CHEMICAL TESTS FOR LIPID

They have the common property of being:

(1) Relatively insoluble in water and

(2) Soluble in polar solvents such as ether and chloroform.

Fatty acids are aliphatic carboxylic acids. If the aliphatic chain contains no double bond
then it is called saturated and if it contains one or more double bond it is called
unsaturated. Most naturally occurring unsaturated fatty acids have cis-double bonds.
Some of the most common fatty acids are palmitic acid and stearic acid. Palmitic has 16
carbon atoms and stearic has 18 carbon atoms.

As it is clear from the formulae, both are saturated fatty acids. Some fatty acids like
oleic acid may be unsaturated. Naturally occurring animal fats consist largely of mixed
glyceride of oleic, palmitic and stearic acids. They are usually mixture of individual fats.
Fats have more saturated fatty acids whereas oils have more of unsaturated ones.

Lipids are simple, complex or derived. Simple lipids are esters of fatty acids with various
alcohols, e.g., fats (esters of fatty acids with glycerol) and waxes (esters of fatty acids
with higher molecular weight of monohydric alcohols). Complex lipids are esters of fatty
acids containing groups in addition to an alcohol and a fatty acid, e.g., phospholipids or
glycolipids etc. Derived lipids include fatty acids, glycerol, steroids, other alcohols, fatty
aldehydes, and ketone bodies, lipid soluble vitamins, and hormones.

Phospholipids yield in addition to alcohol and fatty acids, phosphate and a nitrogenous
base like choline, ethanolamine, etc. Lecithin’s and cephalous are representatives of the
phospholipids. Similarly glycolipids contain carbohydrates, and sulpholipids contain
sulphate. Lipoproteins are combinations of lipids with proteins.
Now we will consider some qualitative and quantitative tests for lipids.

Qualitative Tests:
I. Physical Test:
1. Grease spot test:

Description of the test:

In the test, some oil and some water are smeared onto a piece of paper. Some time
later, the water smear would become not translucent. But the smear of oil would keep
translucent for a long time. This is known as the grease spot test.

Working principle:

The working principle is that most grease or fat have a high boiling point. So, they are
non-volatile. In room temperature, the spot of water can absorb enough heat from the
air and evaporize. But the spot of grease can never absorb enough heat to evaporize.
When the liquid is inside the sheet of paper, it diffracts light. So, light can pass from one
side of the paper to another side. This gives the phenomenon of "translucent". When
there is no liquid in the paper, there is no diffraction. So, light cannot pass it through.

Procedure:

Take a small amount of oil on a piece of paper, a greasy spot penetrating the paper will
be formed. This happens because lipid does not wet paper unlike water.
Positive Result:

2. Test for free fatty acids:

Principle of the test:

Free fatty acids of the sample, at pH<7.0, react with a chromogenous compound and
decrease its color. The decreasing of color, read at 630 nm, is proportional to the acid
concentration of the sample, expressed as % of oleic acid.

The acid content of edible fats is given by the quantity of free fatty acids deriving from
the hydrolytic rancidity of triglycerides. As this alteration occurs in unsuitable conditions
for the processing and preservation of fats, acidity represents a basic indicator of the
genuineness of the product. The test is particularly important during the refining of oils
and fats, for the assessment of the processing cycle and for the definition of product
categories.

Method:

Take a few drops of phenolphthalein solution in a test tube and add to it one or two
drops of very dilute alkali solution, just sufficient to give the solution a pink color. Now
add a few drops of the oil and shake. The color will disappear as the alkali is neutralized
by the free fatty acids present in the oil.

Positive Result:
3. Emulsification:

Principle:

The Ethanol Emulsion Test is a food test which determines the presence of a broad
group of naturally occurring compounds known as lipids. Lipids consist of fats and oils.
Other lipid tests include the Grease Spot Test and the Sudan Stain Test. The Grease
spot test is performed on fats - lipids which are solid at room temperature. Sudan stain
colors lipids red, but is a less common bench reagent than ethanol. The Ethanol
Emulsion Test is the most common test amongst the three.

Oil or liquid fat becomes finely divided and is dispersed in water when shaken with
water to form emulsification. Emulsification is permanent and complete in the presence
of emulsifying agent. The important emulsifying agents are bile salts, proteins, soaps,
mono- and diglycerides. Emulsification is important in the processes of fat digestion in
the intestine. Emulsifying agents lower surface tension of the liquid.

Procedure:

Take 2 clean and dry test tubes, in one test tube added 2 ml water and in other 2ml
dilute bile salt solution. Now to each tube added 2 drops of mustard oil and shaken
vigorously for about one minute. Allow the tubes to stands for two minutes and note that
the water, oil is broken in small pieces and floats on the surface; where as in the bile
salt solution, the oil can be seen in minute droplets suspended in the liquid (permanent
emulsification).
Solid sample 

1. Crush the food sample and place in a dry test tube. 

2. Add ethanol to about 2 cm3 above the level of the sample and shake
thoroughly. 
3. Allow the solid to settle (about 3 min) to allow the lipid to be extracted. 
4. Decant the ethanol into another test tube. 
5. Add 2 cm3 of deionized water to the second test tube
6. Make observations.

Liquid sample

1. Add a few drops of the liquid food sample to a dry test tube.
2. Add 2 cm3 ethanol and shake it thoroughly
3. Add 2 cm3 of deionized water.
4. Make observations.

Positive Result:

A layer of cloudy white suspension forms at the top of the solution. (Upon close
inspection you can see the tiny globules of fat suspended in the solution. This an
emulsion. Foods with high lipid content have a ‘higher’ layer than foods with less).
4. Saponification test:

Principle:

Esters can be hydrolysed by alkali to yield the parent alcohol and salt. When the fatty
acid possesses a long chain the salt formed is a soap which we commonly use. This
process is called saponification. Oils and fats usually contain long chain fatty acids and
are, therefore, the starting materials for the preparation of soap.

Procedure:

Take 1 ml of the oil in a test tube and add an equal amount of alcoholic KOH solution,
mix them thoroughly and keep the mixture during the course of warming and shake up
gently with a little distilled water. Appearance of some oil drops will indicate the
incomplete saponification. After complete saponification no oil drops will appear.

Positive Result:

The formation of bubbles (which indicate that a soap was formed). 

5. Tests for unsaturation of fatty acids:

Principle:

Unsaturated fatty acids like oleic acid can react with halogens like bromine and iodine
due to presence of double bonds as shown below.

CH3 (CH2)7CH = CH (CH2)7COOH + Br2 → CH3 (CH2)7CHBr-CHBr (CH2)7COOH

The amount of Br2 or I2 taken up will indicate the amount of unsaturation present in a
particular acid. Approximate idea about the unsaturation in a different oils and fats can
 be obtained by the following test. Set up four clean and dry test tubes each containing 5
ml of CCl4.
To the first, add one drop of shark liver oil, to the second, one drop of coconut oil, to the
third, a drop of vegetable ghee and add nothing to the fourth tube. Now test for the
unsaturation of the added oil by adding bromine water drop by drop to each tube
followed by shaking.

Record the number of drops required to obtain a permanent yellowish red color in each
tube and infer the relative unsaturation in the three samples used. It may be mentioned
here, vegetable ghee is prepared by hydrogenating vegetable oil. Hydrogenation means
saturation of unsaturated fatty acid by hydrogen.

Procedure:

Orange bromine water can be used to test for unsaturation. When it is added to a

sample of the fat or oil:
 the bromine water stays orange with a saturated fat or oil
 the bromine water goes colourless with an unsaturated fat or oil
This is similar to the test for alkenes (which are unsaturated).
Positive Result:

6. Isolation of free fatty acids from soap:

Principle:

Soap is produced by the saponification (hydrolysis) of a triglyceride (fat or oil). (See

Figure 1.) In this process the triglyceride is reacted with a strong base such as sodium
or potassium hydroxide to produce glycerol and fatty acid salts. The salt of the fatty acid
is called a soap.

Fatty acids are straight-chain monocarboxylic acids. The most common fatty acids
range in size from 10-20 carbons and most often have an even number of carbon atoms
including the carboxyl group carbon. The carbon-carbon bonds in saturated fatty acids
are all single bonds, while unsaturated fatty acids have one or more carbon-carbon
double bonds in their chains. One example of a saturated fatty acid is palmitic acid,
CH3-(CH2)14-CO2H.

Fatty acids are seldom found as free molecules in nature but are most often a part of a
larger molecule called a triglyceride. Triglycerides consist of a three-membered carbon
chain (glycerol backbone) with a fatty acid bonded to each of the three carbon atoms in
the glycerol backbone. The bond between the fatty acid and the glycerol backbone is
referred to as an ester linkage. In the saponification process the ester linkage is broken
to form glycerol and soap.

Procedure:

Take a few ml of 20% H2SO4 in a test tube and gradually add 5 ml of some soap
solution. The fatty acids will separate out in a distinct layer due to the hydrolysis of the
soap.
RCOONa + H2O → RCOOH + NaOH
Cool the solution which will become hot and skim off the surface layer and wash it
several times with water till free from H 2SO4. Then dissolve it in some water and add
alkaline phenolphthalein solution and shake. The pink color will be discharged indicating
the presence of free fatty acids.

 Calcium soap formation:

To a small amount of the soap solution in a test tube add CaCl 2 solution. A white
precipitate will be formed. The white precipitate is due to insoluble calcium salt of fatty
acid. This is referred to as calcium soap.
  Lead soap formation:
To a small amount of the soap solution in a test tube add lead acetate solution, a white
precipitate will appear. The white ppt is due to insoluble lead salt of fatty acids. This is
referred to as lead soap.

7. Tests for Glycerol:

I. Acrolein test:

Principle:

When glycerol is heated with potassium bisulphate or concentrated H2SO4,dehydration

occurs and aldehyde Acrolein formed which has characteristic odour. This test responds
to glycerol free or linked as an ester.
Procedure:

Take pure glycerol in a dry test tube; add to it a few crystals of potassium hydrogen
sulphate. Warm gently to mix and then heat strongly. A very pungent odour of acrolein
is produced. Acrolein is formed due to removal of water from glycerol by potassium
hydrogen sulphate.

Positive Result:

II. Dichromate Test:

Procedure/Principle:

Take in a dry test tube 3 or 4 ml of glycerol solution, to it add a few drops of 5%

potassium dichromate solution and 5 ml of conc. HNO 3, mix well and note that the
brown color is changed to blue. This test is given by the substances containing primary
and secondary alcohol groups. The chromic ions oxidize the glycerol and in this process
they are reduced to chromous ions which give the blue colour. This test is also given by
reducing sugars, so before confirming glycerol be sure that the reducing sugars are not
present.

Positive Result:

Quantitative Tests:
1. Determination of Iodine Number:

Principle and Procedure:

The iodine number of a fat is the amount in gm. of iodine taken up by 100 gm. of fat. Not
only iodine but also equivalent amounts of other halogens will add at double bonds; so
bromine is often used instead of iodine because it is more reactive. The halogenating
reagent used in this method is pyridine sulphate di-bromide. This reagent can be
prepared by adding carefully 8.1 ml pyridine in 20 ml glacial acetic acid and making the
volume up to 1 litre with glacial acetic acid.

Weigh the bottle containing sample of oil plus a medicine dropper and then transfer
about 0.1 to 0.3 gm. of oil to a flask. Reweigh the bottle containing oil and dropper to
find out the exact quantity of the sample transferred. Add 10 ml of chloroform and then
25 ml of the pyridine sulphate di-bromide reagent.

Shake thoroughly; allow standing for 5 minutes and then determining the residual
bromine. To do this, add 10 ml of 10% KI and titrate the equivalent amount of iodine
liberated by the residual bromine with the help of 0.1 (N) Na 2S2O3 (sodium thiosulphate).
The titration can be done by adding sodium thiosulphate solution through a burette to
the flask.
When the colour of the solution in flask becomes light yellow add 1 ml of starch solution.
It will become blue. Slowly add the thiosulphate solution again till it becomes colourless.
Note the total volume of thiosulphate used.

The total amount of bromine originally added is found by titrating 25 ml of the pyridine
sulphate di-bromide reagent with thiosulphate after adding KI as in the previous case.
The amount of bromine taken up by the fat sample can be determined by the difference
between the two titers and then the iodine number can be calculated.

Suppose with a sample of 0.2 gm. oil the data obtained are as follows:
0.1 (N) Na2S2O3 used for titration of blank = 47.0 ml
0.1 (N) Na2S2O3 used for titration of sample = 27.0 ml
0.1 (N) Na2S2O3 equivalent to iodine absorbed by the sample = 20.0 ml
As 1 ml 0.1 (N) Na2S2O3 = 1.0 ml of 0.1 (N) Bromine = 1 ml of 0.1 (N) Iodine
Hence, 20 ml of 0.1 (N) Na 2S2O3 = 20 ml of 0.1 (N) Iodine = 20×12.7/1000 gm Iodine =
0.254 gm Iodine.
Thus 0.2 gm of oil can take up 0.254 gm of iodine.

Therefore, iodine number of oil used = 127.

Qualitative Test of Cholesterol:

Cholesterol is a lipid with a structure quite different from that of phospholipids. It is a
steroid, built from four linked hydrocarbon rings. A hydrocarbon tail is linked to the
steroid at one end, and a hydroxyl group is attached at the other end. In membranes,
the molecule is oriented parallel to the fatty acid chains of the phospholipids, and the
hydroxyl group interacts with the nearby phospholipid head groups.

Cholesterol is absent from prokaryotes but is found to varying degrees in virtually all
animal membranes. It constitutes almost 25% of the membrane lipids in certain nerve
cells but is essentially absent from some intracellular membranes.

It is found in bile and a common constituent of gall stones.

The main test for cholesterol is known as Liberman-Burchard test. This is carried in the
following way. In a dry test tube take a small amount of solution of cholesterol in
chloroform. Add 1 ml of acetic anhydride and 1 drop of conc. H 2SO4. Mix and observe
that a purple colour is formed which soon changes to green. It may take 15-30 min for
full development and it is advisable to put the tube in dark during this time.
Quantitative Estimation of Cholesterol:
The above mentioned qualitative test has been developed quantitatively for the
estimation of cholesterol.

Follow the following protocol for the purpose:

Shake the tubes well and keep them at room temperature for 30 minutes. Blue colour
will develop in all the tubes except blank tube. Measure the absorbencies at 625 m|a.
against the blank tube and plot these against the amount of cholesterol.

Note: Acetic anhydride-sulphuric acid reagent.

This reagent has to be freshly prepared before use. Acetic anhydride (20 ml) is taken in
a glass stoppered flask which is then chilled in ice water. When cold, add 1 ml of conc.
H2SO4 to it drop by drop. The contents are mixed and cooled during the addition. After
completion of the addition the flask is stoppered and shaken vigorously for a few
minutes. The solution has to be kept cold in ice and should be used within an hour.

Enzymatic Methods:
Assays have been developed in which cholesterol oxidase obtained from the bacterium
Nocardia erythropolis is used to convert cholesterol into cholest-4-en-3-one with the
formation of Hydrogen peroxide. The cholest-4en-3-one formed has been measured by
reading at 240 nm after extracting into isopropanol. Alternatively, hydrogen peroxide
has been quantified by formation of chelate complex with quadrivalent titanium and
xylenol orange.

QULITATIVE TEST FOR FATS AND OILS

BAUDOUIN TEST

Principle:

This test is used to detect the presence of added seasame oil, which is characterized by
the presence of certain phenolic substances. The principle of the test is based on the
reaction of phenols with furfurals in acid solution. Furfurals are formed by the
dehydration of the monosaccharides in concentrated mineral acids.
Hence a solution of sucrose in concentrated hydrochloric acid will be hydrolysed to an
equimolar mixture of glucose and fructose,both of which give rise to hydroxymethyl
furfural by dehydration. The latter reacts with the phenols found in seasame oil to give a
compound with a pink colour.

Material and apparatus:

1. ethanol 90% v/v

2. ammonia sol.
3. 1% sucrose sol. concentration hydrochloric acid.

Method:

1- put 5ml of sample in conical flask and add to it 5ml of ethanol/ammonia mixture (9:1)
2- shake well and heat on a water bath until the sol. Gets free from both ammonia and
ethanol.
3- add 1ml of the acid sucrose mixture, skake and allow the sol. To stand for 5min.
4- the development of apink colour indicates the presence of seasame oil.

B-halphen test

Principle:

This test is used to detect in the presence of added cotton seed oil, which is
characterized by the presence of the toxic pigment gossypol. The latter reacts with
giving a reddish colour.

Material:

1-stoppered glass test tubes.

2-water bath.
3-amyl alchhol.
4-precipitated sulpher 1% w/v sol. Dissolved in carbon disulphide.
5-halphen sol. (prepared by mixing equal volume of amyl alchol and precipitated
sulpher)

Methods:

1) put 2-3 ml of the sample in a glass stoppered test tube(20ml capacity)

2) add 3ml of halphen solution and close the tube.
3) immense the tube in boiling water for 30min.
4) development of a red colour indicates the presence of cotton seed oil.
Detection of mineral oils

Edible fats and oils are usually saponifiable , whereas mineral oils are not . in this test,
the ability of the sample to be saponified is determined.

Objectives:

This test is established to detect the presence of mineral oils contaminated with edible
oils.

Material:

1-reflux air condenser.

2-alcocholic potassium hydroxide sol.

Method:

1) place 1ml of the oil in a conical flask and add to it 25ml of alcoholic potassium
hydroxide sol.
2) boil under the reflux contents of the flask about 25ml distilled water and mix
thoroughly, if turbidity appears, the mineral oil is present of more than 0.5%.

Other Tests for Cholesterol:

1. Salkowski’s Test (H2SO4 Test):

Principle:

Salkowski's test is used to detect cholesterol in a chlorophyll sample, according to

Academic Medical Dictionary, and is named after Ernst Leopold Salkowski, a German
biochemist. This test is also used to detect the presence of a chemical compound called
an indole in certain plant species.

Indoles are chemical substances found in dark green and root vegetables, such as
broccoli, kale, turnips and rutabagas. Some types of indoles are used to treat cancer or
fibromyalgia. According to WebMD, diets rich in these types of vegetables are
associated with a decreased risk of developing certain types of cancer. Researchers
suggest that an indole is only one type of substance in vegetables that may prevent
cancer.
Researchers at Oregon State University suggest that chlorophyll can form tight bonds to
cell-damaging substances, including tobacco or charred meat. Tight bonding of the
chlorophyll helps to interfere with the absorption of these damaging substances and
reduce the amount of carcinogen that reaches healthy tissue. Indoles found in
vegetables also help counteract carcinogens by increasing the activity of detoxification
enzymes.
The ability to detect indoles in plant species is important to future research of cancer
prevention. According to WebMD, researchers are interested in a specific substance
called indole-3-carbinol for the prevention of breast, cervical and colorectal cancer.

Dissolve cholesterol in 2 ml of chloroform in dry test tube. Add equal amount of con.
H2SO4. Shake gently. The upper layer turns red and the sulphuric acid layer shows a
yellow colour with a green fluorescence.

Materials:
• 0.5 % cholesterol in chloroform
• Concentrated H2SO4

Procedure:
1. Place 1ml of 0.5 % cholesterol in chloroform in a dry test tube.
2. Add 1 ml of conc. H2SO4
3. Mix carefully and allow to stand and separate two layers, note the red color of the
upper layer (chloroform) and green fluorescence in the lower layer.

Positive Result:
2. Formaldehyde-H2SO4 Test:

Procedure:

Add 2 ml of formaldehyde-sulphuric acid solution (1 part of 40% formaldehyde to 50

parts of the acid) to 2 ml of chloroform solution in a dry test tube. The cherry colour is
developed in the chloroform. Pour off the chloroform in another test tube and add 2-3
drops of acid anhydride. The blue colour develops.

3. Test of Triglycerides

Principle:

This test can be used to distinguish between triglycerides and fatty acids.
Triglycerides are chemically neutral while fatty acids are acidic due to their free
carboxylic group and can decolorize the alkaline red color of phenolphthalein.

Materials:

• 20% Glycerin in chloroform

• 0.5% borax solution
• 1% phenolphthalein in 50% ethanol

Procedure:

1. Place 5ml of Borax solution (0.5%) in test tube

2. Add appropriate volume of Phenolphthalein till red color is appearing.
3. Add 20% glycerin drop by drop till red color is disappearing.
4. warm the test tube, the red color will appear again.
5. fatty acids are not responding to this test.

4. Test of cupric acetate for detecting fatty acids

Principle:

This test is used for detecting saturated and unsaturated fatty acids, fatty acids reacts
with cupric acetate to form cupric salts of fatty acids. The cupric salts of saturated fatty
acids are not dissolved in water or petroleum ether; therefore, it’s precipitated at the
bottom of test tube. The cupric salts of unsaturated fatty acids are dissolved in
petroleum ether with blue-green color.

Materials:

• Saturated fatty acid ( stearic acid or palmitic acid)

• Unsaturated fatty acid ( Oleic acid)
• 10% aqueous cupric acetate
• Petroleum ether

Procedure:

1. Dissolve small amount of fatty acid in petroleum ether.

2. Add appropriate amount of aqueous cupric acetate solution.
3. Shake the test tube carefully and observe green precipitate is appear at lower
aqueous layer ( cupric acetate layer) where this is happened with saturated fatty acids
while at upper organic layer ( petroleum ether layer) , blue or green color is appear
when unsaturated fatty acid is used.

5. Rancidity

Principle:

Rancidity is a process which is accompanied by the formation of unpleasant odour,

taste, as a result of the action of moisture, air (O2) and enzymes.To guard against
rancidity, we have to protect lipid from moisture and direct light as well as its storage
must be in a cold place to deactivate lipase.

Types of rancidity:

1- Hydrolytic rancidity (or lipolytic rancidity) refers to the odor that develops
when triglycerides are hydrolyzed and free fatty acids are released. This
reaction of lipid with water sometimes requires a catalyst, but results in the
formation free fatty acids and salts from free fatty acids (soaps). In particular,
short chain fatty acids, such as common butter fats, are odorous. Rancidity in
foods may be very slight, indicated by a loss of freshness to very severe,
indicated by objectionable odors and/or flavors. Slight degrees of rancidity are
much more common in foods than severe rancidity, yet slight rancidity is a
much more a practical concern. A slight degree of rancidity may not be
objectionable to consumers, but products which do not seem fresh will not
attract repeat purchases. If customers do not return to a product, the long-
term effects of a slight degree of rancidity can be very serious. Even though
meat is held under refrigeration or in a frozen state, the poly-unsaturated fat
will continue to oxidize and slowly become rancid. The fat oxidation process,
potentially resulting in rancidity, begins immediately after the animal is
slaughtered and the muscle, intra-muscular, intermuscular and surface fat
becomes exposed to oxygen of the air. This chemical process continues
during frozen storage, though more slowly at lower temperature. Air tight
packaging will slow rancidity development.

2- Oxidative rancidity of fats such as lard, shortenings, salad and cooking oils
refers to the undesirable odors and flavors which develop when such
products are exposed to the oxygen in the air. Products containing these fats,
 including but not limited to food products such as fish, poultry, meat, frozen
vegetables and dry milk can become rancid as the fats in the products react
to air. The poly-unsaturated fatty acid portions of these foods react with
oxygen to form peroxides. The peroxides decompose to yield a complex of
mixtures, including aldehydes, ketones, and other volatile products. These
products are responsible for "rancid" odors and flavors. It is important to note
that fish contain highly unsaturated (poly-unsaturated and mono-unsaturated)
fatty acids which make some fish products particularly susceptible to oxidative
deterioration. Highly saturated products, such as butter, are not as prone to
oxidative rancidity due to the absence of polyunsaturated fatty acid
compounds. These products also tend to be more solid at room temperature.

 Kreis-Kerr test

Principle:

The term "rancidity" is used to describe the development of bad flavours and odours in
fats and oils. It may result either from hydrolysis of the triacylglycerol present in fats and
oils or from oxidation of the unsaturated fatty acids present in the triacylglycerols. The
former cause may be detected by an increase in the acid value of the sample .
Autooxidation at fatty acid double bonds occurs by reaction with molecular oxygen
present in the atmosphere, causing the formation of labile peroxides.

The peroxides formed during autooxidation are unstable and decompose into free
radicals .These initiate chain reactions which lead to eventually to decomposition of the
fatty acid into various low molecular weight aldehydes and ketones.

Aldehydes and ketones react with phloroglucinol developing a red colour.

Reagent:

1% of Phloroglucinol in alcohol or ether.

Procedure:

1 ml of rancid Oil is mixed with 1 ml of conc. HCl and then add 2 ml of 1% of

Phloroglucinol , Shake and leave to stand for one hour. Appearance of red colour
indicates the presence of aldehydes. The color intensity is increased with the increasing
in the degree of rancidity in lipid.

 Thiobarbituric acid test

Used for detection of aldehydes, Rancid oils give red colour when shaken with
trichloroacetic acid and thiobarbituric acid.
6. Saponification of Fats and Oils

Principle:

Oils can be saponified with alkali soda to form soap and glycerol and it’s as follows:

Materials:

• Oil
• Alcoholic sodium or potassium hydroxides(0.5 N)
• Sodium Chloride
• Concentrated hydrochloric acid

Procedure:

1. Place 5 g of oil or fat, 1.0 g of sodium hydroxide and 25 ml of alcohol in round

flask.
2. Place the boiling stone at round flask for regulating the heating and reflux the
solution for 30 minutes at boiling water bath.
3. Warm the solution for evaporating the alcohol.
4. Cool the solution in a beaker of cold water.
5. Remove a small piece of the solid product (about the size of a pea) using a glass
stirring rod and dissolve it with 20 ml DW and observe the foam formed between soap
and water.

7. Ammonium Molybdate Test for Phosphate

Principle:

The presence of free phosphate in acidic solution can be detected by adding a

molybdate to the solution. Equation illustrates the pertinent reaction between phosphate
and ammonium molybdate solution in the presence of nitric acid (HNO3).

HPO42–(aq) + 12MoO42–(aq) + 3 NH4+(aq) + 23 H3O+(aq)

(NH4)3[P(Mo3O10)4] (yellow,s) + 35 H2O(l)

After a few minutes, the yellow ammonium molybdo-phosphate precipitates from the
reaction mixture. 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
free phosphate. The free phosphate then reacts as in Equation, forming a yellow
precipitate.

Materials/Procedure:

1. Prepare an ice-water bath

2. Place 0.2 g of lecithin (ege yolk) in a washed , drained test tube
3. Add 3 ml of 6M nitric acid solution (HNO3).
4. Stir the reaction mixture by clean glass stirring rod.
5. Place the test tube containing the reaction mixture in ice-water bath for 5-10
mins.
6. Decant the clear solution into cleaned, drained test tube and add to it 1m of 0.2
M ammonium molybdate solution and mix the reaction mixture using a clean glass
stirring rod.
7. Place the test tube containing the reaction mixture into a 65 ͦ C water bath for
approximately 5 minutes.
8. Formation of yellow precipitate is observed due to existence of phosphate.
9. Record the observations.

8. Tests of Cholesterol

Liebermann–Burchard Test for Cholesterol

Principle:

This test is used in the estimation of blood cholesterol.Cholesterol produces a

characteristic green color when it is mixed with the Liebermann–Burchard reagent, a
mixture of acetic anhydride and sulfuric acid. The change in color may be gradual,
initially pink, then blue-purple, and finally deep green.

Material:

• 0.5 % cholesterol in chloroform

• Acetic anhydride
• Concentrated H2SO4

Procedure:

1. Place 1ml of 0.5 % cholesterol in chloroform in a dry test tube.

2. Add 5 drops of acetic anhydride.
3. Add 1 drop of conc. H2SO4 and mix.
4. Observe the appearance of pink color which gradually turns into deep green.

Zak test for cholesterol

Principle:

This test is used for determination of cholesterol in blood

Material:
• 0.2 g cholesterol in 1ml of conc. acetic acid
• Ferric chloride
• Conc. Acetic acid
• Conc. Sulfuric acid

Procedure:

1. Place 0.5 ml of prepared cholesterol solution in a dry test tube.

2. Add 2 ml of colored solution ( mixture of 10% ferric chloride , Conc. CH3COOH
and Conc. H2SO4)
3. Observe appearance deep red which refers to existence of cholesterol

Other Tests for Lipids:

Tschugajen test: Chloroform solution of the extract with excess of acetyl chloride and
pinch of zinc chloride and warming on water bath gives Eosin red colour.

Mayer's test (Potassium Mercuric Iodide): The acid layer with few drops of Mayer's
reagent gives a creamy white precipitate.

Wagner's Tests (Solution of Iodine in Potassium Iodide): The acid layer with few
drops of Wagner's reagent gives reddish brown coloured precipitate.

Hager's Test (Saturated solution of picric acid): The acid layer with Hager's reagent
gives yellow precipitate.

Dragendroff's test (Solution of Potassium Bismuth Iodide): Acid layer with few
drops of Dragendroff's reagent gives reddish brown precipitate.

Ferric chloride test: Extracts mixed with 1 % ferric chloride solution gives blue, green
or brownish green colour.

Gelatin test: Extracts mixed with few drops of 1% solution of gelatin containing 10%
sodium chloride gives white precipitate.

Ferric chloride test: Alcoholic solution of the extracts mixed with few drops of neutral
ferric chloride solution gives green color.

Lead acetate tests: Alcoholic solution of the extracts mixed with few drops of 10 % lead
acetate gives yellow precipitate.

Legal's test: The extracts mixed with mixture of sodium nitro prusside and pyridine and
treatment with methanol alkali gives deep red colour.
Feigel's test: The acidified extracts when shaken with solvent ether, the ether extract
with few drops of saturated alcoholic solution of potassium hydroxide in a porcelain
crucible heated over a flame until cooling it gives light pink colour with 1% ferric chloride
solution.

Baljel's tests: The extracts mixed with solution of sodium picrate gives yellow orange
colour

Copper acetate test: The extracts, mixed with solution of copper acetate gives green
colour.

Sodium hydroxide reagent: Dissolve a small amount of alcoholic extract in 1 ml water

and add sodium hydroxide solution. A yellow colour indicates the presence of
glycosides.

Kellar Killani's test: Dissolve the extract in water with Glacial acetic acid and ferric
chloride and concentrated sulphuric acid. They give brown ring at the junction.

Picrate paper tests (for cyanogenic glycosides): Extracts are added with few drops
of chloroform and concentrated sulphuric acid. The test tube is tightly stopped with
picrate paper protruding into the test tube when kept on water bath, positive sample
turns yellow picrate paper into red.(Bark,1963)

Foam test: A small amount of extract is shaken with little quantity of water. The foam
produced persists for 10 min. It confirms the presence of saponins.

Haemolysis test: To 2 ml normal saline in 2 test tubes, 2 ml distilled water is added to

one and two ml of 1 % extract to the other five drops of blood is added to each test tube
and gently mixed with the content. Haemolysis observed under the microscope in the
tube containing the extracts indicates the presence of saponin.
 Bicol University
College of Science
Department of Biology

RESEARCH #3
IN
BIOCHEMISTRY
LAB
NICOLA FAYE BRON
BS BIOLOGY II-A

Dr. NOEMI MADRID

Professor