LIPIDS
Group 7 INur-1
Mustafa, Sean Justin
Nalagon, Alyza Jane
Padua, Michelle
Pajado, Christine Mae
SPOTTING EFFECT
• This test for lipids has been used in centuries. Lipids that
are derived from glycerol nd sphyngposine, a long-chain
base that is the backbone of sphingolipids, will produce
translusent “spots” or “stains” on the paper. If the lipid is
not a derivative of glycerol or sphingosine, it will not
produce a transluscent spot.
• Objective
➢Test for the presence of lipids by means of locating translucent
spots.
SPOTTING EFFECT
• Results
Cheeck tint Vegetable oil Lotion Hairwax Lip Butter
- + - + -
SPOTTING EFFECT
SOLUBILITY
• Lipids are non-polar organic compounds. The physical
properties of fatty acids, and of compounds that contain
them are largely determined by the length and degree of
unsaturation of the hydrocarbon chain. The nonpolar
hydrocarbon chain accounts for the poor solubility of fatty
acids in water. Solubility of a substance depends on the
simple rule of “like dissolves like”.
SOLUBILITY
• Samples:
SOLUBILITY
SOLUBILITY
• Results
METHYLENE
WATER ETHER TOLUENE
CHLORIDE
VEGETABLE OIL Immiscible Miscible Miscible Miscible
LECITHIN Immiscible Miscible Miscible Miscible
SOLUBILITY
• To get a crude idea of the structure of the lecithin molecule,
imagine a balloon with two long paper streamers attached
to it. The balloon or "head" region corresponds to the polar
portion of the molecule, the negatively-charged phosphate
group, and the positivelycharged choline, which readily
dissolve in water. The streamers or "tails" represent the
nonpolar part, the long chain of 12 to 18 carbon atoms in
the two fatty acids, which are insoluble in water. As a result
of the nature of its head and tail groups, lecithin molecules
tend to disperse themselves in water with their nonpolar
tails back-to-back to form bilayers, or double layers, in
which the polar heads project outward into the water. This
arrangement sequesters, or conceals, the nonpolar tails
away from the water, forming a structural barrier to the
•
passage of polar and ionic molecules as shown.
SOLUBILITY
• Vegetable oil
SOLUBILITY
• Reasons for the results (Vegetable Oil)
• Water
• vegetable oil is immiscible with water even at room temperature
since water is polar solvent, it will not dissolve coconut oil which
is non-polar
• Ether
• vegetable oil is miscible with ether since both are organic and
non-polar solvents.
• Toluene
• vegetable oil is nonpolar. Therefore, solvents such as toluene
(non-polar), can dissolve non-polar substances.
• Methylene Chloride
• like vegetable oil, Methylene Chloride is also non-polar, thus the
principle “like dissolves like” applies.
SOLUBILITY
• Lecithin
SOLUBILITY
• Reasons for the results (Lecithin)
➢Water
▪ Lecithin is immiscible with water, since water is polar solvent, it will not
dissolve with lecithin which is non-polar
➢Ether
▪ Lecithin is miscible with ether since both are organic and non-polar
solvents.
➢Toluene
▪ Lecithin is nonpolar. Therefore, solvents such as toluene (non-polar), can
dissolve non-polar substances.
➢Methylene Chloride
▪ Like Lecithin, Methylene Chloride is also non-polar, thus the principle
“like dissolves like” applies.
BROMINE WATER TEST
• Iodine tests if a lipid is saturated or unsaturated by adding
iodine to various substances. If the iodine changes from
orange to clear the lipid is unsaturated. If the iodine did
not change colour the lipid is saturated.
BROMINE WATER TEST
• Saturated vegetable fats are solid at room temperature,
and have a higher melting point than unsaturated oils.
Unsaturated vegetable oils can be ‘hardened’ by reacting
them with hydrogen, a reaction called hydrogenation.
• During hydrogenation, vegetable oils are reacted with
hydrogen gas at about 60ºC. A nickel catalyst is used to
speed up the reaction. The double bonds are converted to
single bonds in the reaction. In this way unsaturated fats
can be made into saturated fats – they are hardened.
BROMINE WATER TEST
BROMINE WATER TEST
BROMINE WATER TEST
• Results
Vegetable Oil Lecithin
Iodine orange- yellow remained orange
BROMINE WATER TEST
ACROLEIN TEST
• Acrolein is the simplest unsaturated aldehyde. It is a
colourless liquid with a piercing, disagreeable, acrid smell
or has a characteristic sharp irritating odor. (CH2=CH–
CHO)
• Acrolein test is a test for the presence of glycerin or fats.
ACROLEIN TEST
• Objective:
➢To detect the presence of fats or glycerin.
• Principles
➢When a fat is heated strongly in the presence of a dehydrating
agent such as potassium bisulfate (KHSO). the glycerol portion of
the molecule is dehydrated to form the unsaturated aldehyde,
acrolein (CH2=CH–CHO).
ACROLEIN TEST
• Reagents
➢Potassium bisulfate (KHSO)
▪ is a dehydrating agent
o Whenever fat is heated in the presence of a dehydrating agent, the fat
molecule will shed its glycerol in the form of the unsaturated aldehyde –
acrolein.
➢ Concentrated sulphuric acid (H2SO4)
▪ With strong dehydrating agents, as concentrated sulphuric acid,
glycerol can be converted to acrolein that has very irritating odour.
ACROLEIN TEST
• Reaction
2COOR + PHO3C2H4N+(CH3)3
ACROLEIN TEST
• Results
Odor of the Vapor
Glycerol Burned fat odor
Cooking Oil Burned fat odor
Lecithin Burned fat odor
ACROLEIN TEST
• Positive visible result
➢The odor of an acrolein, which has an unpleasant odor, tells us
that a sample is positive in Acrolein test. It resulted with the three
samples used in the experiment, which are the glycerol, cooking oil
and lecithin.
ACROLEIN TEST
ACROLEIN TEST
• Negative visible result
➢There is no presence of fat or glycerine in a sample if there is no
unpleasant odor.
➢Cholesterol gives a negative result in Acrolein test
▪ Cholesterol is a sterol, a lipid molecule, and a waxy, fat-like
substance that’s found in all cells of the body.
▪ In short, Cholesterol does not contain any fats or glycerine.
EXTRACTION OF
BRAIN LIPIDS
• The aim of all extraction procedures is to separate cellular
or fluid lipids from the other constituents, proteins,
polysaccharides, small molecules (amino acids, sugars...)
but also to preserve these lipids for further analysis.
• Neutral lipids or generally storage lipids are extracted with
relatively non-polar solvents such as diethyl ether or
chloroform but membrane-associated lipids are more
polar and require polar solvents such as ethanol or
methanol to disrupt hydrogen bondings or electrostatic
forces.
EXTRACTION OF
BRAIN LIPIDS
Hemoginized brain +
Ether
Decantate (ether Brain Residue + 95%
+acetone) hot ethanol
Filter Filter
Residue B Residue C Filtrate
Soda Lime Test Ninhydrin Test Ammonium Molybdate Leiburman-Burchard Molisch Test
Test Test
EXTRACTION OF
BRAIN LIPIDS
• Brain submerged in Ether
➢E x t r a c t s c e r a m i d e s , s p h i n o g o m y e l i n s , c h o l i n e ,
glycerophospholipids, ethanolamine and
phosphatidylserines.
• Decantation (Non-Polar, Liquid)
➢Add acetone until clear (acetone is used as solvent for
glycophingolipids)
• Filtrate
➢Residue C (Evaporated to dryness over steam bath)
➢Cholesterol
▪ Leibermann-Buschard Test
•
EXTRACTION OF
BRAIN LIPIDS
• Filtrate
➢Residue B
▪ Ninhydrin Test
▪ Soda Lime Test
▪ Ammonium Molybdate Test
MOLISCH TEST
➢The Molisch test is a qualitative test for determining the
presence of carbohydrates in a solution.
➢This test is based on the reaction of a carbohydrate with
concentrated sulfuric acid to form a furfural or
hydroxyfurfural which then reacts with the Molisch’s
reagent (alpha-naphthol) to yield a colored product,
generally a purple ring at the interface of the two layers.
MOLISCH TEST
• Principles
➢C a r b o h y d r a t e s , w h e n t r e a t e d w i t h
concentrated sulphuric acid, undergo
dehydration to give furfural derivatives. These
compounds condense with alpha-naphthol to
form purple or violet colored products.
MOLISCH TEST
• Reagents
➢Molisch’s reagent
▪ is a solution of alpha-naphthol dissolved in 95% ethanol. The
alpha-naphthol reacts with the cyclic aldehydes to form purple
colored condensation products.
➢ Concentrated sulphuric acid
▪ causes dehydration of all carbohydrates to give “furfural”
compounds, that react with α-naphthol (Molisch's reagent)
giving a violet or purple colored complex.
MOLISCH TEST
• Reaction
MOLISCH TEST
• Positive visible results
➢The purple-coloured compound appears as a ring layer at the
interface between the sulphuric acid and test solution. The
sulphuric acid is denser than the test solution and therefore the
reaction will occur at the junction where both substances meet.
MOLISCH TEST
decantate A and
Molisch reagent
concentrated
sulphuric acid
violet ring
MOLISCH TEST
• Negative visible result
➢No violet ring formed at the junction of the liquids, which means
there is an absence of carbohydrates.
NINHYDRIN TEST
• The Ninhydrin test is often used to detect alpha-amino
acids and free carboxyl and amino groups on proteins,
amino acids and peptides, in which ninhydrin reacts with
such compounds to produce a blue-violet colored solution.
NINHYDRIN TEST
• Principles
➢This test is given by the free amino acids, small peptides and
protein will react to give purple color. Ninhydrin reacts with amino
acids to form hyrindantin, a chemical intermediate formed during
the ninhydrin test for amines, and then it further forms Rheumman's
purple by reacting with ammonia and another ninhydrin. Imino
acids, the secondary amino acids, give yellow color owing to
absence of alpha amino acids.
NINHYDRIN TEST
• Reagents
➢Ninhydrin reagent
▪ is an effective reagent for thin-layer chromatography (TLC),
which is a chromatography a chromatography technique used to
separate non-volatile mixtures. Ninhydrin reacts with primary
and secondary amines producing a blue or purple reaction
product (diketohydrindylidene-diketohydrindamine).
NINHYDRIN TEST
• Reaction
NINHYDRIN TEST
• Positive visible result
➢The color of the resulting solution is blue-violet, which means an
alpha-amino group was detected by the Ninhydrin test.
NINHYDRIN TEST
Heated mixture
of residue B,
water and
ninhydrin
reagent
blue-violet solution
NINHYDRIN TEST
• Negative visible result
➢no violet/purple product formed.
➢Proline gives a yellow color because it does not give the ninhydrin
reaction as this reagent requires free alpha amino group (-NH2) but
proline has an imino group (-NH).
SODA LIME TEST
• The Soda Lime test is used to determine if the organic
compound is basic or acid, and if the moistened litmus
paper done along with it results in blue coloration, then
the compound is base, on the other hand, if it turns red,
then the compound is acid.
SODA LIME TEST
• Reagents
➢Soda Lime
▪ is a white or grayish granular mixture of calcium hydroxide and
sodium or potassium hydroxide.
▪ used to absorb carbon dioxide and water vapour in situations in
which rebreathing occurs.
SODA LIME TEST
• Reaction
SODA LIME TEST
• Positive visible result
➢If the colors of the moistened red and blue litmus paper, after
testing the vapor, turned to blue, then the heated mixture of soda
lime and residue B is a base.
➢on the other hand, if the colors of the moistened red and blue
litmus paper became red, then the mixture is an acid.
SODA LIME TEST
heated mixture of
soda lime and residue
B
moistened litmus
paper
SODA LIME TEST
• Negative visible result
➢There is no negative visible result here because in the Soda Lime
test, we are just determining if an organic compound or mixture is
an acid or a base.
AMMONIUM
MOLYBDATE TEST
• The Ammonium Molybdate test is a test for the presence
of phosphate group.
AMMONIUM
MOLYBDATE TEST
• Purpose
➢o determine the phosphate in water
• Principles
➢Hydrolysis
➢Double Decomposition
AMMONIUM
MOLYBDATE TEST
• Reagents
➢(HNO3) Nitric Acid
▪ to break the bonds to reveal the free phosphate group.
AMMONIUM
MOLYBDATE TEST
• Reaction
AMMONIUM
MOLYBDATE TEST
• Positive visible result
➢Yellow precipitate
AMMONIUM
MOLYBDATE TEST
AMMONIUM
MOLYBDATE TEST
• Negative visible result
➢no reaction
LIEBERMANN–
BURCHARD TEST
• Leibermann-Burchard test is also called as acetic
anhydride test.
LEIBERMANN–
BURCHARD TEST
• Principles
➢the cholesterol is react as a typical alcohol with a strong
concentrated acids and the product are colored substances.
LIEBERMANN–
BURCHARD TEST
• Reagents
➢acetic anhydride
▪ the acetic anhydride are used as solvent and dehydrating
agents
➢conc. H2SO4 (sulfuric acid)
▪ the sulfuric acid is used as dehydrating and oxidizing agent
LIEBERMANN–
BURCHARD TEST
• Reaction
LIEBERMANN–
BURCHARD TEST
• Positive visible result
• Red---blue– Bluish green/ dark green solution
LIEBERMANN–
BURCHARD TEST
LIEBERMANN–
BURCHARD TEST
• Negative visible result
➢no reaction
