Isolation and Characterization of Complex Lipids from Egg Yolk

Kirsten Martinez Charmaine Pua Carla Samson Ronald Ty Ara Vertudez 3Bio-1

INTRODUCTION

INTRODUCTION
Lipids - hydrophobic (water insoluble nonpolar) - form aggregrates (non polymeric) - can be extracted from the cell by organic solvents
Major lipid groups include fats, phospholipids, steroids and waxes.

INTRODUCTION
Functions: 1. Component of cell membrane a. Phospholipids (Glycerophospholipids and Sphingolipids) b. Glycolipids (Sphingolipids) 2. Energy storage and Insulation (Triacylglycerol) 3. Signaling molecules

INTRODUCTION
Egg yolk - a rich source of a variety of biochemically important compounds such as proteins and lipids - makes up about 33% of the liquid weight of the egg

INTRODUCTION
The lipids of the egg yolk are divided into: 1. Phosphorylated Lipids

2. Non-Phosphorylated Lipids

INTRODUCTION
OBJECTIVES: 1. To isolate the lipids from egg yolk and to separate them into phosphorylated and non-phosphorylated lipids 2. To characterize these lipids using various chemical tests

MATERIALS

MATERIALS
A. Isolation of Complex Lipids from Egg Yolk

Egg yolk 250-mL beaker 100-mL solvent mixture Filter paper Separatory funnel Funnel 1% NaCl Solution Anhydrous Na2SO4

Erlenmeyer Flask Hydroquinone Evaporating dish Ice bath Acetone solution Test tube Cork Stopper Boiling water bath

MATERIALS
B. Characterization of Complex Lipids

10 drops Acetic Anhydride Concentrated H2SO4 Boiling water bath Test tubes 15 drops Krauts Reagent Distilled water 5 drops Ninhydrin Solution Ethanol 2 drops Molisch Reagent

METHODOLOGY

METHODOLOGY
A. Isolation of Complex Lipids from Egg Yolk

Egg yolk
-Placed in 250 ml beaker -Stirred well -+ 100 ml solvent mixture - Stood for 10 mins -Filtered

Residue
Discarded

Filtrate

METHODOLOGY A. Isolation of Complex Lipids from Egg Yolk

Filtrate
-Volume was noted -Placed in separatory funnel -Extracted with an equal amount of 1% NaCl solution -swirled, opened, released pressure, closed (3-4 mins.)

Aqueous layer
Discarded

Organic layer

METHODOLOGY A. Isolation of Complex Lipids from Egg Yolk

Organic layer
-Placed in separatory funnel -Extracted with an equal amount of 1% NaCl solution

Aqueous layer
Discarded

Organic layer

METHODOLOGY A. Isolation of Complex Lipids from Egg Yolk

Organic layer

-dried with anhydrous Na2SO4 -filtered -+ pinch of hydroquinone -transferred to evaporating dish -evaporated to dryness Sticky yellow residue
-+ 15 ml acetone -cooled in ice bath for 15 mins. -filtered -+ 15 ml cold acetone -decanted Precipitate (Phosphorylated) Filtrate (NonPhosphorylated)

METHODOLOGY A. Isolation of Complex Lipids from Egg Yolk

Precipitate (Phosphorylated) -washed with 5 mL cold acetone -decanted Filtrate (NonPhosphorylated) -evaporated to dryness -+ 5mL CH3 :MeOH mixture

NPL (Cholesterol) Filtrate Precipitate -+ 5mL CH3 :MeOH mixture -+ pinch of hydroquinone PL

Discarded

METHODOLOGY B. Characterization of Complex Lipids

B.1 Liebermann-Burchard test
Lipid sample Standards

Half ml of each lipid solution was placed in a test tube Ten drops Acetic Anhydride was added -gently swirled Four drops conc. H2SO4 was added -mixed well Color produced was noted

METHODOLOGY B. Characterization of Complex Lipids

B. 2 Salkowski Test
Lipid sample Standards

Ten drops of each lipid solution was placed in a test tube

Four drops conc. H2SO4 was added -not mixed Color produced at the interphase was noted

METHODOLOGY B. Characterization of Complex Lipids

B. 3 Krauts Test
Lipid sample Standards

Ten drops of each lipid solution was placed in a test tube

Tubes were put in the boiling water bath in the fume

Dried lipid was suspended in 10 drops distilled water - + 15 drops Krauts reagent Color produced and the precipitate formed was noted

METHODOLOGY B. Characterization of Complex Lipids

B. 4 Ninhydrin Test
Lipid sample Standards

Ten drops of each lipid solution was placed in a test tube

Five drops of ninhydrin in ethanol was added

Tubes were warmed for 1-2 mins.

Color produced was noted

METHODOLOGY B. Characterization of Complex Lipids

B. 5 Molisch Test
Lipid sample Standards

Tubes were put in a boiling water bath to evaporate solvent from lipid solution
-+ 20 drops distilled water Two drops of Molisch reagent was added

-mixed well
Twenty drops of conc. H2SO4 was added -not mixed Color produced at the interphase was noted

RESULTS AND DISCUSSION

RESULTS
Group Vol of filtrate Observation of filtrate NPL PL

1 2 67.5 ml Clear yellow solution Dark yellow solution Clear yellow solution

3
4 5

66 ml
76 ml 75 ml

Clear yellow solution

Clear yellow solution Clear yellow solution

Clear orange solution

Dark yellow solution Dark orange solution

Clear yellow solution

Yellow solution Clear yellow solution

RESULTS
Group Vol of filtrate Observation of filtrate Clear yellow solution Clear yellow solution Clear yellow solution Clear yellow solution Clear yellow solution NPL PL 6 78 ml Dark orange solution Yellow solution (2 layers) Dark orange solution Yellow orange solution Dark yellow solution Clear yellow solution Clear yellow solution Yellow solution

7 8

74 ml 73 ml

73 ml

Clear yellow solution Clear yello solution

10

72.5 ml

A. ISOLATION OF COMPLEX LIPIDS FROM EGG YOLKS

Solvent mixture (Chloroform- Methanol) - dissolves/ miscible with lipids - polar lipids dissolve in methanol
1 % NaCl- salting out method

Anhydrous Na2SO4 drying agent - readily adsorbs moisture

A. ISOLATION OF COMPLEX LIPIDS FROM EGG YOLKS

Hydroquinone- antioxidant (no peroxide formation occurs

Acetone- does not dissolve phosphorylated lipids

Use of hood- toxic and flammable solvents

A. ISOLATION OF COMPLEX LIPIDS FROM EGG YOLKS

Ice bath- lowers solubility by lowering the temperature ** Cold acetone also helps in lowering solubility

Characterization of Complex Lipids

A. Liebermann-Burchard Test
Group Cholesterol Lecithin Galactocerebroside Phosphorylated NonPhosphorylated

1 2 Clear blue green solution

Clear blue green solution Emerald green solution Clear emerald green solution

Clear moss green solution Clear dark brown solution

Dark brown solution Clear dark brown solution

Clear colorless solution

Clear light yellow solution Clear colorless solution Clear colorless liquid solution

Clear light Clear dark green solution green solution

Turbid light Clear blue brown solution green solution Tea brown solution Turbid brown solution Emerald green solution Clear emerald green solution

4 5

Characterization of Complex Lipids

A. Liebermann-Burchard Test
Group Cholesterol Lecithin Galactocerebroside Phosphorylated NonPhosphorylated

Emerald green solution Green solution Green solution

Dark moss green solution Moss green solution Brownish green solution

Clear colorless solution

Dark maroon solution

Dark moss green solution

7 8

Clear colorless Turbid moss Green solution solution green solution Colorless solution Reddish brown Deep green turbid solution solution

9
10

Blue green solution Mass green solution

Clear green solution Clear brown solution

Colorless solution Dark green Emerald green turbid solution solution

Clear colorless solution Turbid reddish Clear dark brown solution green solution

Characterization of Complex Lipids

B. Salkowski Test
Group Cholesterol Lecithin Galactocerebroside Phosphorylated NonPhosphorylated

1 2 Dark red interphase Dark reddish White interphase brown interphase Red interphase Orange interphase Orange interphase Orange interphase Colorless interphase Turbid colorless interphase White interphase Dark reddish brown interphase Orange interphase Dark brown interphase Brown interphase Reddish brown interphase Red interphase

Turbid red interphase Red interphase

Red interphase

Red interphase

Characterization of Complex Lipids

B. Salkowski Test
Group Cholesterol Lecithin Galactocerebroside Phosphorylated NonPhosphorylated

Red interphase

Dark red orange interphase

Grayish turbid interphase

Dark brown interphase

Red interphase

7
8

Red interphase
Clear pinkish interphase

Violet interphase Yellow interphase Dark violet interphase

Red violet interphase

White interphase
White interphase

Orange Clear brown interphase interphase Red interphase Red interphase

Red interphase

Colorless interphase
Colorless interphase

Brown interphase
Brown color interphase

Moss green interphase

Orage to brown interphase

10

Cherry red interphase

Characterization of Complex Lipids

C. Krauts Test
Group 1 2 Reddish brown solution with black precipitate Reddish brown solution with orange precipitate Red solution Reddish brown solution Reddish bworn with black precipitate solution with brownish black precipitate Dark orange solution with black precipitate Dark turbid red solution with orange precipitate Reddish brown solution with black precipitate Dark orange solution Cholesterol Lecithin Galactocerebroside Phosphorylated NonPhosphorylated

Dark orange solution

Dark red solution with black precipitate

Orange solution with orange precipitate

Red orange solution with orange precipitate

Dark red solution with black precipitate

Red solution with orange precipitate

Red solution with black precipitate

Dark red solution with black precipitate

Red solution with black precipitate

Red solution with orange precipitate

Red solution with black precipitate

Characterization of Complex Lipids

C. Krauts Test
Group Cholesterol Lecithin Galactocerebroside Phosphorylated NonPhosphorylated

Dark red orange solution with black precipitate

Red orange solution with black precipitate

Dark orange solution with black precipitate Red orange solution with black precipitate

Red orange solution with orange precipitate Red orange solution with orange precipitate Orange solution with orange precipitate
Orange solution with orange precipitate Clear red solution with orange precipitate

Dark red orange solution with black precipitate

Red orange solution with black precipitate

Dark red orange solution with dark red precipitate

Red orange solution with black precipitate Red orange solution with orange precipitate Red orange with black precipitate

Red orange solution with black precipitate

Red orange solution with black precipitate Dark red orange with black precipitate Red orange with black precipitate

Red orange with black precipitate Red orange solution with black precipitate

10

Dark red solution with moss green to black precipitate

Clear dark red solution with dark green precipitate

Dark red solution with red precipitate

Red orange solution with moss green to black precipitate

Characterization of Complex Lipids

D. Ninhydrin Test
Group Cholesterol Lecithin Galactocerebroside Phosphorylated NonPhosphorylated

1 2 Clear colorless solution Turbid violet solution Clear colorless solution Turbid whitish solution on top; turbid yellowish solution on the botton Turbid purple solution Dark brown interphase Blue violet solution Turbid orange solution

3 4 5

Clear colorless solution Clear colorless solution Clear colorless solution

Turbid purple solution Purple solution Purple turbid solution

Clear colorless solution Clear colorless solution Clear colorless solution

Clear orange solution Red interphase Clear orange solution

Characterization of Complex Lipids

D. Ninhydrin Test
Group Cholesterol Lecithin Galactocerebroside Phosphorylated NonPhosphorylated
Orange solution

Colorless solution

Violet solution Colorless solution

violet solution

7 8

Colorless solution Colorless solution

Turbid violet solution Light violet solution

Colorless solution Colorless solution

Clear light orange solution Violet solution

Yellow solution Reddish brown solution

Colorless solution

Purple turbid solution

Violet turbid solution

Colorless solution

Light yellow turbid solution

Clear violet solution

Dark yellow clear solution

Turbid red orange solution

10

Colorless solution

Colorless solution

Characterization of Complex Lipids

E. Molisch Test
Group Cholesterol Lecithin Galactocerebroside Phosphorylated NonPhosphorylated

1 2 Yellow interphase Red violet interphase Light purple interphase Dark purple interphase Purple interphase

Clear orange interphase

Dark yellow interphase Light pink interphase

Dark purple interphase

White interphase White interphase

Violet interphase

Brown interphase
Turbid white interphase White interphase

Violet interphase

Light purple interphase Light purple interphase

Purple interphase Clear purple interphase

Characterization of Complex Lipids

E. Molisch Test
Group
6

Cholesterol
Violet interphase

Lecithin

Galactocerebroside

Phosphorylated
Orange interphase Dark brown interphase Red interphase

NonPhosphorylated
Purple interphase Light brown to purple interphase Brown to violet interphase Violet interphase

Golden yellow Light purple interphase interphase Red interphase Purple interphase Violet interphase

Yellow interphase

Yellow interphase

Violet interphase

Light violet interphase Dark violet interphase Yellow color interphase Red violet color interphase

Light violet interphase Pale violet color interphase

Light red interphase Reddish brown color interphase

10

Dark red violet color interphase

Liebermann-Burchard Test
Test for: Presence of unsaturated sterols (cholesterol) and triterpenes

Positive Result: Terpenoid saponins tend to produce a red, pink or purple shade and steroid saponins a blue-green coloration

 Principle: Oxidation reaction of Acetic anhydride with concentrated sulphuric acid to give green to blue solutions Explantion: The sulphuric acid would ensure acetylation of the OH of cholesterol,

 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 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.

 This will tend to cluster with each other forming a visible red interface. Like in the Liebermann Burchard test, both the standard cholesterol and the non phosphorylated egg lipid showed a positive result.

 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 Positive result: orange-red solution and precipitate 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.

Phosphorylated lipid sample (+)

Nonphosphorylated lipid sample (-) Cholesterol (-)

Lecithin (+) Galactocerebroside (-)
3/11/12

Ninhydrin Test
Test for: molecules with a free amino group that reacts together with the ninhydrin reagent Positive result: Blue to blue violet color

 Principle: Ninhydrin is a powerful oxidizing agent, it undergoes oxidative deamination that releases ammonia and CO2

Phosphorylated lipid sample (-)

Nonphosphorylated lipid sample (+) Cholesterol (-)

Lecithin (+) Galactocerebroside (-)
3/11/12

Molisch Test
Test for: Carbohydrates that give a rapid positive test on monosaccharides

Positive Results: Appearance of a purple ring at the interface between the acid and test layers

 Principle: Dehydration and condensation of furfural with naphthol forming violet ring
Explanation: The purple ring indicates the presence of carbohydrates. The reaction is due to the formation of furfural derivatives, such as hydroxymethyl furfural

CONCLUSION

1. Complex lipids were isolated from the egg yolk and were separated into phosphorylated and nonphosphorylated lipids.

2.

Non-phosphorylated lipid and cholesterol gave a positive result for Liebermann-Burchard and Salkowski tests which means that cholesterol is present in the nonphosphoylated lipid sample.

Phosphorylated lipid and lecithin reacted positively for both Krauts and Ninhydrin tests which means that lecithin is present in the phosphorylated lipid sample.
For Molisch test, both nonphosphorylated lipid and galactocerebroside yielded a positive result which means that they contain a carbohydrate.

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.

REFERENCES
http://www.infoplease.com/cig/biology/lipids. html Phosphorylated and non-phosphorylated lipids- Retrieved March 9, 2012 http://en.wikipedia.org/wiki/Egg_yolk Lipids- Retrieved March 9, 2012