ANALYTICAL ARTICLE 651

inform October 2003 Volume 14 (10)

Peroxide value determination

Hans-Jochen Fiebig

Over a period of many years, various

methods have been developed for the
determination of peroxides in fats and
oils. The general principle of most of the
methods is the liberation of iodine from
potassium iodide in an acid medium. The
method according to D.H. Wheeler was
standardized more than 50 years ago by
different standardization bodies and is
widely used by producers, receivers, and
official laboratories to control commodities.
In national and international food
legislation (including the Codex
Alimentarius), limit values for peroxide
values (PV) are often fixed. Owing to
anomalies in the reproducibility of the
results, slight differences between the
standardized methods have been noted.
Interestingly, the cause of these problems
had already been reported in the literature of the 1940s and 1950s.
C.H. Lea in 1931 published a method
for the determination of peroxides in fats
and oils. He heated 1 g of fat with 12 g
of powdered potassium iodide in a mixture of chloroformacetic acid and then
titrated the liberated iodine with 0.002 N
sodium thiosulfate solution. The method
gave reproducible results at a given sample weight but was inconvenient and even
somewhat hazardous to use since the solvent mixture was heated to its boiling
point. A year later, D.H. Wheeler published a greatly simplified, rapid, and
more convenient method for the determination of peroxides or active oxygen
in vegetable oil. He suggested that the
sample size and the normality of the thiosulfate solution should be varied if the
levels of peroxides differed greatly. The
following working protocol is taken from
his publication in Oil and Soap (9:8997,
1932):
Three to ten grams of oil are dissolved in 50 cc. of solvent mixture (60
per cent glacial acetic acid, 40 per cent
chloroform) and 1 cc. of saturated potassium iodine solution [is] added. The mixture is stirred by giving a rotary motion

to the flask. After exactly one minute from

the time of the addition of the potassium
iodine, 100 cc. of water are added and
the liberated iodine is titrated with 0.1
N or 0.01 N sodium thiosulfate, depending on the amount of iodine liberated.
This method was examined by several other workers, who found that sample size strongly influenced the PV
obtained, producing a reduction of 3050%
in PV when the sample size was increased.
Decreasing the sample size increased the
PV by up to 140150%.
To achieve a more reliable method,
R.F. Paschke and D.H. Wheeler modified
the original Wheeler method in 1944 (Oil
and Soap 21:5257, 1944). Sample weight
was reduced to one gram, reaction time
increased from one minute to one hour,
and CO 2 was used to minimize further
oxidation during the analyses.
In 1946, Lea revised his earlier
method, simplified many details, and
adopted the same solvent and one-hour
reaction time from the Paschke and
Wheeler method. He suggested that the
increase in PV observed in earlier procedures, when the quantity of fat taken
for the determination was reduced, was
due to an increasing error resulting from
further oxidation of the fat by dissolved
oxygen. He introduced new procedures
for a cold method as well as a hot method
in which oxygen exclusion was adequate.
Today, the hot method is known as the
method according to Sully and is still
used for the determination of the PV in
fatty acids that are difficult to dissolve
(e.g., German Standard Methods for the
Analysis of Fats and Other Lipids, edited
by the German Society for Fat Science
[DGF], Wissenschaftliche Verlagsgesellschaft GmbH, Stuttgart, 2002). But
Leas modified method gave generally
lower results than the other methods.
In 1958 A. Seher published a survey
on the determination of PV (Fette Seifen
Anstrichmittel 60:264267, 1958). From
many tests, he concluded that several fac-

tors such as sample size, reaction temperature, and reaction time as well as secondary reactions influenced the results
of the determination. However, the first
three factors should normally have no
effect when the methods are standardized. But these factors are often responsible for the differences when the results
obtained by different methods are compared.
According to Sehers investigations,
the sample size is not the critical factor,
provided there are no problems with
the solubility of the oil or fat. Depending
on the quantity of the test portion, he
determined a PV of 9.3 meq O 2 /kg for
0.6 g of linseed oil, and 4.8 meq O 2 /kg
with a test portion of 2.7 g of the same
oil. According to his investigations, the
difference was based only on the solubility of the oil. Furthermore, he found a
reaction time of 5 minutes was necessary
rather than the 3 to 4 minutes proposed
earlier by other workers.
The method for the determination of
PV according to Wheeler was adopted
early on by several international organizations including the Association of
Analytical Communities (AOAC
International), AOCS, the International
Union of Pure and Applied Chemistry
(IUPAC), and national bodies such as the
French standardization system
Association franaise de normalisation
(AFNOR), British Standards Institute
(BSI), and DGF. Whereas the original
method used a mixture of acetic acidchloroform, newer versions have replaced this
solvent by an acetic acidisooctane mixture for safety reasons. Unfortunately, all
these standardized methods differ slightly,
above all in the sample size that is required
to perform the method. On the other hand,
it is interesting to see that all standardized methods stick to the reaction time
of 1 minute, despite other recommendations.
Today, there are numerous analytical procedures for the measurement of

652 ANALYTICAL ARTICLE

inform October 2003 Volume 14 (10)

PV. In all cases the results and the accuracy of the methods depend on the experimental conditions, as the method is highly
empirical. Below, the most commonly
used methods are discussed, together with
the principle on which they are based.
AOCS has standardized a method for
PV determination with chloroform (Cd
8-53) and with isooctane (Cd 8b-90). The
latter method uses a mixture of acetic
acidisooctane and a sample size of 5
g. The reaction time is 1 minute, and the
reaction mixture is titrated either with a
0.1 N sodium thiosulfate solution (for PV
10150) or with 0.01 N sodium thiosulfate if the titration is less than 0.5 mL.
For a full validation, AOCS prescribes
the use of 0.001 N sodium thiosulfate
solution. In the scope, it is stated that the
method is applicable to all normal fats
and oils, including margarine, but that it
is highly empirical, and any variation in
the test procedure may produce erratic
results.
The International Organization for
Standardization (ISO) in 1977 published
the first edition of ISO 3960: Animal and
Vegetable Fats and OilsDetermination
of Peroxide Value. This version used an
acetic acidchloroform mixture and a
sample size dependent upon the expected
PV, as shown in Table 1.
The reaction time is 1 minute. This
table for different sample weights is also
used in IUPAC method 2.501. Titration
is performed with 0.01 N and 0.002 N
sodium thiosulfate for PV from 012 and
1290, respectively.
In 1998, ISO published the second
edition of ISO 3960 using acetic
acidisooctane, and the third edition was
published in 2001. ISO 3960:2001 still
prescribes different sample sizes depend-

Table 1
Mass of test portion according to
ISO 3960:1977
Expected
peroxide value

Mass of
test portion (g)

0 to 12
12 to 20
20 to 30
30 to 50
50 to 90

5.0 to 2.0
2.0 to 1.2
1.2 to 0.8
0.8 to 0.5
0.5 to 0.3

ing on the expected PV (see Table 1). For

the titration, 0.01 N sodium thiosulfate
is used. The reaction time is 1 minute.
A national German standard method
(DGF C-VI 6a), published in 1984, prescribes a sample size of 1 g for PV >2 and
5 g for PV 2. After a reaction time of 1
minute, the liberated iodine is titrated
with 0.1 N sodium thiosulfate or with
0.01 N sodium thiosulfate if less than 0.5
mL is used.
The determination of PV according
to the Olive Oil Regulation (EEC:
European Economic Community, forerunner of the European Union) No. 2568/91
is identical to IUPAC 2.501 and still uses
the acetic acidchloroform mixture as
solvent. The International Olive Oil
Council (IOOC) Trade Standard Applying
to Olive Oil and Olive-Pomace Oil prescribes the methods ISO 3960 and IUPAC
2.501 for PV, whereas the Draft Revised
Codex Standard for Olive Oils and Olive
Pomace Oils (Report of the 18th Session
of the Codex Committee on Fats and Oils,
London, 2003) quotes ISO 3960:2001
and AOCS Cd 8b-90.
As can be seen from this discussion,
the main difference in these standardized
methods is the quantity of the test por-

Table 2
Dependence of peroxide value on sample size
Sample size

5g

Peroxide valuea

3
5
5
8
9
16
27
41

3g

2g

1.2 g

10
16

a Titration with 0.01 N Na S O .

2 2 3
bDeviation related to a sample size of 5 g.

19
31

1g
5
7
8
10
14
20
33
77

0.8 g 0.6 g

0.4 g

Dev.b

92

158%
150
149
123
149
123
122
222

33
83

tion prescribed for the determination.

The following unpublished table
(Table 2) from the Institute for Lipid
Research of the Federal Center for Cereal,
Potato and Lipid Research in Mnster
(Germany) shows this influence of the
sample size on PV.
It can be seen that PV increases with
decreasing sample size. In further work,
the reaction time was doubled from 1 to
2 minutes. In this case, the result for
the determination of the PV with 5 g is
67 instead of 41. These results are in good
agreement with the literature-quoted values. Therefore, determination of the PV
is mainly dependent on the sample size
and the reaction time. Fortunately, the
reaction time has been standardized within
international methods to 1 minute, even
if lower PV are obtained.
PV is a commonly used parameter.
The problem of different quantities of
sampleprobably also the reason for bad
reproducibility limits in ring testsneeds
to be addressed by the different standardization bodies and must be harmonized soon. This is the only way to avoid
problems, and long debate, on the results
obtained by different methods for the
determination of PV. ISO/TC 34/SC 11,
the subcommittee of ISO TC 34 responsible for ISO 3960, is prepared to take
the lead in a global harmonization process.

Background resources
AOAC International Methods, Gaitherburg,
Maryland.
AOCS, Official Methods and Recommended Practices of the American Oil
Chemists Society, 5th edn., AOCS Press,
Champaign, Illinois, 1997.
International
Organization
for
Standardization, Geneva, Switzerland.
International Union of Pure and Applied
Chemistry, Research Triangle Park,
North Carolina.
German Standard Methods for the Analysis of Fats and Other Lipids,
Wissenschaftliche Verlagsgesellschaft,
Stuttgart, Germany.
Hans-Jochen Fiebig is chairman of
ISO/TC34/SC11: Animal and Vegetable
Fats and Oils. He may be contacted at
the Institute for Lipid Research, Federal
Center for Cereal, Potato and Lipid
Research, Piusallee 68-76, 48147
Mnster, Germany; e-mail: hjfiebig@uni
muenster.de