FOOD MICROBIOLOGY LABORATORY

(STKM2411)

TITLE :
DETERMINATION OF IODINE VALUE
(CYCLOHEXANE METHOD)

NAME :
ZAINAH NAJIHAH BINTI ZAINAN

MATRIC NO. :
A190670

LECTURER :
ASSOC PROF. DR. LIM SENG JOE
Objectives
 To evaluate the quality of fres h s unflow er oils , us ed of s unflow er oils ,
fres h palm oil and us ed of palm oil by us ing titration techniques.
 To determine the iodine value of fresh sunflower oils, used of sunflower oils, fresh
palm oil and used of palm oil by cyclohexane method.

Introductions
Unsaturated fatty acids can be converted into saturated by the process of hydrogenation.
Depending upon the degree of unsaturation, the fatty acids can combine with oxygen or halogens
to form saturated fatty acids. So it is important to know the extend to which a fatty acid is
unsaturated. There are different methods for checking the unsaturation level in fatty acids, one
among them is by determining the iodine value of fats. Iodine value or number is the number of
grams of iodine consumed by 100g of fat. A higher iodine value indicates a higher degree of
unsaturation. Different methods are available for determining the unsaturation level in fatty
acids, one of which is by determining the iodine value of fats. In this experiment, cyclohexane
method was used to determine the iodine value of fresh sunflower oils, used of sunflower oils,
fresh palm oil and used of palm oil.

Method
1. Weigh 0.5g of fresh sunflower oils, used of sunflower oils, fresh palm oil and used of
palm oil in the different conical flask.
2. Placed the scoop in a 500 mL flask.
3. Added 20 mL of cyclohexane to dissolve the fat.
4. Added 25 mL of the Wijs solution, insert the stopper, shaked gently and placed the bottle
in the dark for 1 hour.
5. After standing, added 20 mL of the KI solution and 100 mL of water.
6. Titrate with 0.1N sodium thiosulfate pentahydrate solution until the yellow colour due to
iodine has almost disappeared.
7. Added 1 to 2 mL of the starch indicator solution and continue the titration until the blue
colour just disappears after very vigorous shaking. Carry out two determinations on the
same test sample.
8. Carry out a blank test simultaneously under the same conditions.
Result
Replication 1

Sample Titration volume (ml)

Initial volume Final volume Volume of sodium
thiosulphate
Fresh sunflower oil 0 3.5 3.5
Used sunflower oil 3.5 6.9 3.4
Fresh palm oil 6.9 19.7 12.8
Used palm oil 19.7 33.2 13.5
Blank 33.2 81.5 48.3

Replication 2

Sample Titration volume (ml)

Initial volume Final volume Volume of sodium
thiosulphate
Fresh sunflower oil 0.0 6.4 6.4
Used sunflower oil 6.4 13.5 7.1
Fresh palm oil 13.5 24.9 11.4
Used palm oil 24.9 34.0 9.1
Blank 0.0 49.2 49.2

Replication 3

Sample Titration volume (ml)

Initial volume Final volume Volume of sodium
thiosulphate
Fresh sunflower oil 1 5.6 4.6
Used sunflower oil 5.6 8.4 2.8
Fresh palm oil 0.7 15.3 14.6
Used palm oil 14.3 32.4 18.1
Blank 0.6 49.0 48.4
 Calculation:
12.69 x N x (V 2−V 1)
Iodine value =
Weight

Fresh of sunflower oil

12.69 x 0.1 x (3.5−0)
R1 : = 8.89
0.5
12.69 x 0.1 x (6.4−0.0)
R2 : = 16.24
0.5
12.69 x 0.1 x (5.6−1)
R3 : = 11.67
0.5
8.89+16.24+11.67
Mean = = 12.27
3

Standard deviation = √(8.89−12.27) +( 16.24−12.27 ) +( 11.67−12.27)²

2 2
= 13.77
3−1
Mean ± SD = (12.27 ± 13.77)

Used of sunflower oil

12.69 x 0.1 x (6.9−3.5)
R1 : = 8.63
0.5
12.69 x 0.1 x (13.5−6.4)
R2 : = 18.02
0.5
12.69 x 0.1 x (8.4−5.6)
R3 : = 7.12
0.5
8.63+18.02+7.12
Mean = = 11.26
3

Standard deviation = √(8.63−11.26) +( 18.02−11.26 ) +(7.12−11.26)²

2 2
= 5.91
3−1
Mean ± SD = (11.26 ±5.91)

Fresh of palm oil

12.69 x 0.1 x (19.7−6.9)
R1 : = 32.49
0.5
12.69 x 0.1 x (24.9−13.5)
R2 : = 28.93
0.5
12.69 x 0.1 x (15.3−0.7)
R3 : = 37.05
0.5
32.49+28.93+37.05
Mean = = 32.82
3
Standard deviation = √
2 2
(32.49−32.82) + ( 28.93−32.82 ) +(37.05−32.82)²
= 4.07
3−1
Mean ± SD = (32.82 ± 4.07)
Used of palm oil
12.69 x 0.1 x (33.2−19.7)
R1 : = 34.26
0.5
12.69 x 0.1 x (34.0−24.9)
R2 : = 23.1
0.5
12.69 x 0.1 x (32.4−14.3)
R3 : = 45.93
0.5
34.26+23.1+45.93
Mean = = 34.43
3

Standard deviation = √(34.26−34.43) +( 23.1−34.43 ) +(45.93−34.43)²

2 2
= 11.42
3−1
Mean ± SD = (34.34±11.42)

Blank
12.69 x 0.1 x (81.5−33.2)
R1 : = 122.59
0.5
12.69 x 0.1 x (49.2−0)
R2 : = 124.87
0.5
12.69 x 0.1 x (49.0−0.6)
R3 : = 122.84
0.5
122.59+ 124.87+122.84
Mean = = 123.43
3

Standard deviation = √(122.59−123.43) +( 124.87−123.43 ) +(122.84−123.43) ²

2 2
= 1.25
3−1
Mean ± SD = (123.43±1.25)

Discussion

Iodine value (IV) is a measure of the total number of double bonds present in fats and oils.
Iodine value is used to measure unsaturation or the average number of double bonds in fats and
oils. Decrease in iodine value shows decrease in the number of double bonds and it indicates
oxidation of the oil. In this experiment, the result shown that sunflower oil had the lowest iodine
number and throughout the testing sunflower oil had the lowest numbers. Compared to palm oil
which had the highest numbers throughout the testing and palm oil has the highest iodine
number. This is a clear representation of the amount of double bonds present in each fat,
sunflower oil doesn’t have many double bonds therefore it is a saturated. Palm oil has lots of
double bonds making it an unsaturated fat. Therefore palm oil has more double bonds because
the greater the iodine value the more unsaturated the fat is.
 Referring to Replication 3 above there were a few errors in the lab results as they were not
in range with the iodine numbers sourced from literature apart from fresh sunflower oil and fresh
palm oil sample. The most crucial error that could have been made in the lab was not shaking the
formulas constantly enough or hard enough during the titration process, shaking during titration
is so important to combine the two separate layers, the sodium thiosulphate which is a water
based layer and a lipid and Iodine layer, which is the chloroform layer (aqueous).

Unsaturated compounds contain molecules with double bonds, therefore saturated

compounds don’t contain molecules with no double bonds. Unsaturated compounds with double
bonds are very reactive with iodine in the dark. The more double bonds that are attached to the
iodine, the higher the iodine value, this demonstrates the difference in iodine numbers between
sunflower oil and palm oil. Because sunflower oil has a lower iodine number it shows that the
molecular structure is different to palm oil.

Conclusion

In the conclusion, sunflower oil had the lowest iodine number confirming that it was the
most saturated fat. Palm oil had the highest iodine number showing it was the most unsaturated
fat. The results from the lab were backed up and confirmed by literature. The biggest error that
was made in the lab was not swirling the titration’s enough therefore the two layers were not
being mixed together, during the titration process the two layers have to stay together.

Question
1. What is the relationship between iodine value and the stability of fats and oils?
Unsaturated compounds contain molecules with double or triple bonds, which are very
reactive toward iodine. The more iodine is attached, the higher is the iodine value, and the
more reactive, less stable, softer, and more susceptible to oxidation and rancidification is
the oil, fat, or wax.

2. Explain how IV is used to monitor the progress of hydrogenation.

In the field of oil hydrogenation, IV is used for monitoring catalyst activity and measuring
hydrogenation conversion. IV declines during hydrogenation as a result of C = C
saturation in which the decline is related to the nature of the oil, operating conditions as
well as catalyst type and concentration. For different food and nonfood applications, soft
oils are usually hydrogenated until IV 70. Trans fatty acids (TFAs) are particular fats with
specific physical properties, are produced as a by product of the vegetable oil
 hydrogenation process in a geometrical isomerization process for the C = C double bond
of the oil subjected to hydrogenation.

3. Besides IV, name other methods used in industry to monitor hydrogenation process.
Catalytic Hydrotreating, Ultra-compact Near-Infrared Spectrometer and Chemometrics.
FOOD MICROBIOLOGY LABORATORY
(STKM2411)

TITLE :
DETERMINATION OF PEROXIDE VALUE

NAME :
ZAINAH NAJIHAH BINTI ZAINAN

MATRIC NO. :
A190670

LECTURER :
ASSOC PROF. DR. LIM SENG JOE
Objectives
 To determine the peroxide value of fresh sunflower oils, used of sunflower oils, fresh
palm oil and used of palm oil by cyclohexane method.

Introduction
The peroxide value is used to measure the extent of oxidation of lipids, fats, and oils. It is a
parameter specifying the content of oxygen as peroxide, especially hydro peroxides in a
substance. The peroxide value is a measure of the oxidation present. The oxidation of food lipids
is undesirable due to off-flavours, toxins and loss of fat-soluble vitamins known as rancidity. The
analysis of the peroxide content of oil samples is a very analytical task because high peroxide
levels in oils have been a threat to human health. The sample is treated in solution with a mixture
of acetic acid and a suitable organic solvent and then with a solution of potassium iodide. The
liberated iodine is titrated with a standard solution of sodium thiosulfate using soluble starch to
indicate the endpoint. In this experiment, to determine the peroxide value we used sample fresh
sunflower oils, used of sunflower oils, fresh palm oil and used of palm oil.

Method
1. Weigh 5g of the sample into the 250 mL conical flask.
2. Add 30 mL of the acetic acid-chloroform solution. Swirl the flask until the sample is
dissolved in the solution.
3. Add 0.5 mL of saturated potassium iodide using a pipette. Swirl the solution for 1 minute
4. Then add 30 mL of distilled water. Add a few drops of starch solution.
5. Titrate with 0.01N sodium thiosulphate solution, adding it gradually and shake. Continue
the titration, shaking the flask vigorously near the end point to liberate all the iodine from
the chloroform layer.
6. Add the thiosulphate dropwise until the blue colour disappeared.
Result
Replication 1

Sample Titration volume (ml)

Initial volume Final volume Volume of sodium
thiosulphate
Fresh sunflower oil 0 6.6 6.6
Used sunflower oil 6.6 16.9 10.3
Fresh palm oil 16.9 22.5 5.6
Used palm oil 19.4 70.6 51.2
Blank 24.5 25 0.5

Replication 2

Sample Titration volume (ml)

Initial volume Final volume Volume of sodium
thiosulphate
Fresh sunflower oil 16.7 17.6 0.9
Used sunflower oil 17.6 25.5 7.9
Fresh palm oil 0.4 2.2 1.8
Used palm oil 2.2 16.7 14.5
Blank 0.0 0.4 0.4

Replication 3

Sample Titration volume (ml)

Initial volume Final volume Volume of sodium
thiosulphate
Fresh sunflower oil 14.4 17.3 2.9
Used sunflower oil 5.1 14 8.9
Fresh palm oil 15.5 34.6 3
Used palm oil 18.5 34.6 16.1
Blank 12.2 14.4 2.2
Calculation:
(Vs−Vb) x N x 1000
PeroxideValue (meq. peroxide/kg fat) =
W

Fresh of sunflower oil

(6.6−0) x 0.01 x 1000
R1 : = 13.2
5
(17.6−16.7)x 0.01 x 1000
R2 : = 1.8
5
(17.3−14.4) x 0.01 x 1000
R3 : =6
5
13.2+1.8+ 6
Mean = =7
3

Standard deviation = √(13.2−7) +( 1.8−7 ) +(6−7)²

2 2
= 3.99
3−1
Mean ± SD = (7±3.99)

Used of sunflower oil

(16.9−6.6)x 0.01 x 1000
R1 : = 20.6
5
(25.5−17.6)x 0.01 x 1000
R2 : = 15.8
5
(14−5.1) x 0.01 x 1000
R3 : = 17.8
5
20.6+15.8+17.8
Mean = = 18.07
3

Standard deviation = √(20.6−18.07) +( 15.8−18.07 ) +(17.8−18.07)²

2 2
= 2.41
3−1
Mean ± SD = (18.07±2.41)

Fresh of palm oil

(22.5−16.9) x 0.01 x 1000
R1 : = 11.2
5
(2.2−0.4) x 0.01 x 1000
R2 : = 3.6
5
(34.6−15.5) x 0.01 x 1000
R3 : = 38.2
5
11.2+ 3.6+38.2
Mean = = 17.67
3
Standard deviation = √
2 2
(11.2−17.67) + ( 3.6−17.67 ) +(38.2−17.67) ²
= 18.18
3−1
Mean ± SD = (17.67±18.18)
Used of palm oil
(70.6−19.4) x 0.01 x 1000
R1 : = 102.4
5
(16.7−2.2) x 0.01 x 1000
R2 : = 29
5
(34.6−18.5) x 0.01 x 1000
R3 : = 32.2
5
102.4+29+32.2
Mean = = 54.53
3

Standard deviation = √(102.4−54.53) +( 29−54.53 ) +(32.2−54.53) ²

2 2
= 41.48
3−1
Mean ± SD = (54.53±41.48)

Blank
(25−24.5) x 0.01 x 1000
R1 : =1
5
(0.4−0.0) x 0.01 x 1000
R2 : = 0.8
5
(14.4−12.2) x 0.01 x 1000
R3 : = 4.4
5
1+ 0.8+4.4
Mean = = 2.07
3

Standard deviation = √(1−2.07) +( 0.8−2.07 ) +( 4.4−2.07) ²

2 2
= 2.02
3−1
Mean ± SD = (2.07±2.02)

Discussion

Detection of peroxide gives the initial evidence of rancidity in unsaturated fats and oils.
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-flavours and off-odours. Peroxide value, concentration of peroxide
in an oil or fat, is useful for assessing the extent to which spoilage has advanced.

Peroxide value is defined as the amount of peroxide oxygen per kilogram of oil, which is
reported in units of milliequivalents or meq. The peroxide value tells us a lot about the quality of
the oil. Unsaturated oils and fats become rancid by oxidation, forming peroxides. In this
experiment, the result shown that fresh sunflower oil and fresh palm oil had the lowest peroxide
number than used sunflower oil and used palm oil. In the result on replication 3, it shown that
fresh sunflower oil get 2.9ml of sodium thiosulphate. Meanwhile, used sunflower oil shown
8.9ml of sodium thiosulphate. The fresh palm oil shown that peroxide value is 3ml of sodium
thiosulphate and used palm oil is more higher of peroxide value which is 16.1ml of sodium
thiosulphate.

High peroxide levels that used palm oil shown indicate that oil has been damaged by free
radicals and will give rise to aldehydes and ketones that can cause oil to smell musty and rancid.
These reactions are accelerated by heat, light, and air. A lower number of peroxide value that
fresh palm oil and fresh sunflower palm oil indicates a good quality of oil and a good
preservation status. Unsaturated free fatty acids react with oxygen and form peroxide value,
which determine a series of chain reactions that generate the production of smelling volatile
substances.

Conclusion

In the conclusion, peroxide value is very important because it measure the oxidationpresent
in oil. Oxidation oil is very bad for human health. It can give disease such as highblood pressure,
high cholesterol and cancer if it consumed in long term. So, food handlermust avoid using
oxidized oil in their product. Oil has limit number to be use. Ministry ofHealth Malaysia
suggest cooking oil can be use 3 times only to avoid the oil becomeoxidized. We
can know the amount of quality of cooking oil after 5 times usage with different oil and no of
frying.

Question

1. Explain what are oxidative and hydrolytic rancidity?

Hydrolytic rancidity is the result of the hydrolysis of fats with the liberation of one or
more volatile fatty acids, whereas in oxidative rancidity the unsaturated fatty acid
fragments of glycerides are oxidisedat their double bonds with the ultimate production of
aldehydes, ketones and acids.

2. Describe the steps taken to overcome the rancidity problem in food.

Rancidity can be prevented using the following methods:
 Adding antioxidants (substances which prevent oxidation) to food.
 Storing food in airtight containers to slow the process of rancidification.
 Refrigerating food also helps to slow down rancidification.
 Replacing oxygen in the containers with another gas. For example, to prevent chips from
turning rancid, chips manufacturers flush the bags with nitrogen gas

3. A sample of 5.0g of food-grade oil was reacted with excess KI to determine peroxide
value. The free iodine was titrated with a standardized solution of 0.10N Na2S2O3. The
amount of titrant required was 0.60 mL (blank corrected). Calculate the peroxide value of
the oil.
0.60 ml x 0. 10 N x 1000
Peroxide value = = 12
5 .0 g