BRAIN LIPID EXTRACTION

SAPONIFICATION
- Hydrolysis of fats or oils, in the presence of alkali to produce glycerol and a soap. The
commonly used bases are NaOH and KOH.
Reaction:

The hydroxyl ions, a strong nucleophile, will attack the ester link of the fatty acid and glycerol,
cleaving it into two parts. The sodium ions will then bind with the free fatty acid, forming a soap.
Tests on Soap
Salting Out
Results: Formation of white precipitate soluble in water
Explanation:
The sodium ions are more attracted to water. (They will form a stronger bond). As NaCl is added,
the sodium molecules will bind to water. In excess, these will be precipitated out (saturation of
solution). When more water is added, the sodium ions will have available water molecules to
bind with (once bound, it will dissolve in water).

After saponification is complete, most of the soap is dissolved in the solution. By adding sodium
chloride, the metal ion of the salt reacts with the fatty acids forming back the soap and glycerol.
This is called salting out, an effect based on the electrolyte-nonelectrolyte interaction. Following
this equilibrium principle, additional sodium ions would cause the reaction to shift to the left
forming the salt precipitate.
Insoluble Salts
Results: For both, formation of white precipitate insoluble in water
Explanation:

BRAIN LIPID EXTRACTION

Calcium, magnesium, lead or iron ions displace the sodium or potassium present in the soap.
There will be deposition of the soap and it will be insoluble in water. Two molecules of the soap
will bind to the divalent cations, forming the compound below:

Water cannot bind to the ion. Moreover, it is difficult to break the bonds formed. This is why the
precipitate will be insoluble in water.
Surface Tension
Results: It should SINK in the soap solution; and FLOAT in Water and 2% NaCl
-

Defined as the force with which surface molecules of a liquid are held together. In the
molecule in the surface of a liquid, there is resultant attraction inwards because the
number of molecules per unit volume is greater in bulk than in the vapor. Hence, there is
less freedom in its movement which causes the surface of the liquid to pull itself together
tending to occupy the least possible area. The surface then behaves as if it were in a state
of tension.
It leads to efficient absorption: particularly in enzymatic reactions, formation of complex
compounds, and action of drugs and poisons.

Gibbs-Thomsons principle
Solutes, dispersing in a liquid instead of concentrating on its surface, raise its surface tension;
while those concentrating on the liquid surface, lowers its surface tension. Soaps, oils, proteins
and bile salt reduce the surface tension of water. Due to the lower surface tension, the sulfur
powder sinks into the solution. Inorganic salts, on the other hand, increase it, which is why the
powder stated afloat. Water, inherently has a high surface tension due to the H bonds.
Explanation:
Water inherently has a high surface tension due to H-bonds, which are strong IMFs
(intermolecular forces of attraction). As mentioned, the ionic interaction between water and
sodium is very strong (ionic bonds>H-bonds) which is why the sulphur powder cannot easily
break it causing it to float on top. The fatty acids, on the other, hand, from its structure, has a
hydrophilic head and a hydrophobic tail. The tendency of the molecule is to orient itself in such a
way that the head will be the ones aggregated towards the water molecules. Hence, the tails,
which do not really exhibit strong IMF, are near the surface. The sulphur powder may then
penetrate the surface and sink.
Application:
Hays test for bile salts in urine
Solubility
Results: Insoluble in Water, Soluble in chloroform- FATTY ACIDS
Soluble in Water, Insoluble in chloroform- GLYCEROL

BRAIN LIPID EXTRACTION

NO
N
POL
AR
Explanation:
The long hydrocarbon chain of a fatty acid is highly nonpolar due to the low difference in the
electronegativity of carbon and hydrogen. This is responsible for the regional non-polarity of the
fatty acid. From the principle, like dissolves like, indicating that the solutes will dissolve In
solvents with similar properties. Water is a polar compound, and the fatty acid is nonpolar.
Hence, it will not dissolve in water. It is expected to be soluble in nonpolar solvents, like
chloroform. Glycerol is soluble in water due to the presence of the three hydroxyl or alcohol
groups. These are hydrophilic which permits the solubility of glycerol in water.
Translucent Spot Test
Results: Presence of translucent spot that remains even after long standing for FATTY ACIDS
Presence of translucent spot that eventually fades for GLYCEROL
Explanation:
Fats and oils have higher boiling points so at room temperature they cannot absorb enough heat
to evaporate. When fat or oil is placed on a sheet of paper, it diffracts light. The diffracted light
can pass from one side of the paper to another side to produce the translucent spot. The
difference between the two lies in the length of the hydrocarbon chain.
Iodine Absorption Test
Result: Decolorization of iodine [violet to colorless or slightly pink (xs)] for both LECITHIN
and the FATTY ACID
Explanation:

BRAIN LIPID EXTRACTION

This test is used to identify the degree of unsaturation in oil or fat. During the reaction, the violet
color of iodine fades away if the oil or fat is unsaturated. If it is saturated, the color will not fade
away. Iodine (or other halogens) add at the double bonds and thus cause the decolorization of the
solution indicating the presence of double bonds.
Acrolein Test
Result: Pungent odor for both GLYCEROL and LECITHIN
Explanation:

This is a test for the presence of glycerin or fat. When potassium bisulfate, a strong acid and
dehydrating agent, is heated with fat, hydrolysis occurs and the glycerol portion is dehydrated to
form acrolein. This compound is responsible for the strong pungent odor
Benedicts Test
Results: Presence of brick red precipitate
Explanation:

Benedicts test is a test for the presence of reducing sugars. This is based on their ability to
reduce the cupric ions to cuprous oxide at basic pH. Cuprous oxide ranges from green to brick
red. A green solution would indicate the presence of small amounts of reducing sugar. The
formation of brick red precipitate would indicate an abundance. Almost all carbs are reducing
agents with the exception of sucrose.
Lecithin
Aka phosphatidylcholines
Most abundant phospholipids of the cells membrane
Amphipathic
-

Since the molecule is amphipathic, it can form micelles and more importantly, it can also
form lipid bilayers which are the primary component of cell membranes. It is a major
component of biological membranes.

BRAIN LIPID EXTRACTION

Lecithin should test positive for all the tests. This is due to its structure. It will test
positive for the acrolein test since it contains a glycerol backbone. It will test positive for
the iodine absorption test since the R2 chain often contains an unsaturation. It should test
for the phosphorus test since it also contains a phosphate group.

Phosphate test
Result: formation of yellow precipitate; yellow crystals upon cooling
Explanation:

When lipids with phosphate groups are added to an acidic environment, the lipid is hydrolysed
producing the free phosphate which would react in the reaction above. The ammonium
phosphomolybdate would be responsible for the yellow precipitate observed. Upon cooling, this
would be observed as crystals.
Cholesterol
- Precursor of some hormones, vitamins, and steroids.

Salowski Test
Results: In Chloroform layer- rose red color (tho usually found dapat sa interface of the two
layers yung color na ito); acid- yellowish with fluorescent green color
Explanation:

BRAIN LIPID EXTRACTION

This is a test for the presence of cholesterol. Cholesterol is reacted with concentrated sulfuric
acid, wherein the sulfuric acid will remove two molecules of water from two molecules of
cholesterol causing a connection at position 3 which would form bi-cholestadiene. At the same
time, the sulfuric acid will sulfonate the bi-cholestadien at positions 7, 7 of aromatic ring and the final
product will be a bisulfonic acid of bi-cholestadien whichi is responsible for the rose red color.

Liebermann-Burchard Test
Result: Green solution

It undergoes a series of reactions which is shown by the color changes that occur during the
experiment. This is a quantative test (quantifiable through spectrophotometry).