3. theoretical analysis 3.1. extraction antioxidant
Post on 12-Feb-2022
5 Views
Preview:
TRANSCRIPT
92
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
93
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
95
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
96
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
97
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.
98
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.
99
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
100
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.
101
• 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
102
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.
103
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
REFERENCES
1. Julkunen-Tiitto, R. Phenolic constituents in the leaves of northern willows:
methods for the analysis of certain phenolics. J Agric Food Chem 33, 1985, pp
213-217.
2. Houghton, P.J and Raman, A. Laboratory Handbook for the fractionation of
natural Extracts 1st Ed. New York, NY: Chapman and Hall, 1998.
3. Diaz-Reinoso B, Moure A, Dominguez H and Parajo JC. Supercritical CO2
extraction and purification of compounds with antioxidant activity. J Agric
Food Chem 54, 2006, pp 2441-2469.
4. Raaman.N. Phytochemical Techniques, New India Publishing Agency, New
Delhi, 2005, pp7-18.
5. Folin, O and Ciocalteu, V. Tyrosine and tryptophan determinations in proteins,
J Biol Chem 73, 1927, pp 672-649.
6. Prior RL, Wu X, Schaich K. Standardized methods for the determination of
antioxidant capacity and phenolics in foods and dietary supplements. J Agric
Food Chem 53, 2005, pp 4290-4302.
7. Singleton, V.L and Rossi, J.A .Colorimetry of total phenolics with
phosphomolybdic-phosphotungstic acid reagents. Am J Enol Vitic 16, 1965,
pp 144-158.
8. Cao. G.H, Alessio, H.M and Cutler, R.G. Oxygen-radical absorbency capacity
assay for antioxidants. Free Radical Biol Med 14(3), 1993, pp 303-311.
9. Ghiselle, A, Serafii, M, Maiane, G, Azzini, E and Ferro-Luzzi, A.A
fluorescence - based method for measuring total plasma antioxidant capacity.
Free Rad Biol Med 18, 1995, pp 29-36.
10. Miller, N.J, Rice-Evans, C.A, Davies, M.J, Gopinath, V and Milner, A. A
Novel method for measuring antioxidant capacity and its application to
monitoring antioxidant status in premature neonates, Clin. Sci 84, 1993, pp
407-412.
11. Brand-Williams, W, Cuvelier, M.E and Berset, C. Use of a free radical method
to evaluate antioxidant activity. Lebensm Wiss Technol 28, 1995, pp 25-30.
114
12. Re, R, Pellergrini, N, Proteggente, A, Pannala A, Yang, M and Rice-Evans, C.
Antioxidant activity applying an improved ABTS radical cation decolorization
assay. Free Rad Biol Med 26, 1999, pp 1231-1237.
13. Plumb, G.W. DE Pascual-Teresa, S.Santos-Buelga, C, Cheynier, V. and
Williamson, G. Antioxidant properties of catechins and proanthocyanidins:
effect of polymerization, galloylation and glycosyalation. Free Rad Res 29,
1996, pp 351-358.
14. Hatano T, Kagawa H, Yasuhara T and Okudu T, Two new flavonoids and
other constituents in licorice root; their relative astringency and radical
scavenging effects. Chem Pharm Bull 36, 1988, pp 2090-2097.
15. Aruna Prakash, Antioxidant Activity, Medallion Laboratories Analytical
Progress, Playmouth Ave North, Minneapolis, Minnnesota 19(2), 2001 pp
763-767.
16. Sanchez- Mareno C, Review: Methods to evaluate the free radical scavenging
activity in foods and biological systems. Food Sci Technol Intern Vol.8, 2002,
pp 121-137.
17. Yildirim, A, Oktay, M and Bülaloúlu, V, The Antioxidant Activity of the
Leaves of Cydonia vulgaris, Turk J Med Sci 31: 2001, pp 23-27.
18. Oyaizu, M. Studies on products of browning reaction: antioixdative activities
of products of browning reaction prepared from glucosamine. Jap J Nut. 44,
1986, pp 307-315.
19. Blázovics A, Lugasi A, Szentmihályi K, Kéry A, Reducing power of the
natural polyphenols of Sempervivum tectorum in vitro and in vivo+, Acta Biol
Szeged 47, 2003, pp 99-102.
20. Kim J.S and Kim M.J, In vitro antioxidant activity of Lespedeza cuneata
methanolic extracts, J Med Plants Res Vol. 4(8), 2010, pp. 674-679.
21. Antioxidant activities of roselle (Hibiscus sabdariffa) calyces and fruit
extracts, Ramakrishna BV, Jayaprakasha GK, Jena BS, Singh RP, J Food Sci
and Technol, 45(3), 2008, pp 223-227.
22. Backett, A.H. and Stenlakc, J.B., Practical Pharmaceutical chemistry 4th Ed,
CBS publishers and distributors, 1997. pp 162 – 164 and 275 - 305.
115
23. Sharma, B.K., Instrumental methods of chemical analysis 18th Ed Krishna
Prakasham Media Pvt. Ltd., Meerut 1999. pp S10 - S30.
24. Gurudeep Chatwal, Sham Anand, Instrumental methods of chemicals
analysis. 5th Ed. Himalaya publishing house, New Delhi, 2002. p567.
25. Connors, K.A., A text book of pharmaceutical analysis. 3rd Ed Wiley –
Interscience publication, New York, 1982. pp 638 – 639.
26. Avinash Upadhyay, Kakoli Upadhyay and Nirmalendu Nath, Biophysical
Chemistry, Principles and techniques, Himalaya Publishing House,
Hyderabad, Geetanjali Press 2007, pp 344-421.
27. Skoog, Donald West. Principles of Instrumental Analysis. 6th Ed. Thomson
Brooks/Cole. 2007, pp 169-173.
28. Tan, C.P, Che Man, Y.B. Selemat, J and Yusoff, M.S.A., Comparative studies
of oxidative stability of edible oils by differential scanning Calorimetry and
oxidative stability index methods. Food Chem 76, 2002, pp 385-389.
29. Guillen, M.D and Cabo, N. Fourier transform infrared spectral data versus
peroxide and anisidine values to determine oxidative stability of edible oils.
Food Chem 77, 2002, pp 503-520
30. Che, Y and Hsu, H. Effect of antioxidants on peanut oil stability. Food Chem
66, 1999, pp 29-34.
31. Rudnik, E, Szczucinska, A, Gwardiac, H, Szulk, A and Winiarska, A.
Comparative studies of oxidative study of linseed oil. Thermochimica Acta,
37, 2001, pp 135-140.
32. Abou- Gharbia, H.A, Shehata, A.A.Y and Shahidi, F. Effect of processing on
oxidative stability and lipid classes of sesame oil. Food Res Intern 33, 2000,
pp 331-340.
33. Abdalla, A.E and Roozen, J.P. Effect of plant extracts on the oxidative
stability of sunflower oil and emulsion. Food Chem 64, 1999, pp 323-329.
34. O’Brien, R.D., Fats and Oils: Formulating and Processing for Applications.
2nd Ed. London: CRC Press. 2004, pp 192-199.
116
35. Rossell, J.B. Classical Analysis of oils and fats. In: Hamilton, R.J and Rossell,
J.B. (Eds). Analysis of oils and fats. England: Elsevier Applied Science, 1986,
pp 1-32.
36. Codex Alimentarius Commission. Codex standard for named vegetable oils.
Codex Alimentarius 8, 2001.pp 11-25.
37. Joaquin Velasco, Mogens L.Andersen, Leif H.Skibsted. Evaluation of
oxidative stability of vegetable oils by monitoring the tendency to radical
formation. A comparison of electron spin resonance spectroscopy with the
Rancimat method and differential scanning Calorimetry. Food Chem 85, 2004,
pp 623-632.
38. Study of the Rancimat Test Method in Measuring the Oxidation Stability of
Biodiesel Ester and Blends NRC project.
top related