Determination of Lipids-2

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BIOC 201
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Determination of Lipids
The term lipids applies to a class of compounds that are soluble in
organic solvents (e.g. alcohol and chloroform) and insoluble in water.
Lipids contain carbon, hydrogen and oxygen but have far less oxygen
proportionally than carbohydrates.
Lipids are an important part of living cells. Together with carbohydrates
and proteins, lipids are the main constituents of plant and animal cells.
Classification of lipids
Lipids are classified into:
I. Simple lipids: They are esters of fatty acids with various alcohols. They
include oils and fats which are esters of fatty acids with glycerol (i.e.
triglycerides).
II. Complex lipids: They contain (in addition to fatty acids and alcohols)
additional groups as phosphate (e.g. phospholipids), carbohydrates (e.g.
glycolipids) and proteins (e.g. lipoproteins).
III. Precursor and derived lipids: They include fatty acids, cholesterol,
steroid hormones, fat-soluble vitamins (vitamin A, D, E & K) and
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eicosanoids, which
thromboxanes.
include
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prostaglandins,
leukotrienes
and
Biological functions of lipids:

1. They provide energy storage and metabolic fuels.
2. Cholesterol is a precursor of steroid hormones, bile acids (which help in
digestion of dietary fats) and vitamin D.
3. They act as functional and structural components of the cell membrane.
4. Lipoproteins (e.g. LDL & HDL) are a mean for transporting lipids in
blood.
5. Imbalance in lipid metabolism can lead to major clinical
Problems, such as Obesity and atherosclerosis.
I- Chemicals reactions of lipids:
A- Test for solubility of lipids in polar & non-polar solvents:
How to perform the test:
1-Add about 2 ml of the provided oil sample to 5 ml of water in a test
tube and try to mix oil with water. By shaking, oil and water mix
initially; however, they gradually separate out to form 2 layers.
2- Repeat the experiment using chloroform instead of water.
A positive test is indicated by:

Oil dissolves in the organic solvent chloroform but not in water
because oils contain long hydrocarbon tails which are hydrophobic in
nature.
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B- Saponification of oils:
Saponification of oils means the hydrolysis of triglycerides in the oil in
presence of an alkaline medium (e.g. NaOH) into glycerol and fatty acids
with the production of sodium salt of fatty acids or soap.
1- Place 5 ml of the provided oil sample in a 250 ml beaker.
2- Add 15 ml of alcoholic NaOH solution.
3- Cover the beaker with a watch glass and heat on a boiling W.B.
4- Stir the reaction mixture from time to time using a glass rod until a
semi-solid mass is formed.
5- Put a small amount of this mass in a test tube containing about 5 ml of
water and shake well; froth is obtained indicating the presence of soap.
6- In another test tube, prepare a soap solution by dissolving a small
amount of the formed soap in water. Add few drops of CaCl 2 or MgSO4 to
this solution and shake well.
Boiling water bath

Note the formation of insoluble Ca or Mg soaps without any froth. This
explains why soap is not effective in hard water which contains calcium
and magnesium; for soap to be effective, it must be soluble in water.
2 RCOO- + Ca2+ (RCOO) 2Ca ppt
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D- Comparison of the surface tension of soap solution and water:

1- Fill two 10 ml syringes, one with soap solution (prepare it
yourself) and the other with water.
2- Fix both syringes vertically using the available stands.
3- Remove pistons and start collecting soap solution and water in
10 ml cylinders placed below each syringe.
4- Record the time for collection of 10 ml soap solution or water.
Note that the time for collection of soap solution is less than that for
collection of water because soap lowers the surface tension of water
making it flow easily through the syringe needle.
II- Chemical reaction of fatty acids
- Cupper acetate experiment for differentiation between saturated
and unsaturated fatty acids:
1- Add I ml of each fatty acids (palmitic, stearic and oleic acids) in 3 test
tubes.
2- Each of test tube contain add 3 ml of petroleum ether, shake tubes
until fatty acid dissolve.
The green precipitate is appearing in the bottom of tube if fatty acid is
saturated (palmitic, stearic) . But if it is unsaturated fatty acids (oleic) the
blue color in the upper solution will appear.
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III- Chemical reaction of glycerol
Glycerol (or glycerin, glycerine) is a simple polyol compound. It is a

colourless, odourless, viscous liquid that is widely used in pharmaceutical
formulations. Glycerol has three hydrophilic hydroxyl groups that are
responsible for its solubility in water and its hygroscopic nature. The
glycerol backbone is central to all lipids known as triglycerides. Glycerol
is sweet-tasting and of low toxicity.
A- Acrolein test:
1- Put half ml of glycerol in test tube and add drops of (KHSO4)
2- Heat the test tube.
Smell the pungent Acrolin.
B- Denestan experiment:
1234-
Put 1 ml of borax solution in clean test tube.

Add one drop of phenolphthalein, the red color is formation.
Add dilute glycerol until the red colour disappears.
Heat in the boiling water bath until red colour produced again.
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VI- Cholesterol
A- Salkowski test:
1- Add 2 ml of the provided chloroform to cholesterol.
2- Add an equal volume of concentrated sulfuric acid (H2SO4).
A yellow to brick-red colour is formed indicating the presence of
cholesterol.
B-Liebermann-Burchard test:
1- Add 10 drops of acetic anhydride.
2- Add 2 drops of concentrated sulfuric acid to 2 ml chloroform to
cholesterol.
A bluish-green colour is formed indicating the presence of cholesterol.
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When you are finished collecting data, determine the identity of

each unknown. Record chemical properties of your test, observation and
results in the tables below.
Unknown (1)
Test
Observation
Results
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Unknown (2)
Test
Observation
Results
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Unknown (3)
Test
Observation
Results

Determination of Lipids-2

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Biological functions of lipids:

A positive test is indicated by:

Boiling water bath

A positive test is indicated by:

D- Comparison of the surface tension of soap solution and water:

III- Chemical reaction of glycerol

Glycerol (or glycerin, glycerine) is a simple polyol compound. It is a

Put 1 ml of borax solution in clean test tube.

When you are finished collecting data, determine the identity of

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