Biological Chemistry

Isolation and Characterization of Complex Lipids from Chicken Brain

Ellicia Vern Mendoza, John Michael Joseph Napa, Nicanor Olanka, *Maria Christina Paine
Group 6, 3Bio-3
Department of Biological Sciences, College of Science,
University of Santo Tomas, España, Manila 1055

Date Submitted: February 19, 2011

Abstract
The brain tissues contain complex lipids with different chemical composition and solubility.
These include cholesterol, galactocerebrocide, and sphingolipid. This experiment discusses how
the complex lipids are being isolated and characterized. To explain how this happens,
microscale method, Liebermann-Burchard test, Salkowski test, Test for phosphate, Kraut’s test,
Ninhydrin test, & Molisch test were performed. A table of color reactions in the end shows the
results.

I. Introduction

Lipids are natural substances that are arbitrarily grouped together based on their solubility

in fat solvents, and their solubility in water (Hein, Peisen & Ritchey, 2005). Lipids are different

among organic molecules because their identity is defined based on their physical property and

not by a particular functional group present in them. This characteristic makes them come in a

wide variety of structures and functions (Smith, 2010). Lipids are divided into two subunits:

simple and complex. Complex lipids include phospholipids, sphingolipids, and glycolipids.

Phospholipids are classified according to the alcohol esterified to the phosphate group. For

example, phosphatidylcholines are also known as lecithin (Milio & Lofreddo, 1994). Lecithin

can be found in animal tissues like brain tissues. Galactocerebrosides are found in cell

membranes of the brain (McKee, J. & McKee, T., 2003). Sphingolipid is another lipid

component of biological membranes. Sphingosine is the backbone of sphingolipids (McKee, J. &

McKee, T., 2003). Cholesterol is a type of steroid. These substances possess a 17-carbon unit

structure containing four fused rings known as the steroid nucleus (Hein, Peisen & Ritchey,

2005). Steroids having an –OH group attached to the ring are known as sterols (Hein, Peisen &

Ritchey, 2005). Cholesterol is the most abundant steroid in the body and is found in foods from

animal sources such as eggs or chicken brain. Little amounts of cholesterol can be detected by

the Lieberman-Burchard test wherein treating a chloroform solution of the steroid with acetic

anhydride and concentrated sulfuric acid is involved. The formation of blue-green color is a

positive test (Hein, Peisen & Ritchey, 2005). Kraut’s test tests the presence of alkaloids or amine

oxide bases and yields a maroon solution with brown precipitate for positive results. Phosphate

test determines the presence of phosphate where the appearance of yellow color is a positive

result. Salkowski’s test is a test that determines the presence of cholesterol. The appearance of a

red interface is a sign of positive result. Ninhydrin test indicates the reaction of amines with the

ninhydrin which gives a blue to violet color. Molisch test is a general test for carbohydrates, and

any monosaccharide will give a positive test (Henrickson, Byrd & Hunter, 2005). The

experiment has the objectives of isolating the lipids from the chicken brain and separating them

into phosphorylated and non-phosphorylated lipids, isolating non-saponifiable lipids from the

chicken brain using the microscale method, and characterizing isolated lipids using various

chemical tests. The lipids present in the chicken brain are being extracted through microscale

method and characterized through chemical tests such as Liebermann-Burchard test, Salkowski

test, Test for phosphate, Kraut’s test, Ninhydrin test, & Molisch test.

II. Materials

Chicken brain

Acetone
Sand

95% Ethanol

Methanol-chloroform mixture

Filter paper

50-mL Erlenmeyer flask

Acetic anhydride

Conc. H2SO4

Fusion mixture (KNO3:Na2CO3, 3:1)

3 M HNO3

2.5% (NH4)2 MoO4

Kraut’s reagent

Molisch reagent

Ninhydrin solution

Standards: cholesterol

Lecithin

Galactocerebroside

Sphingolipid

Phosphorylated lipid

Non-phosphorylated lipid

III. Methodologies

The experiment was divided into two parts: isolation and characterization. The first part

to be conducted was the isolation of complex lipids from the chicken brain. A medium-sized

brain was triturated with 10.0 mL acetone using sand to aid in the disruption of the cells. The
system was then transferred to the smallest Erlenmeyer flask and 20.0 mL of acetone was added

with it. After that, the Erlenmeyer flask was covered with aluminum foil and kept in the

refrigerator until the following meeting. The following meeting, the mixture was filtrated. The

residue was washed with 10 mL acetone and was mixed with the filtrate. It was evaporated over

a steam bath until almost dry. The recrystallized cholesterol was then dissolved to CHCl 2. On the

other hand, the residue of the acetone - washed residue was extracted with 20 mL hot ethanol. It

was then boiled in 10 minutes through steam bath. The product was filtered and the filtrate was

cooled. The precipitate was collected as the sphingosine phosphatides. Next, the filtrate was

evaporated. 10 mL acetone was poured and stirred. It was decanted and the supernatant was

discarded. The residue was then dissolved in 5 mL CHCl3.

The other part of the experiment was the characterization of complex lipids using various

tests. Six tests were performed in this experiment: 1. Liebermann-Burchard test, 2. Salkowski

test, 3. Test for phosphate, 4. Kraut’s test, 5. Ninhydrin test, & 6. Molisch test. In Liebermann-

Burchard test, 0.5 mL of each lipid solution was placed in a separate small test tube. Next, 10

drops of acetic anhydride were added to each and swirled gently. After this, 4 drops of conc.

H2SO4 were carefully added down the side of the tube. Finally, it was mixed well and the color

was noted. In Salkowski test, 10 drops of the lipid solution were placed in a small test tube. 20

drops of conc. H2SO4 were then carefully added down the side of the tube. The two layers

formed were not mixed and the color of the interphase was finally noted. In the test for

phosphate, 0.5 mL of the lipid was mixed with the fusion mixture in a crucible. It was then

ignited over a free flame until all organic matter was burnt away and the mixture had turned to a

grayish or colorless liquid, or a white or gray ash was obtained. It was cooled and dissolved in 3

mL of warm water. The contents were transferred to a test tube and were acidified with 3 M
HNO3. The solution was then heated to 65 oC. 3 mL of 2.5% ammonium molybdate was added

and the tube was warmed. The color of the precipitate and solution were observed. In Kraut’s

test, 10 drops of the lipid solution were put in a small test tube. The tube was then put in a

boiling water bath in the fume hood to evaporate off the solvent from the lipid solution. The

dried lipid was suspended in 10 drops of distilled water. 15 drops of Kraut’s reagent were added.

The tube was finally warmed for 1-2 minutes and the color of the solution and the precipitate

formed were noted. In Ninhydrin test, 10 drops of the lipid solution were put in a small test tube.

Then, 5 drops of ninhydrin reagent were added in ethanol. The tube was finally warmed for 1-2

minutes and the color of the solution was noted. Finally, in the Molisch test, 10 drops of the lipid

solution was put in a small test tube. The tubes were then put in a boiling water bath in the fume

hood to evaporate off the solvent from the lipid solution. Next, the lipid was suspended in 20

drops of distilled water. 2 drops of Molisch reagent was added and mixed well. After that, 20

drops of conc. H2SO4 was carefully added down the side of the tube. The two layers formed were

not mixed and the color of the interface was noted.

IV. Data & Results

A. Chemical Test Results

Chemical Cholesterol NPL Lecithin PL Galactocerebroside Sphingolipid

Test
Salkowski Red Red --------- ---- -------------------- ----------------
test interface interface
Liebermann- Emerald- Emerald- --------- ---- -------------------- ----------------
Burchard green green
Test solution solution
Test for Yellow Yellow -------------------- ----------------
PO43- -------------- ------------- solution solutio
n
Kraut’s test Maroon Maroon
solution solutio
w/ n w/ ----------------------- ----------------
-------------- ------------- brown brown
 ppt ppt
Ninhydrin Violet -------------------- ----------------
test -------------- ------------- ---------- solutio
n
Molisch test ----- Interface present Interface
-------------- ------------- ---------- present

V. Discussion

Salkowski test is a test that indicated the presence of cholesterol. Non-phosphorylated

lipid (NPL) is also the crude cholesterol. Appearance of red interface on the upper layer of the

solution is a sign of positive result. As cholesterol increases, the red interface shows on the

upper layer. The presence of a double bond in none of cholesterol rings is responsible for its

ability to form color products in the presence of concentrated inorganic acids. The action of

concentrated sulfuric acid results in dehydration of cholesterol molecule with a formation of a

red interface. The standard cholesterol showed slightly brighter red interface than the standard

cholesterol because the standard cholesterol has higher purity than the crude one. In

Liebermann-Burchard test, which is also a cholesterol-detection test, the presence of acetate

anhydride forms an emerald-green color. Normally, this color begins as a purplish, pink color

and progresses through to a light green then very dark green color. The color is produced 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. Traces of water make this reaction

possible. Phosphate test is a qualitative method wherein the presence of phosphate ions is being

determined. The positive color for this is a bright yellow. When lipids containing phosphate

groups in their structures are added to a strong acid solution, the lipid hydrolyses, producing

free phosphate. The free phosphate then reacts forming a yellow color (Milio & Lofreddo,

1994). Lecithin or phosphatidylcholine is a kind of lipid which has an alcohol esterified to a

phosphate group. Kraut’s test is used to identify presence of alkaloids or amine oxide bases. It is

an adaptation of "Dragendorff's reagent" which is a solution of potassium bismuth iodide.

Lecithin contains alkaloids which gave it a positive result. Phosphorylated lipids present in the

brain have amide bonds which are cleaved by specific enzymes which results in compounds

which have primary amine groups and will therefore produce a positive ninhydrin test result. In

the Molisch test, galactocerebroside and sphingolipid contain carbohydrates which made an

interface formed by reaction with alpha-naphthol in the presence of sulfuric acid.

Galactocerebroside is a cerebroside consisting of a ceramide with a galactose residue. Type of

sphingolipid called glycolipids contains one or more molecules of sugar (galactose & glucose).

Glycolipids are abundant in the nervous system. Hence, we can infer that it’s present in the

brain.

The purpose of acetone in the isolation of cholesterol from chicken brain is to separate

one step polar lipids (i.e phospholipids & glycolipids) from all neutral or non-polar lipids (i.e.

cholesterol). Acetone and 95% ethanol dissolve the lipids readily and overcome the interactions

between the lipids and the tissue matrix. Recrystallization is done on the isolated to cholesterol

to purify it. Hexane or petroleum is usually used to isolate glycerophosphatides in the brain. It is

because it is non-polar and thus separates the lipid from more polar lipids.

VI. Conclusion & Recommendation

In conclusion, complex lipids can only be characterized through their chemical

composition. The chicken brain was separated into phosphorylated and non-phosphorylated

lipids through using certain chemical to promote differences in solubility. Microscale method has

used to isolate non-saponifiable lipids from the chicken brain. The isolated lipids have been
characterized through different chemical test to determine their chemical composition and

therefore, determine their identity.

During the experiment, the measurements and time requires should be done properly. The

laboratory glasswares should be maintained clean to avoid mixing of unwanted chemical that

may affect the results. Accuracy and precision should be observed to avoid wrong results.

VII. References

Hein, M., Peisen, J.N., & Ritchey, J.M. Introduction to General, Organic, & Biochemistry in the

Laboratory, 8th ed. Hoboken, NJ: John Wiley & Sons, Inc., 2005. Page used: 347-349

Henrickson, C.H., Byrd, L.C., & Hunter, N.W. A Laboratory for General, Organic, &

Biochemistry, 4th ed. New York: McGraw-Hill, 2005. Page used: 127

McKee, T. & McKee, J.R. Student Study Guide, Solutions Manual for use with Biochemistry:

The Molecular Basis of Life, 3rd ed. New York: McGraw Hill, 2003. Page used: 328-329

Milio, F. R. & Loffredo, W. M. Qualitative Testing for Lipids.USA: Chemical Education

Resources, Inc, 1994. Page used: 2, 4

Smith, J. G. General, Organic, & Biological Chemistry. New York: McGraw Hill, 2010. Page

used: 570