DETERMINATION OF PEROXIDE VALUE

Detection of Peroxide gives the initial evidence of rancidity in unsaturated fats and oils. It
gives a measure of the extent to which an oil sample has undergone primary oxidation. The
method of Lea and Wheeler was used to determine the peroxide value (Gray.J.I 1978 and
Robards.K 1988). 1 gram of the sample was weighed with an accuracy of 1mg into a 100ml
conical flask. 10ml of the solvent (3/2 mixture glacial acetic acid/chloroform V/V) was added
and dissolved with the sample. 0.2ml of the Potassium Iodide (KI) solution was added with a 1ml
pipette and kept in the dark for exactly 1minute. Then 20ml of diluted starch solution added with
20ml measuring cylinder. Titration was carried out with 0.01N Sodium thiosulphate (Na2S2O3)
solution until the colour changes from purple to colourless (Make simultaneously a blank
determination). It was measured in milliequivalent peroxide per kg of fat or oil.
Expression of result:
1000 X (V1-V2) X N
Peroxide value = ---------------------------------
W
W - Weight of the sample
V1- Number of ml Na2S2O3 solution used in the test
V2 - Number of Na2S2O3 in blank
N - Normality of the Na2S2O3 solution

5.5 DETERMINATION OF P-ANISIDINE VALUE

The p–anisidine value is defined by convention as 100 times the optical density measured
at 350 nm in a 1 cm cuvette. Anisidine value measures the secondary oxidation of fats and oils. It
represents the levels of non-volatile aldehyde in the fats. The AOCS Official Method Cd 18-90
(Firestone, 1997) was used to determine the p–anisidine value.
Clear and dry sample was taken for analysis of pAV. 0.5 - 4.0g of sample was measured
into a volumetric flask. It was dissolved and diluted to volume with iso-octane. Absorbance (Ab)
of the solution was measured at 350nm in a cell with the spectrophotometer, using the reference
cell filled with solvent as a blank. Exactly 5ml of the fat solution was pipette out into one test
tube and exactly 5ml of the solvent into a second test tube. By means of an automatic pipette
exactly 1ml of the p-anisidine reagent was added to each tube and mixed well. After exactly
10minutes the absorbance (As) of the solution was measured in the first test tube in a cell at
350nm, using the solution from the second test tube as a blank in the reference cell.
Expression of results:
The p-anisidine value is given by,

25 X (1.2As – Ab)
p-A.V = -----------------------
m

As – absorbance of the fat solution after reaction with p-anisidine reagent

Ab – absorbance of the solution of the fat
m - mass in g of the test portion

5.6. DETERMINATION OF ACID VALUE

The acid value is the number of mg of potassium hydroxide (KOH) required to neutralize
the free fatty acids in 1g of the fat. Acid value indicates the proportion of free fatty acid present
in oil or fat.
The sample was homogenised and separated (if necessary remove insoluble substances or
eliminate water by filtration over anhydrous sodium sulphate (Na2SO4)). Size of the sample was
determined by the Table 6 (weigh between 5 and 10g if there is no specific expectation)
(Paquot.C, Hautfenne.A 1984 and Sallee.E.M 1964)
TABLE 5.1
Determination of sample size in Acid value
Expected acid value Mass of test portion (g) Weighing accuracy (g)
>1 20 0.05
1-4 10 0.02
4-15 2.5 0.01
15-75 0.5 0.001
>15 0.1 0.0002
 The sample was weighed into a conical flask. It was dissolved with 50ml of the solvent
mixture (1/1 (V/V) of 95% ethanol and diethylether). A few drops of phenolphthalein indicator
was added and titrated with NaOH until the pink colour of phenolphthalein turned into
colourless. An increased amount of FFA in a sample indicates hydrolysis of triglycerides. Such
reaction occurs by the action of lipase enzyme and this is an indicator of inadequate processing
and storage conditions (i.e., high temperature and relative humidity). Free fatty acid is a source
of flavors and aromas.

Expression of results:
The acid value is given by,
56.1 X T X V
AV = --------------------
m
V – number of ml of the KOH solution
T – normality of the NaOH solution
m - mass(g) of the test portion