Analysis of Lipids

 5.1. 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

  5.2. 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.

5.4. Determination of Total Lipid Concentration

5.4.1. 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.

5.4.2. 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).
 5.5 Determination of Lipid Composition

5.5.1. Introduction

In the previous lecture analytical methods to measure total concentration of lipids in foods
were discussed, without any concern about the type of lipids present. Lipids are an extremely
diverse group of compounds consisting of tri-, di- and monoacylglycercols, free fatty acids,
phospholipids, sterols, caretonoids and vitamins A and D. In addition, most of these sub-groups
are themselves chemically complex. All triacylglycerols are esters of glycerol and three fatty
acid molecules, nevertheless, the fatty acids can have different chain lengths, branching,
unsaturation, and positions on the glycerol molecule. Thus even a lipid which consists of only
triacylglycerols may contain a huge number of different chemical species. It is often important
for food scientists to either know or to be able to specify the concentration of the different types
of lipid molecules present, as well as the total lipid concentration. Some of the most important
reasons for determining the type of lipids present in foods are listed below:

 Legal. Government regulations often demand that the amounts of saturated, unsaturated
and polyunsaturated lipids, as well as the amount of cholesterol, be specified on food
labels.
 Food Quality. Desirable physical characteristics of foods, such as appearance, flavor,
mouthfeel and texture, depend on the type of lipids present.
 Lipid oxidation. Foods which contain high concentrations of unsaturated lipids are
particularly susceptible to lipid oxidation, which can lead to the formation of undesirable
off-flavors and aromas, as well as potentially toxic compounds e.g., cholesterol oxides.
 Adulteration. Adulteration of fats and oils can be detected by measuring the type of lipids
present, and comparing them with the profile expected for an unadulterated sample.
 Food Processing. The manufacture of many foods relies on a knowledge of the type of
lipids present in order to adjust the processing conditions to their optimum values, e.g.
temperatures, flow rates etc.

5.5.2. Sample Preparation

It is important that the sample chosen for analysis is representative of the lipids present in the
original food, and that its properties are not altered prior to the analysis. Analysis of the types of
lipids present in a food usually requires that the lipid be available in a fairly pure form. Thus
foods which are almost entirely lipids, such as olive oil, vegetable oil or lard, can usually be
analyzed with little sample preparation. Nevertheless, for many other foods it is necessary to
extract and purify the lipid component prior to analysis. Lipids can sometimes be extracted by
simply applying pressure to a food to squeeze out the oil, e.g., some fish, nuts and seeds. For
most foods, however, more rigorous extraction methods are needed, such as the solvent or
nonsolvent extraction methods described in the previous lecture. Once the lipids have been
separated they are often melted (if they are not liquid already) and then filtered or centrifuged to
remove any extraneous matter. In addition, they are often dried to remove any residual moisture
which might interfere with the analysis. As with any analytical procedure it is important not to
alter the properties of the component being analyzed during the extraction process. Oxidation of
unsaturated lipids can be minimized by adding antioxidants, or by flushing containers with
nitrogen gas and avoiding exposure to heat and light.

5.5.3. Separation and Analysis by Chromatography

Chromatography is one of the most powerful analytical procedures for separating and
analyzing the properties of lipids, especially when combined with techniques which can be used
to identify the chemical structure of the peaks, e.g., mass spectrometry or NMR. A
chromatographic analysis involves passing a mixture of the molecules to be separated through a
column that contains a matrix capable of selectively retarding the flow of the molecules.
Molecules in the mixture are separated because of their differing affinities for the matrix in the
column. The stronger the affinity between a specific molecule and the matrix, the more its
movement is retarded, and the slower it passes through the column. Thus different molecules can
be separated on the basis of the strength of their interaction with the matrix. After being
separated by the column, the concentration of each of the molecules is determined as they pass
by a suitable detector (e.g., UV-visible, fluorescence, or flame ionization). Chromatography can
be used to determine the complete profile of molecules present in a lipid. This information can
be used to: calculate the amounts of saturated, unsaturated, polyunsaturated fat and cholesterol;
the degree of lipid oxidation; the extent of heat or radiation damage; detect adulteration;
determine the presence of antioxidants. Various forms of chromatography are available to
analyze the lipids in foods, e.g. thin layer chromatography (TLC), gas chromatography (GC), and
high pressure liquid chromatography (HPLC).

5.7. Characterization of Physicochemical Properties

5.7.1. Introduction

In addition to their nutritional importance lipids are also used in foods because of their
characteristic physicochemical properties, such as mouthfeel, flavor, texture and appearance.
They are also used as heat transfer agents during the preparation of other foods, e.g. for frying. It
is therefore important for food scientists to have analytical techniques that can be used to
characterize the physicochemical properties of lipids.

Melting point

In many situations, it is not necessary to know the SFC over the whole temperature range,
instead, only information about the temperature at which melting starts or ends is required. A
pure triacylglycerol has a single melting point that occurs at a specific temperature. Nevertheless,
foods lipids contain a wide variety of different triacylglycerols, each with their own unique
melting point, and so they melt over a wide range of temperatures. Thus the "melting point" of a
food lipid can be defined in a number of different ways, each corresponding to a different
amount of solid fat remaining. Some of the most commonly used "melting points" are:

 Clear point. A small amount of fat is placed in a capillary tube and heated at a controlled
rate. The temperature at which the fat completely melts and becomes transparent is called
the "clear point".
  Slip point. A small amount of fat is placed in a capillary tube and heated at a controlled
rate. The temperature at which the fat just starts to move downwards due to its weight is
called the "slip point".
 Wiley melting point. A disc of fat is suspended in an alcohol-water mixture of similar
density and is then heated at a controlled rate. The temperature at which the disc changes
shape to a sphere is called the "Wiley melting point".

5.7.4. Cloud point

This gives a measure of the temperature at which crystallization begins in a liquid oil. A fat
sample is heated to a temperature where all the crystals are known to have melted (e.g., 130oC).
The sample is then cooled at a controlled rate and the temperature at which the liquid just goes
cloudy is determined. This temperature is known as the cloud point, and is the temperature where
crystals begin to form and scatter light. It is often of practical importance to have an oil which
does not crystallize when stored at 0oC for prolonged periods. A simple test to determine the
ability of lipids to withstand cold temperatures without forming crystals, is to ascertain whether
or not a sample goes cloudy when stored for 5 hours at 0oC.

5.7.5. Smoke, Flash and Fire Points

These tests give a measure of the effect of heating on the physicochemical properties of
lipids. They are particularly important for selecting lipids that are going to be used at high
temperatures, e.g. during baking or frying. The tests reflect the amount of volatile organic
material in oils and fats such as free fatty acids.

 The smoke point is the temperature at which the sample begins to smoke when tested
under specified conditions. A fat is poured into a metal container and heated at a
controlled rate in an oven. The smoke point is the temperature at which a thin continuous
stream of bluish smoke is first observed.
 The flash point is the temperature at which a flash appears at any point on the surface of
the sample due to the ignition of volatile gaseous products. The fat is poured into a metal
container and heated at a controlled rate, with a flame being passed over the surface of
the sample at regular intervals.
 The fire point is the temperature at which evolution of volatiles due to the thermal
decomposition of the lipids proceeds so quickly that continuous combustion occurs (a
fire).