Exp 9 Egg Lipids GRP 8

Isolation and Characterization
of Complex Lipids from Egg

Yolk and Pig Brain
Gumatay, Sheena S.
Daleja, Jose Alfonso A.
Amurao, Gail Louise L.
Fredeluces, Mariah Mae E.
Leogardo, David Emmanuel R.
3Bio5
INTRODUCTION
LIPIDS
Lipids
- hydrophobic (water insoluble - nonpolar)
- form aggregrates (non polymeric)
- can be extracted from the cell by
organic solvents
◦ There are various ways of classifying

lipids. Major lipid groups include
fats, phospholipids, steroids and waxes.
LIPIDS
Lipids may be classified according to function.
Storage Lipids
Triacylglycerol
Fatty acid
Glycerol
Fatty acid
Fatty acid
LIPIDS
Lipids may be classified according to function.
Membrane
Lipids
Phospholipids Glycolipids
Glycerophospholipids Sphingolipids Sphingolipids Galactolipids
Fatty acid Fatty acid
Glycerol
Glycerol
Sphingosine
Sphingosine
Fatty acid Fatty acid Fatty acid Fatty acid
PO4 alcohol PO4 choline sugar sugar

LIPIDS
Lipids are conveniently classified as:
◦ Simple lipids
– lipids which yield fatty acids and alcohols on
hydrolysis
◦ Complex lipids
– lipids which may contain phosphate, various
carbohydrates, and nitrogenous components
such as sphingosine, ethanolamine, serine,
hexosamines
LIPIDS
 Lipids may also be classified by the
presence of phosphoester group
(phosphorylated lipids) or absence
of such group (nonphosphorylated
lipids)
 Biological functions:
◦ energy source, energy storage, cell
membrane structural components,
hormones, vitamins, vitamin
adsorption, protection, and insulation.
Saponifiable Lipids
 Can undergo saponification or base
hydrolysis
 Also known as: Simple lipids
 Examples:
◦ Triacylglycerols
◦ Glycolipids
◦ Sphingolipids
◦ Waxes (some)
Non - Saponifiable Lipids
 Cannot be hydrolyzed by aqueous base
 Cannot be transformed into soap
 Examples:
◦ Phospholipides Steroids
◦ Prostaglandins
◦ Leukotrienes
◦ Terpenes
Nonsaponifiable lipids
Cholesterol
 Compound of the sterol type found in
body tissues, blood and nerves.
 Important constituent of cell membrane.
 Precursor of other steroid compounds.
 High concentration in blood; promotes
atherosclerosis.
PIG BRAIN
 Contains Docosahexaeonic acid (DHA),
among other fatty acids, as in most
mammalian brains.
 Contains high levels of cholesterol.
EGG YOLK
- a rich source of a variety of
biochemically important compounds
such as proteins and lipids
- makes up about 30 to 33% of the
liquid weight of the egg
- Composition of yolk lipid is
- 65.5% triglyceride
- 28.3% phospholipid
- 5.2% cholesterol
LIPIDS IN EGG YOLK
Why egg yolk not egg white?
Egg Component % Lipid
Egg White 0.03%
Egg Yolk 31.8-35.5%
The lipids of the egg yolk are divided

into:
1. Phosphorylated Lipids
2. Non-Phosphorylated Lipids
OBJECTIVES
 For egg yolk lipids, to isolate the lipids
from egg yolk and to distinguish
them into phosphorylated and non-
phosphorylated lipids
 For, pig brain lipids, to isolate the
lipids from pig brain and to
distinguish them into different types
of lipids.
 To characterize these lipids using
various chemical tests
METHODOLOGY
METHODOLOGY
A. Isolation of Complex Lipids from Egg Yolk
Egg yolk
Placed in 250 ml beaker
Stirred well
+ 100 ml CHCl3: CH3OH (2:1)
Let it stand for 10 mins
Filter
Residue Filtrate
Discard
METHODOLOGY
Filtrate
Note volume
Place in separatory funnel
Extract with an equal

amount of 1% NaCl
solution
Aqueous Organic
layer layer
Discard
METHODOLOGY
Organic layer
Place in separatory funnel
Extract with an equal

amount of 1% NaCl solution
Aqueous Organic
layer layer
Discard
METHODOLOGY
Organic layer
dry with anhydrous Na2SO4

filter
+ pinch of hydroquinone
transfer to evaporating dish
evaporate to dryness
Sticky yellow residue
+ 15 ml acetone
cool in ice bath (15 mins.)
filter
Precipitate Filtrate
(Phosphorylated) (Non-Phosphorylated)
METHODOLOGY
A.1 Isolation of Complex Lipids from Egg
Yolk
Precipitate Filtrate (Non-
(Phosphorylated) Phosphorylated)
wash with 5 mL evaporate to dryness

cold acetone
+ 5mL CHCl3 :MeOH
decant mixture
NPL (Cholesterol)
Filtrate Precipitate
Discard + 5mL CHCl3 :MeOH mixture
+ pinch of hydroquinone
PL
METHODOLOGY
A.2 Isolation of Complex Lipids from Pig
Brain Pig’s Brain
Triturate 40g of pig’s brain

with 20 ml acetone and sand
Residue
Transfer to an Erlenmeyer
Flask
Add 100 ml of acetone
Allow to stand overnight

in the refrigerator
Filter and extract the system

METHODOLOGY
A.2 Isolation of Complex Lipids from Pig
Brain
Residue Extract
Filter the
Wash with 20 ml of
extract
acetone
Save for the Filtrate

preparation of:
Glycero
Sphingolipids
phosphatides
METHODOLOGY
A.2 Isolation of Complex Lipids from Pig Brain
Cholesterol
Filtrate
Evaporate the filtrate to

10ml over a steam bath
Cool with cold water
Crude Crystallized Cholesterol
Collect product in a funnel
Dissolve using hot Ethanol,

drop wise
Filter while hot
Allow to cool
Recrystallized Cholesterol
METHODOLOGY
A.2 Isolation of Complex Lipids from Pig Brain
Glycerophosphatides
Residue in E. Flask
(transferred to beaker)
+30 ml hexane or pet.

ether
Swirl
Let stand for 30 mins. (w/

occasional stirring
Filter
Residue Filtrate
For 5.322
METHODOLOGY
5.321 Glycerophosphatide
Residue Filtrate
Pre-concentrate w/
steam bath
+30 mL acetone; stir
filter
Filtrate Residue
Glycerophosphatides
Residue from
extraction of
Gylcerophosphatides
Transfer to beaker
Extract with 50mL hot ethanol
Boil over water bath for 10

minutes
Filter
Residue Filtrate
Discard Cool
Filter
Filtrate Residue
+10 mL MeOH: CHCl3 (1:3)

Sphingosine phosphatides
Sphingosine glycosides
METHODOLOGY
B. Characterization of Complex Lipids
B.1 Liebermann-Burchard test
Lipid sample/Standard
Place .5 ml in test tube
Add 10 drops of
Acetic Anhydride
gently swirl
Add 4 drops of
conc. H2SO4
Mix well
Note color produced
Emerald Green solution
METHODOLOGY
B. 2 Salkowski Test
Place 10 drops in test tube
Add 4 drops of H2SO4
Note color produced at

interphase
Red interphase
METHODOLOGY
B.3 Test for Phosphate
Place 1.5 ml of sample with

fusion mixture (5 times more
than sample) in a crucible
Subject to free flame until it

turns gray or into a
colorless liquid or obtains a
white or gray ash
Transfer contents to a test
tube and add 2-3 drops of
3M nitric acid
Heat to 65ºC
Add 3 ml of 2.5% ammonium
molybdate and warm test tube
Note color produced
Yellow Solution and

ppt
METHODOLOGY
B. 4 Kraut’s Test
Put in boiling water bath

in fume hood
Suspend dried lipid in
10 drops distilled water
Add 15 drops of Kraut’s
reagent
Note color produced

and precipitate formed
Red – Orange Solution and ppt
METHODOLOGY
B. 5 Ninhydrin Test
Add 5 drops of ninhydrin

in ethanol
Warm test tube for 1-2
minutes
Note color produced
Violet Solution
METHODOLOGY
B. 6 Molisch Test
Evaporate solvent from

solution via boiling water bath
Add 20 drops of distilled

water
Add 5-6 drops of
Molisch reagent
Mix well
Add 20 drops of conc.
H2SO4
Note color produced at
interphase
Violet Solution and Violet
Interphase
RESULTS AND
DISCUSSION
A. ISOLATION OF COMPLEX
LIPIDS FROM EGG YOLKS
 Chloroform:Methanol (2:1)
◦ Chloroform – denatures the proteins, eliminates
protein-lipid complexes in egg yolk
◦ Methanol – weakens H-bonds between lipids and
proteins
 1 % NaCl - salting out method

- Makes the solution more polar and separates polar
compounds from the lipids which are nonpolar
compounds
- removes some water from lipid extract
 Anhydrous Na2SO4 – drying agent

- readily adsorbs moisture
A. ISOLATION OF COMPLEX
LIPIDS FROM EGG YOLKS
 Hydroquinone - antioxidant;
- i.e. prevents oxidation of lipids (no
peroxide formation occurs)
 Acetone - does not dissolve

phosphorylated lipids
Characterization of Complex Lipids
A. Liebermann-Burchard Test
Cholesterol Lecithin Galactocerebroside Phosphorylated Non-

Phosphorylated
(cholesterol)
Emerald green Dark moss

solution green solution
B. Salkowski Test

Phosphorylated
Dark red interphase Dark red

interphase
C. Phosphate Test

Phosphorylated
Yellow to Yellow to yellow-

yellow-green green solution
solution with with precipitate
precipitate
D. Kraut’s Test

Phosphorylated
Reddish orange Reddish orange

solution with solution with
orange orange precipitate
precipitate
E. Ninhydrin Test

Phosphorylated
Violet solution
F. Molisch Test

Phosphorylated
Light purple
interphase
B. ISOLATION OF COMPLEX LIPIDS
FROM PIG BRAIN
 Ethanol - dissolves sphingolipids and
denatures proteins and disrupts lipoprotein
complexes
 Sand – help for disruption of cells
 Acetone – mild but rapid method of
dehydrating tissues; for the extraction of
cholesterol, acetone was used because it is the
only solvent where sterols are soluble
 Hexane – dissolves glycerophosphatides
 Chloroform:Methanol – to dissolve residue
to suspend lipids.
Group Cholesterol Lecithin Galactocerebros Cholesterol Glyceropho Sphingolipids
ide sphatides
1 Dark Green Murky, green,

sol’n w/ brown,
orange orange sol’n
bottom
2 Dark teal Light
sol’n orange/brow
n sol’n
3 Dark blue- Light
green sol’n orange sol’n
4 Emerald Emerald
green green
solution solution
5 Dark Clear
Green Orange
Sol’n sol’n
ide sphatides
6 Moss-like Colorless
dark green liquid
sol’n
7 Blue-green Green upper
layer and
sol’n emerald green
lower layer in
sol’n
8 Blue-green Slightly
to emerald yellowish
green sol’n liquid
9 Blue green Orange
to dark sol’n
green sol’n
B. Salkowski Test
Group Cholesterol Lecithin Galactocerebros Cholesterol Glycerophos Sphingolipids
(standard) ide phatides
1 Red upper Murky,

layer and
orange lower
brown sol’n
layer in sol’n
2 Orange sol’n Yellow sol’n w/
w/ dark band yellow band at
at interphase interphase
3 Orange Yellowish
sol’n sol’n
4 Red orange Orange

solution solution
with red with red-
interphase orange
interphase
5 Red-orange Orange
sol’n sol’n
B. Salkowski Test
(standard) ide sphatides
6 Dark orange Light yellow

sol’n w/ red 2nd layer,
interphase orange upper
layer
7 Orange Cloudy sol’n
sol’n w/ w/ faded
red-orange orange
interphase
interphase
8 Red Orange to
interphase red
interphase
9 red-orange Cloudy
interphase orange upper
layer and
clear lower
C. Test for Phosphate
ide phatides
1 Gray, clear Yellowish, clear Gray, clear Gray, clear

sol’n w/ B&W sol’n w/ white sol’n w/ B&W sol’n w/ B&W
ppt. ppt. ppt. ppt.
2 Gray, clear sol’n Yellowish, clear Oily liquid w/ Black ppt. in

w/ B&W ppt. sol’n w/ white B&W clear sol’n
ppt. suspension
3 Gray, clear Yellowish, clear Clear sol’n Yellow sol’n
sol’n w/ B&W sol’n w/ white w/ white ppt. w/ yellow
ppt. ppt. ppt.
4 Gray, clear Yellowish, clear Gray solution Yellowish

sol’n w/ B&W sol’n w/ white with solution with
ppt. ppt. precipitate precipitate
5 Gray, clear Yellowish, clear White White

sol’n w/ B&W sol’n w/ white sediment in sediment in
ppt. ppt. clear sol’n clear sol’n
C. Test for Phosphate
ide phatides
6 Yellowish Yellowish, Light pink White,

clear sol’n w/ clear sol’n w/ ppt. crystal clear
B&W ppt. white ppt. sol’n
7 Gray, clear Yellowish, Yellow Yellow

sol’n w/ clear sol’n w/ faded sol’n faded
B&W ppt. white ppt. w/ B&W solution w/
ppt. white ppt.
8 Gray, clear Yellowish, Gray, clear Colorless
sol’n w/ clear sol’n w/ sol’n w/ sol’n
B&W ppt white ppt. B&W ppt.
9 Gray, clear Yellowish, Light yellow Yellow sol’n

sol’n w/ clear sol’n w/ sol’n
B&W ppt. white ppt.
D. Kraut’s Test
ide phatides
1 Red-orange Dark red orange Orange Red orange

solution solution sol’n w/ sol’n w/
crystal ppt. black ppt.
2 Red-orange Red-orange Red sol’n w/ Red sol’n w/
solution with red- solution w/ black
orange precipitate black ppt. black ppt.
ppt.
3 Red-orange Red solution with Dark Dark red

solution with red-orange
red-orange precipitate
orange red orange sol’n
precipitate sol’n w/ w/ black
black ppt. ppt.
4 Red-orange Red solution with Dark red Dark orange
solution with red-orange solution with solution with
red-orange precipitate red-orange ppt. orange ppt.
precipitate
5 Dark, red sol’n Dark, red sol’n Dark, red Dark, red
w/ black ppt. w/ black ppt. sol’n w/ black sol’n w/ black
ppt. ppt.
D. Kraut’s Test
6 Dark orange Dark orange Dark orange Dark orange

sol’n w/ red sol’n w/ red sol’n w/ red sol’n w/ red
orange ppt. orange ppt. orange ppt. orange ppt.
7 Red-orange Orange w/ Red-orange Blackish-

solution black ppt. sol’n w/ orange
black ppt. liquid w/
black ppt.
8 Dark orange Red ppt. Dark red Red ppt.
ppt. ppt.
9 Red orange Dark orange Dark Dark orange

sol’n w/ sol’n w/ green orange sol’n sol’n w/ dark
metallic green ppt. w/ metallic metallic
ppt. green ppt.
green ppt.
E. Ninhydrin Test
1 Murky
orange-
brown sol’n
2 Liquid
evaporated,
light purple
stain/ residue
3 Light pink
sol’n
4 Red-violet
liquid
5 Orange
sol’n w/
crystal ppt.
E. Ninhydrin Test
6 Light pink
ppt.
7 Pink sol’n
w/ pink ppt.
8 Violet sol’n
9 Pinkish/
light purple
sol’n
F. Molisch Test
1 Turbid sol’n Gray sol’n w/

dark red ppt.
and purple
red
interphase
2 Pinkish-white Turbid sol’n
interphase
3 Pink solution Light pink
sol’n
4 Pink solution Pink
solution
5 Yellow White/
interphase colorless
interphase
F. Molisch Test
6 Light pink sol’n Turbid

peach sol’n
7 Cloudy light Cloudy,
pink sol’n w/ pomegranat
light e-colored
interphase interphase
8 Naked Violet/purpl
pink/purplish ish
interphase interphase
9 Peach sol’n in Cloudy peach
upper layer, UL, dark blue
yellow-green LL and green
interphase in
lower layer w/ sol’n
black ppt.
Liebermann-Burchard Test
 Test
for: Presence of
cholesterol
 Reagents: acetic anhydride

and H2SO4
 Positive
Result: blue green to
emerald green solution
 Principle:
Acetylation of OH
group in cholesterol, then
reaction with conc. H2SO4
 Positive
in: cholesterol
(nonphosphorylated lipid)
 The 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.
Salkowski Test
 Test for: Cholesterol
 Reagent: conc. H2SO4
 Positive result: red interphase
 Principle: Sulfuric acid react
with cholesterol to form bi-
cholestadiene disulphonate,
then dehydration forming
bisteroid
 Similar with the Liebermann–
Burchard test. The only difference is
that an acetic anhydride was not
used because an esterification is
not the main concern in this test.
 Only H2SO4 was added on the lipid
sample. The oxidative property of
H2SO4 triggers the formation of
additional double bonds between
two cholesterol molecules.
 The formation of double bond gave
rise to a red bisteroid product.
 Thiswill tend to cluster with
each other forming a visible
red interface.
 Like in the Liebermann –
Burchard test, both the
standard cholesterol and the
nonphosphorylated egg lipid
showed a positive result.
Test for Phosphate
 Test for: Free phosphate groups
 Reagent: fusion mixture (Na2CO3,
KNO3), 2.5% ammonium
molybdate and HNO3
 Positive result: presence of yellow
precipitate (ammonium
phosphomolybdate)
 Principle: oxidation, conversion of
inorganic phosphate,
precipitation reaction
 Principle:formation of
additional double bonds or
condensation of two
cholesterol molecules to form
bisteroids.
◦ Phosphorylated lipid sample (-)
◦ Nonphosphorylated lipid sample
(+)
◦ Cholesterol (+)
◦ Lecithin (-)
◦ Galactocerebroside (-)
Kraut's Test
 Test for: choline
 Reagents: Kraut’s reagent
(potassium iodide, bismuth
subnitrate, HNO3)
 Positive result: orange-red solution
and precipitate
Kraut's Test
 Principle: Complexation of choline
with bismuth potassium iodide
 Further explanation: The color

orange precipitate indicates the
presence of an alkaloid. The
reaction is due to the heavy metal
iodide which is the potassium
bismuth iodide.
Ninhydrin Test
 Test for: molecules with a free α
amino group that reacts together
with the ninhydrin reagent
 Reagents: Ninhydrin
(triketohydrindenehydrate)
 Positive result: Blue to blue violet

color
Ninhydrin Test
 Principle: Ninhydrin acts as
powerful oxidizing agent, it
undergoes oxidative deamination;
followed by condensation
 Positive in: glycerophosphatides

Ninhydrin Test
Molisch Test
 Test for: Carbohydrates
 Reagents:alpha-naphthol in
ethanol, conc. H2SO4
 Positive
Results: Appearance
of a purple ring at the
interface
Molisch Test
 Principle:Dehydration forming
furfural and its derivative
 Positive
in: Galactocerebroside
(a glycolipid)
CONCLUSION
• Complex lipids were isolated from
the egg yolk and were separated
into phosphorylated and non-
phosphorylated lipids.
• Non-phosphorylated lipid and

cholesterol gave a positive result for
Liebermann-Burchard and
Salkowski tests which means that
cholesterol is present in the non-
phosphorylated lipid sample.
 Phosphorylated lipid reacted
positively for Kraut’s test.
 Lipids were extracted from pig’s
brain based on solubility of different
lipids to different solvents.
 Cholesterol gives a positive result for
Liebermann Burchard Test and
Salkowski Test.
 Lecithin, Galactocerebroside, Sphingolipids
and glycerophosphatides give a positive
result for Kraut’s test because of the
presence of choline.
 For Molisch test, galactocerebroside
yielded a positive result which means
it contains a carbohydrate in both
the egg yolk and brain lipid.
REFERENCES
 Patil, U. & Muskan, K. (2010). Essentials of
Biotechnology. I. K. International Pvt Ltd.
 Smith, J. G. General, Organic, & Biological
Chemistry. New York: McGraw Hill, 2010.
 Pratt, C.W. and Cornely, K. (2011).Essential
Biochemisty (2nd Edition). New Jersey, USA: John
Wiley & Sons, Inc.
 http://www.infoplease.com/cig/biology/lipids.html
Phosphorylated and non-phosphorylated lipids-
Retrieved March 9, 2015
 http://en.wikipedia.org/wiki/Egg_yolk
Lipids- Retrieved March 9, 2015
 Horrocks, LA. & Yeo, YK. (1999). Health benefits of
docosahexaenoic acid (DHA). Retrieved
November 11, 2015

Exp 9 Egg Lipids GRP 8

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Exp 9 Egg Lipids GRP 8

Uploaded by

Copyright:

Available Formats

Isolation and Characterization

of Complex Lipids from Egg

◦ There are various ways of classifying

Glycerophospholipids Sphingolipids Sphingolipids Galactolipids

Fatty acid Fatty acid

Fatty acid Fatty acid Fatty acid Fatty acid

PO4 alcohol PO4 choline sugar sugar

The lipids of the egg yolk are divided

Place in separatory funnel

Extract with an equal

Place in separatory funnel

Extract with an equal

dry with anhydrous Na2SO4

Sticky yellow residue

wash with 5 mL evaporate to dryness

Discard + 5mL CHCl3 :MeOH mixture

Triturate 40g of pig’s brain

Add 100 ml of acetone

Allow to stand overnight

Filter and extract the system

Save for the Filtrate

Evaporate the filtrate to

Cool with cold water

Crude Crystallized Cholesterol

Collect product in a funnel

Dissolve using hot Ethanol,

Filter while hot

+30 ml hexane or pet.

Let stand for 30 mins. (w/

+30 mL acetone; stir

Extract with 50mL hot ethanol

Boil over water bath for 10

+10 mL MeOH: CHCl3 (1:3)

Place .5 ml in test tube

Place 10 drops in test tube

Add 4 drops of H2SO4

Note color produced at

Place 1.5 ml of sample with

Subject to free flame until it

Note color produced

Yellow Solution and

Place 10 drops in test tube

Put in boiling water bath

Note color produced

Place 10 drops in test tube

Add 5 drops of ninhydrin

Evaporate solvent from

Add 20 drops of distilled

 1 % NaCl - salting out method

 Anhydrous Na2SO4 – drying agent

 Acetone - does not dissolve

Cholesterol Lecithin Galactocerebroside Phosphorylated Non-

Emerald green Dark moss

Cholesterol Lecithin Galactocerebroside Phosphorylated Non-

Dark red interphase Dark red

Cholesterol Lecithin Galactocerebroside Phosphorylated Non-

Yellow to Yellow to yellow-

Cholesterol Lecithin Galactocerebroside Phosphorylated Non-

Reddish orange Reddish orange

Cholesterol Lecithin Galactocerebroside Phosphorylated Non-

Cholesterol Lecithin Galactocerebroside Phosphorylated Non-

1 Dark Green Murky, green,

1 Red upper Murky,