experiment 7 biochem

Isolation and Characterization of Complex Lipids from Egg Yolk

Kirsten MartinezCharmaine PuaCarla Samson

Ronald TyAra Vertudez

3Bio-1

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

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

INTRODUCTIONThe 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

MATERIALSA. 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

MATERIALSB. Characterization of Complex Lipids

• 10 drops Acetic Anhydride• Concentrated H2SO4

• Boiling water bath• Test tubes• 15 drops Kraut’s Reagent• Distilled water• 5 drops Ninhydrin Solution• Ethanol• 2 drops Molisch Reagent

METHODOLOGY

METHODOLOGYA. 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 FiltrateDiscarded


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.)

Organic layer

Aqueous layer

Discarded


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

Organic layer

Aqueous layer

Discarded


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

Filtrate (Non-Phosphorylated)

Precipitate (Phosphorylated)


Filtrate (Non-Phosphorylated)

Precipitate (Phosphorylated)

-washed with 5 mL cold acetone-decanted

-evaporated to dryness-+ 5mL CH3 :MeOH mixture

NPL (Cholesterol)

PrecipitateFiltrate

Discarded-+ 5mL CH3 :MeOH mixture-+ pinch of hydroquinone

PL

METHODOLOGYB. Characterization of Complex Lipids

B.1 Liebermann-Burchard test

Half ml of each lipid solution was placed in a test tube

StandardsLipid sample

Ten drops Acetic Anhydride was added

Four drops conc. H2SO4 was added

Color produced was noted

-gently swirled

-mixed well


B. 2 Salkowski Test

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


Four drops conc. H2SO4 was added

Color produced at the interphase was noted

-not mixed


B. 3 Kraut’s Test



Tubes were put in the boiling water bath in the fume

Dried lipid was suspended in 10 drops distilled water

Color produced and the precipitate formed was noted

- + 15 drops Kraut’s reagent


B. 4 Ninhydrin Test



Five drops of ninhydrin in ethanol was added

Tubes were warmed for 1-2 mins.

Color produced was noted


B. 5 Molisch Test

Tubes were put in a boiling water bath to evaporate solvent from lipid solution


Two drops of Molisch reagent was added

Twenty drops of conc. H2SO4 was added

Color produced at the interphase was noted

-+ 20 drops distilled water

-not mixed

-mixed well

RESULTS AND DISCUSSION

RESULTSGroup Vol of filtrate Observation of

filtrateNPL PL

1

2 67.5 ml Clear yellow solution

Dark yellow solution

Clear yellow solution

3 66 ml Clear yellow solution

Clear orange solution




Yellow solution


Dark orange solution


RESULTSGroup Vol of filtrate Observation of

filtrateNPL PL





Yellow solution (2 layers)




Yellow solution


Yellow orange solution


10 72.5 ml Clear yellow solution


Clear yello solution

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


• Hydroquinone- antioxidant (no peroxide formation occurs

• Acetone- does not dissolve phosphorylated lipids

• Use of hood- toxic and flammable solvents


• Ice bath- lowers solubility by lowering the temperature

** Cold acetone also helps in lowering solubility

Characterization of Complex LipidsA. Liebermann-Burchard Test

Group Cholesterol Lecithin Galactocerebroside Phosphorylated Non-Phosphorylated

1

2 Clear blue green solution

Clear moss green solution

Clear colorless solution

Clear light green solution

Clear dark green solution

3 Clear blue green solution

Clear dark brown solution

Clear light yellow solution

Turbid light brown solution

Clear blue green solution

4 Emerald green solution

Dark brown solution


Tea brown solution

Emerald green solution

5 Clear emerald green solution

Clear dark brown solution

Clear colorless liquid solution

Turbid brown solution

Clear emerald green solution

Characterization of Complex LipidsA. Liebermann-Burchard Test

6 Emerald green solution

Dark moss green solution


Dark maroon solution

Dark moss green solution

7 Green solution Moss green solution


Turbid moss green solution

Green solution

8 Green solution Brownish green solution

Colorless solution Reddish brown turbid solution

Deep green solution

9 Blue green solution Mass green solution

Colorless solution Dark green turbid solution

Emerald green solution

10 Clear green solution Clear brown solution


Turbid reddish brown solution

Clear dark green solution


Characterization of Complex LipidsB. Salkowski Test


1

2 Dark red interphase Dark reddish brown interphase

White interphase Dark reddish brown interphase

Reddish brown interphase

3 Red interphase Orange interphase

Colorless interphase

Orange interphase

Red interphase

4 Turbid red interphase

Orange interphase

Turbid colorless interphase

Dark brown interphase

Red interphase

5 Red interphase Orange interphase

White interphase Brown interphase

Red interphase

Characterization of Complex LipidsB. Salkowski Test

6 Red interphase Dark red orange interphase

Grayish turbid interphase


Red interphase

7 Red interphase Violet interphase

White interphase Orange interphase

Clear brown interphase

8 Clear pinkish interphase

Yellow interphase

White interphase Red interphase Red interphase

9 Red interphase Dark violet interphase


Brown interphase

Moss green interphase

10 Cherry red interphase

Red violet interphase


Brown color interphase

Orage to brown interphase


Characterization of Complex LipidsC. Kraut’s Test


12 Reddish brown solution

with black precipitateReddish brown solution with orange precipitate

Reddish brown solution with black precipitate

Reddish bworn solution with brownish black precipitate

Reddish brown solution with black precipitate

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

Dark turbid red solution with orange precipitate


4 Dark red solution with black precipitate

Orange solution with orange precipitate

Dark red solution with black precipitate

Red solution with orange precipitate

Red solution with black precipitate

5 Dark red solution with black precipitate

Red orange solution with orange precipitate


Red solution with orange precipitate


Characterization of Complex LipidsC. Kraut’s Test

6 Dark red orange solution with black precipitate


Dark red orange solution with black precipitate

Dark red orange solution with dark red precipitate

Red orange solution with black precipitate

7 Red orange solution with black precipitate





8 Dark orange solution with black precipitate


Red orange with black precipitate


Dark red orange with black precipitate

9 Red orange solution with black precipitate





10 Dark red solution with moss green to black precipitate

Clear red solution with orange 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 LipidsD. Ninhydrin Test


1

2 Clear colorless solution

Turbid violet solution


Turbid whitish solution on top; turbid yellowish solution on the botton

Turbid orange solution


Turbid purple solution


Turbid purple solution



Purple solution



Red interphase


Purple turbid solution


Blue violet solution


Characterization of Complex LipidsD. Ninhydrin Test

6 Colorless solution Violet solution Colorless solution violet solution Orange solution

7 Colorless solution Turbid violet solution

Colorless solution Clear light orange solution

Yellow solution

8 Colorless solution Light violet solution

Colorless solution Violet solution Reddish brown solution

9 Colorless solution Purple turbid solution

Colorless solution Light yellow turbid solution

Dark yellow clear solution

10 Colorless solution Violet turbid solution

Colorless solution Clear violet solution

Turbid red orange solution


Characterization of Complex LipidsE. Molisch Test


1

2 Yellow interphase Red violet interphase

Light purple interphase

Dark purple interphase

Purple interphase

3 Clear orange interphase

Dark purple interphase

Violet interphase Brown interphase

Violet interphase

4 Dark yellow interphase

White interphase


Turbid white interphase

Purple interphase

5 Light pink interphase White interphase


White interphase

Clear purple interphase

Characterization of Complex LipidsE. Molisch Test

6 Violet interphase Golden yellow interphase


Orange interphase

Purple interphase

7 Yellow interphase Red interphase Violet interphase Dark brown interphase

Light brown to purple interphase

8 Yellow interphase Purple interphase

Violet interphase Red interphase Brown to violet interphase

9 Light violet interphase Dark violet interphase

Light violet interphase

Light red interphase

Violet interphase

10 Yellow color interphase

Red violet color interphase

Pale violet color interphase

Reddish brown color interphase

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.

3/11/12

Phosphorylated lipid sample (+)

Nonphosphorylated lipid sample (-)

Cholesterol (-)

Lecithin (+)

Galactocerebroside (-)

Ninhydrin Test

• Test for: molecules with a free α 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

3/11/12

Phosphorylated lipid sample (-)

Nonphosphorylated lipid sample (+)

Cholesterol (-)

Lecithin (+)

Galactocerebroside (-)

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 non-phosphorylated 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 non-phosphoylated lipid sample.

Phosphorylated lipid and lecithin reacted positively for both Kraut’s and Ninhydrin tests which means that lecithin is present in the phosphorylated lipid sample.

For Molisch test, both non-phosphorylated 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.htmlPhosphorylated and non-phosphorylated lipids- Retrieved March 9, 2012

• http://en.wikipedia.org/wiki/Egg_yolkLipids- Retrieved March 9, 2012

experiment 7 biochem

Documents

additional

boiling water

5ml ch3

dark orange

dark red solution

egg yolkorganic

nonphosphorylated

phosphorylated