Food Analysis (PC 524)

Level III - 2nd Semester

Dr. Mohamed El-Awady

Lecture 7

6-Dec-2014

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=C→CH-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 + I2 Remaining

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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

sample containing 20 molecules per 1 gram of lipid.

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:

- Halphen’s test for cotton-seed oil.

- Halphen’s insoluble bromine test for fish oil.

- Baudouin’s 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

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

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1. Chromatography.

2. Oxygen Uptake.

3. Peroxide value.

4. Conjugated dienes method.

5. Thiobarbituric acid (TBA).

6. Accelerated Oxidation Tests.

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.

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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

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. Halphen’s test for cotton-seed oil,

Halphen’s insoluble bromine test for fish oil, Baudouin’s 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