Biochemistry Laboratory (BCM 362L), Experiment No.

9, © March 9, 2006
3nd Quarter A.Y. 2005-2006

Determination of Saponification and Iodine Numbers of Some

Lipids

Mr. *****1, *****2, *****2

1
Professor, School of CHE-Chm, Mapua Institute of Technology
2
Student, BCM 362L/ A31, School of CHE-Chm, Mapua Institute of Technology
______________________________________________________________________________________

ABSTRACT

Olive oil was used as a source of triacylglycerol one with the sample and one as a blank. The
analysis. The experiment focuses on the iodine number was 3.55348 g for the sample, and
determination of iodine and saponification 1.2691 g for the blank solution.
number. The saponification of olive oil was
0.672 which matches with the theoretical range. Keywords: Olive oil, triacylglycerol, iodine,
Two trials were made for the Iodine experiment, saponification.
______________________________________________________________________________________

INTRODUCTION with a water-cooled reflux condenser. Each

Acylglycerols can be hydrolyzed by
heating with acid or base or by treatment with
lipases. Hydrolysis with alkali is called
saponification and yields salts of free fatty acids
ad glycerol. This is how our ancestors made
soap. One method used potassium hydroxide
leached from wood ashes to hydrolyze animal
fat. When the fatty acids esterified at the first and
third carbons of glycerol are different, the second
carbon is asymmetric. The various acylglycerols
are normally soluble in benzene, chloroform,
ether, and hot ethanol.
Saponification value of a fat or oil is
defined as the number of milligrams of KOH
needed to saponify 1g of fat. It basically involves
the heating of fat in alkali leading to the
formation of glycerol and potassium of the fatty
acids.The saponification value is an indication of
the sized or nature of the fatty acid chanins
esterified to glycerol.
METHODOLOGY
A. Samponification Number
Two clean and dry round bottom flasks
were prepared. 1 g sample of olive oil were
placed into a round bottom flask. The second
round bottom flask was served as a blank. 25 mL
of 5% alcoholic KOH was delivered into each of
the two flasks from a burette. A few boiling
chips were added to each flask, and was fitted
sample was refluxed for an hour in a water bath
on an electric mantle. The flasks were then
cooled to room temperature. The refluxed
solutions were transferred into Erlenmeyer flasks
using small amounts of hexane to thoroughly
rinse off the round bottom flask. To each flask, 3
drop of 1% phenolphthalein and was titrated
with standard 0.5 M HCl to colorless endpoint.
The saponification number and molecular mass
of the cooking oil was then computed.
B. Iodine Number
Two clean and dry 250 mL Erlenmeyer
flasks were prepared. About 0.1 g of cooking oil
was weighed into a flask; the other flask was
served as a blank. 5 mL ether and 15 mL Hanus
reagent was added to each flask. The flasks were
allowed to stand for 30 minutes in the dark and
were occasionally shaked. After 30 minutes, the
flasks were brought out and 5 mL of 15% KI was
added to each. The mixture was shaked
thoroughly to extract any remaining free iodine
into the KI solution. 12.5 mL of distilled water
was added to each flask and was titrated with
standard 0.1 N Na2S2O3 until the color of the
solution was pale yellow. 1 mL of 1% starch
solution was added to both flasks. The titration
was continued with 0.1 N Na2S2O3 until the blue-
violet color just disappears. The iodine number
was then calculated.
RESULTS AND DISCUSSION

By heating a triglyceride in aqueous
potassium hydroxide (KOH) the fatty acyl esters
can be cleaved off (hydrolysis) leaving behind
glycerol and the potassium salt of the fatty acid.
For saponification, the data are as
follows:
For trial 1, I.N. = (0.014 L Na2S2O3 x 0.09918
mol Na2S2O3/1L) x (1 mol I2/0.09918 mol
Na2S2O3 x 253. 82 g/ 1 mol) = 3.55348 g
For blank, I.N. = (0.005 L Na2S2O3 x 0.09918
mol Na2S2O3/1L) x (1 mol I2/0.09918 mol
Na2S2O3 x 253. 82 g/ 1 mol) = 1.2691 g

Titrant volume (mL) 24 CONCLUSION

Blank titrant volume (mL) 30.8
Net titrant volume (mL) 6.8 The smaller the length of fatty acid
Moles KOH consumed 0.012 chains the higher the saponification number.
Saponification Number 0.672 The value of saponification is a
Molecular Mass 250 g/mole measurement of the ml of KOH required to
complete the hydrolysis of one gram of fat or oil.
The computations for saponification The iodine mumber is the number of
number are: grams of iodine absorbed by 100 g of fat or oil.

Moles KOH = (0.5 M HCl)(24 mL HCl)(1 L / LITERATURE CITED

1000 mL)( 1 mole KOH / 1 mole HCl) = 0.012
mole. 1. Biochemistry, Garrett and Grisham, 3rd Edition

SN = (0.012 mol x 56 g/mol) / 1 g = 0.672 2. © 1993-2003 Microsoft Corporation. All

rights reserved.
MM = (3 x 1 g) / 0.012 mol = 250 g /mol

Fatty acids (component of triglycerides)

can be saturated (no C=C double bonds) or
unsaturated (C=C double bonds are present).
Treatment of a triglyceride (or fatty acid) with a
halogen will result in the addition of the halogen
at the C=C double bond site:
HC=CH + HI  HIC-CH2
The result is simple, more double bonds
mean more iodine (or other halogens) will be
absorbed. The data for the determination of
iodine number is located below:

Trial 1 Blank

Titrant vol. 35.5 47

Net Titrant vol. 11.5

Meq I2 Consumed 0.00138852 0.0004559

moles moles

Iodine No. 3.55348 g 1.2691 g

The computation for iodine number are

as follows:

I.N. = 100 x (eq. I2 x E.W. of I2) / 1 g sample