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 litera-
ture 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 mix-
ture of chloroformacetic acid and then
titrated the liberated iodine with 0.002 N
sodium thiosulfate solution. The method
gave reproducible results at a given sam-
ple weight but was inconvenient and even
somewhat hazardous to use since the sol-
vent mixture was heated to its boiling
point. A year later, D.H. Wheeler pub-
lished a greatly simplified, rapid, and
more convenient method for the deter-
mination of peroxides or active oxygen
in vegetable oil. He suggested that the
sample size and the normality of the thio-
sulfate 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 dis-
solved in 50 cc. of solvent mixture (60
per cent glacial acetic acid, 40 per cent
chloroform) and 1 cc. of saturated potas-
sium iodine solution [is] added. The mix-
ture 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, depend-
ing on the amount of iodine liberated.
This method was examined by sev-
eral other workers, who found that sam-
ple 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 pro-
cedures, 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 Verlagsge-
sellschaft 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-
ANALYTICAL ARTICLE 651
tors such as sample size, reaction tem-
perature, and reaction time as well as sec-
ondary reactions influenced the results
of the determination. However, the first
three factors should normally have no
effect when the methods are standard-
ized. But these factors are often respon-
sible for the differences when the results
obtained by different methods are com-
pared.
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 solu-
bility 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 organ-
izations 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 acidchlo-
roform, newer versions have replaced this
solvent by an acetic acidisooctane mix-
ture 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 standard-
ized methods stick to the reaction time
of 1 minute, despite other recommen-
dations.
Today, there are numerous analyti-
cal procedures for the measurement of
652 ANALYTICAL ARTICLE
PV. In all cases the results and the accu-
racy of the methods depend on the exper-
imental 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 thiosul-
fate 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 2
Dependence of peroxide value on sample size
Sample size 5g 3g 2g
Peroxide valuea 3 5
5 7
5 8
8 9 10
9 14
16 16
27
41
a Titration with 0.01 N Na S O .
2 2 3
bDeviation related to a sample size of 5 g.
inform October 2003 Volume 14 (10)
tion prescribed for the determination.
Table 1 The following unpublished table
Mass of test portion according to (Table 2) from the Institute for Lipid
ISO 3960:1977 Research of the Federal Center for Cereal,
Expected Mass of Potato and Lipid Research in Mnster
peroxide value test portion (g) (Germany) shows this influence of the
sample size on PV.
0 to 12 5.0 to 2.0 It can be seen that PV increases with
12 to 20 2.0 to 1.2 decreasing sample size. In further work,
20 to 30 1.2 to 0.8 the reaction time was doubled from 1 to
30 to 50 0.8 to 0.5 2 minutes. In this case, the result for
50 to 90 0.5 to 0.3 the determination of the PV with 5 g is
67 instead of 41. These results are in good
ing on the expected PV (see Table 1). For agreement with the literature-quoted val-
the titration, 0.01 N sodium thiosulfate ues. Therefore, determination of the PV
is used. The reaction time is 1 minute. is mainly dependent on the sample size
A national German standard method and the reaction time. Fortunately, the
(DGF C-VI 6a), published in 1984, pre- reaction time has been standardized within
scribes a sample size of 1 g for PV >2 and international methods to 1 minute, even
5 g for PV 2. After a reaction time of 1 if lower PV are obtained.
minute, the liberated iodine is titrated PV is a commonly used parameter.
with 0.1 N sodium thiosulfate or with The problem of different quantities of
0.01 N sodium thiosulfate if less than 0.5 sampleprobably also the reason for bad
mL is used. reproducibility limits in ring testsneeds
The determination of PV according to be addressed by the different stan-
to the Olive Oil Regulation (EEC: dardization bodies and must be harmo-
European Economic Community, fore- nized soon. This is the only way to avoid
runner of the European Union) No. 2568/91 problems, and long debate, on the results
is identical to IUPAC 2.501 and still uses obtained by different methods for the
the acetic acidchloroform mixture as determination of PV. ISO/TC 34/SC 11,
solvent. The International Olive Oil the subcommittee of ISO TC 34 respon-
Council (IOOC) Trade Standard Applying sible for ISO 3960, is prepared to take
to Olive Oil and Olive-Pomace Oil pre- the lead in a global harmonization process.
scribes the methods ISO 3960 and IUPAC
2.501 for PV, whereas the Draft Revised
Codex Standard for Olive Oils and Olive
Background resources
Pomace Oils (Report of the 18th Session AOAC International Methods, Gaitherburg,
of the Codex Committee on Fats and Oils, Maryland.
London, 2003) quotes ISO 3960:2001 AOCS, Official Methods and Recom-
and AOCS Cd 8b-90. mended Practices of the American Oil
As can be seen from this discussion, Chemists Society, 5th edn., AOCS Press,
the main difference in these standardized Champaign, Illinois, 1997.
methods is the quantity of the test por- International Organization for
Standardization, Geneva, Switzerland.
International Union of Pure and Applied
Chemistry, Research Triangle Park,
North Carolina.
German Standard Methods for the Analy-
1.2 g 1g 0.8 g 0.6 g 0.4 g Dev.b sis of Fats and Other Lipids,
158% Wissenschaftliche Verlagsgesellschaft,
150 Stuttgart, Germany.
149
10 123 Hans-Jochen Fiebig is chairman of
149 ISO/TC34/SC11: Animal and Vegetable
19 20 123 Fats and Oils. He may be contacted at
31 33 33 122 the Institute for Lipid Research, Federal
77 83 92 222 Center for Cereal, Potato and Lipid
Research, Piusallee 68-76, 48147
Mnster, Germany; e-mail: hjfiebig@uni
muenster.de