Investigations in food science and technology, whether by the food industry, governmental

agencies, or universities, often require determination of food composition and characteristics.

Trends and demands of consumers, the food industry, and national and international
regulations challenge food scientists as they work to monitor food composition and to ensure
the quality and safety of the food supply. All food products require analysis as part of a
quality management program throughout the development process (including raw
ingredients), through production, and after a product is in the market. In addition, analysis is
done of problem samples and competitor products. The characteristics of foods (i.e., chemical
composition, physical properties, and sensory properties) are used to answer specific
questions for regulatory purposes and typical quality control. The nature of the sample and
the specific reason for the analysis commonly dictate the choice of analytical methods.
Speed, precision, accuracy, and ruggedness often are key factors in this choice. Validation of
the method for the specific food matrix being analyzed is necessary to ensure usefulness of
the method. Making an appropriate choice of the analytical technique for a specific
application requires a good knowledge of the various techniques.

STEPS IN ANALYSIS

 Select and Prepare Sample

 Perform the Assay
 Calculate and Interpret the Results

Proximate analysis of foods refers to determining the major components of moisture, ash
(total minerals) , lipids , protein and carbohydrates. The performance of many analytical
methods is affected by the food matrix (i.e., its major chemical components, especially lipid,
protein, and carbohydrate).

CLASSIFICATION OF LIPIDS
The general classification of lipids that follows is useful to differentiate lipids in foods.

Simple Lipids

Ester of fatty acids with alcohol:

• Fats: Esters of fatty acids with glycerol –triacylglycerols

• Waxes: Esters of fatty acids with long-chain alcohols other than glycerols (e.g.,
myricylpalmitate, cetyl palmitate, vitamin A esters, and vitamin D esters)

Compound Lipids

Compounds containing groups in addition to an ester of a fatty acid with an alcohol:

• Phospholipids: Glycerol esters of fatty acids,phosphoric acids, and other groups containing
nitrogen (e.g., phosphatidyl choline, phosphatidyl serine, phosphatidyl ethanolamine and
phosphatidyl inositol)

• Cerebrosides: Compounds containing fattyacids, a carbohydrate, and a nitrogen moiety

(e.g., galactocerebroside and glucocerebroside)

• Sphingolipids: Compounds containing fattyacids, a nitrogen moiety, and phosphoryl group

(e.g., sphingomyelins)

Derived Lipids

Derived lipids are substances derived from neutral lipids or compound lipids. They have the
general properties of lipids – examples are fatty acids, long chain alcohols, sterols, fat-soluble
vitamins, and hydrocarbons.

Analysis of Lipids
Introduction

Lipids are one of the major constituents of foods, and are important in our diet
for a number of reasons. They are a major source of energy and provide essential
lipid nutrients. Nevertheless, over-consumption of certain lipid components can be
detrimental to our health, e.g. cholesterol and saturated fats. In many foods the
lipid component plays a major role in determining the overall physical
characteristics, such as flavor, texture, mouthfeel and appearance. For this reason,
it is difficult to develop low-fat alternatives of many foods, because once the fat is
removed some of the most important physical characteristics are lost. Finally,
many fats are prone to lipid oxidation, which leads to the formation of off-flavors
and potentially harmful products. Some of the most important properties of
concern to the food analyst are:

 Total lipid concentration

 Type of lipids present
 Physicochemical properties of lipids, e.g.,  crystallization, melting point, smoke point,
rheology, density and color
 Structural organization of lipids within a food
Properties of Lipids in Foods

Lipids are usually defined as those components that are soluble in organic
solvents (such as ether, hexane or chloroform), but are insoluble in water. This
group of substances
includes triacylglycercols, diacylglycercols, monoacylglycercols, free fatty acids,
phospholipids, sterols, caretonoids and vitamins A and D. The lipid fraction of a
fatty food therefore contains a complex mixture of different types of molecule.
Even so, triacylglycercols are the major component of most foods, typically
making up more than 95 to 99% of the total lipids present. Triacylglycerols are
esters of three fatty acids and a glycerol molecule. The fatty acids normally found
in foods vary in chain length, degree of unsaturation and position on the glycerol
molecule. Consequently, the triacylglycerol fraction itself consists of a complex
mixture of different types of molecules. Each type of fat has a different profile of
lipids present which determines the precise nature of its nutritional and
physiochemical properties. The terms fat, oil and lipid are often used
interchangeably by food scientists. Although sometimes the term fat is used to
describe those lipids that are solid at the specified temperature, whereas the
term oil is used to describe those lipids that are liquid at the specified temperature.

Sample Selection and Preservation

As with any analytical procedure, the validity of the results depends on proper
sampling and preservation of the sample prior to analysis. Ideally, the composition
of the sample analyzed should represent as closely as possible that of the food from
which it was taken. The sample preparation required in lipid analysis depends on
the type of food being analyzed (e.g. meat, milk, margarine, cookie, dairy cream),
the nature of the lipid component (e.g. volatility, susceptibility to oxidation,
physical state) and the type of analytical procedure used (e.g. solvent extraction,
non-solvent extraction or instrumental). In order, to decide the most appropriate
sample preparation procedure it is necessary to have a knowledge of the physical
structure and location of the principal lipids present in the food. Since each food is
different it is necessary to use different procedures for each one. Official methods
have been developed for specific types of foods that stipulate the precise sample
preparation procedure that should be followed. In general, sample preparation
should be carried out using an environment that minimizes any changes in the
properties of the lipid fraction. If lipid oxidation is a problem it is important to
preserve the sample by using a nitrogen atmosphere, cold temperature, low light or
adding antioxidants. If the solid fat content or crystal structure is important it may
be necessary to carefully control the temperature and handling of the sample.
Determination of Total Lipid Concentration

Introduction

It is important to be able to accurately determine the total fat content of foods

for a number of reasons:

 Economic (not to give away expensive ingredients)

 Legal (to conform to standards of identity and nutritional labeling laws)
 Health (development of low fat foods)
 Quality (food properties depend on the total lipid content)
 Processing (processing conditions depend on the total lipid content)

The principle physicochemical characteristics of lipids (the "analyte") used to

distinguish them from the other components in foods (the "matrix") are their
solubility in organic solvents, immiscibility with water, physical characteristics
(e.g., relatively low density) and spectroscopic properties. The analytical
techniques based on these principles can be conveniently categorized into three
different types: (i) solvent extraction; (ii) non-solvent extraction and (iii)
instrumental methods.

Solvent Extraction

The fact that lipids are soluble in organic solvents, but insoluble in water,
provides the food analyst with a convenient method of separating the lipid
components in foods from water soluble components, such as proteins,
carbohydrates and minerals. In fact, solvent extraction techniques are one of the
most commonly used methods of isolating lipids from foods and of determining
the total lipid content of foods.

Sample Preparation

The preparation of a sample for solvent extraction usually involves a number of

steps:

Drying sample. It is often necessary to dry samples prior to solvent

extraction, because many organic solvents cannot easily penetrate into foods
containing water, and therefore extraction would be inefficient.

Particle size reduction. Dried samples are usually finely ground prior to

solvent extraction to produce a more homogeneous sample and to increase the
surface area of lipid exposed to the solvent. Grinding is often carried out at low
temperatures to reduce the tendency for lipid oxidation to occur.
 Acid hydrolysis. Some foods contain lipids that are complexed with proteins
(lipoproteins) or polysaccharides (glycolipids). To determine the concentration
of these components it is necessary to break the bonds which hold the lipid and
non-lipid components together prior to solvent extraction. Acid hydrolysis is
commonly used to release bound lipids into easily extractable forms, e.g. a
sample is digested by heating it for 1 hour in the presence of 3N HCl acid.

Solvent Selection. The ideal solvent for lipid extraction would completely

extract all the lipid components from a food, while leaving all the other
components behind. In practice, the efficiency of solvent extraction depends on
the polarity of the lipids present compared to the polarity of the solvent. Polar
lipids (such as glycolipids or phospholipids) are more soluble in polar solvents
(such as alcohols), than in non-polar solvents (such as hexane). On the other
hand, non-polar lipids (such as triacylglycerols) are more soluble in non-polar
solvents than in polar ones. The fact that different lipids have different
polarities means that it is impossible to select a single organic solvent to extract
them all. Thus the total lipid content determined by solvent extraction depends
on the nature of the organic solvent used to carry out the extraction: the total
lipid content determined using one solvent may be different from that
determined using another solvent. In addition to the above considerations, a
solvent should also be inexpensive, have a relatively low boiling point (so that
it can easily be removed by evaporation), be non-toxic and be nonflammable
(for safety reasons). It is difficult to find a single solvent which meets all of
these requirements. Ethyl ether and petroleum ether are the most commonly
used solvents, but pentane and hexane are also used for some foods.

Batch Solvent Extraction

These methods are based on mixing the sample and the solvent in a suitable
container, e.g., a separatory funnel. The container is shaken vigorously and the
organic solvent and aqueous phase are allowed to separate (either by gravity or
centrifugation). The aqueous phase is then decanted off, and the concentration of
lipid in the solvent is determined by evaporating the solvent and measuring the
mass of lipid remaining: %Lipid = 100 � (Mlipid/Msample). This procedure may have
to be repeated a number of times to improve the efficiency of the extraction
process. In this case the aqueous phase would undergo further extractions using
fresh solvent, then all the solvent fractions would be collected together and the
lipid determined by weighing after evaporation of solvent. The efficiency of the
extraction of a particular type of lipid by a particular type of solvent can be
quantified by an equilibrium partition coefficient, K = csolvent/caqueous,
where csolvent and caqueous are the concentration of lipid in the solvent and aqueous
phase, respectively. The higher the partition coefficient the more efficient the
extraction process.
Semi-Continuous Solvent Extraction

Semi-continuous solvent extraction methods are commonly used to increase the

efficiency of lipid extraction from foods. The Soxhlet method is the most
commonly used example of a semi-continuous method. In the Soxhlet method a
sample is dried, ground into small particles and placed in a porous thimble. The
thimble is placed in an extraction chamber, which is suspended above a flask
containing the solvent and below a condenser. The flask is heated and the solvent
evaporates and moves up into the condenser where it is converted into a liquid that
trickles into the extraction chamber containing the sample. Eventually, the solvent
builds up in the extraction chamber and completely surrounds the sample. The
extraction chamber is designed so that when the solvent surrounding the sample
exceeds a certain level it overflows and trickles back down into the boiling flask.
As the solvent passes through the sample it extracts the lipids and carries them into
the flask. The lipids then remain in the flask because of their low volatility. At the
end of the extraction process, which typically lasts a few hours, the flask
containing the solvent and lipid is removed, the solvent is evaporated and the mass
of lipid remaining is measured (Mlipid). The percentage of lipid in the initial sample
(Msample) can then be calculated: %Lipid = 100 � (Mlipid/Msample). A number of
instrument manufacturers have designed modified versions of the Soxhlet method
that can be used to determine the total lipid content more easily and rapidly
(e.g. Soxtec).

Continuous Solvent Extraction

The Goldfish method is similar to the Soxhlet method except that the extraction

chamber is designed so that the solvent just trickles through the sample rather than
building up around it. This reduces the amount of time required to carry out the
extraction, but it has the disadvantage that channeling of the solvent can
occur, i.e., the solvent may preferentially take certain routes through the sample
and therefore the extraction is inefficient. This is not a problem in
the Soxhlet method because the sample is always surrounded by solvent.

Accelerated Solvent Extraction

The efficiency of solvent extraction can be increased by carrying it out at a

higher temperature and pressure than are normally used. The effectiveness of a
solvent at extracting lipids from a food increases as its temperature increases, but
the pressure must also be increased to keep the solvent in the liquid state. This
reduces the amount of solvent required to carry out the analysis, which is beneficial
from a cost and environmental standpoint. Special instruments are available to
carry out solvent extraction at elevated temperatures and pressures.
Supercritical Fluid Extraction

Solvent extraction can be carried out using special instruments that use
supercritical carbon dioxide (rather than organic liquids) as the solvent. These
instruments are finding greater use because of the cost and environmental problems
associated with the usage and disposal of organic solvents. When pressurized
CO2 is heated above a certain critical temperature it becomes a supercritical fluid,
which has some of the properties of a gas and some of a liquid. The fact that it
behaves like a gas means that it can easily penetrate into a sample and extract the
lipids, while the fact that it behaves like a fluid means that it can dissolve a large
quantity of lipids (especially at higher pressures). Instruments based on this
principle heat the food sample to be analyzed in a pressurized chamber and then
mix supercritical CO2 fluid with it. The CO2 extracts the lipid, and forms a separate
solvent layer, which is separated from the aqueous components. The pressure and
temperature of the solvent are then reduced which causes the CO 2 to turn to a gas,
leaving the lipid fraction remaining. The lipid content of a food is determined by
weighing the percentage of lipid extracted from the original sample.

 Non solvent Liquid Extraction Methods.

A number of liquid extraction methods do not rely on organic solvents, but use
other chemicals to separate the lipids from the rest of the food. The Babcock,
Gerber and Detergent methods are examples of nonsolvent liquid extraction
methods for determining the lipid content of milk and some other dairy products.

Babcock Method

A specified amount of milk is accurately pipetted into a specially designed flask

(the Babcock bottle). Sulfuric acid is mixed with the milk, which digests the
protein, generates heat, and breaks down the fat globule membrane that surrounds
the droplets, thereby releasing the fat. The sample is then centrifuged while it is hot
(55-60oC) which causes the liquid fat to rise into the neck of the Babcock bottle.
The neck is graduated to give the amount of milk fat present in wt%. The Babcock
method takes about 45 minutes to carry out, and is precise to within 0.1%. It does
not determine phospholipids in milk, because they are located in the aqueous phase
or at the boundary between the lipid and aqueous phases.

Gerber Method

This method is similar to the Babcock method except that a mixture of sulfuric
acid and isoamyl alcohol, and a slightly different shaped bottle, are used. It is
faster and simpler to carry out than the Babcock method. The isoamyl alcohol is
used to prevent charring of the sugars by heat and sulfuric acid which can be a
problem in the Babcock method since it makes it difficult to read the fat content
from the graduated flask. This method is used mainly in Europe, whilst the
Babcock method is used mainly in the USA. As with the Babcock method, it does
not determine phospholipids.

Detergent Method

This method was developed to overcome the inconvenience and safety concerns
associated with the use of highly corrosive acids. A sample is mixed with a
combination of surfactants in a Babcock bottle. The surfactants displace the fat
globule membrane which surrounds the emulsion droplets in milk and causes them
to coalesce and separate. The sample is centrifuged which allows the fat to move
into the graduated neck of the bottle, where its concentration can then be
determined.

Instrumental methods

The are a wide variety of different instrumental methods available for

determining the total lipid content of food materials. These can be divided into
three different categories according to their physicochemical principles: (i)
measurement of bulk physical properties, (ii) measurement of adsorption of
radiation, and (iii) measurement of scattering of radiation. Each
instrumental methods has its own advantages and disadvantages, and range of
foods to which it can be applied.

Measurement of bulk physical properties

 Density: The density of liquid oil is less than that of most other food components, and
so there is a decrease in density of a food as its fat content increases. Thus the lipid
content of foods can be determined by measuring their density.
 Electrical conductivity: The electrical conductivity of lipids is much smaller than that
of aqueous substances, and so the conductivity of a food decreases as the lipid
concentration increases. Measurements of the overall electrical conductivity of foods
can therefore be used to determine fat contents.
 Ultrasonic velocity: The speed at which an ultrasonic wave travels through a material
depends on the concentration of fat in a food. Thus the lipid content can be
determined by measuring its ultrasonic velocity. This technique is capable of rapid,
nondestructive on-line measurements of lipid content.

Measurement of adsorption of radiation

 UV-visible: The concentration of certain lipids can be determined by measuring the

absorbance of ultraviolet-visible radiation. The lipid must usually be extracted and
diluted in a suitable solvent prior to analysis, thus the technique can be quite time-
consuming and labor intensive.
 Infrared:  This method is based on the absorbance of IR energy at a wavelength of
5.73 m due to molecular vibrations or rotations associated with fat molecules: the
 greater the absorbance the more fat present. IR is particularly useful for rapid and on-
line analysis of lipid content once a suitable calibration curve has been developed.
 Nuclear Magnetic Resonance: NMR spectroscopy is routinely used to determine
the total lipid  concentration of foods. The lipid content is determined by measuring
the area under a peak in an NMR chemical shift spectra that corresponds to the lipid
fraction. Lipid contents can often be determined in a few seconds without the need for
any sample preparation using commercially available instruments.
 X-ray absorption: Lean meat absorbs X-rays more strongly than fat, thus the X-ray
absorbance decreases as the lipid concentration increases. Commercial instruments
have been developed which utilize this phenomenon to determine the lipid content of
meat and meat products.

Measurement of scattering of radiation

 Light scattering: The concentration of oil droplets in dilute food emulsions can be

determined using light scattering techniques because the turbidity of an emulsion is
directly proportional to the concentration of oil droplets present.
 Ultrasonic scattering: The concentration of oil droplets in concentrated food
emulsions can be determined using ultrasonic scattering techniques because the
ultrasonic velocity and absorption of ultrasound by an emulsion is related to the
concentration of oil droplets present.

A number of these instrumental methods have major advantages over the

extraction techniques mentioned above because they are nondestructive, require
little or no sample preparation, and measurements are usually rapid, precise and
simple.

A major disadvantage of the techniques which rely on measurements of the

bulk physical properties of foods are that a calibration curve must be prepared
between the physical property of interest and the total lipid content, and this may
depend on the type of lipid present and the food matrix it is contained in. In
addition, these techniques can only be used to analyze foods with relatively simple
compositions. In a food that contains many different components whose
concentration may vary, it is difficult to disentangle the contribution that the fat
makes to the overall measurement from that of the other components.

Comparison of Methods

Soxhlet extraction is one of the most commonly used methods for

determination of total lipids in dried foods. This is mainly because it is fairly
simple to use and is the officially recognized method for a wide range of fat
content determinations. The main disadvantages of the technique are that a
relatively dry sample is needed (to allow the solvent to penetrate), it is destructive,
and it is time consuming. For high moisture content foods it is often better to
use batch solvent or nonsolvent extraction techniques. Many instrumental methods
are simple to operate, rapid, reproducible, require little sample preparation and are
nondestructive. Nevertheless, they are often expensive to purchase and can only be
used for certain types of foods, i.e., where there is no interference from other
components. In addition, calibration curves prepared for instrumental methods
usually require that the fat content be measured using a standard method.

Extraction techniques tend to be more accurate and more generally applicable

and are therefore the standard methods for official analysis of many food materials
(e.g., for labeling or legal requirements). Instrumental methods are most useful for
rapid measurements of fat content on-line or in quality assurance laboratories of
food factories where many samples must be measured rapidly.

Oil Refining
1.  Introduction

The quality of fried foods depends not only on the type of foods and frying conditions, but
also on the oil used for frying. Thus, the selection of stable frying oils of good quality is of
great importance to maintain a low deterioration during frying and consequently a high
quality of the fried foods.

Many refined oils and fats are used for frying and the ideal oil composition may be different
depending on technical or nutritional considerations. In general, the decision is influenced by
many factors amongst which functionality, nutritional properties, cost and availability stand
out. Palm olein and partially hydrogenated oils have been considered the most stable oils for
frying although, in the last decades, development of genetically modified seeds containing
oils with a lower degree of unsaturation than those of the traditional oils has significantly
increased the availability of oils of high thermostability in the marketplace.

However, whatever the oil or fat used, its initial quality may vary significantly and affect the
rate of deterioration during frying. Thus, extraction of good-quality seeds and the appropriate
development of the different steps in the refining process to fulfill frying oil specifications are
necessary. This is the only guarantee for obtaining the best frying performance of the selected
oil.

In this article, the main steps of the refining process are discussed briefly with special
reference to the changes in the crude oils and their importance in the production of high-
quality oils. For complete information on the different conditions and equipments used in the
different steps, a wide literature is available. Also, specifications for refined frying oils are
given and justified.

2.  The Refining Process

Refining of crude oil is done to remove unwanted minor components that make oils
unappealing to consumers, while trying to cause the least possible damage to the neutral oil
as well as minimum refining loss. The components to be removed are all those glyceridic and
nonglyceridic compounds that are detrimental to the flavour, colour, stability or safety of the
refined oils. They are primarily phosphoacylglycerols, free fatty acids, pigments, volatiles
and contaminants.

On the other hand, not all the minor compounds in fats and oils are undesirable. For example,
phytosterols are considered of nutritional interest, and tocopherols with vitamin E
activity, protecting the oil against oxidation are highly appreciated. Consequently, to reach
the maximum oil quality all the steps of the refining process should be carried out with the
minimum losses of desirable compounds.

The major steps involved and the main components removed are shown in Table 1. As can
be observed, alkali (or chemical) and physical refining are the standard processes used. The
main difference between the processes is that alkali refining procedure includes caustic soda
treatment to neutralise the oil while, following physical refining, free fatty acid are eliminated
by distillation during deodorization. Physical refining reduces the loss of neutral oil,
minimises pollution and enables recovery of high-quality free fatty acids. Nevertheless, not
all oils can be physically refined.

Table 1. Basic steps of the refining process

Alkali or chemical Main groups of compounds
refining removed Physical Refining
Degumming    Phospholipids Degumming
Neutralization    Free fatty acids -
Bleaching    Pigments/metals/soaps Bleaching
Winterization    Waxes/saturated triacylglycerols Winterization
Deodorization/
Deodorization    Volatiles/free fatty acids deacidification
 

2.1  Degumming

The purpose of degumming is to remove phospholipids or gums from the crude oil. Two
types of phospholipids are present in crude oils according to their level of hydration, i.e.
hydratable and nonhydratable ones, the latter mainly present as calcium and/or magnesium
salts of phosphatidic acid and phosphatidylethanolamine. After addition of water (1-3%),
most of the phospholipids are hydrated and are insoluble in the oil. The hydrated compounds
can be efficiently separated by filtration or centrifugation. For the elimination of the
nonhydratable fraction, the oil is usually treated with phosphoric acid (0.05 to 1%), which
chelates the Ca and Mg converting the phosphatides into the hydratable forms (the acid
treatment has the additional function of chelating trace prooxidant metals). Due to the
variable content of phospholipids in crude oils, analysis of phosphorus prior to acid treatment
is necessary to ensure that the acid dosage is correct, especially when the content of Ca and
Mg salts is high.

Depending on the oil composition, the degumming step can be eliminated as the phosphatides
are also removed along with the soaps during the next step of neutralization. However,
degumming is mandatory for physical refining and the content of phosphorus after
degumming should be lower than 10 mg/kg.

2.2  Neutralization

In this step, the oil is treated with caustic soda (sodium hydroxide) and free fatty acids are
converted into insoluble soaps, which can be easily separated by centrifugation. Thus, the
main objective of this step is the removal of free fatty acids, although, as commented above,
residual phospholipids in degummed oils or all the phospholipids in the crude oils are also
removed as insoluble hydrates. Also, caustic neutralization improves significantly the oil
colour partly by reacting with polar compounds (gossypol, sesamol, sterols, hydroxy fatty
acids, etc) and partly by solubilization. Alkali refining of oil is compulsory in crude oils of
high acidity and pigment contents.

The free fatty acid content of the oil is the main factor that determines the amount and
concentration of the caustic soda and also its excess (5 to 20%) for a minimum oil loss. After
a reaction time of around 30 minutes at slow stirring and temperature around 80ºC, the water
phase is eliminated by centrifugation and the oil washed with water to remove the remaining
soap.

2.3  Bleaching

In this step, which is common to both physical and alkali refining, the hot oil (around 100ºC)
is slurried with acid-activated bleaching earth (1-2%), normally calcium montmorillonite or
natural hydrated aluminium silicate (bentonite). Under these conditions adsorption of colour
bodies, trace metals and oxidation products as well as residual soaps and phospholipids
remaining after washing neutralized oils takes place. For optimum adsorption of both colour
bodies and oxidation products to be achieved, the reaction time has to exceed 15 minutes and
no more than 30 minutes at usual bleaching temperatures. The removal of chlorophyllic
pigments is very important since they are not eliminated in any other stage of refining, as
carotenoid compounds are in deodorization. On the other hand, final filtration must eliminate
completely the activated earths as residual traces act as prooxidants during oil storage
because of their iron content.

Acid-activated clays are the major adsorbent used, although active carbons and synthetic
silicas are also applied industrially with more specific goals. Thus, active carbons are used
specifically to eliminate polycyclic aromatic hydrocarbons (PAH) from some oils, especially
fish oils and pomace oils, while synthetic silicas are quite efficient in adsorbing secondary
oxidation products, phospholipids and soaps.

This is a critical step to obtain high-quality oils, because two types of adsorption occur
between the compounds to be adsorbed and the absorbent: on one hand, reversible physical
adsorption based on intermolecular forces of low strength and, on the other hand, irreversible
chemisorption with a strong interaction, which causes chemical reactions.

Chemical changes taking place at this stage have been well studied in olive oil, because of the
need to control the presence of refined oils in virgin oils. The two main reactions found
extensively in all the vegetable oils are the following:

 Decomposition of hydroperoxides. Previous steps do not modify the peroxide value

and it may even increase if air is available in the earlier stages. However, during
bleaching, hydroperoxides decompose to form volatiles and oxidized triacylglycerols
containing keto and hydroxy functions. After bleaching, peroxide value should be
zero or close to zero, but the presence of aldehydes and ketones is clearly detected by
the significant increase in the anisidine value.

 Dehydration of alcohols. Hydroxy acids formed from hydroperoxides undergo a

partial dehydration by earth catalysis. As the function is at an allylic position, a rapid
increase in UV absorption at 232 nm is observed because of the formation of
conjugated dienes from oleic acid hydroperoxides and in UV absorption at 268 nm
due to formation of conjugated trienes from linoleic acid hydroperoxides. Also,
sterols undergo significant dehydration and the formation of the hydrocarbon 3,5-
stigmastadiene from the major sterol (β-sitosterol) is considered a proof of the
presence of refined oil in virgin olive oil.

2.4  Winterization

This step, also called dewaxing, is only applied when the oil is not clear at room temperature
because of the presence of waxes or saturated triacylglycerols. It is important to note that
these compounds do not affect negatively the oil performance or functionality, but the
appearance of the oil is not acceptable to consumers.

Thus, the objective of this step is the removal of high temperature melting components
present in small quantities. The crystallization process normally used consists of cooling the
oil down gradually to temperatures of 5 to 8ºC in a maturing tank. After increasing the crystal
size at this temperature for 24 to 48 h, the solids are separated by centrifugation at 15-16ºC.
This treatment ensures excellent clarity of oils when stored at either room or refrigeration
temperatures.

2.5  Deodorization/deacidification

Deodorization of fats and oils normally consists of steam distillation at elevated temperature
under reduced pressure, although nitrogen has also been used. The purpose of this step is to
remove volatile compounds (mainly ketones and aldehydes) contributing to oil taste and
odour, total free fatty acids in physical refining and the residual free fatty acids from
neutralized bleached oils. The deodorization conditions also contribute to the removal of
contaminants (light PAH, pesticides, etc.) and to the reduction of colour of the oil due to the
breakdown of the remaining carotenes at high temperature. The efficiency of deodorization is
a function of pressure (1 to 5 torr), temperature (200 to 260ºC), residence time (0.5 to 3 h)
and volume of stripping gas (1 to 3%). However, differences in the deodorization equipment
used also have a major impact on efficiency. After the deodorization, the oil is cooled and
addition of citric acid (100 mg/kg of 20% citric acid) is recommended to chelate metal traces
and increase its stability during storage.

Apart from the physical changes, chemical reactions taking place in the triacylglycerols due
to the drastic conditions of this step have been studied in detail and are summarized as
follows:

 Decomposition of oxidation compounds. Even if hydroperoxides were destroyed

during bleaching, some new primary and secondary oxidation products formed
decompose during heat treatment to form volatile and nonvolatile compounds.

 Dimerization of triacylglycerols. Acyclic dimers of triacylglycerols, i.e. nonpolar

dimers (C–C bridges) as well as oxygenated dimers (C–O–C), are detected in
significant amounts, which may involve the formation of alkyl and alkoxyl radicals at
high temperature even in the absence of oxygen.

 Geometrical and positional isomers induced by heat are also formed in this step. Thus,
more trans isomers and also more dienoic conjugation are found. However, in oils
containing linolenic acid, a decrease in the trienoic conjugation is observed, which is
attributed to the formation of cyclic fatty acids and the concurrent elimination of
double bonds.

 Finally, an inter esterification reaction is detected in vegetable oils deodorized at

temperatures above 240ºC by an increase in saturated fatty acids in the 2-position of
the triacylglycerols.

The importance of these reactions is higher, as expected, as the temperature and the
deodorization time increases, being dramatic in highly unsaturated oils. It is also remarkable
that hydrolytic reactions have not been observed as the content of diacylglycerols remains
unchanged, not only in this step but throughout the complete process.

Finally, it is important to take into account that long deodorization times and/or too high
temperatures can have a devastating effect on the quality of the oil due not only to the
chemical changes commented above but also to the distillation of a significant part of the
natural tocopherols (20 to 40%), which would decrease the stability of the refined oil. In this
respect, the by-product obtained from the deodorization, i.e. deodorizer distillate, contains
significant amounts of compounds of high-added value like tocopherols, sterols and
hydrocarbons, and a great effort is being made for their recovery.

HYDROGENATION OF OILS
 Hydrogenation of vegetable oil has been practiced for over a century. The process was
originally introduced to convert some of the unsaturated fatty acids in vegetable oils, as well
as marine or animal fats to make them more stable to oxidation.

Refined oil on storage becomes, rancid due to deteriorative changes in unsaturated fatty acids
(USFA). Therefore refined oils are hydrogenated by passing H2 under pressure through hot
oil in presence of catalysts like nickel.

The quality of final products depends on

 Temperature

 Rate of mixing of H2

 Nature of catalysts

 Pressure of H2

 The fat will be neutral in flavour

 High smoking temperature, therefore will have good shortening power

 High stability

 Low undesirable flavour

Hydrogenation is a heterogeneous reaction process and complex in nature where the reaction
occurs between the hydrogen (gaseous phase) and the unsaturated fatty acids (liquid phase),
converting some or all of the unsaturated fatty acids into saturated fatty acid (stearic acid).
This involves the following physical and chemical reactions:

 Mixing and dispersion of the catalyst by a mechanical mixer.

 Diffusion of hydrogen gas through the mass of oil to the catalyst surface.
 Adsorption of the reactants (hydrogen and unsaturated fatty acids) on the catalyst
surface.
 Partial or complete saturation of the unsaturated fatty acids on the catalyst surface.
 Desorption of the products of reaction and the unreacted fatty acids and oil molecules
from the catalyst surface.
 Release of heat due to the exothermic nature of the hydrogenation reaction.

Hydrogen gas and the unsaturated fatty acids diffuse through the bulk of the oil and reach the
catalyst surface. Migration of these reactants through the bulk of the oil is facilitated by
mechanical agitation.

The two reactants, hydrogen and the unsaturated fatty acids, diffuse through a stagnant
microfilm of oil on the surface of the catalyst to reach the active sites on the catalyst.
The reaction between the unsaturated fatty acids and hydrogen gas occurs on the catalyst
surface. This is sometimes referred to as the chemisorption process, implying a chemical
reaction aided by the adsorption process.

The products of reaction, which are typically saturated fatty acids, unsaturated fatty acids (cis
and trans isomers), and triglyceride molecules, leave the catalyst surface via the desorption
process. More fresh reactants are adsorbed on the surface of the catalyst and the
hydrogenation reaction continues. Fig. 7.1 is a pictorial concept of the hydrogenation
reaction.

Heat is generated in the reaction. The average heat of reaction in hydrogenation is 1.65 Btu/lb
of oil for a drop in the iodine value (IV) by one unit. This is a valuable source of process heat
and is recovered in many plants via a heat-recovery system.

7.3.1 Effects of Hydrogenation

The following events occur in the hydrogenation process:

 The unsaturated fatty acids become more saturated.

 The IV of the oil decreases.
 The melting point of the oil increases.
 The oxidative stability of the oil improves.
 The solid content of the oil increases.
 A multitude of side reactions take place, including the formation of certain alcohols
and acids.
 Isomerization of some of the polyunsaturated fatty acids takes place.
Adulteration of Fats and Oils

Some edible oils and fats such as olive oil, cocoa butter and milk fat are so expensive
which makes tempting to adulterate them with other lower price vegetable oils and
fats to achieve more profit. The need for authentication is a necessity of the food
industry. Today, adulterations are more sophisticated. Therefore, it is necessary to use
advanced and suitable methods to detect adulteration. Adulteration can cause several
problems in edible oils application and industry. To detect edible oils and fats
adulteration, it is possible to use both major and minor components as detection tool.
Types of adulteration
There are two major adulterations in edible oils and fats namely 1) admixing cold
press oil with refined one and 2) replacement of more expensive oils and fats with
cheaper one.
Detection of cold press oil adulteration
All crude oils obtained by solvent extraction contain variable amounts of non-TAG
components such as fatty acids, mono-and diacylglycerols, phosphatides, and etc.
The amount of the non-TAG varies with the oil source, extraction process, season and
geographical source. Removal of non-TAG constituents from the oil with the least
possible damage to the TAG and minimal loss of desirable constituents is the
objective of the refining process. Low quality vegetable oils are also refined to
produce higher quality edible oil.
During refining processes, particularly during deodorisation and bleaching, Trans
fatty acids and steradienes are also formed which are generally absent in cold press
vegetable oils &fats (VOFs).
Virgin olive oil adulteration with refined vegetable oils can be detected using trans
fatty acid or steradienes as markers. Detection of stigmastadienes in virgin olive oil at
levels in excess of 0.15 mg/kg is regarded under EC regulations as evidence of the
presence of refined oils. It should be noted that detection of steradienes and trans fatty
acids isomer in other cold press oils are also a sign of adulteration with refined VOFs.
Stigmastadiene level in unrefined cocoa butter are well below 0.1 mg/kg whereas in
refined butters it may present up to several hundred mg/kg. Determination of trans
fatty acid in cocoa butter can be a useful tool to detect hydrogenated VOFs in cocoa
butter since some cis fatty acid isomers are converted to trans fatty acid isomers
during hydrogenation.
Adulteration of admixing oil and fats
As mentioned before, the replacement of expensive oil by lower price one is usual
respecting economic point of view. Some oils are more prone to be adulterated due to
their higher price and limited accessibility. To make it easy for readers, three of the
most common adulteration would be investigated in the next sections. Virgin olive oil
is obtained by the fruit of the olive tree solely by mechanical or other physical means
under certain thermal conditions that do not alter the oil, and the oil will not undergo
any treatment other than washing, decantation, centrifugation and filtration.
Because of the high price of virgin olive oil, there is a great temptation to adulterate it
with oils with similar fatty acid and sterol profiles. Olive oil adulteration with most
vegetable oils can be detected by conventional methods. Fatty acid composition is
useful to detect adulteration of olive oil with the following vegetable oils including
soybean, walnut, canola, rapeseed, peanut and mustard, even at level of adulteration
below 5% .
Olive oil adulteration with sunflower, soybean, cotton, corn, walnut, sesame,
safflower and canola oils can also be detected based on the differences in
triacylglycerol and fatty acid composition between the olive oil and these vegetable
oils.
Hazelnut oil has been used to adulterate olive oil due to its similar composition of
TAG, fatty acids and major sterols.
It is difficult to detect olive oil adulteration with hazelnut oil lower than 20%
concentration using conventional methods.
Different methods have been proposed to detect adulteration of olive oil with hazelnut
oil. The sterol profile can be used as a mean of differentiating between vegetable oils
or detecting their authenticity.
Detection of cocoa butter adulteration
Cocoa butter is derived from the Theobroma cacao tree, which grows in several
tropical areas, including Indonesia, the Ivory coast, Malaysia, New Guinea and
Brazil. Cocoa butter is used mainly in the manufacture of chocolate confectionery, but
it has also applications in cosmetics and pharmaceuticals. Because of the high price of
cocoa butter, there is a place of adulteration for defrauders. The non-cocoa fats also
used in confectionery are known as cocoa butter alternatives, of which the most
important are cocoa butter equivalents, cocoa butter replacers and cocoa butter
substitutes.
Cocoa butter has identical fatty acid composition. Despite of the other edible oils and
fats which has several fatty acids with different ratio, cocoa butter has three major
fatty acids, palmitic acid (16:0) 25- 30%, stearic acid (18:0) 24-37% and oleic acid
(18:1) 29- 38% and in minor amount linolenic acid (18:3) 0-4%. Changes and
variation from this identical composition can be a sign of adulteration. TAG is
different in VOFs and therefore, it can be as a useful tool to detect adulteration. TAG
analysis can be used to detect adulteration of cocoa butter with other VOFs and even
it can be used to determine its origin. Phytosterol composition can also be used to
detect cocoa butter adulteration. Cocoa butter has a high proportion of stigmasterol
compared with other confectionery fats. Significant difference were in the ratio of
stigmasterol to campesterol when comparing cocoa butter (mean ratio for three
samples=3.18) with ten CBAs and fats used in cocoa butter formulation (range=0.38
to 1.47).
Detection of milk fat adulteration
Dairy products have great importance in our diet. Milk fat is present in dairy products
such as milk, cream, butter, whole milk powder and cheese. Milk fat is more
expensive than VOFs; therefore, it is tempting to admix it with VOFs. Milk fat has
special fatty acid composition which can be vary by changes in factors such as breed
of cow, diet and stage of lactation. Fatty acid composition can be used to detect VOFs
with different fatty acid composition in dairy products. However, differences in fatty
 acid of VOFs and milk fat should be very distinct to be applicable to use as a
detection tool. Soybean and canola oils have high amount (7-10%) of linolenic acid
(18:3) but milk fat has very low amount (0.9-1.2). Therefore, detection of higher
amount of linolenic acid in dairy products can be a sign of adulteration with soybean
or canola oils.
Cottonseed, sunflower and corn oils have high amount (40-70%) of linoleic acid
(18:2), but milk fat has low amount of this fatty acid (1-2%). Therefore, linoleic acid
can also be used to detect some VOFs in dairy products. In some cases, it is difficult
to use fatty acid composition to detect VOFs such as palm oil in dairy products,
because there is similarity in their fatty acid composition and also variation in fatty
acid composition of palm oil and milk fat can make the detection of adulteration
difficult (Edem, 2002). Sterols are very useful tool to detect almost all types of VOFs
in dairy products. Main sterol in milk fat is cholesterol, and phytosterols are present in
trace amount (1-3% of total sterols) in milk fat. Phytosterols are present in all VOFs at
high amount (1000-12000 ppm), therefore detection of phytosterols in higher level in
dairy products can be an indication of adulteration with VOFs. Therefore, in cases
fatty acids are not suitable marker, such as detection of palm oil in dairy products, it is
possible to use phytosterols as a detection tool.

Spoilage of fats and oils

1. Flavor reversion
1. An objectionable flavour found before the onset of rancidity in
refined oils when exposed to UV light, visible light or heating.
2. The reaction is catalysed by O2 and small amounts of metals such as
iron and copper
3. Fats containing nucleic acid are most susceptible to reversion.
4. Soya bean oil is mostly subjected to flavor reversion.

Prevention of reversion

5. Hydrogenation
6. Small amount of linolenic acid prevents reversion.
7. Metallic activators or sequestrants tie up to iron and copper and
prevents revertion in soya oil.

2. Rancidity
1. Occurs mostly in fats containing unsaturated fatty acids.

Hydrolytic rancidity

2. Occurs due to enzymes that decomposes fat in to free fatty acids

(FFA) and glycerol.
3. Butyric acid and caproic acids are the volatile fatty acids,
 predominately present in butter are responsible for rancid flavour or
odour in butter and makes butter inedible.
4. Long chain fatty acids such as stearic, palmitic and oleic acids do not
produce rancidity unless oxidation occurs.
5. Heating thoroughly to destroy lipase catalyses hydrolysis of trans
fats and prevents hydrolytic rancidity.
6. Certain microorganisms also produces lipase.

Oxidation

7. Unsaturated fats have lipoxygenase and are susceptible to oxidative

changes.
8. Highly hydrogenated saturated fatty acids are resistant to oxidation.
9. Hydro peroxides that are formed readily producing smaller volatile
substances will give characteristic odours of rancid fat.

Characteristics of Rancidity

10. Undesirable changes in odour, flavour, colour and consistency

11. Inactivates vitamin A & E
12. Oxidative rancidity may be a problem in dry foods containing only
small quantities of fat such as prepared cereals.

Prevention of rancidity

13. Storage at refrigerator temperature prevents rancidity.

14. Light coloured glass containers absorb active rays and gives
protection against spoilage.
15. Certain shades of green bottles, cora papers, yellow transparent
cellophane etc. prevents rancidity.
16. Vacuum packaging
17. Anti-oxidants naturally present in food such as vitamin ‘C’, β-
carotene and vitamin E.
18. Added antioxidants such as
1. Butylated hydrogen anisole (BHA)
2. Tertiary butly hydro quivous (TBHQ)
3. Propyl gallate
19. synergists or sequestering agents – citric acid – bind or chelate the
metals and prevents oxidation process.

1.IODINE VALUE:

 It is defined as the weight of iodine absorbed by 100 parts by weight of the sample of fat
or oil.

 Iodine value is a measure of the extent of unsaturation.

 Susceptibility to rancidity increases for the oil or fat having higher iodine values.
Significance:

 Iodine number directly proportional to content of unsaturated fatty acids.

 Lower Iodine number, less is the degree of saturation.

 Iodine number is useful to analyze the degree of adulteration.

2. SAPONIFICATION VALUE:

 It is defined as the number of milligrams of potassium hydroxide required to neutralize the

fatty acids resulting from complete hydrolysis of 1 g of the sample of oil or fatty acid present
in the oil.

 This value is normally applied for butterfat, coconut oil in which lower fatty fat.

 Saponification value occurs in an inverse proportion to the average molecular weights of

acids glycerides occur in high content.

Significance:

 It is measure of the average molecular size of constituent fatty acids of given fat/oil.

 Higher saponification number for fats containing short chain fatty acids.

3. HYDROXYL VALUE:

 It is defined as number of milligrams of potassium hydroxide required to neutralize the

acetic acid capable of combining by acetylation with 1 g sample of fat or oil.

4. ACETYL VALUE:

 It is the number of milligrams of potassium hydroxide required to neutralize acetic acid

obtained when 1g value 150), most of the oils and fats have low acetyl value (3 -15).

Significance

 Acetyl number is a measure of number of hydroxyl groups present.

 It is used for studying the natural properties of the fat and to detect adulteration and
rancidity.

5. UNSAPONIFIABLE MATTER:

 It is the matter present in fats and oil, which after saponification by caustic alkali and
subsequent extraction with an organic solvent, remains non-volatile on drying at 8o°C.

 It includes sterols (phytosterol and cholesterol), oil soluble vitamins, hydrocarbons and
higher alcohols.

 Paraffin hydrocarbons can be detected by this method as adulterants.

6.ACID VALUE:

 It is defined as the number of milligrams of potassium hydroxide required to neutralize the

free acids present in 1 g sample of fat or oil.

 Generally, rancidity causes free fatty acids liberation, hence acid value is used as an
indication of rancid state.

Significance:

 Higher acid number mean it stored for longer duration.

7. PEROXIDE VALUE:

 It is a measure of peroxides present in oil.

 A peroxide value is generally less than 10 mEqkg in fresh samples of oil.

 Due to temperature or storage, rancidity occurs causing increase in peroxide values.

8. KREIS TEST (RANCIDITY INDEX):

 Due to rancidity, epihydrin aldehyde or malonaldehyde are increased which are detected
by Kreis test using phloroglucinol which produces red colour with the oxidized fat.

 It is defined as number of milligrams of potassium hydroxide required to combine with

fatty acids which are present in glyceride form in 1 g sample of oil or fat.

9.ESTER VALUE:

 Difference between saponification value and acid value is ester value.

10. REICHERT MESSLE VALUE:

 This value is a measure of volatile water soluble acid contents the fat.

 It is defined as number of milli litres N/10 potassium hydroxide solution required to

neutralize the volatile water soluble fatty acids obtained by 5 g fat.

Significance:

 Higher content of volatile fatty acids of butter responsible for its higher reichert-meissl
number.

 It is useful in testing purity/adulteration of butter.

11.POLENSKI VALUE:

 It is defined as the number of millitres of N/10 potassium hydroxide solution required to

neutralize water-insoluble, steam - distillable acids liberated by hydrolysis of 5 gm of fat.

Significance:
 The Polenski value is an indicator of how much volatile fatty acid can be extracted from
fat through saponification.

Test for presence of Rancidity:

 In routine work apart from the free fatty acids determination, the analysis should include
the determination of Peroxide Value, Kries Test, Ultra-violet Absorption at 234 nm and 268
nm to establish rancidity.

PEROXIDE VALUE

 This is an indication of the extent of oxidation suffered by an oil.

Reagents:

i) Acetic acid - chloroform solvent mixture (3: 2). Mix 3 volumes of glacial acetic acid with 2
volumes of chloroform.

ii) Freshly prepared saturated potassium iodide solution.

iii) 0.I N and 0.0I N sodium thiosulphate solutions.

Weigh 25 g of sodium thiosulphate and dissolve in 1 L of distilled water. Boil and cool, filter
if necessary. Standardize against standard potassium dichromate solution.

iv) Starch solution - 1% water-soluble starch solution

Procedure:

Weigh 5 g (±50 mg) sample into a 250 ml stoppered conical flask.

 Add 30 ml acetic acid chloroform solvent mixture and swirl to dissolve.

 Add 0.5 ml saturated potassium iodide solution with a mohr pipette. Let stand for 1min in
dark with occasional shaking, then add about 30 ml of water.

 Slowly titrate the liberated iodine with 0.1 N sodium thiosulphate solution, with vigorous
shaking until yellow color is almost gone.

 Using Add about 0.5 ml starch solution as indicator and continue titration shaking
vigorously to release all I 2 from CHCl3 layer until blue color disappears. If less than 0.5 ml
of 0.1 N Na2S2O3 is used repeat using 0.01 N Na2S2O3. Conduct blank determination
( must be less than 0.1 ml 0.1 N Na2S2O3).

Calculation:

 Peroxide value expressed as milli equivalent of peroxide oxygen per kg sample (meq/kg):

Peroxide Value = Titre Value X N X 100/ Weight Of The Sample

Where,
Titre value= ml of Sodium Thiosulphate Used (blank corrected)

N = Normality of sodium thiosulphate solution.

 Fresh oils usually have peroxide values well below 10 meq/kg.

A rancid taste often begins to be noticeable when the peroxide value is above 20 meq/kg.
(between 20 – 40 meq / Kg).

 In interpreting such figures, however, it is necessary to take into account the particular oil
or fat.

KRIES TEST

 TWO TESTS

1.Qualitative

2.Quantitative

1.Qualitative

 Shake 5 ml of the oil vigorously with 5 ml of 0.1% phloroglucinol solution in diethyl ether
and add 5 ml of conc. hydrochloric acid.

 A pink colour indicates incipient rancidity.

2.Quantitative: (method:1)

Weigh 0.8 – 1.02 gm of oil or fat into a 100 ml beaker.

Melt sample of fat and add slowly with stirring 20 ml of phloroglucinol ( 0.1 gm in100 ml
of diethyl ether, freshly prepared) until sample dissolved.

 Transfer solution to a separating funnel, add 10 ml conc HCl, shake well and allow to
separate.

Run off acid layer into a 1inch (2.54mm) Lovibond cell and match the colour using red ,
yellow and blue glasses.

Express result as red Lovibond units.

Up to 3 red units indicates incipient rancidity, between 3and 8 units indicates the end of
induction period, over 8 units indicates definite rancidity.

Quantitative (method:2)

Shake 5 ml of oil and 5 ml chloroform in a stoppered test tube.

 Add 10 ml of a 30% solution of trichloroacetic acid in glacial acetic acid and 1 ml of 1

percent solution of phloroglucinol in glacial acetic acid.
 Incubate the test tube at 45ºC for 15 min. After incubation, add 4 ml of ethanol and
immediately measure the absorbance at 545 nm.

 Absorbance values below 0.15 indicate no rancidity.

Absorbance values greater than 0.2 denote incipient rancidity, and absorbance values
around 1.0 show that the sample is highly rancid.

ULTRA-VIOLET ABSORPTION

 Oxidised fatty acids containing conjugated double bonds absorb UV strongly between 230
and 375 nm, dienes absorbing at 234 nm and trienes at 268 nm.

 Conjugated trienes may be formed by industrial processing, E.g. decolorising with

bleaching earths.

 A secondary absorption by trienes occurs at about 278 nm. In the early stages of oxidation
the UV absorption increases somewhat proportionately to the uptake of oxygen and the
formation of peroxides.

 The UV absorption curve forms plateau just before the end of the induction period.

 The magnitude of UV absorbance is not readily related to the amount of oxidation; so the
method is best applicable to detecting relative changes in oxidation of an oil in comparison
experiments or stability tests.

Procedure :

 Weigh accurately into a 25 ml volumetric flask, an amount of the oil sample so that the
absorbance of its solution in isooctane in a 10 mm quartz cell lies between 0.2 and 0.8.

Trace the absorption curve against iso-octane between 220 and 320 nm and select the
wavelength l max of maximum absorption near 230, 268 and 278 nm, and the absorbance (A)
at these points.

 The specific absorbance

E1 cm1% (λ max) = A = c x d

 Where,

‘c’ is the concentration of the sample solution (g/100 ml)

‘d” is the cell length in c.