O6 +2NaI --------------------3.8

A wide range of PVs have been reported which could lead to a fat or oil

developing signs of rancidity resulting from the decomposition of the hydro

peroxides. Rossell35 indicated that a freshly refined fat should have a PV of less than 1

milliequivalent /kg of the fat and fats that have been stored for some time after

refining could have a PV of up to 10meq/kg before off-flavours are encountered. The

Codex Alimentarius Commission36 (2001) similarly allows up to a maximum PV of

10meq/kg for refined oils in general.

3.5.2. Oxidative Stability Index (OSI)

Oxidative stability is known as the resistance to oxidation under defined

conditions and is expressed as the period of time required to reach an end point which

can be selected according to different criteria (e.g. development of rancidity), but

usually corresponds to a sudden increase in oxidation rate. An oxidation normally

proceeds very slowly until this point is reached, this time period is known as the

induction period.

The OSI analysis method was developed as a means of measuring the natural

stability of fats and oils. This method involves inducing oxidation of oils using heat

and air while collecting the volatile organic acids produced during oxidation in a

water trap and measuring the increase in conductivity in the water. Natural oils

110

containing varying levels of different unsaturated fatty acid and other natural

antioxidants resulting in variability among test, so by use of a standardized fatty acid,

the oxidation reaction mechanisms are concise and defined. Increase in OSI value;

suggest that a more concentrated level of antioxidant or a more efficient antioxidant

compound is present to inhibit the peroxy radical reactions under high temperature

and oxygen content.

Numerous methods, using accelerated oxidation conditions, have been

developed for the evaluation of oxidative stability. Elevated temperatures in the

presence of oxygen or air, in excess, are applied to obtain results in reasonably short

periods of time. The OSI method, also commonly known as the Rancimat method,

allows oxidative stability to be determined automatically under standardized

conditions14. This method is widely used in the fats and oils, industry and it can be

applied by using two commercially available instruments: the Rancimat from

Metrohm Ltd. (Heriasau, Switzerland) and the Oxidative Stability Instrument from

Omnion Inc. (Rackland, MA). The end point is that corresponding to a sudden rise of

volatile acids generated from the oil samples heated at high temperature under

constant aeration. These compounds are trapped in water and monitored by electro-

conductivity 37.

3.5.3. Rancimat38

The 743 Rancimat is equipped with two heating blocks each with 4 measuring

positions. Each block can be individually heated, i.e. 2 sets of 4 samples can be

measured at two different temperatures or 8 samples can be measured at the same

temperature. Measurements at the individual measuring positions can be started

individually.

111

The complete operation of the rancimat is carried out by a PC connected to the

RS 232 interface with the aid of the rancimat control and evaluation program. The

evaluation algorithm of the PC program determines the point of inflection of the

Rancimat curve and therefore the induction time can be obtained automatically or can

be evaluated manually. Apart from the induction time, the so-called stability time, i.e.,

the time taken until a certain alteration in the conductivity is reached, can also be

determined. The results of the determinations are stored in a database together with all

the data connecting the method and determination. Apart from the graphical display

of single and multiple curves it is also possible to carry out recalculations with altered

parameters and to extrapolate the results to a particular temperature.

The operational sequence has been designed to be easy to use:

• Switch on the heater. The instrument will indicate when the set temperature

has been reached.

• Insert the reaction vessels with the samples.

• Switch on the air stream.

• Start the determination by means of the GO key.

• The instrument will now print out the curves in real-time and supply the

evaluation.

Today, the Rancimat method developed by Hador and Zurcher has to a large

extent has replaced the time-consuming AOM or Swift methods. The Rancimat's fully

automatic determination of 'induction time' also allows large numbers of

determinations to be carried out in less time. In the Rancimat method, a stream of air

is blown through the sample at a temperature between 500C–2200C. This oxidises the

fatty acids in several stages. In principle, oxidation takes place according to a radical

112

chain mechanism, in which easily volatile oxidation products (chiefly formic acid) are

finally formed. These are transferred by the stream of air into a measuring vessel

containing deionised water, whose conductivity is continually being measured.

Plotting conductivity against time produces oxidation curves, whose point-of-

inflection is known as the induction time. These induction times are correlated with

values determined by the more complicated AOM. The Rancimat principle is suitable

for the determination of the oxidative stability of a range of natural oils and fats.

These are normally mixed triglycerides of both saturated and unsaturated fatty acids.

This treatment results in oxidation of the oil or fat molecules in the sample,

with peroxides initially being formed as the primary oxidation products. After some

time the fatty acids are completely destroyed; the secondary oxidation products

formed include low-molecular organic acids like formic acid in addition to other

volatile organic compounds. These are transported in the stream of air to a second

vessel containing distilled water. The conductivity in this vessel is recorded

continuously. The organic acids can be detected by the increase in conductivity. The

time that elapses until these secondary reaction products appear is known as the

induction time, induction period or OSI.

113

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