Soran University – Faculty of Science

Chemistry department
Biochemistry
3rd stage – Group (A)

Experiment No. (6):

Qualitative analysis of Lipids part II

Prepared

by: Zhino

Ali Azhy
Azad Yusra
Rzgar
Rawen Hassan
Sima Sherzad
Belal
Muhammad
Saadulla Salm
Introduction
Copper acetate test

This test is used to distinguish between Oils [neutral fats] and Fatty acids [Saturated and
Unsaturated]. The Copper acetate solution does not react with Oils or Fats. The Copper acetate
solution react with fatty acids (saturated and unsaturated) to form the Copper salt of the fatty
acid. Copper salt formed in the case of fatty acids can only be extracted by Petroleum ether.

Unsaturation test (Huble’s Iodine test)

This test is used to know the degree of unsaturation in the given sample. Oils on reaction with
Huble’s reagent fads the violet color of iodine then it is unsaturated and if the color persists
then the given fat or oil is saturated.

Most neutral fats contain glycerides of some unsaturated fatty acids. These unsaturated fatty
acids become saturated by taking up halogens like Iodine or Bromine. If the Fat (Oil) contains
more unsaturated fatty acids, it will take up more halogen (Iodine) molecules. Halogens (I, Br)
will add across the double bonds of fatty acyls chains and induce the decolorization of the
solution. The decolorization of Iodine or Bromine solution indicates the presence of
unsaturated fatty acids.
Procedure & Material
Material & Equipment

 Pipet
 Diethyl ether
 Conical flask
 Distilled water
 Beaker
 Stearic acid
 Test tube
 Copper acetate monohydrate
 Stirrer
 Chloroform
 Filter paper
 Huble's iodine reagent
 Balance
 Olive oil

Procedure
Copper acetate test
• Dissolve a piece of fatty substances (e.g. butter) in about 3 mL of petroleum ether. Use Olive
oil or Shea butter (as a neutral fat) and a solution of Palmitic acid (as a source of fatty acid).

• Add 3 mL of Copper acetate solution.

• Shake the mixture, then leave it for a few minutes. The mixture separate into two layers on
standing.

In the case of Oleic acid: Notice that The petroleum ether-upper layer becomes green as a
result of Copper oleate. The lower layer becomes Pale blue color (less colored).

Unsaturation test

• Take 5 mL of Chloroform and 5 mL of Huble’s iodine reagent in a beaker which will give pink
color to the solution.

Note: Huble’s iodine reagent is alcoholic solution of Iodine containing some Mercuric chloride
(HgCl2).

• Add lipid samples (Olive oil and Butter) drop by drop and shake the flask vigorously, until the
pink color disappears.
Discussion
Copper acetate Test

The aim of this test to distinguish between oil and fatty acid, and to know that fatty acid
saturated or unsaturated.

At first, we brought two test tube, first test tube contains of (diethyl ether and cupric acetate
monohydrate) and we add olive oil and shake it produced two layers that cupper acetate didn’t
react with oil because oil didn’t have free fatty acid and not produced precipitate (salt) that
mean this test (-) VE. But if we hydrolysis olive oil by heating in this step can react with copper
acetate and produced precipitate but in the laboratory, we did not hydrolysis it.

Second test tube contains (die ethyl ether and cupric acetate monohydrate) and we add stearic
acid if we shake it reacted with copper acetate and produced precipitation (copper stearate
salt) because stearic acid has free fatty acid (saturated) that mean this test (+) VE.

Unsaturation test (Huble’s Iodine test)

The aim of this test determination the degree of saturation by the amount of double bond in
different type of Oils (fats).

In the first, we prepare the Huble's iodine solution by mix an amount of alcoholic solution
(Ethanol), with Mercuric chloride (HgCl2).

After we add 5 ml of Chloroform with 5 ml of Olive oil as a sample of lipid, we use Chloroform
due to that it proved to be the best solvent for Oils (Fats). While we add reagent drop by drop
to the lipid solution, we observe that the pink color disappears as quick as possible which mean
to (+) VE result, also the number of disappeared drop equal to the number of double bonds of
the lipid sample (Fat or Oil).

To observe the (-) VE result we add some drop of the reagent to a sample of Water, while
Water does not have double bond in its structure (Saturate molecule), by the first drop of the
reagent pink color appear, it could mean (-) VE result.

As the lipid sample the number of drops reach 300 drop and the pink color could disappear, we
have to use a small amount of lipid by some drop not by a (mL) of the solution as our assistant
say to us. And this because to see the last double bond, and measure the amount of saturation.