BIOLS 315

Biochemistry Lab. No. 6

Lipids Reactions and Characterization

1. Unsaturation Test
Unsaturated fatty acids and fats containing them will readily take up iodine (or bromine)
at double bonds to form additional compounds. The decoloration of Hubl's reagent
(containing iodine) is used as a qualitative test for differentiating unsaturated from
saturated fatty acids. The amount of iodine taken up indicates the degree of
unsaturation.

R-CH=CH (CH2) n-COOH + I2 R-CH-CH- (CH2) n -COOH

Materials
5 ml oleic acid
5 g stearic acid
5 ml olive oil
500 ml chloroform

Hubl's reagent: dissolve 15 g of mercury (II) chloride into 250 ml of 95% ethanol and
dissolve 13 g of powdered iodine into 250 ml of 95% ethanol. Mix the two solutions
and filter.
Test tubes
10 each of 0.2 ml and 5 ml pipettes
Stop-clock

Method
Set up the system described in the following table:

Tube Chloroform Hubl's Oleic acid Stearic Olive Butter

No. (ml) reagent (ml) acid (ml) oil (ml) (g)
(ml)
1 5 2.5 0.05 0 0 0
2 5 2.5 0 0.05 0 0
3 5 2.5 0 0 0.05 0
4 5 2.5 0 0 0 0.05

Start the reaction in each tube (at 2-minute intervals) by adding the reagent and mixing.
Observe the decoloration of the reagent after exactly 10 minutes.

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 Discussion
The experiment shows the addition of iodine across the double bonds of the unsaturated
oleic acid compared to the weak reaction of its saturated counterpart, stearic acid. The
addition of iodine to the double bonds of fatty acids may be quantified as the "iodine
number". This constant is defined as "the iodine absorbed (in grams) per 100g of
sample".

2. The Determination of the Acid Value of a Fat

Materials

Olive oil, butter and margarine (use a fresh sample and one that has stood for several
days at room temperature)
Fat solvent (equal volumes of 95% alcohol and ether neutralized to phenolphthalein)
Phenolphthalein (10 g/liter in alcohol)
Potassium hydroxide (0.1 mol/liter)
Burettes (5 ml and 25 ml)

Method
Accurately weigh or measure out 2g or 2ml of the test compound and suspend the
melted fat in about 10 ml of fat solvent. Add 1 ml of phenolphthalein solution, mix
thoroughly, and titrate with 0.1 mol/liter KOH until the faint pink color persists for 20-
30 s. Note the number of milliliters of standard alkali required and calculate the acid
value of the fat. Note: 0.1 mol/liter KOH contains 5.6 g/liter or 5.6 mg/ml.
*Phenolphthalein is an organic compound often used as an pH indicator.
Phenolphthalein turns pink in pH greater than 8.3 and colorless in acidic solutions.

Discussion
During storage, fats may become rancid as a result of peroxide formation at the double
bonds by atmospheric oxygen and hydrolysis by microorganisms with the liberation of
free acid. The amount of free acid present therefore gives an indication of the age and
quality of the fat.
It is suggested that each pair of students choose one lipid and assay (a) a fresh sample
and (b) one that has been exposed to the atmosphere. Results should then be compared
with other groups who have determined other lipids. The acid value is the number of
milligrams of KOH required to neutralize the free fatty acid present in1g of fat.

Calculation of the acid value:

MKOH = 0.1 mole/liter

Remember that 0.1 mole/liter KOH contains 5.6 g/liter or 5.6 mg/ml.
Acid value = V x N
W
Acid value = V x N x 5.6 mg/ml = mg KOH/g oil or fat
W

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 Where:
V = volume of KOH.
N = normality of KOH.
W = wt of sample.

Calculation of the Iodine No.:

Example
Pure olive oil (MW 884) was reacted with iodine. Exactly 578 mg of 12 were absorbed by 680 mg of the
oil. (a) On the average, how many double bonds are present in a molecule of triglyceride? (b) What is
the iodine number of the oil? The average molecular weight of triacylglycerol in olive oil is 884.

Solution

(a) Calculate the number of moles of 12 absorbed by a mole of the oil. Each mole of 1 2 adds across a
double bond.

0.578 g I 2 wt g I 2

0.680 g oil 884 g oil

wt g I 2 absorbed 
884  0.578  751.4 g I 2 per mole of oil
 0.680
MW of I2 = (2) (126.9) = 253.8

751.4 g I 2
 2.96 mole I 2 / mole of oil
253.8 g / mole

Thus, on the average, there are three double bonds per molecule of triglyceride.

(b) Iodine number is defined as g iodine absorbed per 100 g of oil or fat.

751.4
Iodine number =  100  85
884

Assignment
1. Compare between the followings, which one is fast in discoloring I2 and why:
Oleic and stearic acids
Palmitic and palmitoleic acid
EPA and DHA
Olive oil and butter
Canola and nutmeg oil
2. If linoleic acid is used instead of oleic acid, will the decoloration rate be faster or
slower?
3. Find out the iodine number ranges of 8 different commonly used oils including
(sunflower oil, olive oil, canola oil, coconut oil, corn oil, palm oil, peanut oil, and
soybean oil) and their indications (explain your findings).

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4. Which of these oils will decolorize I2 faster? Why?

Palmitic acid Stearic acid Oleic acid Linoleic acid

16:0 18:0 18:1 18:2
Coconut oil 10.7% 1.7% 24.6% 57.4%
Palm oil 42% 4.3% 9% 0.8%

5. Find out the value of 8 different commonly used oils including (sunflower oil, olive
oil, canola oil, coconut oil, corn oil, palm oil, peanut oil, and soybean oil) and their
indications (explain your findings).

6. Complete the table below.

Test sample Sample Amount of KOH Acid value (mg
type used in titration KOH/g oil or fat)
(ml)
Olive oil Fresh
Old
Corn oil Fresh
Old
Margarine Fresh
Old

List the references that you have used to find out this information
following this style:

References

Mayo Clinic. (2019). How eating fish helps your heart. Available at
https://www.mayoclinic.org/diseases-conditions/heart-disease/in-
depth/omega-3/art-20045614.

Lindshield, B. (2018). Kansas State University Human Nutrition (FNDH

400) Flexbook. 1st edition. Manhattan, New York City: New Prairie Press,
pp. 103.

Smith, S. (2009). Mechanism of chain length determination in biosynthesis

of milk fatty acids. Journal of Mammary Gland Biology and Neoplasia,
14(3), 245-260.

Samiee, K., Rustaiyan. A (2015). Omega-3 Fatty Acids Composition and

Lipid Content from Liver and Muscle Tissues of Scomberomorus
commerson (Lacepede, 1800) in the Persian Gulf. Journal of Food and
Nutrition Research 3(9):620-623. doi: 10.12691/jfnr-3-9-10.

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 Geleijnse, J. M., Giltay, E. J., Grobbee, D. E., Donders, A. R., Kok, F. J.
(2002). Blood pressure response to fish oil supplementation:
metaregression analysis of randomized trials. Journal of
hypertension, 20(8), 1493–1499.