Identifying Lipids Using Chemical tests

1. Acrolein Test
Principle
When glycerol is heated with potassium bisulphate or concentrated H2SO4
dehydration occurs and aldehyde Acrolein !ormed which has characteristic odour. This
test responds to glycerol !ree or lin"ed as an ester.
#H2 $ OH #H2

#H $ OH #H % 2H2O

#H2 $ OH #HO
&lycerol Acrolein
'aterials
1. Test compounds ( Oil or !at Oleic acid)
2. Potassium bisulphate or conc. H2SO4
Procedure
1. Place * drops o! test compound in a clean and dry test tube
2. Add 1 ml o! conc. H2SO4 care!ully. Or 1.+ g o! ,HSO4
-. Heat the test tube directly.
4. .ote the characteristic pungent odour o! Acrolein.

2. Test o! Triglycerides
Principle
This test can be used to distinguish between triglycerides and !atty acids. Triglycerides
are chemically neutral while !atty acids are acidic due to their !ree carbo/ylic group and can
decolori0e the al"aline red color o! phenolphthalein.
'aterials
2+1 &lycerin in chloro!orm
+.*1 bora/ solution
11 phenolphthalein in *+1 ethanol
Procedure
1. Place *ml o! 2ora/ solution (+.*1) in test tube
Heat
,HSO4 or
#onc.H2SO4
2. Add appropriate 3olume o! Phenolphthalein till red color is appearing.
-. Add 2+1 glycerin drop by drop till red color is disappearing.
4. warm the test tube the red color will appear again.
*. !atty acids are not responding to this test.
-. Test o! cupric acetate !or detecting !atty acids
This test is used !or detecting saturated and unsaturated !atty acids !atty acids
reacts with cupric acetate to !orm cupric salts o! !atty acids. The cupric salts o!
saturated !atty acids are not dissol3ed in water or petroleum ether4 there!ore it5s
precipitated at the bottom o! test tube. The cupric salts o! unsaturated !atty acids
are dissol3ed in petroleum ether with blue6green color.
'aterials
Saturated !atty acid ( stearic acid or palmitic acid)
7nsaturated !atty acid ( Oleic acid)
1+1 a8ueous cupric acetate
Petroleum ether
Procedure
1. 9issol3e small mount o! !atty acid in petroleum ether.
2. Add appropriate amount o! a8ueous cupric acetate solution.
-. Sha"e the test tube care!ully and obser3e green precipitate is appear at
lower a8ueous layer ( cupric acetate layer) where this is happened
with saturated !atty acids while at upper organic layer ( petroleum
ether layer) blue or green color is appear when unsaturated !atty acid
is used.
4. Test !or the 9egree o! 7nsaturation o! :atty Acids (;odine test)
:atty acids in animal !ats are usually saturated whereas those in 3egetable
oils are generally unsaturated. Halogens ( ; 2r ) will add across the double bonds
and thus the decolori0ation o! an iodine or bromine solution will indicate the
presence o! unsaturated !atty acids. ;odine test is used !or distinguish between
saturated and unsaturated !atty acids as well as between oils and !ats.
'aterials
1. Oil !at or oleic and stearic acids.
2. Organic sol3ents (chloro!orm or ethanol)
-. Hubl5s reagent ( alcoholic solution o! iodine which contains some mercuric
chloride)
Procedure
1. 9issol3e small amount o! unsaturated !atty acid (Oleic acid) in small 3olume
o! chloro!orm.
2. Add Hubl5s reagent drop by drop and sha"ing the tube a!ter each addition.
The halogen solution should be added <ust until it !ails to be decolori0ed.
3. =ecord the number o! drops needed to bring about !ull decolori0ation.
4. =epeat the same procedure but by using saturated !atty acid ( stearic acid)
and #ompare your results.
5. Rancidity
=ancidity is a process which is accompanied by the !ormation o! unpleasant odour taste as a
result o! the action o! moisture air (O2) and en0ymes.To guard against rancidity we ha3e to
protect lipid !rom moisture and direct light as well as its storage must be in a cold place to
deacti3ate lipase.
Types of rancidity:
1- Hydrolytic rancidity (or lipolytic rancidity)
2- Oxidative rancidity
Kreis-Kerr test
Reagent: 1% of Phloroglucinol in alcohol or ether.
Procedure
1 ml o! rancid Oil is mi/ed with 1 ml o! conc. H#l and then add 2 ml o! 11 o! Phloroglucinol
Sha"e and lea3e to stand !or one hour. Appearance o! red colour indicates the presence o!
aldehydes. The color intensity is increased with the increasing in the degree o! rancidity in lipid.
6. Saponification of Fats and Oils
Oils can be saponi!ied with al"ali soda to !orm soap and glycerol and it5s as !ollows>

O
# =
O
O
# =
# =
O
H
2
# O
H# O
H
2
# O
,OH
H
H
H
H
2
# O
H# O
H
2
# O
= # O,
+ 3
+
3
'aterials
Oil
Alcoholic sodium or potassium hydro/ides(+.* .)
Sodium #hloride
#oncentrated hydrochloric acid
Procedure
1. Place * g o! oil or !at 1.+ g o! sodium hydro/ide and 2* ml o! alcohol in round
!las".
2. Place the boiling stone at round !las" !or regulating the heating and re!lu/ the
solution !or -+ minutes at boiling water bath.
-. Warm the solution !or e3aporating the alcohol.
4. #ool the solution in a bea"er o! cold water.
*. =emo3e a small piece o! the solid product (about the si0e o! a pea) using a glass
stirring rod and dissol3e it with 2+ ml 9W and obser3e the !oam !ormed between
soap and water.
7. Ammonium Molybdate Test for hosphate
The presence o! !ree phosphate in acidic solution can be detected by adding a molybdate
to the solution. ?8uation illustrates the pertinent reaction between phosphate and ammonium
molybdate solution in the presence o! nitric acid (H.O-).
HPO4
2$
(a8) % 12'oO4
2$
(a8) % - .H
4%
(a8) % 2- H-O
%
(a8) (.H4)-@P('o-O1+)4A (yellows) % -* H2O(l)
A!ter a !ew minutes the yellow ammonium molybdo6phosphate precipitates !rom the
reaction mi/ture. When lipids containing phosphate groups in their structures are added to a
strong acid solution such as the solution used here the lipid hydrolyses producing !ree
phosphate. The !ree phosphate then reacts as in ?8uation !orming a yellow precipitate.
'aterials
1. Prepare an ice6water bath
2. Place +.2 g o! lecithin (ege yol") in a washed drained test tube
-. Add - ml o! B' nitric acid solution (H.O-).
4. Stir the reaction mi/ture by clean glass stirring rod.
*. Place the test tube containing the reaction mi/ture in ice6water bath !or *61+ mins.
B. 9ecant the clear solution into cleaned drained test tube and add to it 1m o! +.2 '
ammonium molybdate solution and mi/ the reaction mi/ture using a clean glass stirring
rod.
C. Place the test tube containing the reaction mi/ture into a B* # water bath !or
appro/imately * minutes.
D. :ormation o! yellow precipitate is obser3ed due to e/istence o! phosphate.
E. =ecord the obser3ations.
8. Tests of Cholesterol
Liebermann!"urchard Test for Cholesterol
This test is used in the estimation o! blood cholesterol.#holesterol produces a
characteristic green color when it is mi/ed with the Fiebermann$2urchard reagent a
mi/ture o! acetic anhydride and sul!uric acid. The change in color may be gradual
initially pin" then blue6purple and !inally deep green.
'aterial
+.* 1 cholesterol in chloro!orm
Acetic anhydride
#oncentrated H2SO4
Procedure
1. Place 1ml o! +.* 1 cholesterol in chloro!orm in a dry test tube.
2. Add * drops o! acetic anhydride.
-. Add 1 drop o! conc. H2SO4 and mi/.
4. Obser3e the appearance o! pin" color which gradually turns into deep green.
Salkowski test for cholesterol
'aterial
+.* 1 cholesterol in chloro!orm
#oncentrated H2SO4
Procedure
1. Place 1ml o! +.* 1 cholesterol in chloro!orm in a dry test tube.
2. Add 1 ml o! conc. H2SO4
-. 'i/ care!ully and allow to stand and separate two layers note the red color o! the
upper layer (chloro!orm) and green !luorescence in the lower layer.
Zak test for cholesterol
This test is used !or determination o! cholesterol in blood
'aterial
+.2 g cholesterol in 1ml o! conc. acetic acid
:erric chloride
#onc. Acetic acid
#onc. Sul!uric acid
Procedure
1. Place +.* ml o! prepared cholesterol solution in a dry test tube.
2. Add 2 ml o! colored solution ( mi/ture o! 1+1 !erric chloride #onc. #H-#OOH and
#onc. H2SO4)
-. Obser3e appearance deep red which re!ers to e/istence o! cholesterol