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EXPERIMENT 6:

Analysis of Lipids

TJ ABION
Isolation of Lipids
from an Egg Yolk
Chicken Egg Yolk
Rich in lipids such as phospholipids, triglycerides /
triacylglycerols + cholesterol:
Phospholipid: Lecithin
Fatty acids in acylated form:
Unsaturated: Oleic, Linoleic, Palmitoleic, &
Linolenic
Saturated: Palmitic, Stearic & Myristic
Yellow color is due to a carotenoid,
Zeaxanthine
Isolation of Lipids
Ethanol
- denature the proteins and disrupt the lipoprotein complexes of
the egg yolk
Hexane
- was used to remove phosphoacylglycerol
Acetone / anhydrous Na2SO4
- provides a mild dehydration condition
- as the H2O decreases, it extracts fatty acids, sterol and other
simple lipids
- the remaining polar lipids with neutral lipids will further precipitate
Isolation of Lipids
Involves the use of selective solvent extraction:
Hexane I
N
- Cholesterol ester C
- TAG (as fatty acid ester) R
E
Acetone A
- Cholesterol S
I
- Glycerophosphatides/ phosphoacylglycerol N
e.g. lecithin, cephalin, cardiolipin G

Ethanol POLARITY
- Sphingolipids, sphingomyelin
- Glycolipids (cerebroside, ceramide)
Thin-Layer
Chromatography
Thin Layer Chromatography of Lipids
Thin Layer Chromatography of Lipids
Based on adsorption phenomenon
Ascending & Normal
Two-dimensional chromatography (uses 2 solvent system)
Stationary phase: Silica G-250
Mobile phase:
Petroleum ether: methanol: H2O (65:25:4)
Petroleum ether: methanol: NH4OH (65:25:4)
Thin Layer Chromatography of Lipids
Visualizing agents: b.) Ninhydrin Spray
a.) Iodine crystals Lipids containing free amino
groups
phosphotidylethanolamine
(PE) and
phosphotidylserine (PS)
Purple spots

Detects unsaturated lipids c.) Phosphoric acid


Yellow-brown spots All lipids
Charred spots
d.) Rhodamine B
Specific for lipids only
Less polar
1st development: more 1. Sterol ester
polar (H2O)
2. Fatty acid ester

3. Triglycerides

4. Free fatty acids

5. Diglycerides
6. Sterol

7. Phosphatidic acid

8. Phosphoglycerides

9. Sphingolipids
Origin Egg extract
Less polar
2nd development:
Less polar (NH4OH)
More analytical

1. Sterol ester
2. Fatty acid ester
3. Triglycerides
4. Free fatty acids
5. Diglycerides
6. Sterol
7. Phosphatidic acid
8. Phosphoglycerides
9. Sphingolipids

9 8 7 6 5 4 3 2 1
Column
Chromatography
of Lipids
Column Chromatography of Lipids
Based on adsorption phenomenon
Descending
Stationary phase: Silica gel
Mobile phase/eluent:
1. Petroleum ether: ethyl ether (9:1) Least polar
2. 5% MeOH in DCM
3. DCM:MeOH:H2O (1:3:1) Most polar
Column Chromatography of Lipids
Liebermann-
Eluates Ester Glycerol
Burchard

Deep burgundy Burnt fat odor Emerald green


1st (least polar)
(++) (++) (++)

Deep burgundy
2nd Burnt fat odor (+) Emerald green (+)
(+)
No deep
Burnt fat odor No emerald green
3rd (most polar) burgundy (-)
(+/-) (-)
Column Chromatography of Lipids
Liebermann-
Eluates Ester Glycerol
Burchard
Due to FA ester of
Presence of Due to
1st (least polar) TAG and
glycerol in TAG cholesterol
cholesteryl ester
Due to glycerol Due to
Due to FA ester in
2nd backbone of cholesterol
phoshoglycerides
phosphoglycerides
Glycerol backbone
No fatty acid (can be absent); No cholesterol
3rd (most polar)
ester present sphingosine present
backbone only
Qualitative tests
for Lipids
Test for Ester (Hydroxamic acid)
oReagents : EtOH:ButOH (3:1), NH2OH.HCl,
NaOH, FeCl3, HCl
oPurpose : detect presence of carboxylic
acid ester
oPositive result : burgundy color
oPrinciple involved : complexation reaction

Positive for:
Triacylglycerides (TAGs)
Cholesteryl esters
Glycerophospholipids
Acrolein Test
oReagents : KHSO4 Positive for:
Triacylglycerides (TAGs)
oPurpose : detect presence of glycerol Glycerophospholipids
oPositive result : burnt fat/acrid odor
oPrinciple involved : dehydration and oxidation
Leibermann-Burchard test
oReagents : conc. H2SO4, acetic anhydride
oPurpose : detect presence of unsaturated sterols (cholesterol)
oPositive result : blue-green / emerald green solution
oPrinciple involved : Acid-catalyzed dehydration & stepwise oxidation of SO3

The blue-green product in the


L-B reaction with max near
680 nm is a pentaenylic cation
Krauts test
o Reagents : Bismuth subnitrate, 3 M HNO3, KI (Krauts reagent)
o Purpose : detects presence of choline
o Positive result : dark orange to red precipitate
o Principle involved : complexation reaction of Choline with bismuth potassium
iodide

Positive for:
Lecithin & Sphingomyelin
Phosphate test (Ascorbate method)
Ammonium molybdate and potassium antimonyl tartrate react in acid
medium with orthophosphate to form a heteropoly acid -
phosphomolybdic acid - that is reduced to intensely colored
molybdenum blue by ascorbic acid.
Test for Lipid Unsaturation
o Reagents : 5% Bromine in DCM solution
o Purpose : detects presence of double bonds in Lipids
o Positive result : reddish brown/deep burgundy color
o Principle involved : addition of bromine to double bonds (Halogenation)
o Iodine can be a used as an alternative
Lipid index Details Significance Lipid index Details Significance
Saponification Number of mg of KOH Indicates Polenske Number of ml of 0.1 Indicates the
number required to saponify molecular weight number normal KOH required to presence of non
the free and and is inversely neutralize the insoluble volatile fatty acids in
combined fatty acids proportional to it. fatty acids from 5 gram a given fat
in 1 g of a given fat of fat

Iodine number Number of grams of It is a measure of Reichert-Meissl Number of ml of 0.1 N Measures the
iodine absorbed by degree of Number alkali required to amount of volatile
100 mg of fat unsaturation of a neutralize the soluble soluble fatty acids.
fat fatty acids distilled from
5 G of fat

Acid number Number of mg of KOH Indicates the Acetyl Number Number of mg of KOH Measures the
required to neutralize degree of rancidity required to neutralize number of OH
the fatty acids in a of a fat the acetic acid obtained groups present in a
gram of a fat by saponification of 1 g fatty acid
of fat after it has been
acetylated.
Test for Lipid
Unsaturation
Salkowski test (Sterol test)
o Reagents : conc. H2SO4, CHCl3
o Purpose : presence of sterol
o Positive result : red to red-orange interphase (chloroform layer)
o Principle involved : dehydration and condensation to form a bisterols (red
bicholestadien disulphonate)
THANK YOU!
General Biochemistry Lab ends here