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Food Analysis

(PC 524)
Level III - 2nd Semester

Lecture 7
Dr. Mohamed El-Awady
6-Dec-2014

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Analysis of
Lipids (Fats & Oils)
Continued

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(B) Determination of Lipid Composition


(A) Chemical Methods.
(B) Instrumental Methods.

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Introduction

In the previous lecture, we discussed analytical methods to


determine the total lipid content in food, without any
concern about the type of lipids present.

Lipids include a complex mixture of compounds


consisting of tri, di- and monoacylglycercols, waxes,
phospholipids, glycolipids, fat-soluble vitamins, steroids like
cholesterol, free fatty acids and others.

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In addition, most of these sub-groups are themselves


chemically complex which means that even a lipid which
consists of only triacylglycerols (fats & oils) may contain a

huge number of different chemical species.

Therefore, it is important to develop analytical method to


specify and determine the concentration of the different
types of lipid molecules present, as well as the total lipid
concentration.

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(A) Chemical Methods


Several chemical methods have been developed to provide
information about the type of lipids present in edible fats and
oils. These methods are characterized by being simple to
perform. In addition, they do not require expensive apparatus,
and so they are widely used in industry and research.
Before testing, we should ensure that the samples are
visually clear and free of sediment.

Examples of chemical methods used for determination of


lipid composition can be summarized as follows:
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(1) Iodine Value


Definition:
It is the number of grams of iodine absorbed per 100 g of lipid.

Significance:
It is a measure of degree of unsaturation of lipid. The higher
the iodine value, the greater the number of double bonds

(C=C) in the lipid.


Iodine value can be used in the following applications:
1. To characterize some oils.
2. To follow the hydrogenation process (C=CCH-CH)
during oil refining.
3. To indicate lipid oxidation because lipid oxidation is
associated with a decrease in the degree of unsaturation.

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Principle:

The

most

commonly

used

methods

for

the

determination of iodine value are:

(A) Wijs method (pronounced like Ves) & (B) Hanus method.

Wijs method: (Back titration)


It depends on dissolving a known amount of lipid in a suitable organic
solvent Add known excess of iodine monochloride (ICl) Part of
ICl reacts with the sample by addition reaction on the double bond:
I Cl
I I
R-CH=CH-R + ICl R-CH-CH-R
Known excess

Then the remaining unreacted ICl is determined by addition of


excess KI which reacts with the remaining ICl to produce I2:

ICl + KI KCl +
Remaining

I2
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Then the liberated I2 is titrated with sodium thiosulfate


(Na2S2O3) using starch as indicator. The end point is equivalent
to the remaining ICl. By subtraction from the total added ICl,

the reacted ICl can be calculated.


Principle of Hanus method: (Back titration)

It depends on the same principle as Wijs method but IBr


instead of ICl. Wijs method is preferred over Hanus method
because: ICl reacts faster than IBr with C=C double bonds.

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(2) Acid Value


Definition:
It is the number of milligrams of KOH required to neutralize

the free fatty acids present in 1 gram of lipid.


Significance:
It is a measure of the amount of free fatty acids present in lipid.

Acid value can be used in the following applications:


1. To test the efficiency of fat refining steps designed to remove
fatty acids. A high acid value means a poorly refined fat.
2. To measure the degree of hydrolysis of lipid. A high acid
value means poor quality or bad storage conditions of lipids.
N.B. The acid value may be over-estimated if any acidic additives
are present in the sample, e.g. citric acid added as preservative.

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Principle:
The

lipid

sample

is

dissolved

in

ethanol

containing

phenolphthalein indicator (ph.ph). Then the sample is titrated


with standard KOH. Finally the acid value can be calculated from
the obtained end point.
Free fatty acid (-COOH) + KOH K-salt of the fatty acid + H2O

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(3) Saponification Value


Definition:
It is the number of milligrams of KOH required to saponify

1 gram of lipid.
Significance:
It is a measure of

the mean molecular weight of the

triacylglycerols (triglycerides) in the lipid sample.


Saponification value can be used in the following applications:
For detection of adulteration. For example, adulteration of

lipid with mineral oils like liquid paraffin (very cheap) leads
to a decrease in the saponification value because mineral oils
do not contain esters and so are not saponifiable.
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Principle: (back titration)


The lipid sample is mixed with a known excess of ethanolic KOH

solution of high concentration (at least 0.5 N) heat to saponify


the lipid then the unreacted KOH is back-titrated with
standardized HCl using ph.ph indicator. Finally the saponification

value can be calculated from the obtained end point.


Triacylglycerol + 3 KOH

Glycerol + 3 K-salt of fatty acid


(soap)

The higher the number of combined fatty acids per 1 gram of the
lipid sample, the higher will be the saponification value.

N.B. The calculation of saponification value is not applicable if the


sample contains high amounts of free fatty acids >0.1%
(because they will also interact with KOH).

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Question: Complete the following:


The higher the saponification value, the .. will be the mean
molecular weight of the triacylglycerols present.
Answer:

The

higher the saponification value, the lower will be the mean

molecular weight of the triacylglycerols present.

Because high saponification value means presence of high number


of triacylglycerol molecules per 1 gram of the lipid sample which
means lower molecular weight.
For example, if 100 molecules are present in 1 gram of lipid, that
means that the molecular weight in this case is lower than the
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sample containing 20 molecules per 1 gram of lipid.

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N.B:
- Determination of iodine value, acid value and saponification value
are examples of chemical techniques used for the analysis of lipid
composition. In addition, there are also other less important
chemical methods used for analysis of lipids.
- Beside the above mentioned methods, there are also several
specific color tests for some specific oils or fats. For example:
- Halphens test for cotton-seed oil.
- Halphens insoluble bromine test for fish oil.
- Baudouins test for sesame oil.
- and others
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(B) Instrumental Methods


A variety of instrumental methods can also be used to provide
information about lipid composition. The most powerful of
these are: Chromatography (TLC, GC, HPLC) and nuclear
magnetic resonance (NMR) spectroscopy.
Chromatography including thin layer chromatography (TLC),
gas

chromatography

(GC),

and

high

pressure

liquid

chromatography (HPLC) can be used for both separation and

analysis of lipid in food samples.


NMR spectroscopy can be used for both identification and
quantitation of different lipid components in food samples.
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(C) Analysis of Lipid Oxidation

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Introduction

Foods which contain high concentrations of unsaturated


lipids are particularly susceptible to lipid oxidation. Lipid
oxidation is one of the major forms of spoilage in foods,
because it leads to the formation of bad flavors and
potentially toxic compounds. Lipid oxidation is a complex
process involving numerous reactions that give rise to
a variety of chemical and physical changes in lipids.

Rancidity

is the condition at which the lipid has

unpleasant odor and flavor as a result of lipolysis or


lipid hydrolysis (hydrolytic rancidity) or lipid oxidation
(oxidative rancidity).

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Methods of Analyzing Lipid Oxidation in Foods


1. Chromatography.
2. Oxygen Uptake.
3. Peroxide value.
4. Conjugated dienes method.
5. Thiobarbituric acid (TBA).
6. Accelerated Oxidation Tests.

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1. Chromatography
Chromatography is the most powerful method of monitoring lipid
oxidation because it provides a detailed profile of the fatty acids and

other molecules present in lipids (e.g., aldehydes, ketones or


hydrocarbons) especially when peaks are identified using mass
spectrometry or NMR.

2. Oxygen Uptake
Lipid oxidation depends on the reaction between unsaturated fatty
acids and oxygen. Thus it is possible to monitor the rate at which it

occurs by measuring the uptake of oxygen by the sample. Usually,


the lipid is placed in a sealed container and then we measure the
amount of oxygen that must be entered into the container to keep the
oxygen pressure in the head-space above the sample constant.

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3. Peroxide Value
Definition:
It is the number of milliequivalents of peroxide per kilogram of sample.

Significance:
Peroxides (R-O-O-H) are transient products of lipid oxidation (i.e. after
being forming, peroxides are broken down to form other products).
Low peroxide value may represent either the beginning of oxidation or
advanced oxidation.

Principle:
The method is based on the ability of peroxides to liberate iodine

from potassium iodide. The lipid is dissolved in a suitable organic


solvent and an excess of KI is added:

ROOH + KI ROH + KOH + I2


Then the liberated I2 is titrated with Na2S2O3 using starch indicator.

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4. Conjugated dienes method


During lipid oxidation, the non-conjugated double bonds (C=C-C-C=C)
are converted to conjugated double bonds (C=C-C=C). Conjugated

dienes strongly absorb UV light at 233 nm. Thus oxidation can be


followed by dissolving the lipid in a suitable organic solvent and
measuring the change in its absorbance with time using a UV-visible

spectrophotometer.
In the later stages of lipid oxidation the conjugated dienes (primary
products) are broken down into secondary products (which do not
adsorb UV light strongly) which leads to a decrease in absorbance.
This method is therefore only useful for monitoring the early stages
of lipid oxidation.
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5. Thiobarbituric acid (TBA)


This is one of the most widely used tests for determining the extent of
lipid oxidation. It measures secondary products of lipid oxidation, mainly

malonaldehyde. It involves reaction of TBA with malonaldehyde to


yield a colored compound that is measured spectrophotometrically.

6. Accelerated Oxidation Tests


It measures susceptibility of lipid to oxidation. Normally, oxidation can
take a long time to occur, e.g., a few days to a few months, which is

impractical for routine analysis. For this reason, a number of accelerated


oxidation tests have been developed to speed up this process. These
methods depend on increasing the rate of lipid oxidation by exposing the
lipid to heat, oxygen, metal catalysts, light or enzymes.
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(D) Analysis of Physicochemical


Properties

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Different analytical techniques can be used to characterize the


physicochemical properties of lipids.
Examples:
Determination of (1) solid fat content, (2) melting point,
(3) cloud point (the temperature at which a cloud is formed in
a liquid lipid due to the beginning of crystallization), (4)
smoke, flash and fire points (smoke point = temperature at
which the sample begins to smoke when tested under specified
conditions, flash point = temperature at which a flash appears at
any point on the surface of the sample, fire point = temperature at
which evolution of volatiles by decomposition of sample proceeds
with enough speed to support continuous combustion).

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Summary
Analysis of Lipids (Fats & Oils)
(A) Determination of of Lipid Composition
(A) Chemical methods:
1- Iodine Value.. Definition, significance and principle (Wijs method-Hanus method).
2- Acid value .. Definition, significance and principle.
3- Saponification value. .. Definition, significance and principle.
+ examples of specific color tests (e.g. Halphens test for cotton-seed oil,
Halphens insoluble bromine test for fish oil, Baudouins test for sesame oil).
and others
(B) Instrumental methods: Chromatography (TLC, GC, HPLC) amd NMR spectroscopy.
(C) Analysis of Lipid Oxidation
(1) Chromatography, (2) Oxygen Uptake, (3) Peroxide value, (4) Conjugated dienes
method, (5) Thiobarbituric acid and (6) Accelerated Oxidation Tests.
(D) Analysis of Physicochemical Properties
(1) solid fat content, (2) melting point, (3) cloud point & (4) smoke, flash and fire points.
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Questions
I- Complete the following sentences:
1- Iodine value is defined as . and used as a measure of ..
2- Acid value is defined as . and used as a measure of ..
3- Saponification value is defined as . and used as a measure of ..
4- The higher the saponification value, the .. will be the mean molecular weight of the
triacylglycerols present.
5- Peroxidene value is defined as . and used as a measure of ..

II- Write the principle of Wijs method.


III- Mention the names of two specific color tests used for lipid analysis.
IV- Enumerate 3 methods used for analyzing lipid oxidation and discuss one of them
V- Define each of the following: cloud point, smoke point, and fire point.

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Thank You
6-Dec-2014