Isolation and Characterization of Saponifiable and Non-Saponifiable Lipids

from Calf’s Brain using various chemical tests

Dino, Catherine, Duzon, Jhoanna Rein*, Elsisura, Rjan and Gamboa, Alissa Jane
College of Science, University of Santo Tomas,
Espana Blvd., Manila

Abstract

The main objective for this experiment is to be able to isolate and characterize the two classifications of lipids
present in the calf’s brain. The calf’s brain is rich in saponifiable and non-saponifiable lipids, a fact that indicates the
importance of these compounds to life processes. The first experimental procedure that was conducted involve the
isolation of cholesterol, by the filtration, collection, and recrystallization of the crude cholesterol. The recrystallized
cholesterol, then, will be used for the second part of the experiment which involves the isolation of
glycerophosphatides, by precipitation, filtration, and collection of the precipitate that will be produced. The
dissolved precipitate (dissolved in 1 methanol: 3 chloroform) will be used for the isolation of sphingosine
phosphatides and sphingosine glycosides. This part will involve filtration, and collection of the precipitate that will
be produced. And after which will be dissoloved (in a 3 mL 1 methanol: 3 chloroform mixture)

Introduction
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 used by living organisms such as humans for energy storage and can also be

found in different parts of the body, especially the brain. Calf’s brain or Bos Taurus 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 calf’s brain are complex lipids such as

Cholesterol, Phosphatides, Cerebrosides and Sphingosines. Figure 1 shows the different types of

lipids.
 Aside from this, they can also function as heat, and is often found associated with the

human skin. Lipids can be classified accordingly, namely fatty acids, glycerophospholipid,

Sphingolipids, waxes, terpenes, and steroids. Their reaction for strong bases gave them a

distinction for two major groups; the saponifiable lipids and nonsaponifiable lipids.

Saponifiable lipids are those lipids that can be hydrolyzed under basic conditions (usually

Sodium Hydroxide is used) due to it possessing an ester group. The major saponifiable lipids are

triacylglycerides, glycerophospholipids, and the sphingolipids. The former two use glycerol as the

backbone. Triacylglycerides is composed of three fatty acids esterified to the three hydroxides on

glycerol. Glycerophospholipids are composed of two fatty acids esterified at carbon numbers 1

and 2. On the other hand, non-saponifiable lipids are those lipids which does not contain an ester

group, which is the reason why they will never be hydrolyzed when exposed to strong base (like

Sodium Hydroxide). Cholesterol is one of the major examples of a non-saponifiable lipid.

Lipids have two principal functions in the body: as repositories of chemical energy in

storage fat, primarily triglycerides, and as structural components of cell membranes. Some lipids,

such as inositides and phosphatidylcholine, which were believed to have possessed only a

structural role, also have vital functions for signal transduction via neurons in the nervous system

of a living organism. In addition, lipids covalently coupled to proteins play a major role in

anchoring marker proteins within bio membranes.

The objective of this experiment is to isolate saponifiable and non-saponifiable lipids from

calf’s brain and to characterize the isolated complex lipids using various chemical tests.
Figure

1. Different types of lipids

Methodology

The first part of the experiment is the Isolation of saponifiable and non-saponifiable lipids.

The cholesterol was isolated from 20 g of calf’s brain that was triturated with 20 mL acetone with

the use of sand to aid in the disruption of cells. The mixture was transferred to a 125 mL

Erlenmayer flask and 50 mL of acetone was added. A cork was used as a stopper to the flask. The

mixture was allowed to stand overnight in the refrigerator. The extract was filtered and the residue

was washed with 15 mL acetone. The residue was saved for the preparation of

glycerophosphatides, sphingosine phosphatides and sphingosine glycosides. The filtrate was

evaporated to 10 mL over a steam bath inside the fume hood and then the evaporating dish was

cooled over an ice bath. The cholesterol was crystallized from the solution. The crude cholesterol

was then filtered and collected. The crude cholesterol was recrystallized by dissolving it in a

minimal value (2-3 mL) of hot ethanol. The solution was filtered while it is hot and the filtrate was
allowed to cool. The recrystallized cholesterol was collected by decantation and was dissolved in

3 mL of methanol:chloroform mixture (1:3).

The glycerophosphatides was isolated from the residue that was washed with 15 mL

acetone in the isolation of cholesterol. The residue was transferred to a 100 mL beaker and was

extracted with 20 mL hexane or petroleum ether. The beaker was covered with a watch glass and

was allowed to stand for 30 min with occasional stirring every 5 minutes. The product was filtered

and the residue was saved for the isolation of sphingosine phosphatides and sphingosine

glycosides. The extract was concentrated into 20 mL acetone and was stirred well until a precipitate

was formed. The mixture was then put in an ice bath and the precipitate was filtered off and

dissolved at once in a 3 mL of methanol:chloroform mixture (1:3). The filtrate was disregard and

the precipitate was used in the isolation of sphingosine phosphatides and sphingosine glycosides.

The sphingosine phosphatides and sphingosine glycosides was isolated from the residue of

the isolation of glycerophosphatides. The residue was transferred to a 100 mL beaker and was

extracted with 40 mL of warm ethanol. The mixture was boiled for 8 minutes over a water bath

using a 400 mL beaker with small volume of water. The hot mixture was filtered and the residue

was discarded. The filtrate was cooled and the resulting precipitate by filtration was collected. The

collected precipitate was dissolved in 3 mL of methanol:chloroform mixture (1:3).

The second part of the experiment is the characterization of isolated lipids using various

chemical tests. For the Liebermann-Burchard test, a 0.5 mL of each lipid solution (cholesterol,

glycerophosphatides and sphingosine phosphatides and sphingosine glycosides) and standards

(cholesterol, lecithin, galactocerebroside) was placed in small test tubes. 10 drops of acetic

anhydride was added and each test tube was swirled gently. 4 drops of concentrated sulfuric acid
(H2SO4) was very carefully added down the side of the tube. The tube was mixed well and the

color produced was noted.

For the Salkowski test, 10 drops of each lipid solution (cholesterol, glycerophosphatides

and sphingosine phosphatides and sphingosine glycosides) and standards (cholesterol, lecithin,

galactocerebroside) was placed in a small test tube. 20 drops of concentrated sulfuric acid

(H2SO4) was very carefully added down the side of the tube. The 2 layers were not mixed and

the color of the interphase was noted.

For the Test for Phosphate which was not done in the laboratory, 1.0 mL of lipid

(cholesterol, glycerophosphatides and sphingosine phosphatides and sphingosine glycosides) and

standards (cholesterol, lecithin, galactocerebroside) with fusion mixture (5 times its bulk) was

placed in a crucibles. The mixture was ignited over a free flame until all organic matter is burned

away and until the mixture turned to a grayish or colorless liquid, or a white or gray ash was

obtained. It was then cooled and dissolved in 3mL of warm water. The contents was transferred

to test tubes and acidified with 3M Nitric acid (HNO3). Then, the solution was heated to 65° C

and 3 mL of 2.5% ammonium molybdate was added and the tube was warmed. The color of the

precipitate and the solution was then observed.

For the Kraut’s test, 10 drops of each lipid solution (cholesterol, glycerophosphatides and

sphingosine phosphatides and sphingosine glycosides) and standards (cholesterol, lecithin,

galactocerebroside) was placed in small test tubes. The test tubes was placed in a boiling water

bath in the fume hood and the solvent from the lipid solution was evaporated. The dried lipid was

suspended in 10 drops of distilled water and then 15 drops of Kraut’s reagent was added. The

tubes were warmed for 1-2 min and the color of the solution and precipitate was noted.

For the Ninhydrin test, 10 drops of each lipid solution (cholesterol, glycerophosphatides
and sphingosine phosphatides and sphingosine glycosides) and standards (cholesterol, lecithin,

galactocerebroside) was placed in small test tubes. 5 drops of ninhydrin in ethanol was added in

each tube. The tubes were warmed for 1-2 min and the color of the solution was noted.

Lastly, for the Molisch test, 10 drops of each lipid solution (cholesterol,

glycerophosphatides and sphingosine phosphatides and sphingosine glycosides) and standards

(cholesterol, lecithin, galactocerebroside) was added in small test tubes. The tubes was then

placed in a boiling water bath in the fume hood and the solvent from the lipid solution was

evaporated. Then, 20 drops of lipid was suspended in 20 drops of distilled water. 2 drops of

Molisch reagent was mixed well and 20 drops of concentrated sulfuric acid (H2SO4) was very

carefully added down the side of the tube. The 2 layers were not mixed and the color of the

interphase was noted.

Results and Discussion

Table 1: Appearance of Isolated Complex Lipid Samples

Sample Appearance of Filtrate

Cholesterol Clear colorless solution

Glycerophosphatides Brownish yellow solution

Sphingosine Phosphatides and Sphingosine Slightly turbid with suspended precipitate

Glycosides

Table 1 describes the appearance of isolated lipid samples. The appearance of cholesterol

is a clear colorless solution, the glycerophosphatides’ appearance is a brownish yellow solution

and Sphingosine Phosphatides and Sphingosine Glycosides are slightly turbid and have suspended
precipitates. Figure 2 shows the appearance of isolated cholesterol, glycerophosphatides and

sphingosine phosphatides and sphingosine glycosides.

In the isolation of lipid samples, different concepts and reagents

were involved. For the isolation of cholesterol, the acetone, which is a

highly nonpolar molecule that is less reactive to different lipid

components compared to alcohol and easier to evaporate after being used

as a solvent. Also, it provides a mild but rapid method of extracting fats,

sterols and other lipids. The role of the acetone is to separate the
Figure 2. Isolated
cholesterol, phosphorylated lipids from non-phosphorylated lipids. Non-
glycerophosphatides
and phosphorylated lipids such as sterols will dissolve in acetone because
sphingosine
glycosides both of them have high non-polarity. Another concept involved is the

recrystallization which is a procedure to purify non-volatile organic

compounds. Its principle involves that as the temperature is increased, the solubility of solutes

increases also which means that the amount of solute that can be dissolved increases also.

The cholesterol belongs to the group of steroids, the sterols. Steroids is a fused-ring

system of 3 six-membered rings & 1 five-membered ring. The general structure of steroids,

shown in Figure 3 could contain ketones, alcohols, double bonds and hydrocarbon chains. The

cholesterol is a very hydrophobic compound that is a precursor to bile acids, steroid hormones

and vitamin D. Figure 4 shows the structure of a cholesterol.

 Figure 3. General Figure 4. Structure of a cholesterol

steroid structure

For the isolation of glycerophosphatides, hexane a non-polar, colorless liquid that was used

to extract lipids from mixtures of water with alcohols. It has a low boiling point of 62 ° C and a

good solvent for lipids with low polarity. It acts as a selective solvent to separate

glycerophosphatides and sphingolipids.

The glycerophosphatides also called phosphoacylglycerol and glycerophospholipid is the

most abundant class of membrane lipid. It has range of compounds with glycerol as backbone. In

the structure of phosphatidic acid in figure 4, the R3 groups can be Phosphatidic acid (H),

Phosphatidylserine (Serine), Phosphatidylethanolamine (Ethanolamine), Phosphatidylcholine

(lecithin) and Phosphatidylinositol (inositol). Figures 5.6.7,8 and 9 shows these structures with its

net charges.
Figure 5. General structure of phosphatidic Figure 6. Structure of Phosphatidylethanolamine

acid (net charge -1) (net charge 0) (contains free amino group)

Figure 7. Structure of Phosphatidylcholine

(net charge 0) (contains quaternary amine)

 Figure 8. Phosphatidylserine (net charge -1)
Figure 9. Phosphatidylinositol net charge: -1)
(contains amino acid serine) (inositol is NOT a SUGAR)

For the Sphingosine Phosphatides and Sphingosine Glycosides, a hot ethanol was used in

extraction because the residue is more soluble in hot solvents than in cold solvents. As the

temperature increases, in ethanol, the solubility also increases making it easier to dissolve residues

of glycerophosphatides. The mixture was then boiled over a water bath to evaporate excess

solvents. The mixture was filtered while it is hot because filtering it off will prevent sudden

precipitation. In dissolving the collected precipitate, methanol:chloroform mixture is a general

extraction solvent used to dissolve other interfering agents such as glycerophosphatide

components. The methanol is a polar solvent used to dissolve membrane-associated lipids, which

is also polar, and disrupts hydrogen bonding or electrostatic forces. The chloroform mixture is a

nonpolar solvent used to extract neutral or storage lipids.

The sphingolipids, also called the membrane lipids is a group of lipids that is built on

amino alcohol, the sphingosine. It has a long chain of hydrophobic tail, an amino group ((if fatty

acid is linked then sphingosine becomes ceramide) and an Alcohol (can be attached with

phosphocholine, or saccharide groups [sugar]). Figures 10 and 11 shows the structure of

sphingosine and ceramide.

Figure 10. Structure of sphingosine Figure 11. Structure of ceramide

The sphingolipids used in this experiment are sphingosine phosphtides and sphingosine

glycosides. The sphingosine phosphatides or the sphingomyelin are phospholipids that contain

sphingosine and phosphocholine/ ethanolamine. This is found in large quantities in nerve tissues,

particularly, the myelin. Figure 12 shows the structure of sphingosine phosphatides.

Figure 12. Structure of sphingosine phosphatides

 The Sphingosine glycosides are glycolipids containing a phospho group and sphingosine.

It contains a lipids part bound with sugars (Galactose or Glucose) and a hydrocarbon, R. Figure

13 shows the structure of a cerebroside.

Figure 13. Structure of cerebroside

 Table 2: Characterization of Complex lipids using various chemical tests

TESTS
Standards Liebermann Salkowski Kraut’s test Ninhydrin test Molisch test
-Burchard test
test
From From From colorless From colorless From colorless
colorless colorless solution to solution with solution to light
Cholesterol solution to solution to solution with no color pink solution.
blue-green golden yellow black change No interphase
solution solution precipitate

Lecithin From yellow From yellow From yellow From yellow From yellow
solution to solution to solution to red solution to solution with
mossy green golden yellow orange solution violet solution very light brown
solution solution with with orange upper layer.
dark red upper precipitate No interphase
layer
From From From colorless From colorless From colorless
Galctocerebroside colorless colorless solution to red solution to solution to
solution solution to orange solution colorless
with no color very light with black solution with
change brown precipitate white
solution precipitate
Samples
Cholesterol From From From colorless From colorless From colorless
colorless colorless solution to red solution to light solution to
solution to solution to orange solution violet solution
very light turbid with black
green solution with precipitate
solution peach
interphase
Glycerophosphatides From yellow From yellow From yellow From yellow Yellow solution
solution to solution to solution to solution to dark to brown
mossy green brown bright orange violet solution solution with red
solution solution with solution with precipitate
bloody red orange
interphase precipitate
Sphingosine From From From colorless From colorless From colorless
Phosphatides/ colorless colorless solution to red solution to dark solution to light
Sphingosine solution to solution to orange solution violet solution brown solution
Glycosides pinkish solution with with whitish
solution with purple orange
light pink interphase precipitate
precipitate
 Table 2 shows the appearance of the standards and samples in the 5 chemical tests. The

first test is the Liebermann-Burchard test, which is a general test for sterols such as cholesterol and

non-phosphorylated lipids. The reagents used in this test were Acetic anhydride and concentrated

Sulfuric acid (H2SO4). The principle involved in this test is condensation of Acetic anhydride with

the carbon 3 of alcohol (OH) and carbon 5 double bond. A positive result for this test is an emerald

green solution. (See figure 2)

The second test is the Salkowski test, which is a specific test for cholesterol such as

cholesterol itself and non-phosphorylated lipids. The reagents used in this test are Chloroform

(CHCl3) and concentrated Sulfuric acid (H2SO4). The principle involved is condensation of 2

cholesterol molecules to form bisteroids. A positive result for this test is a red to purple interphase.

(See figure 3)

The third test, the Kraut’s test is a specific test for Choline (Lecithin) such as

glycerophosphatides, sphingolipids and phosphorylated lipids. The reagents used in this test are

Krauts’s reagent (KBiI4) which is composed of Potassium iodide and Bismuth subnitrate. The

principle involved is complexation reaction of Choline with KBi4 (formation of colored salt). A

positive result for this test is a brick-red precipitate. (See figure 4)

The fourth test, which is the Ninhydrin test is a general test for the presence of amino acids

such as glycerophosphatides, Sphingolipids, and Phosphorylated lipids. The principle involved is

oxidative deamination and condensation reactions. A positive result for this test is a violet solution.

(See figure 5)

The fifth test, which was not done in the laboratory is the Phosphate test or Nitrogen test.

It is a general test for the presence of phosphate or nitrogen. The reagents used include the fusion
mixture (KNO3/Na2CO3, 3:1), 3M HNO3 and 2.5% ammonium molybdate. The principle involved

is the oxidation reaction. The positive result is a yellow precipitate. (See figure 6)

Lastly, the Molisch test is a general test for the presence of sugar moiety (carbohydrates)

such as the sphingolipids, galactocerebrosides and non phosphorylated lipids. The principles

involved were, 1. Hydrolysis of glycosidic bond forming the reduced sugar, 2. Dehydration of the

monosaccharide into hydroxymethyl furfurals and 3. Condensation of the furfural with a-naphtol.

The positive result is a violet/ring interphase. (See figure 7)

Figure 2. Results of Liebermann- Figure 5. Results of Ninhydrin test

Burchard test

Figure 3. Results of Salkowski test Figure 6. Results of Molisch test

 Figure 4. Results of Kraut’s test

Conclusion

Based on the results of this experiment, in the characterization, the samples and standards

that obtained a positive test in Liebermann-Burchard test are cholesterol (standard and sample),

lecithin

In Salkowski test are

In Ninhydrin test are

In Molisch test

And lastly, in Krauts test are

The possible sources of error includes

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