CHAPTER 3

LIPID CHEMISTRY

GROUP 4 SEC. A1
BALANSAG, JOANNE
BALBASTRO, ROWENA
BALAORO, FRANCE
BANTUAS, ALMAIRA
BARCELONA, LULUBELLE
BARLOLONG, ROLDAN

PROCTOR:
NICO D. CALINGACION, M.D.

EXPERIMENT 1: EXTRACTION, FRACTIONIZATION AND IDENTIFICATIONOF BRAIN

LIPIDS
The brain has a high content of lipid compared to all other tissues of the body. It contains
a variety of lipids. Complex lipids such as the phosphatides, cerebrosides, and sphingosides
contain fatty acid components of the C-16 to C-24 carbon chains, chiefly in the form of stearic,
oleic, linoleic and arachidonic acids are found in the brain. The steroids alcohol, cholesterol,
occurs abundantly in the brain.
This experiment may be divided into four major parts:
1. Extraction of the total lipids.
2. Isolation of the triglycerides and saponificaiton of the triglycerides make way for
the study of soaps, fatty acid and glycerol.
3. Isolation and tests on lecithin
4. isolation and tests on cholesterol
Objectives:
1. To be able to study the properties of various lipids.
2. To be able to classify and identify the lipids.
Procedure:
30 grams of pigs brain
(homogenized in ether-alcohol solution)
TOTAL LIPID EXTRACT

2/3 portion
Isolation of Triglycerides

20 ml portion
Test on Soaps
1. Salting Out
2. Insoluble Salts
3. Surface Tension

30 ml portion
A. Test on Fatty Acids
1. Solubility
2. Translucent Spot
3. Iodine Absorption
B. Test on Glycerol
1. Solubility
2. Translucent Spot
3. Acrolein
4. Benedicts Test

1/3 portion
Lecithin-Cholesterol Isolation

A. Test on Lecithin
1. Acrolein
2. Iodine Absorption
3. Phosphorus
B. Test on Cholesterol
1. LiebermannBurchard Test
2. Salkowski Test

20 ml Portion
Test on Soap

Salting Out
5 ml of
Soap Solution

Add
NaCl

Precipitate

Add
Water

Observe

Insoluble Soaps
5 ml
5 ml
Distilled Water

Add
CaCl2

Add
MgSO4

Precipitate

Add
Water

Observe

Surface Tension
5 ml
5 ml Soap
5 ml Solution
Solution 2% NaCl

Add
Sulfur Powder

Precipitate

Add
Water

Observe

Observe

Results and Observations

Test
Salting Out
Insoluble salts

Surface Tension

Obtained Results
A white precipitate was
formed, and dissolved upon
addition of water.
CaCl2 and MgSO4 formed
white precipitates and upon
contact with water, it did
not dissolve.
In water: The sulfur
powder stayed at the water
surface (floated).

Expected Results
White precipitate which
dissolves in water.

In soap solution: The

sulfur powder went at the
bottom of the tube (sank).

In soap solution: The sulfur

powder sinks.

In 2% NaCl: The sulfur

powder stayed at the water
surface (floated).

In 2% NaCl: The sulfur

powder floats.

White precipitates which

are insoluble in water
In Water: The sulfur
powder floats.

Discussion:
Lipids that contain fatty acid ester linkages can undergo hydrolysis, a reaction that is
catalyzed by strong acid or base. Alkaline hydrolysis of fatty acid esters is known as
saponification. Saponification of triglycerides produces glycerol and fatty acids salts. High
molecular mass of fatty acid salts are commonly referred to as soap. The chemical reaction
between a triglyceride and a base to form glycerol and fatty acids salts is shown below.

When the hydrolyzed solution is saturated with NaCl, the soap separates. This principle is
used in manufacturing soaps. Insoluble soaps are formed when calcium and magnesium react
with the higher fatty acids of soaps and forms an insoluble white curd like precipitate of
magnesium or calcium salts (scum).

Surface tension expresses the force with which the surface molecules attract each other. It
is also a property of liquids arising from unbalanced molecular cohesive forces at or near the
surface, as a result of which the surface tends to contract and has properties resembling those of a
stretched elastic membrane. Water has a high surface tension because of its hydrogen bonding as
shown below.

Substances that break hydrogen bonding of water that causes to lower its surface tension
are called surfactants. Soap is an example of surfactant. Soaps have an unusual behavior in
water because it comprises both hydrophilic and hydrophobic regions. The cleaning action of
soaps is due to the arrangement of the hydrophilic (polar) heads, which are the carboxyl groups;
and hydrophobic (nonpolar) tails are the hydrocarbon chains. When in water the surfactant
molecules reversibly assemble into polymolecular aggregates called micelles. By gathering the

hydrophobic chains together in the center of the micelle, the grease or oil present in the solution
will be emulsified by the hydrophobic tail inside of the micelles and it is rinsed away.
Hydrophilic heads
Hydrophobic tails

Hydrophobic
(nonpolar) tail

Hydrophilic
(polar) head

30 ml Portion
A. Test on Fatty Acids

Solubility
Heat the solution

Add HCl

Translucent Spot Test

Small amount of precipitate

Iodine Absorption Test

Small amount of
precipitate

Ordinary writing paper

Add 5 ml of CHCl3

Precipitate

Observe

Place in water

Observe

Place in CHCl3

Add Hubls iodine

Observe

30 ml Portion
B. Test on Glycerol

Solubility
Precipitate

Acrolein Test
0.5 grams of powdered
KHSO4

Translucent
Spot Test
Precipitate

Benedicts Test
Litmus paper
If acidic

Water

CHCl3

Add 10 drops of
Glycerol solution

Ordinary writing
paper

Heat

Observe

Note the odor

Add dilute
Na2CO3

in 5 ml
Benedicts
Reagent

Observe

Add 5 drops of
Glycerol solution
Boil for
2-3 mins.

Observe

Results and Observations:

FATTY ACID
Obtained Results

Test
Solubility test
H2O
CHCl3
Translucent Spot test

insoluble
soluble
It formed a spot and
remained even after
long standing

Iodine Absorption test

Test
Solubility test
H2O
CHCl3
Translucent Spot test

Acrolein Test
Benedicts test

Expected Results
insoluble
soluble
There is a presence of
translucent spot and
remains even after long
standing

The iodine solution was The iodine solution

decolorized
decolorized
GLYCEROL
Obtained Results

Expected Results

soluble
soluble
insoluble
insoluble
It formed a spot but it did There is a presence of
not remain even after long translucent spot and
standing
remains even after long
standing
pungent odor
negative

Pungent odor
positive

Discussion:
The degree of solubility in water and in nonpolar solvents of fatty acids will depend on
whether it is a saturated or unsaturated and if it is saturated whether it is short-chain, mediumchain or long-chain fatty acids. Below is a table showing the solubility of a short chain saturated
fatty acid, long-chain saturated fatty acids and unsaturated fatty acids in water

Solubility in Water

Short-chain
saturated fatty
acids
Limited solubility

Long-chain
saturated fatty
acids
Insoluble

Unsaturated
fatty acids
Insoluble

However, glycerol behaves differently in water from fatty acids. Glycerol is soluble in
water but insoluble in nonpolar solvents. Glycerol is soluble in water because it contains three
hydroxyl or alcohol groups. These hydroxyl or alcohol groups are hydrophilic and are
responsible the solubility of glycerol in water.

Another test for fatty acids and glycerol is the translucent spot test, which is a simpler test
for the lipids wherein marks on an unglazed paper shows the presence of triglycerides
(fats/glycerol). Below is a figure of a translucent spot test. On the left is a negative result while
on the right shows a spot which is a positive result.

NEGATIVE
(No spot formed)

POSITIVE
(A translucent spot is formed)

The volatility of fatty acids will also depend whether the fatty acids are saturated or
unsaturated and if saturated whether it is a short-chain, medium-chain or long-chain fatty acids.
Below shows the difference of short-chain, long-chain fatty acid and unsaturated fatty acid.

Volatility

Short-chain
saturated fatty
acids
Volatile

Long-chain
saturated fatty
acids
Nonvolatile

Unsaturated fatty
acids
Nonvolatile

Acrolein test is a test for the presence of glycerin or fat. Acrolein (CH 2=CH-CHO), is
released if the test is positive. When a fat is heated strongly in the presence of a dehydrating
agent such as KHSO4, the glycerol portion of the molecule is dehydrated to form the unsaturated
aldehyde. This unsaturated aldehyde is produced by the loss of water by the glycerol portion of
the fat molecule. In biological membrane this will also result in the formation of free radicals, a
process known as lipid peroxidation.

Glycerol

Acrolein (pungent odor)

Another reaction exhibit by fatty acids is halogenation reaction which is the basis of the
chemical procedure known as Iodine Absorption test. It is use for determination of unsaturation
of fats. Halogens ( I, Br ) will add across the double bonds and thus the decolorization of an
iodine or bromine solution will indicate the presence of unsaturated fatty acids. Iodine from

Huble's solution adds up across the unsaturation sites of oil or fat giving a colourless product and
a little more of iodine gives a permanent violet colour to the solution.

Benedict's test identifies reducing sugars based on their ability to reduce the cupric
(Cu2+) ions to cuprous oxide at basic (high) pH. Cuprous oxide is green to reddish orange. In
this carbohydrate test, a green solution indicates a small amount of reducing sugars, and reddish
orange indicates an abundance of reducing sugars. Nonreducing sugars such as sucrose produce
no change in color (the solution remains blue). Monosaccharides such as glucose and fructose
are reducing sugars, meaning that they possess free aldehyde (-CHO) or ketone (-C=0) groups
that reduce weak oxidizing agents such as the copper in Benedict's reagent. Benedict's reagent
contains cupric (copper) ion complexes with citrate in alkaline solution.

1/3 Portion
A. Test on Lecithin
1/3 portion
Heat until syrupy consistency

Add 15 ml of ether

Stir

Add 15 ml acetone

Filter

Precipitate (lecithin)

Filtrate (cholesterol)

Precipitate (lecithin)
Acrolein test
0.5 grams of powdered
KHSO4

Add 10 drops of glycerol

Solution

Iodine Absorption Test

Small amount of
Precipitate

Ordinary writing paper

Heat

Note the odor

Test for Phosphorus

Small amount of
Precipitate

Add 5 ml of 10% HCl

Boil for
10 mins.

Observe

Add equal volume of

Conc. HNO3 and ammonium
molybdate solution
Warm

Yellow crystals

Filtrate (cholesterol)
Evaporate the ether-acetone filtrate
Cool

Add CHCl3
Filter

Clear solution

Salkowski test

Liebermann-Burchard test

2 ml

2 ml

Add equal volume of H2SO4

and CHCL3

Observe

Add 10 drops acetic anhydride

Add 2 drops of conc.

Sulfuric acid
Stand for 15
mins.

Note the end color

Results and Observations:

Lecithin
TEST
Acrolein test
Iodine absorption test

Test for phosphorus

Obtained Result
It produces an unpleasant odor
(pungent)
Pinkish color of precipitate
region upon dropping of
iodine
(decolorization of iodine)
Yellow precipitate formed and
formation of crystals upon
cooling

Expected Result
Pungent
Decolorization of iodine

Yellow precipitate formed and

formation of crystals upon
cooling

Cholesterol
Test

Obtained result

Expected result

Salkowski test

CHCl3: cherry red color

Acid layer: clear

CHCl3: Cherry red color

Acid layer: fluorescent green

Liebermann-Burchard
reaction

Dark green color

Green color

Discussion:
As aforementioned, Acrolein test is a test for the presence of glycerine or fat. Lecithin or
phosphatidyl choline is a phosphoglycerides which, the parent compound is phosphatidic acid
and its structural backbone is glycerol. Lecithin produce acrolein and will yield expected result
in iodine absorption test because the presence of double bonds. As we stated earlier that lecithin
has a phosphatidic acid as its parent compound, it will yield yellow crystal, an expected result in
test for phosphorus. Test for phosphorus detects the presence of free phosphate in acidic solution
can be detected by adding a molybdate to the solution.
HPO42-(aq) + 12MoO42-(aq)+3NH4+(aq)+23H3O+(aq) (NH4)3[P(Mo3O10) 4](yellow,s) + 35 h20(l)
The equation above illustrates the pertinent reaction between phosphate and ammonium
molybdate solution in presence of nitric acid.
When lipids contains phosphate groups in their structures are added to a strong acid solution
such nitric acid, the lipid hydrolyses, producing free phosphate. The free phosphate then reacts as
in equation above, forming yellow precipitate which forms crystals upon cooling.

For cholesterol analysis we perform the Salkowski test and the Liebermann-Burchard Test.
The Salkowski test is a qualitative analysis of cholesterol detection in natural products.
Salkowski reaction is the reaction of cholesterol with concentrated sulphuric acid. Concentrated
sulphuric acid is highly hygroscopic and it removes two molecules of water from two molecules
of cholesterol, it causes a connection at position 3, forming bi- cholestadien (a). Simultaneously
the sulphuric acid sulphonates the molecule of bi-cholestadien at positions 7,7 of aromatic ring
and, as a final product, red colour bi-sulphonic acid of bi-cholestadiene is formed (b).

a.
bi-cholestadien

b.

bisulfonic acid of bi-cholestadiene

Liebermann-Burchard Test on the other hand is a colourimetric test for unsaturated

sterols, notably cholesterol; a blue-green color develops when such substances are added to
acetic anhydride and sulphuric acid in chloroform

Some biological importance of lipids

Surfactant in lungs is dipalmitoyl lecithin which lowers the surface tension in the alveoli.
The result of cohesive forces where molecules pulls each other inward is surface tension. This
collapses the alveoli, but because there is a presence of surfactant in our lungs, it lowers the
surface tension thus preventing it to collapse during expiration also a study is conducted that
surfactant proteins present in the lungs can recognize bacterial, viral and fungal surface
oligosaccharides and thus can opsonize these pathogen
Bile Acids
1. Primary Bile Acids
Cholic Acid and chenodeoxycholic Acid
Synthesized in the liver from cholesterol
2. Secondary Bile acids
Deoxycholic acid and lithocholic Acid
Synthesized in the small intestines from the primary bile acids
FUNCTIONS:
Aid in fat digestion and absorption
Facilitate absorption of fat-soluble vitamins (A,D,E,and K)
Help in solubilization of cholesterol in bile
C21H30O2
Progesterone
Chemical name:Preg-4-ene-3,20-dione
FUNCTION
Intermediate metabolite in biosynthesis of other steroid
hormones
Maintain pregnancy during the first trimester
Involved in menstrual cycle
Drug for threatened abortion
Component of oral contraceptives
C21H30O5
Cortisol
Chemical name: 11beta,17alpha,21-Trihydroxypegn-4-ene-3,20-dione
FUNCTIONS
Regulate intermediary metabolism
Immunosuppressive and anti-inflammatory action
Play an important role in stress response.

C21H28O5
Aldosterone
FUNCTIONS
Regulates water and sodium metabolism
Involved in blood pressure regulation

C19H28O2
Testosterone
Chemical name:17beta-Hydroxyandrost-4-en-3one
FUNCTIONS
Normal male sexual development of the embryo
Develops and maintain male secondary sexual characteristics
Important for male fertility

C18H24O2
Estradiol
Chemical name: estra-1,3,5(10)-triene-3,17beta-diol
FUNCTIONS
Normal female sexual development
Development of female secondary sexual characteristics
Induce differentiation of mammary glands
Involved in menstrual cycle
Affects synthesis of neurotransmitter and receptor protein
in the CNS
Influences lipid metabolism increase HDL, decrease
LDL