Experiment 5b

Qualitative tests for lipids

I. Objectives
- To subject the different types of isolated lipids (Cerebroside, phospholipid, cholesterol)
and other given lipids to various qualitative tests
- Identify the types of tests undertaken in the qualitative tests
- Identify some properties of the different types of lipids from based on the qualitative
tests.

Definition of terms

II. Theory behind the experiment

Acroleins test
- Acrolein test is used to detect the presence of glycerol or fat. When fat is treated
strongly in the presence of a dehydrating agent like potassium bisulphate (KHSO4), the
glycerol portion of the molecule is dehydrated and breaks down to form an unsaturated
aldehyde, acrolein that has a pungent irritating odor.
- When is heated with potassium bisulfate (KHSO4) dehydration occurs to form the
aldehyde acrolein from glycerol or esterified glycerol (example triglycerides)

Test for unsaturation

- The unsaturated fatty acids absorb iodine at the double bonds until all the double bonds
are saturated with iodine. Hence the amount of iodine required to impart its color to
the solution is a measure of the degree of the fatty acids.

Phosphate test

- When a solution containing phosphate ions is heated with a solution of ammonium

molybdate [(NH4)2MoO4] and dilute nitric acid a bright yellow precipitate is formed.
The yellow precipitate is extremely insoluble in nitric acid. Phosphate ions react with
ammonium molybdate to produce a coloured complex ammonium phosphomolybdate
[(NH4)3PO4 • 12MoO4]
- The purpose of the reagents is to break the bonds in order to reveal the free phosphate
group
- Nitric acid doesn't take part in the reaction as a whole, from what I remember it is there
to keep the pH low and to keep redox potential in the solution high to not allow side
reactions.

Emulsification Test

- Principle: Emulsification is the process which stabilizes the water and oil emulsion, by
the help of emulsifying agents.
- The lipid or oil in water appears on the top of the water because of the high surface
tension of water which gets together to form a separate layer. On the addition of
emulsifying agents Emulsifying agents help stabilize the Emulsion of immiscible
- substances slowing the process of immiscible substances to form 2 separate layers. by
which the lipid appears as the tiny droplets suspended in the solution.
- An emulsifier consists of hydrophilic (water-soluble) part and lipophilic (oil-soluble) part.
When an emulsifier is added to a mixture of water and oil, the emulsifier is arranged on
the interface, anchoring its hydrophilic part into water and its lipophilic part into oil.
- On the interface of water/air or oil/air, emulsifiers are arranged, reducing interfacial
tension of them and making them easy to be mixed. In other words, the force to
separate the oil and water turns weaker, ending up with oil and water easily get
emulsified.

-
- Positive result: It gives a permanent or stable emulsion of lipid and water.
- Negative result: Oil in water emulsion will form at the top, due to the high surface
tension of water.
- When oil and water, which are immiscible, are shaken together, the oil is broken up into
very tiny droplets which are dispersed in water. This is known as oil in water emulsion
- The water molecule due to the high surface tensions has a tendency to come together
and form a separate layer. This is why the oil and water emulsion is unstable in the
presence of substances that lower the surface tension of water. Eg: Sodium carbonate,
soap, bile salts, etc.
- The tendency of the water molecule to coalesce is decreased and the emulsion becomes
stable.
- Since bile salts cause the greatest decrease in surface tension they are best emulsifying
agents.
- Not all compounds composed of hydrophilic and lipophilic can be used as emulsifiers. If
hydrophilicity in a compound is greater than lipophilicity in it, the compound will
disperse into water, while one with greater lipophilicity than hydrophilicity will disperse
into oil.
- When the hydrophilicity and lipophilicity are well-balanced, the emulsifier exhibits
sufficient effects.
-
Reference:

https://biologyreader.com/qualitative-analysis-of-lipids.html#EmulsificationTest

https://www.rikenvitamin.com/foodingredients/emulsifier/properties.html#targetText=
When%20an%20emulsifier%20is%20added,them%20easy%20to%20be%20mixed.

Lieberman-Buchard or Acetic Anhydride test

- The Liebermann–Burchard or acetic anhydride test is used for the detection of

cholesterol. The formation of a green or green-blue colour after a few minutes is
positive
- The color is due to the hydroxyl group (-OH) of cholesterol reacting with the reagents
and increasing the conjugation of the un-saturation in the adjacent fused ring.
- cholesterol and its acetate and sulfate derivatives undergo sulfonation at a variety of
positions, often with skeletal rearrangements. Elimination of an SO(3)H group as
H(2)SO(3) generates a new double bond. Repetition of this desaturation process leads to
polyenes and ultimately to aromatic steroids. Linearly conjugated polyene cations can
appear blue but form too slowly to account for the color response.

Modified Furter-Meyer test

- Modified further-meyer Test is used to detect the presence of tocopherols by giving a

bronze-red solution.

Molisch test

- Molisch test is a group test for all carbohydrates, whether free or in combined form.
Despite its limitations, it is routinely used to detect the presence of carbohydrates.
- The reaction is based on the fact that concentrated H2SO4 catalyses the dehydration of
sugars to form furfural (from pentoses) or hydroxymethyl furfural (from hexoses).These
furfurals then condense with sulfonated alpha-naphthol to give a purple or violet
coloured product (furfuryl-diphenyl-methane-dyes).
 -
- Polysaccharides and glycoproteins also give a positive reaction. In the event of the
carbohydrate being a poly- or disaccharide, the acid first hydrolyses it into component
monosaccharides, which then get dehydrated to form furfural or its derivatives.
III. Discussion of results
IV. Sample calculation
V.
VI. Conclusion