LIPIDS

Prepared by:
Janelle Anne D. Shiozawa, RPh
Gabriel louis P. Llacuna, RPh
 Lipids are a vast group of biochemical molecules, and are among one
of the most abundant biomolecule that is to be found in an organism,
alongside carbohydrates and proteins. It has a very limited number of
functional groups in their structures, for this reason, analyzations and
tests which confirms biochemical nature cannot be used for this kind
of biomolecule. It is a class of compound that can be distinguished as
hydrophobic molecules by its insolubility in water and solubility in
nonpolar solvents such as chloroform or methanol.
Lipids- are esters of long chain fatty
acids and alcohols.
 Building block of lipids: Fatty acids
 It can be defined as NON-POLAR organic compound insoluble in
POLAR solvent, but SOLUBLE IN ORGANIC SOLVENTS (Eg. Ether,
chloroform, benzene, acetone and etc.)
 BIOLOGICAL ROLE OF LIPIDS:
*Lipids are found naturally in all living organisms.
1.) It represents in cell structure and has a structural
Function in the cell: it presents in cell membranes.
2.)An essential source of energy, reservoir for energy.
 FATS CAN BE DIVIDED ACCORDING TO
THEIR CHEMICAL COMPOSITION TO:
1. SIMPLE LIPIDS:
- These compounds are: Esters of fatty acids with glycerol. (Eg. Fats and waxes)
- The triacylglycerol (TAG) is the simplest and most common fat. It is the form in which
lipids are stored in the cell. Also known as neutral fat
2. COMPOUND LIPIDS (Conjugated lipids)
-Lipids are linking with the other compounds such as proteolipids, phospholipids
& glycolipids.
- It contains glycerol, fatty acid, Nitrogenous base and Phosphoric acid.
3. Derived lipids
-They are substances that are soluble in lipid or derived from above groups of lipids by
hydrolysis. Example, cholesterol, sphingosine, glycerophosphatides.
SAPONIFIABLE VS NON-SAPONIFIABLE LIPIDS
Saponifiable Lipids
-Easily hydrolyzed by NaOH and those
generally have ester linkages.
Examples: Fats and oils
Non-saponifiable lipids
Cannot be hydrolyzed by NaOH because
they have no ester bonds
Example: Cholesterol & Eicosanoids
SAPONIFIABLE LIPIDS
NON-SAPONIFIABLE LIPIDS
SAPONIFICATION TEST:
 TAG can be hydrolyzed into its component fatty acids and alcohol. This reaction can also
be carried out in the laboratory by a process called saponification where the hydrolysis
is carried out in the presence of a strong base (such as NaOH or KOH).

 Principle: Saponification is a process of hydrolysis of oils or fat with alkaline and result in
glycerol and salts of fatty acids (soap).

 Note: soap is salt of fatty acid. The soap is soluble in water but insoluble in ether. Soap
works on emulsification of oils and fats in the water .
 The overall reaction of triacylglycerol saponification can be thought of as occurring in
two steps. The first step is the
>hydrolysis of the ester linkages to produce glycerol and three fatty acid molecules:

The second step involves a reaction between the fatty

acid molecules and the base (usually NaOH) in the
alkaline solution. This is an acid–base reaction that
produces water plus salts
 10g sample lipid in 250 erlenmeyer flask

Add 25mL 6.0M NaOH and 70mL of 95% ethanol

Heat for 75-80deg.cel.on a steam bath through

reflux
Heat for distillation for
75-80deg.cel.on 30 minutes.
a steam bath through
Shake
reflux distillation forocassionally
30 minutes.

Test for complete saponification by pouring 1mL

NOTE: The purpose of of H2O .If cloudiness appears ,heat the mixture
alcohol in again for 10-15 minutes.
saponification
reaction:
Alcohol is less polar
than water so it helps Add water to make 75ml vol. when saponification
dissolve the non-polar is complete. Mix thoroughly.
fat so it can react with
NaOH. Use the solution for the following test.
Separation of Soap by salting out
OBJECTIVE: To investigate the effect of NaCl on soap
solubility

Principle:
 To get the soap out of solution by salting out, when
added solid sodium chloride to the solution until
saturation; separated soap in the form of insoluble and
floats above the surface.
 The NaCl solution provides Na+ and Cl ions that bind to
the polar water molecules, and help separate the water
from the soap. This process is called salting out the
soap.
Separation of Fatty acids and glycerol
10ml of soap solution
Add H2SO4 in excess
Test with litmus

Set Aside
Fatty layer have come to
Fatty acid on
the top
the top

Filter

Filtrate Residue
Wash with Hot water until
free from H2SO4
Test for glycerol Set aside if it solidify
Compare their physical state with fatty
Pungent odor and save for
acids prepared from different fats.
test for unsaturation.
Principle: Break apart triglycerides which are
the major components of vegetable oil and to
recover the glycerol backbone
 HYDROLYSIS OF TRIACYLGLYCEROL
ACROLEIN TEST-is used to detect the
presence of glycerol or fat.
Filtrate
Note:When fat is treated Add KhSO4 or NaHSO4
strongly in the presence of a
dehydrating agent like potassium
bisulphate (KHSO4), the glycerol
Pungent odor
portion of the molecule is
dehydrated to form an
unsaturated aldehyde, acrolein
that has a pungent irritating
odour.
ACROLEIN TEST

With strong dehydrating agents, as concentrated sulphuric acid,

glycerol can be converted to the aldehyde acrolein that has a very
irritating pungent odour.
Cholesterol gives a negative acrolein test.
Five drops of filtrate from the separated filtrate
in (Procedure 5 of 4.1.2) in an evaporating
Dish and add 1g of KHSO4 of NahSo4.

Heat over a free flame and

Note the odor evolved.
Test for Unsaturation
Objective: To determine the iodine value of fats
and oils and thus estimate the unsaturation of
the fats and oils.
 Principle:
The most important application of the iodine
value is to determine the amount of
unsaturation contained in fatty acids. This
unsaturation is in the form of double bonds
which react with iodine compounds. The higher
the iodine value, the more unsaturated fatty
acid bonds are present in a fat.
Dissolve 1 ml of fat sample (fromexp.4.1.2) in 5ml chloroform.

Add Iodine sol’n drop by drop.Shake the sol’n after each drop
Until the color of iodine persists.

Note the number of drops of Iodine until permanent yellow

to orange color.
 Brain lipids
- Lipids are used by living organisms such as
humans for energy storage and can also be found
in different parts of the body, especially the
brain. Pig’s brain contains a high amount of lipid
content, the docosahexanoic acid (DHA) or
popularly known as omega-3 fatty acid. Different
complex lipids that can be found in Pig’s brain
are complex lipids such as Cholesterol,
Phosphatides, Cerebrosides and Sphingosines
BRAIN LIPIDS
OBJECTIVE: Isolate Non-Saponifiable and Saponifiable
Lipids from Fish/Pig’s Brain
Chemicals:
1. Acetone
2. 95% Ethanol
3. Hexane/Petroleum Ether
4. Methanol-Chloroform Mixture
BRAIN LIPIDS 20g Brain + 20ml Acetone w/ sand (Triturate)

+ 50ml Acetone

SCHEMATIC DIAGRAM: Allow to stand overnight

1. NON-SAPONIFIABLE Filter

A. CHOLESTEROL Filtrate
Residue

Wash w/ 20ml Evaporate to 10 ml

acetone over steam

Save for:
Crystallization
Glycerophosphatides
Sphingosine phosphatide Pipette the
Sphingosine glycoside remaining liquid and
collect product

Recrystallization w/
5ml of Hot 95% Ethanol

Filter while hot

Filtrate Residue

to be
BRAIN LIPIDS
filtrate

SCHEMATIC DIAGRAM: Cool over ice and recrystallize

1. NON-SAPONIFIABLE Collect

A. CHOLESTEROL Dissolve 5ml methanol-chloroform mixture

Residue

2. SAPONIFIABLE
Extract w/ 30ml hexane/petroleum
B. GLYCEROPHOSPHATIDES
ether

Allow to stand for 30 mins. w/

constant stirring

Filter

Residue Filtrate
Place into 30 ml Acetone
Save
Filter

Supernatant(discard) ppt. Dissolve 5ml methanol-chloroform mixture

BRAIN LIPIDS Residue
SCHEMATIC DIAGRAM: Extract w/ 50ml of
Hot/Boiling 95% Eth.

1. SAPONIFIABLE: Extract w/ 30ml hexane/petroleum

ether
B. Sphingosine, phosphatide,
Sphingosine glycoside
Boi for 10 mins. over water bath

Filter while
hot

Residue(discard) Filtrate

cool

collect

Dissolve 5ml methanol-chloroform mixture

BRAIN LIPIDS
Appearance of Isolated Complex Lipid Samples

Sample Appearance of Filtrate

Cholesterol Clear colorless solution

Glycerophosphatides Brownish yellow solution

Sphingosine Phosphatides Slightly turbid with suspended

and Sphingosine Glycosides precipitate

General tests for lipids
 Liebermann-Burchard test
 Krauts test
 Salkowski test
 Ninhydrin test
Molisch test
Acrolein test
CHARACTERIZATION OF LIPIDS

 Separation of cholesterol and Triglycerides

- Precipitate- cholesterol digitonide after digitonin precipitation
- Supenatant – Triglycerides
- > Precipitate tested for salkowski test and Liebermann-Burchard test
- -Test for cholesterol
SALKOWSKI’S TEST

A yellow to brick-red colour is

formed indicating the presence of
cholesterol.
There may be a red color in the
upper layer (chloroform) and green
fluorescence in the lower layer due
to the heavier sulfuric acid settling.
Salkowski test- Presence of a double bond in one cholesterol
rings is responsible for its ability to form color products in
the presence of Sulfuric acid- concentrated inorganic acids.

 results in dehydration of cholesterol molecule with a formation of a

red bicholestadien disulphonate.
 Bluish color between the 1st layer (Chloroform) and 2nd layer (Sulfuric
acid).
 Another test is the Salkowski test. It is used to identify the presence
of the double bond in one cholesterol ring. These rings are the ones
that are responsible in the formation of color products in
the presence of inorganic acids. The cholesterol is reacted
with concentrated sulfuric acid that dehydrates to form the red part
in the results
 10drops of the lipid solution in a test tube

Add 20 drops of conc. H2SO4 down the side of

the tube

Observe the color change at the

interface.
Liebermann-Burchard test
 Deep green color
 Due to hydroxyl group (-OH) of cholesterol reacting with the
reagents (Acetic anhydride and concentrated sulfuric acid) and
increasing the conjugation of the unsaturation in adjacent fused
ring
 The first one is the Liebermann-Burchard test. It is used to
detect the presence of steroids. In this test, the acetic
anhydride reacts with the third carbon of the hydroxyl group of
cholesterol and the steroid is the presence of strong acids that
forms the blue-green color of the solution. This test should be
done without any single amount of water for it will
be contaminated.
 0.5mL lipid solution in a test tube

Add 10 drops of acetic anhydride and

swirl.

Add 4 drops of conc. H2SO4 down the side

Of the tube.

Mix .Note the color produced.

LIBERMANN- BURCHARD TEST
 uses acetic anhydride and sulfuric acid as
reagents, gives a characteristic green color
in the presence of cholesterol. This color is
due to the OH group of cholesterol and the
unsaturation found in the adjacent fused ring.
 The color change is gradual: first it appears
as a pink coloration, changing later to lilac,
and finally to deep green.
 the intensity is roughly proportional to the
amount of steroid present
A positive reaction is indicated by appearance of a purple ring at the interface
between the acid and test layers.
Pentoses are then dehydrated to furfural, while hexoses are dehydrated to 5-
hydroxymethylfurfural. Either of these aldehydes, if present, will condense with
two molecules of α-naphthol to form a purple-colored product

MOLISCH TEST
 10 drops of lipid solution in a test tube

Evaporate off the solvent from the tube Suspend the lipid in 20 drops of
In boiling water bath(in fume hood). distilled water.

Add 2drops of molisch reagent .Mix Add 20 drops of conc.H2SO4

Note the 0color0 in the interphase

Molisch reagent (0.5 g
α
-naphthol in 95% ethanol)
 KRAUT’S TEST

Kraut’s reagent (diluted bismuth

subnitrate in 3M nitric acid [HNO3] with
potassium iodide [KI])
Red- orange solution
Test mainly used for choline due to
complexation.
Kraut’s test is a specific test for Choline
(Lecithin) such as glycerophosphatides,
sphingolipids and phosphorylated lipids
 10 drops lipid solution in a test
tube

Evaporate off the solvent from the Suspend the dried lipid in 10 drops of
tube in boiling water bath(in fume distilled water.
hood)

Add 15 drops of Kraut’s reagent. Warm the tube in water bath for 1-
2 minutes

Note the color of the

precipitate/sol’n.
NINHYDRIN TEST
 The principle involved in this
test is oxidative deamination
followed by decarboxylation
and/ or condensation.
 Ninhydrin reagent (0.1 g
Ninhydrin in 95% ethanol)
 Blue- violet solution indicates a
positive result
 Test mainly used for cephalins,
lecithins, and sphingomyelins
 10 drops of lipid solution in a test tube

Add 5 drops of ninhydrin reagent in ethanol

Warm the tube in water bath for 1-2 minutes Note the color of the solution.