Introduction

The Peroxide value of an oil or fat is used as a measurement of the extent to which
rancidity reactions have occurred during storage. Other methods are available but peroxide value
is the most widely used. Peroxide value (PV) is the most commonly used measurement of lipid
oxidation. The standard iodometric method requires a relatively large sample (5 g) when the lipid
is only slightly oxidized. The ferric thiocyanate method, based on the oxidation of ferrous to
ferric ion, involves colorimetric measurement of ferric thiocyanate. This method is more
sensitive than the iodometric method and requires a relatively small sample (0.1 g). The PV is a
useful measure for samples with low levels of oxidation and when the hydroperoxides are not
decomposed. During prolonged oxidation, a maximum PV is reached and the value then begins
to decrease due to peroxide degradation. This maximum value occurs early for soybean and
rapeseed oil, due to the more rapid decomposition of the hydroperoxides of the polyunsaturated
fatty acids.

The double bonds found in fats and oils play a role in autoxidation. Oils with a high
degree of unsaturation are most susceptible to autoxidation. The best test for autoxidation
(oxidative rancidity) is determination of the peroxide value. Peroxides are intermediates in the
autoxidation reaction.

Autoxidation is a free radical reaction involving oxygen that leads to deterioration of fats
and oils which form off-flavors and off-odors. Peroxide value, concentration of peroxide in an oil
or fat, is useful for assessing the extent to which spoilage has advanced.

The peroxide value is defined as the amount of peroxide oxygen per 1 kilogram of fat or
oil. Traditionally this was expressed in units of mill equivalents, although if we are using SI units
then the appropriate option would be in millimoles per kilogram (N.B. 1 millimole = 2 milli
equivalents). Note also that the unit of milliequivalent has been commonly abbreviated as
mequiv or even as meq.

Equipment and Materials

Distilled Water, Starch Solution (fresh prepared), Analytical Balance, Beaker (100ml), Water
Dispenser, Washing Solution (soap), Burette (25ml), Erlenmeyer flask, Measuring Cylinder
(50ml and 100ml), Droppers, Retort stand, Sodium thiosulphate, Potassium iodide( saturated),
Acetic acid(chloroform solution with ratio 1:4), funnel, oil sample(six types).

Procedure:

1) 0.1 mg, 2.00 ± 0.05 of the oil sample was weighted in the 250 ml flask.

2) 30 ml of the acid-chloroform solution was added to the oil sample.

3) The solution was swirled until the sample completely dissolved.

4) 50 ml of potassium iodide solution was added through graduated pipette.

5) The solution was swirled for 1 minute and then 30 ml of distilled water was added to the
solution. Few drops of starch solution were added for freshly produced oil.

6) The solution was titrated with gradual and vigorous shaking of 0.01 N sodium thiosulphate.

7) The titration was carried out until all the iodine liberated from the from the chloroform layer.

8) Thiosulpahte solution was added drop wise until the blue color of the solution disappeared.

9) For the samples with high peroxides titration was carried out with 0.01 N sodium thiosulphate
until the yellow color just got disappeared.

10) 50 ml of starch indicator was added until the blue color just got disappeared.

Results / Calculation:

Table 1 The amount of 0.1N sodium thiosulphate used in each oil solution
 Oil Initial Vs1 Final Vs1 Vs1 Initial Vs2 Final Vs2 Vs2 Average Vs
1 12.3 14.0 1.7 10.2 13.7 3.5 2.6
2 8.5 10.5 2.0 21.3 23.0 1.7 1.85
3 13.9 21.5 7.6 0 7.8 7.8 7.7
4 0 7.5 7.5 0 7.3 7.3 7.4
The peroxide value can be calculated by using the formula below:

( Vs−Vb ) ×1000
N×
W

Vs = the volume in millilitres (mL), sodium thiosulphate solution of normality N, used for the
determination.

Vb = the volume in millilitres (mL), sodium thiosulphate solution used for the blank test.

W = the weight, in gram (g) of the test portion

N = the normality of thiosulphate solution

Peroxide value of Oil 1

(2.6−0)× 1000
0.01 × = 12.87
2.02

Peroxide value of Oil 2

(1.85−0)× 1000
0.01 × = 9.26
1.997

Peroxide value of Oil 3

(7.7−0)× 1000
0.01 × = 38.31
2.01

Peroxide value of Oil 4

(7.4−0) ×1000
0.01 × = 35.58
2.08

DISCUSSION
 In this experiment, peroxide value is determined by iodometric titration. While the
peroxide value is use to determine the rancidity of the oil sample .During the experiment, oil
sample is dissolved in the acetic acid-chloroform solution to generate hydroperoxide .

R−H + O 2 → ROOH

After that, potassium iodide was added into the mixture to produce iodine.

− +
KI + CH 3 COOH → HI + CH 3 COO K

ROOH + 2 HI → ROH + H 2 O + I 2

The iodine produced will float above ROH and water due to its low density causes 2
layers of solution formed which the upper layer is pale yellow in colour while the bottom layer is
colourless. In order to test the peroxide value, starch solution is added as an indicator to make the
upper layer solution become dark blue from pale yellow. After that, the mixture is ready to titrate
with sodium thiosulphate. During the titration, sodium thiosulphate is added slowly until the blue
colour turn to colourless which mean that iodine is fully oxidized.

I 2 + 2 Na 2 S 2 O 3 → Na2 S 4 O 6 + 2 NaI

Peroxide value is a measure of the concentration of peroxides and hydroperoxides

formed in the initial stages of lipid oxidation. After adding the potassium iodide into the mixture
of the fatty acid and the chloroform-acetic acid, the excess of iodide formed. In this stage, the
peroxides present oxidize the iodide to iodine. In this experiment, the amount of iodine produced
is directly proportional to the peroxide value as the amount of sodium thiosulfate is needed to
decolourise the purple colour starch.

If there are more unsaturated double bonds in the fatty acid, they will allow more
oxygen to react, known as autoxidation. This means that the more the polyunsaturated a fat is,
the faster it will go rancid. In generally, oils will not suddenly do rancid.
 Next, the oxidation of oil will destroys the essential fatty acids and the toxic compound
will be produced. The polymers will also be affected and oxidized. The oxidation of oil is very
important in term of nutritional quality and toxicity of edible oils.

As stated above, the amount of iodine produced is directly proportional to the peroxide
value and it can be used to test the quality of the oils that was used in this experiment. First,

First, the amount of the 0.01N sodium thiosulphate used to decolourise the dark yellow
coloured upper layer of the solution in oil 1 was 2.6 ml. Oil 2 needed 1.85ml to decolourise the
cloudy pink colour of the upper layer of the solution and oil 3 needed 7.7ml of the 0.01N sodium
thiosulphate to decolourise the light yellow iodine layer. Meanwhile, oil 4 changed the bright
yellow layer into pink and finally into colourless solution when it was titrated with 7.4ml of
0.01N sodium thiosulphate solution. Thus, this clearly shown that the peroxide value of oil 3 is
the highest. The peroxide value of oil 2 is lower than that of oil 1 and oil 4. Therefore, oil 2 has
the highest quality while oil 3 has a lowest quality.

One of the methods that can be used to determine the quality of oils and fats is
Thiobarbituric Acid test, which is commonly known as TBA test. The 2- thiobarbituric acid is
able to react with the saturated aldehydes, 2-enals, and 2-dienals which are produced in the
termination phase of lipid oxidation to produce a pink colour mixture. However, TBA test is not
an absolute good indicator of the quality of oils and fats. This is due to the aldehydes may have
not yet formed or lost during or before the terminating phase of lipid oxidation.

The test can be carried out by dissolving the sample lipid in a suitable non-polar solvent.
After that, an aqueous solution of TBA reagent is added to the flask and the mixture is shaken.
After shaking, the mixture is then placed in a test tube and it is allowed to heat in boiling water
for 20 minutes. After the 20 minutes of heating, the pink colour will be produced. The intensity
of its pink colour is directly related to the concentration of TBA- reactive substances in the
sample. It means that the higher the intensity of the pink colour formed, the higher the
concentration, and it illustrates the lower quality of the oils and fats. The intensity of the pink
colour can be determined by measuring the absorbance at 540nm using a UV- visible
spectrophotometer.
 During the experiment, there are several precaution steps that must be considered. Firstly,
the starch indicator must added into the solution only near the end point (the end point is near
when fading of the yellowish iodine occurs) as at high iodine concentration starch is
decomposed to products whose indicator properties are not entirely reversible. Besides, the
solution should be avoided being oxidized or carbon dioxide; CO2 being dissolved in the
solution because it will affect the actual result. Hence, instead of preparing all the solution
together, the tested solution should be prepared one by one. Thirdly, the sodium thiosulphate
must added gradually or drop by drop during titration and shake it vigorously and constantly to
avoid inaccurate value taken.

Conclusion

We have successfully achieved the objective of this experiment which is to investigate the degree
of oxidation of different oils and fats. The quality of oil can be determined by checking their
peroxide value. According to the results, we found that sample oil 2 has the lowest peroxide
value (9.26), followed by sample oil 1 (12.87) and sample oil 4 (35.58) and finally oil 3(38.31).
In other words, sample oil 2 has the best oil quality compared to others.

References

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