V
Vegetable Oils: Composition and Analysis
AJ Dijkstra
ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Edible oils and fats have been produced for centuries, so it is
not surprising that their analysis has a long history. The French
chemist Michel Eugène Chevreul, who can be regarded as the
first lipid chemist, had a book published in 1823 in which
he coined names like stearic acid, oleic acid, and butyric
acid, all compounds he discovered and described. He also
reported melting points, which he used to assess the purity of
compounds obtained by recrystallization – a method he devel-
oped – and to define the titer of fats.
One of the fatty substances that Chevreul investigated was
cholesterol, which he obtained from human gallstones. He
noted that it differed fundamentally from oils and fats in that
he could not saponify the cholesterol. Oils and fats were used
extensively in soap works and left some ‘unsaponifiables.’
Accordingly, oils and fats also became characterized by their
unsaponifiable content.
Subsequent quantitative analysis made use of what methods
were available at the time. Early on, a number of titrimetric
methods were developed, the results of which were reported as
‘values’: acid value, iodine value (IV), etc. Fatty acid composi-
tions started to be determined, but as explained by Gunstone,
this determination took a long time (http://lipidlibrary.aocs.
org/history/beforechrom/index.htm). This became much easier
with the advent of gas–liquid chromatography (GLC), first with
packed columns and later with capillary columns. The latter
enabled separations between isomers, first cis–trans and later
also with respect to positional isomers of unsaturated fatty
acids. GLC was combined with mass spectrometry; HPLC was
used for analysis of the triglyceride composition with silver-ion
HPLC providing a means to separate triglycerides according to
their degree of unsaturation. Liquid chromatography was com-
bined with mass spectrometry, and more sophisticated analytic
techniques were developed for a research environment.
Perhaps, the most useful method to characterize a fat is by
its solid fat content (SFC). Its determination makes use of a
‘time-domain NMR’ technique that is also referred to as ‘pulse
NMR.’ In fact, the equipment used for this determination was
specifically developed for fat characterization and replaced the
laborious dilatometric method. The same equipment can also
be used to determine the oil content of oilseeds.
Encyclopedia of Food and Health http://dx.doi.org/10.1016/B978-0-12-384947-2.00708-X
Composition of Vegetable Oils
Saponifiable Fraction
The saponifiable fraction of vegetable oils, usually at least 98%
of the oil, is characterized in that it forms soaps (salts of fatty
acids), when the oil is treated with an alkali such as sodium
hydroxide. The fraction contains several different classes of
compounds that will be discussed later. The main constituent
of the saponifiable fraction is triglyceride oil, but some of this
oil has been hydrolyzed so that this fraction also contains
partial glycerides and free fatty acids (FFAs).
Fatty acids
Nearly all naturally occurring fatty acids are carboxylic acids
with a straight chain and an even number of carbon atoms. If
they contain double bonds, these are mostly in the cis-
configuration, and in polyunsaturated fatty acids, the double
bonds tend to be methylene-interrupted. This is illustrated by
the molecular structure of linoleic acid shown below:
18 1
O
C H
12 9
O
In this molecule, the carbon atom of the carboxyl group has
number 1. The first double bond starts at carbon atom number
9 and the second one at number 12. The systematic name of
this fatty acid is therefore (9Z,12Z)-octadeca-9,12-dienoic acid.
Because this is rather a mouthful, scientific literature often uses
a shorthand notation: 9c12c-18:2. This notation starts with the
location and configuration of the double bonds. Then, the
number of carbon atoms of the fatty acid chain is given,
which is followed by the number of unsaturated bonds. This
shorthand notation is explained in http://lipidlibrary.aocs.org/
education/names/FAnames1.htm.
When a fatty acid is synthesized in plants, it is elongated at
its carboxyl end. Consequently, the position of a double bond
does not change with respect to the other, methyl end. So,
there are families of fatty acids according to the distance of
the last double bond to the methyl end. This distance can be
indicated with the prefix n or o. The linoleic acid shown in the
preceding text is part of the n-6 or o-6 family of fatty acids.
357
358 Vegetable Oils: Composition and Analysis

Table 1 lists some fatty acids that kept their trivial names, 2-random. This is illustrated by the soybean oil data in
their shorthand notations, and sources. Table 2. The saturated fatty acids 16:0 and 18:0 predominantly
occupy the outer or 1,3-positions in equal amounts: 18:2
Triglycerides mainly occupies the 2-position and 18:1 fills the empty posi-
The official name of a triglyceride molecule is ‘triacylglycerol,’ tions. An example of the fatty acid distributions for a few oils is
indicating that it consists of a glycerol backbone that is ester- given in Table 2.
ified by three fatty acids as shown below:
Partial glycerides
O
The hydrolysis of triglycerides leads to the formation of FFAs
C R1
and di- and monoglycerides: diacylglycerol and acylglycerol.
H2C O
Often, this hydrolysis is catalyzed by a lipase enzyme present in
O CH
the raw material. This is the reason that palm fruit bunches are
R2 C H2C O
sterilized before the oil is extracted from the fruitlets and that
O C R3
rice bran profits from being heated in an expander/extruder as
O
soon as possible. Both partial glycerides have two positional
The fatty acid chains R1, R2, and R3 can be the same but isomers: 1,2- and 1,3-diglyceride and 1- and 2-monoglyceride.
seldom are. Only oils with a very high content of a single fatty The compounds with the fatty acids at the outer position are
acid such as oleic acid in ‘high-oleic sunflower seed oil’ will thermodynamically more stable. So, whereas statistically, the
contain a triglyceride with three identical fatty acids, in this 2-monoglyceride fraction is 1/3, at equilibrium, it is only 10%.
case trioleate. Normally, the fatty acids are different so that a Monoglycerides are so volatile that most will be removed
triglyceride oil contains very many different molecular species. from the oil during vacuum stripping. They can be synthesized
In vegetable oils, the distribution of the fatty acids over the by glycerolysis of triglycerides and then concentrated by
glycerol backbone is not fully random but 1,3-random, molecular distillation. They are used as food grade emulsifiers
and serve as starting material for various other emulsifiers
Table 1 Trivial names, shorthand notations, and occurrence of and oleochemicals. Diglycerides remain in the oil during pro-
several fatty acids cessing. They do not act as emulsifier but can be used in food as
a kind of fat substitute for obese and diabetic patients.
Trivial name Notation Source

Butyric acid 4:0 Butter Vegetable waxes

Caproic acid 6:0 Butter Oils containing waxes comprise sunflower seed oil, rice bran
Caprylic acid 8:0 Coconut oil, palm kernel oil oil, corn germ oil, safflower seed oil, and grape-seed oil. Veg-
Capric acid 10:0 Coconut oil, palm kernel oil etable waxes are esters of fatty acids and fatty alcohols. Fatty
Lauric acid 12:0 Coconut oil, palm kernel oil
alcohols are monohydric long-chain alcohols. If both the fatty
Myristic acid 14:0 Coconut oil, palm kernel oil
acid and the fatty alcohol are saturated, the resulting wax will
Palmitic acid 16:0 Palm oil
Stearic acid 18:0 Cocoa butter, shea butter have a high (>70
C) melting point as a result of which oils
Oleic acid 18:1(n-9) All vegetable oils containing such waxes will throw an unsightly deposit when
Linoleic acid 18:2(n-6) All vegetable oils cooled. When the alcohol is unsaturated, the melting point of
a-Linolenic 18:3(n-3) Linseed oil, soybean oil, CanolaW the wax is much lower and the wax will remain dissolved when
acid the oil is cooled.
g-Linolenic 18:3(n-6) Evening primrose oil, blackcurrant, seed Wax compositions published in the literature are rather
acid oil, borage oil contradictory. They also depend on whether they refer to the
Arachidic acid 20:0 Groundnut oil crystalline wax fraction or the total wax content of the oil.
Gondoic acid 20:1(n-9) High-erucic acid rapeseed (HEAR) oil
However, there seems to be some kind of consensus that the
Behenic acid 22:0
main fatty acids in the crystalline waxes are 20:0, 22:0, and
Erucic acid 22:1(n-9) HEAR
24:0 and that the main alcohols are 24:0, 26:0, and 22:0. This

Table 2 Positional distribution of fatty acids in vegetable oils

Oil Position 8:0 10:0 12:0 14:0 16:0 18:0 18:1 18:2 18:3

Coconuta 1,3 11.4 9.1 32.7 22.2 13.2 4.0 5.6 1.1
2 2.9 1.1 78.2 10.2 5.9 2.0
Palm oilb 1,3 62.0 8.0 27.5 2.5
2 11.0 2.0 65.0 22.0
Soybean oilc 1 16.7 5.4 24.3 46.2 6.4
2 2.2 0.3 23.4 68.1 5.7
3 16.1 4.6 24.6 47.0 7.0
a
Source: Caro, Y. et al. (2004). European Journal of Lipid Science Technology 106, 503–512.
b
Source: Table 3.162 in Gunstone, F. D., Harwood, J. L. and Padley, F. B. (eds.) (1994). The lipid handbook (2nd ed.). London: Chapman & Hall.
c
Source: Table 2.28 in Gunstone, F. D., Harwood, J. L. and Dijkstra, A. J. (eds.) (2007). The lipid handbook (3rd ed.). Boca Raton, FL: CRC Press.
 Vegetable Oils: Composition and Analysis 359

is to some extent confirmed by the carbon number composi-
tion of the waxes themselves that shows a range of 38–52 for
the crystalline waxes and a somewhat lower range for the waxes
that also contain unsaturated waxes and peaks of around 44.
According to some authors, waxes also contain odd-numbered
fatty acid and/or fatty alcohol chain lengths.
Phosphatides
Phosphatides are diglycerides that have been esterified with a
phosphate group at the sn-3 position that itself can again be
esterified with the hydroxyl group of compounds indicated as
X: choline, ethanolamine, or inositol. The resulting phospha-
tides are called phosphatidylcholine (PC), phosphatidylethan-
olamine (PE), and phosphatidylinositol (PI). The structure
in which R1 and R2 represent fatty acid chains is given in the
succeeding text:
O R1
C R1
H2 C O
O CH
O CH3
R2 C R3
H2 C O P O X
O
O In addition to one or more methyl groups, the benzene ring
The phosphate group has a free hydroxyl group left that is
quite acidic (pKa < 3.5). In crude oil, it may have a metal
counterion (potassium, calcium, or magnesium) or hydrogen.
This hydrogen is titrated when the acidity of the oil is mea-
sured. When the group X is a hydrogen atom, the phosphatide
is called phosphatidic acid (PA). It is formed during the drying,
conditioning, and extraction of the oilseeds. If PA is present as
a free acid or potassium salt, it will be removed from the oil by
hydration on water degumming. If PA is present as a calcium or
magnesium salt, this salt will remain in the oil on water
degumming as a so-called nonhydratable phosphatide.
Besides PC, PE, PI, and PA, there are some minor phospha-
tides such as phosphatidylserine in which the phosphate group
has been esterified with the amino acid serine. There are the
lysocompounds in which one of the fatty acids moieties has
been eliminated. There is the acetylphosphatidylethanolamine
in which the amino group has been acetylated. There is the
phosphatidylglycerol in which the phosphate group has been
esterified with a glycerol moiety.
Unsaponifiable Fraction
The unsaponifiable fraction of vegetable oils can be isolated by
extracting the soaps that result from oil saponification with an
organic solvent (diethyl ether).
Table 3 Molecular structures of tocopherols and tocotrienols
Type R1 R2 R3
a CH3 CH3 CH3
b CH3 H CH3
g H CH3 CH3
d H H CH3
The benzene ring on the left has three substituents named
R1, R2, and R3. The extent to which these substituents are
methyl groups determines the type in accordance with Table 3.
Accordingly, a-tocopherol, which has the highest vitamin E
activity, carries three methyl substituents on its benzene ring
and d-tocopherol only one. The same nomenclature is valid for
the tocotrienols illustrated below:
HO CH3 CH3 CH3
R2 O CH3
also carries a hydroxyl group. This group can be esterified, but in
vegetable oils, only the free tocopherols are present. Accordingly,
they act as antioxidants according to the same mechanism as the
synthetic antioxidants butylated hydroxytoluene (BHT) and
butylated hydroxyanisole (BHA), which also have a stereoche-
mically hindered phenol group.
Vegetable oils vary widely in their tocopherol and tocotrie-
nol contents as illustrated by Table 4.
Given their molecular weight of about 400 Da, which is not
that different from that of monoglycerides, it is only to be
expected that some tocopherols will be removed from the oil
during vacuum steam stripping. The amounts depend of course
on the initial concentration of the tocopherol and the stripping
process conditions (relative amount of stripping medium, sys-
tem pressure, and oil temperature). By using a two-stage con-
densing system, a condensate that is rich in tocopherols can be
isolated. It can be used to enrich oils that are poor in tocoph-
erols and thus increase their oxidative stability.
Phytosterols
Phytosterols are the sterols that occur in plants. Their molecu-
lar structure is similar to that of cholesterol, the main sterol in
animals. Its structure has been represented in the succeeding
text together with the position numbers that refer to the double
bonds and substituents:

Tocopherols and tocotrienols 22 24

H3 C
The general structural formula of the tocopherols is given CH3
below: H
1 CH3 H
R1
5 4 H
HO 6 H
3 CH3 CH3 CH3 7
2 HO 5
3
R2 7 O 4⬘ 8⬘ CH3
8 CH3 How these various phytosterols differ from cholesterol has
1
R3 been indicated in Table 5.
360 Vegetable Oils: Composition and Analysis

Table 4 Tocopherol and tocotrienol contents of various vegetable oils

Tocopherols (mg/100 g) Tocotrienols (mg/100 g)

Oil a b g Total a b g d Total

Palm 26 32 7 65 14 3 29 7 53
Soybean 10 59 26 96 0
Canola 17 35 1 53 0
Sunflower 49 5 1 55 0
Coconut 0.5 0.6 1 0.5 2 0.6 3
Corn germ 11 5 60 2 78 0
Wheat germ 121 65 24 25 235 2 17 19
Olive 20 1 1 22 0
Rice bran 12 4 5 21 18 2 57 77

Source: Table 2.48 in Gunstone, F. D., Harwood, J. L. and Dijkstra, A. J. (eds) (2007). The lipid handbook (3rd ed.), Boca Raton, FL: CRC Press.

Table 5 Molecular structures of various phytosterols

Position of additional methyl Position of double bond in ring Position of double bond Substituent in side
Phytosterol group system in side chain chain

Cholesterol – 5 – –
Brassicasterol – 5 22 24-Methyl
Campesterol – 5 – 24-Methyl
Stigmasterol – 5 22 24-Ethyl
b-Sitosterol – 5 – 24-Ethyl
D5-Avenasterol – 5 – 24-Ethylidene
D7-Avenasterol – 7 – 24-Ethylidene
D7-Stigmasterol or – 7 22 24-Ethyl
spinasterol
Citrostadienol 4 7 – 24-Ethylidene

All phytosterols have a hydroxyl group at the 3-position. In
vegetable oils, this group can be esterified with a fatty acid but
if it is not, it is referred to as a free sterol. Like tocopherols, free
phytosterols will also be partially removed from the oil during
vacuum stripping; esterified sterols are insufficiently volatile
and remain in the oil.
When the double bond in the sterol is saturated, the resulting
compound is called a stanol. Cholesterol yields cholestanol on
hydrogenation. Phytostanols are used in dietary fat products like
margarine because they reduce the cholesterol absorption from
food and thus lead to a somewhat reduced blood serum choles-
terol content, which some people consider to be desirable.
Its molecular formula (C40H56) and conjugated polyene
structure show that it is a polyisoprene-based molecule. The
structural formula given above relates to the synthetic, all-trans
isomer. Crude vegetable oils may also contain isomers with
one or more cis double bonds. b, b-Carotene is a precursor of
vitamin A. In addition to this, vegetable oils can also contain
hydroxyl-substituted compounds such as lutein, violaxanthin,
and neoxanthin. All of them are thermally unstable. So, when
oil containing these compounds is heated, they decompose in
the so-called heat bleaching process.
Chlorophyll is an ester of a chlorophyllic acid and a phytol
alcohol. Its structure is given in the next column:

R1

Coloring compounds
Vegetable oils contain two kinds of coloring compounds: N N
carotenes and chlorophyll. The carotenes give the oils an Mg
orange–red color that is very pronounced in crude palm oil.
N N
The chlorophylls and their derivatives are responsible for the
green color in crude oils.
The structural formula of b, b-carotene is shown below:
R2 O
R3
O
O

The chlorophyllic acid moiety on the left contains a mag-

nesium ion. If this has been replaced by two hydrogen ions,
 Vegetable Oils: Composition and Analysis 361

Table 6 Molecular structures of various chlorophyll-related compounds (0.3–0.6%). The latter compound can release sesamol during
oil processing. The molecular structures of these compounds
Derivative Type R1 R2 R3 are given in the succeeding text:
Chlorophyll a CH3 CO2CH3 H O
O
a0 CH3 H CO2CH3 O
O
b CHO CO2CH3 H O
b0 CHO H CO2CH3 O HO
Pheophytin a CH3 CO2CH3 H
a0 CH3 H CO2CH3 O O O
O
b CHO CO2CH3 H O
b0 CHO H CO2CH3 O
Sesamin O Sesamolin O Sesamol
Pyropheophytin a CH3 H H O
b CHO H H
The presence of sesamol can be detected by the reaction of
Source: Zschau, W. (2000). Bleaching. In: O’Brien, R. D., Farr, W. E. and Wan, P. J. Baudouin in which the oil is treated with hydrochloric acid and an
(eds). Introduction to fats and oils technology. Champaign, IL: AOCS Press. ethanolic solution of furfural; a red color results. For a long time,
Belgian margarine had to contain 2% of sesame oil by law to
the resulting compound is called pheophytin. The structure enable the adulteration of butter with margarine to be detected.
contains several groups indicated by R1, R2, and R3. Their Rice bran oil also contains unsaponifiable compounds that
nature determines the chlorophyll derivative in accordance are particular to this oil: oryzanols. They are esters of ferulic
with Table 6. acid (4-hydroxy-3-methoxycinnamic acid) and cycloartenol
In this table, the a- and b-types differ with respect to the R1 (oryzanol A) or 24-methylenecycloartanol (oryzanol B). The
substituent. In the a-type, this is a methyl group, whereas in the structure of oryzanol A is given in the succeeding text:
b-type, this is a formaldehyde group. The difference between H 3C
OH
the types without and with prime is stereochemical with CH3
respect to the dimethyl ether group. H3CO H
H
O
Miscellaneous unsaponifiable compounds CH3
The presence of unsaponifiable compounds necessarily precedes O
their molecular identification, but they could have been given a H3C CH3
name when isolated. So shea butter, ‘beurre de karité’ in French,
Hydrocarbons cannot be saponified. Vegetable oils can
was found to contain a substantial unsaponifiable fraction that
contain various hydrocarbons not all of which have been
did not dissolve in acetone. It was given the name ‘karitene,’ but
identified. A hydrocarbon of which the structure has been
this name does not stand for a compound in true chemical
determined is squalene:
sense; it is the name given to a fraction that is highly unsaturated
and probably consists of a mixture of different polyisoprenes.
An unsaponifiable compound that is specific for cottonseed oil
is gossypol; its molecular structure is shown below:
O O Its systematic name is (6E,10E,14E,18E)-2,6,10,15,19,
OH OH
23-hexamethyl-2,6,10,14,18,22-tetracosahexaene. Squalene is
HO OH
found in several vegetable oils in variable amounts. Olive oil
contains 700–1200 ppm of squalene, which is about half the
HO OH total unsaponifiable. Squalene is the precursor of phytosterols,
which becomes clear when the molecular structure is represented
in a different manner next to cholesterol, as shown below:
The systematic name of this compound is 1.10 ,6,60 7,70 -
hexahydroxy-3,30 -dimethyl-5,50 -bis(1-methylethyl)-[2,20 bi- H3C
CH3
naphthalene]-8,80 -dicarboxaldehyde. Most of the gossypol H
remains in the meal, but the ()-form in cottonseed is CH3 H
toxic to non-ruminants. It is removed from the oil during
H H
the neutralization process since the many phenolic
HO
hydroxyl groups react with the alkali.
Squalene Cholesterol
Cottonseed oil also contains some fatty acids (sterculic acid
and malvalic acid) that contain a cyclopropene ring. This
feature is used for the detection of cottonseed oil by the
so-called Halphen test. Kapok seed oil also contains these Analysis of Vegetable Oils
cyclopropenoid acids and also gives a positive Halphen test.
Sesame oil contains two unsaponifiable compounds that Because oils and fats are traded and often have to meet specifica-
are typical for this oil: sesamin (0.5–1.1%) and sesamolin tions, it is necessary to use standard analytic methods for these
362 Vegetable Oils: Composition and Analysis
specification parameters. Consequently, what is now the American
Oil Chemists’ Society (AOCS) started as the Cotton Products Ana-
lysts. The AOCS therefore publishes ‘AOCS Official Methods and
Recommended Practices’ after having tested proposed methods
or changes, by having them evaluated by several laboratories;
other national societies like the DGF (Deutsche Gesellschaft für
Fettforschung) and countries also publish their own methods.
Points and Values
When early analytic methods became established, their results
were expressed as ‘points’ or ‘values.’ Points refer to tempera-
tures and values have other units.
Slip melting point
Oils and fats are mixtures of a large number of different tri-
glycerides. Therefore, they do not have a sharp melting point
but a melting range. Fats are also polymorphic, and different
polymorphs have different melting points. Consequently, the
temperature at which the fat is completely molten depends on
how this melting point is determined. The agreed method to
determine the ‘slip melting point’ measures the temperature at
which a column of fat of specified length starts to rise in an
open capillary tube under the influence of a hydrostatic pres-
sure by placing the capillary in a water bath that is heated.
Cloud point
The cloud point of an oil is the temperature at which the oil
becomes hazy when it is cooled down. This temperature is
especially important for oils that could solidify during
transport, and it is also used when estimating the wax content
of oil to be winterized. Strict adherence to the method is
essential for attaining a reasonable repeatability.
Smoke point
The smoke point is the temperature at which smoking is first
detected when the oil is heated in a special cabinet with special
illumination. The smoke point decreases from normal values
>200
C when the FFA content of the oil increases as during
deep fat frying.
Acid value
The acid value of an oil is determined by titrating a solution of
the oil in diethyl ether with an alcoholic solution of sodium or
potassium hydroxide. It is expressed as the amount of KOH (in
mg) to neutralize 1 g of oil.
Often, the acid value is converted to an FFA content by
multiplying the acid value with a factor that equals the molec-
ular weight of the fatty acid concerned (usually oleic acid,
MW ¼ 282.4) divided by ten times the molecular weight of the
potassium hydroxide (56.1). This factor ten stems from the fact
that the acid value is expressed as mg/g, whereas the FFA content
is expressed as a percentage. When the FFA content is expressed
as ‘wt% oleic acid,’ this factor therefore equals 0.50.
Saponification value
Whereas the acid value only takes FFAs into account, the
saponification value (SV) refers to the FFA plus any ester
bonds present in the sample. It is determined by saponifying
a known amount of sample with a known amount of ethanolic
potassium hydroxide and titrating the excess of potassium
hydroxide with hydrochloric acid. Like the acid value, the SV
is also expressed as mg KOH per gram of oil.
The SV is an indication of the average molecular weight of
the oil. HEAR with relatively few ester bonds per unit of weight
has therefore a low SV of 174–176. Oils with predominantly
fatty acids with 18 carbon atoms like sunflower seed oil or
soybean oil have an SV of 191–194, whereas lauric oils like
coconut oil and palm kernel oil have SVs of 255–270 mg KOH
per gram of oil. Because of these differences, it is quite under-
standable that the SV determination has been an essential tool
in vegetable oil characterization.
Ester value
The ester value of an oil is not determined directly. It is calcu-
lated as the difference between the SV and the acid value. It is
not much used.
Hydroxyl value
The hydroxyl value (HV) of an oil is an indication of its
hydroxyl group content. The hydroxyl groups can be part of
fatty acid moieties such as ricinoleic acid (9Z,12R)-
12-hydroxyoctadecenoic acid, but they can also be part of the
glycerol moiety when this is not esterified as in partial glycer-
ides. So, in oils without fatty acids with hydroxyl groups, the
HV is an indication of the partial glyceride content.
The HV is determined by acetylating the free hydroxyl
groups with an excess of acetic acid anhydride, hydrolyzing
the excess, and determining the acetic acid content of the final
reaction mixture. As usual in this kind of determination, a
blank is used to avoid systematic errors. The HV is also
expressed as mg KOH per gram of oil.
Iodine value
The IV is perhaps the most often quoted value of an oil. It is a
measure of the degree of unsaturation. It is determined by
weighing an amount of sample, the mass of which depends
on the expected IV. When this value is low (0–5 g I2/100 g oil),
a sample mass of about 3 g is appropriate, whereas when the
IV > 130, a smaller amount of 0.10 g suffices. The sample is
dissolved in 25 ml tetrachloromethane, and then, 25 ml of
Wijs solution is added. This solution of iodine monochloride
(ICl) in glacial acetic acid is commercially available as such.
After mixing, the reaction mixture is allowed to stand in the
dark for 1 or 2 h for the addition of the ICl to the double bonds
to take place.
After standing, an amount of 20 ml of an aqueous 10%
potassium iodide solution is added with some (150 ml) addi-
tional water after which the iodine content of the mixture is
determined by titration with sodium thiosulfate. By carrying
out a blank determination with the same amounts of reagents,
the amount of iodine added to the double bonds in the sample
can be calculated by difference.
The IV is actively used in the hydrogenation of vegetable
oils. Its value describes the extent to which an oil has been
hydrogenated. Fully hydrogenated oils specify an IV of <1. The

IV is also used to characterize an oil so as to have an idea about
its likely stability.
Peroxide value
When oil is stored, it can deteriorate by oxidation. Since one of
the early oxidation products is a peroxide, the peroxide value
(PV) of an oil provides information how fresh the oil is. It is
determined by allowing the peroxides in the oil to liberate
molecular iodine from a saturated solution of potassium
iodide. The liberated iodine is then titrated with sodium thio-
sulfate. The PV is expressed as mmol of active oxygen per 2 kg
of oil (Wheeler method) and of per 1 kg of oil (Sully method).
Anisidine value
When primary oxidation products decompose, they can form
aldehydes and ketones. The former, especially 2,4-dienals and
2-alkenals, can be determined by allowing them to react with
p-anisidine. The reaction product is yellow, so its amount is
determined by measuring its absorbance at 350 nm against a
blank in the reference cell (Eb). The absorbance of the oil itself
is also measured against pure isooctane in the reference cell
(Ea). The anisidine value is then calculated according to
25  ð1:2  Eb  Ea Þ
p-AV ¼
W rather than on triglycerides also affected nutritional studies in
wherein W stands for the mass of the sample in grams. To
distinguish the anisidine value from the acid value, the former
is often written as p-AV so that the AV refers to the acid value.
Totox value
The Totox value is defined as the sum of the anisidine value
and two times the PV (p-AV þ 2 PV). It is an arbitrary value to
indicate the oxidative load an oil has had to suffer.
Miscellaneous values
When it was not yet possible to determine a fatty acid compo-
sition by GLC, other methods were used to get an idea about
this composition. The Reichert–Meissl value indicates the
number of ml 0.1 N lye that is required to neutralize the
water-soluble fatty acids that have been volatilized with water
vapor from a 5 g fat sample. The Polenske value is similar in
that it also indicates how many ml 0.1 N lye are required to
neutralize certain fatty acids from a 5 g sample, but the differ-
ence is that the Reichert–Meissl value is concerned with the
water-soluble fatty acids (butyric acid and caproic acid),
whereas the Polenske value titrates the volatile fatty acids that
do not dissolve in water (caprylic acid and capric acid).
The determinations of these values are difficult to repeat,
and the outcomes are not an accurate representation of the
fatty acids concerned. In fact, these values are well and truly
obsolete, but in the past, they have been useful to establish the
adulteration of butter with margarine.
The Crismer value measures the solubility of an oil in a
standard solvent mixture of t-amyl alcohol, ethanol, and water
(5:5:0.27 by volume). Values are characteristic for each oil
since this solubility depends on degree of unsaturation and
fatty acid chain length. It was used in international trade,
mostly in Europe.
Vegetable Oils: Composition and Analysis 363
Instrumental Analytic Methods
Some instrumental analytic methods have been used for a long
time. The refractive index of an oil can be measured with the
Abbe refractometer to indicate the degree of unsaturation of a
vegetable oil. The higher its IV, the higher the index. It can be
used to follow the progress of a hydrogenation batch.
When UV spectrophotometers became available, they
started to be used in the determination of the fatty acid com-
position. Conjugated dienes and trienes absorb at 232 and
268 nm, respectively. So, after conjugation of the polyunsatu-
rated fatty acids, their content could be quantified. The
spectrophotometric method can also be used to assess oil
deterioration.
Chromatographic Methods
The advent of GLC greatly facilitated lipid analysis. It is fast,
reproducible, and quantitative. In the early days of GLC,
packed columns were used and their performance only
allowed a fatty acid analysis. Consequently, research in many
field focused on the fatty acid composition of lipids rather than
on their triglyceride composition. The hydrogenation process
is a typical example of this focusing. This focus on fatty acids
which insufficient attention was given to the fact that in the
body, fatty acids on the outer position of a triglyceride are
digested through a different mechanism as those on the middle
position.
Later, with capillary columns, GLC could also tackle triglyc-
eride analysis and distinguish between fatty acid isomers and
trans fatty acids. Progress was made in the analysis of triglycer-
ides by GLC for the determination of the carbon number
composition, and this complemented the analysis HPLC deter-
mination of the partition number composition. These and
other chromatographic methods are available from The Lipid
Library (http://lipidlibrary.aocs.org/analysis.html).
Nuclear Magnetic Resonance
Two routine measurements make use of nuclear magnetic res-
onance methods. One is the low-resolution pulse NMR
method to determine the SFC of a mixture of solid, crystalline
fat, and liquid oil. It is used intensively to follow the partial
hydrogenation of vegetable oils and for specifying fat blends to
be used in margarines and shortenings, and it also plays a vital
role in confectionery fats.
The method involves melting the fat sample at 80
C in a
special glass tube that fits the NMR spectrometer, tempering
the sample at 60
C for at least 5 min, cooling it for a set period
of 60 min at 0
C, and transferring it to the measuring temper-
ature and keeping it there for 30–35 min before transferring it
to the NMR instrument. This sounds very labor-intensive, but
the transfers of the glass tubes to the various thermostatic baths
and the measuring instrument can be fully automated by lab-
oratory robotics.
For cocoa butter and other fats like cocoa butter equivalent
having a high content of symmetrical monounsaturated tri-
glycerides, the thermal treatment of the sample is slightly
364 Vegetable Oils: Composition and Analysis
different in that it also comprises a tempering stage. After
having been placed in the 0
C bath for 90 min, the samples
are tempered at 26
C for 40 h. Then, they are cooled again at
0
C for another 90 min before being brought to the measuring
temperature where they are kept for 60 min before being trans-
ferred to the NMR instrument.
Another NMR method used in lipid chemistry uses the
31
P NMR spectrometer for the determination of phosphatide
compositions. It uses triphenyl phosphate as internal standard
and thus arrives at absolute values for the contents of the
various phosphatides. Often, these contents do not account
for the total phosphorus content of the sample as determined
by inductively coupled plasma emission spectroscopy (ICP-ES)
(see later). The reason for this discrepancy is not known, but
the presence of phytates has been suggested and after all, you
do not need much phytates to introduce a lot of phosphorus.
Inductively Coupled Plasma Emission Spectrometry
Originally, the analytic method using ICP was used to deter-
mine the trace element contents of aqueous samples, for
instance, effluents. In 1982, the use of this ICP equipment to
measure the concentration of these elements in oils was
published, and now, the method has become a standard tool
for the control of the degumming process, and it has been
incorporated in AOCS compendium of analytic methods that
can be used when specifying oil in trading contracts.
Oil Stability Index
The oil stability index (OSI) of an oil is determined with a piece
of equipment called Rancimat®. In the equipment, samples of
oil are kept at a set temperature, and air is bubbled through the
sample at a set rate. The air leaving the sample is bubbled
through distilled water, the conductivity of which is moni-
tored. In the early stages of the measurement, this conductivity
hardly changes, but after a certain period, called the ‘induction
period,’ it increases because the air has stripped volatile acids
like formic acid from the oil sample. The conductivity curve
shows an inflection and the point in time where this extrapo-
lated inflection commences is called the ‘OSI’; it is expressed in
hours, and the temperature at which the experiment was car-
ried out should be mentioned as well.
The OSI has been found to be determined by the fatty acid
composition of the sample and its antioxidant content. Exper-
iments have been carried out in which oil was stored under
different conditions and its OSI was measured at set intervals.
Sometimes, the OSI did not alter from one interval to the
other. This allows the conclusion to be drawn that the OSI
does not predict shelf life. All it can do is reveal batches that
deviate from normal.
See also: Carotenoids: Occurrence, Properties and Determination;
Choline: Physiology; Chromatography: Focus on Multidimensional
GC; Chromatography: High-Performance Liquid Chromatography;
Colors: Properties and Determination of Natural Pigments; Fats:
Classification and Analysis; Fatty Acids: Fatty Acids; Fatty Acids: Trans
Fatty Acids; Oxidation of Food Components; Phospholipids: Properties
and Occurrence; Quality Control in Food Processing; Rapeseed Oil/
Canola; Sunflower Oil; Tocopherols: Properties and Determination;
Triacylglycerols: Structures and Properties; Triacylglycerols:
Characterization and Determination.
Further Reading
AOCS (2013) Official methods and recommended practices of the AOCS, 6th ed.
Urbana, IL: AOCS Press.
Byrdwell WC (2005) Modern methods for lipid analysis by liquid chromatography/mass
spectrometry and related techniques. Urbana, IL: AOCS Press.
Chevreul M-E (2009) A chemical study of oils and fats of animal origin, translated and
annotated by Albert J. Dijkstra. Urbana, IL: AOCS Press.
Christie WW (1987) HPLC of lipids. Oxford: Pergamon Press.
Christie WW (2003) Lipid analysis, 3rd ed. Bridgwater: The Oily Press.
Dijkstra AJ and Meert D (1982) Determination of trace elements in oils by plasma
emission spectroscopy. Journal of the American Oil Chemists Society 59: 199–204.
Dijkstra AJ, Maes PJ, Meert D, and Meeussen WLJ (1996) Interpreting the oil stability
index. Oléagineux Corps Gras Lipides (OCL) 3: 378–386.
Gunstone FD and Harwood JL (2007) Occurrence and characterisation of oils and fats.
In: Gunstone FD, Harwood JL, and Dijkstra AJ (eds.) The lipid handbook, 3rd ed.,
pp. 37–142. Boca Raton, FL: Taylor & Francis Group, LLC.
Gunstone FD, Harwood JL, and Dijkstra AJ (2007) The lipid handbook, 3rd ed. Boca
Raton, FL: Taylor & Francis.
Hamilton RJ (2008) Fatty acids: structure, occurrence, nomenclature, biosynthesis and
properties. In: Dijkstra AJ, Hamilton RJ, and Hamm W (eds.) Trans fatty acids,
pp. 1–24. Oxford: Blackwell Publishing.
Katsuragi Y, Yasukawa T, Matsuo N, Flickinger BD, Tokimitsu I, and Matlock MG (2008)
Diacylglycerol oil, 2nd ed. Urbana, IL: AOCS Press.
Ratnayake WMN and Cruz-Hernandez C (2009) Analysis of trans fatty acids of partially
hydrogenated vegetable oils and dairy products. In: Destaillats F, Sébédio J-L,
Dionisi F, and Chardigny J-M (eds.) Trans fatty acids in human nutrition,
pp. 105–146. Bridgwater: The oily Press.
Sébédio J-L and Nimal Ratnayake WM (2008) Analysis of trans mono-and
polyunsaturated fatty acids. In: Dijkstra AJ, Hamilton RJ, and Hamm W (eds.) Trans
fatty acids, pp. 102–131. Oxford: Blackwell Publishing Ltd.
Zschau W (2000) Bleaching. In: O’Brien RD, Farr WE, and Wan PJ (eds.) Introduction to
fats and oils technology, pp. 158–178. Champaign, IL: AOCS Press.