Bicol University

College of Science

Legazpi City

BIOCHEMISTRY
Chemical Laboratory Tests for Lipids

Mikaela Rome C. Bigay

BS Biology 2-A
Dr. Noemi Madrid
 The Grease Spot Test
Shows positive test for:
Triglycerides

How to perform the test:

Two ml of methylene chloride is used to dissolve ~0.1 g of the sample to be tested. If solid material
remains, grind it in a mortar and pestle and then filter to remove the solid from the test solution. If a
liquid sample is insoluble, take the test solution from the methylene chloride layer. Several drops of
the test solution are placed on a sheet of paper and the solvent allowed to evaporate.

A positive test is indicated by:

The formation of translucent spot on the paper.

Negative Test Positive Test

Sudan Red Test

Sudan red is a lipid soluble dye. When Sudan red is added to a mixture of lipids and water, the dye
will move into the lipid layer colouring it red.
Procedure:
1. Add one drop of Sudan red dye (dissolved in alcohol) to each tube.
2. Mix the contents of each tube using the vortex genie
3. Add 0.1 mL of H20. Wait 2 minutes.
4. Examine each tube carefully. Locate where the red color is found.
5. Record your observations.
Results:
The oil will stain red with Sudan III dye since it is a lipid and contains triglycerides. However, since the
oil is less dense than water and insoluble in water, the oil will form a layer or globules above the water
and appear as a red layer above the water in the test tube.
 Ethanol Emulsion Test
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.
Process

 Add the food sample to 2 cm3 of ethanol, shake well.

 Allow to settle in a test tube rack for 2 minutes for food to dissolve in ethanol.
 Empty any clear liquid into a test tube containing 2 cm3 of distilled H2O.
 A MILKY-WHITE EMULSION is a positive result: lipid is present.
 If the mixture remains clear, there are no fats present in the sample

Explanation

 Lipids are insoluble in water and soluble in ethanol (an alcohol).

 After lipids have been dissolved in ethanol and then added to H2O, they will form tiny
dispersed droplets in the water. This is called an emulsion.
 These droplets scatter light as it passes through the water so it appears white
and cloudy.
Illustration

Acrolein Test
Objective: To detect presence of fats or glycerin.
Principle: 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), which has the odor peculiar to burnt cooking grease. Glycerol
Acrolein has odor of burnt cooking grease.
Materials:
1. Test compounds ( Oil or fat ,Oleic acid)
2. Potassium bisulphate or conc. H2SO4

Procedure:
1. Place 5 drops of test compound in a clean and dry test tube2 . A d d 1 ml of
c o n c . H2SO4 carefully or 1 gram of KHSO4.
2. Heat the test tube directly.
3. Note the characteristic pungent odour of Acrolein.
Result:
Acrolein is a compound formed by dehydration of glycerol, so its presence indicates the presence of
a glyceride ester (usually a triglyceride) ie a fat or oil. The smell is a bit like a barbecue.

Copper Acetate Test

Objective: To distinguish between oil or neutral fat and fatty acid saturated and unsaturated.
Principle: The copper acetate solution does not react with the oils, while saturated and unsaturated
fatty acids react with copper acetate to form copper salt. Unsaturated fatty acids can only be
extracted by petroleum ether.

 In the case of olive oil notice that petroleum ether upper layer containing the dissolved oil and
appears colourless, aqueous solution remains blue in the bottom.
 In the case of oleic acid the upper layer of petroleum ether becomes green as a result of
copper oleate. The lower layer becomes less in blue.
 In the case of stearic acid notice that the petroleum ether upper layer remains colourless, while
consists of pale green precipitate of copper stearate at the bottom.
Materials: Olive oil, oleic acid, petroleum ether, copper acetate solution (5%).
Method: Take two test tubes put 1 / 2 g of each sample and then added 3 ml of petroleum ether and
an equal volume of a solution of copper acetate.
Result : Glycerol - 2 layer “ the above layer is ether and the other layer is copper acetate”

Qualitative estimation of Cholesterol by Liebermann - Burchard Test

Objective: To detect the presence of cholesterol.
Principle: The cholesterol reacts as a typical alcohol with a strong concentrated acids and the
product are colored substances. Acetic anhydride is used as solvent and dehydrating agents, and
the sulfuric acid is used as dehydrating and oxidizing agent. A positive result is observed when the
solution becomes red to blue and finally bluish to green color.
Materials: Crystals of cholesterol, Acetic anhydride, Concentrated sulfuric acid, Chloroform.
Method: Dissolve a few crystals of cholesterol in 2 ml of chloroform in a dry test tube, now add 10
drops of acetic anhydride and add 2 to 3 drops of conc. sulfuric acid.
Result: The formation of a green or green-blue colour after a few minutes is positive.
 Unsaturation Test
Objective: To indicates the amount of presence of double bonds in the lipid sample.
Principle: All neutral fats contain glycerides of some unsaturated fatty acids. These unsaturated fatty
acids become saturated by taking up iodine. If the fat contains more unsaturated fatty acids, it will
take up more iodine.
Materials: Hubl’s iodine reagent (alcoholic solution of iodine containing some mercuric chloride),
Chloroform, Mustard oil, coconut oil, olive oil, saturated fat.
Method: Equally into 4 flasks add 10 ml of Chloroform then 10 drops of Hubl’s iodine reagent, the
chloroform shows pink color due to presence of iodine. To one test flask add the oil sample drop by
drop shaking the tube vigorously for about 30 seconds after addition of each until the pink color is
discharged and count the number of drops. The pink color is discharged owing to the taking up of
iodine by the unsaturated fatty acids of the oil. Compare unsaturation, it should be remembered that
more the number of drops required to discharge the pink color, the less is the unsaturation.
Result: Iodine solution —iodine dissolved in an aqueous solution of potassium iodide— reacts with
starch producing a purple - black color. The colour can be detected visually with concentrations of
iodine as low as 0.00002M at 20°C.

Saponification Test
Shows positive test for:
Triglycerides and saponifiable lipids. Saponifiable lipids are those that can be hydrolyzed to under
basic conditions to form fatty acid salts (soaps).

How to perform the test:

Approximately 0.5 ml of a liquid or 0.5 g of a solid is added to 0.5 ml of 3 M NaOH. After the sample is
heated for 15 minutes, 10 ml of water is added and the solution shaken vigorously.

A positive test is indicated by:

The formation of bubbles (which indicate that a soap was formed).
 a negative test (left) and a positive test (right)

Tests for unsaturation of fatty acids:

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.

Procedure:
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 colour 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

Result:

Recording the number of drops required to obtain a permanent yellowish red colour in each tube will
give the result.
 Isolation of Free Fatty Acids from 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 H2SO4. Then dissolve it in some water and add alkaline phenolphthalein solution
and shake.
Result:
The pink colour will be discharged indicating the presence of free fatty acids.

Dichromate Test
This test is given by the substances containing primary and secondary alcohol groups.
Procedure:
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. HNO3, mix well and note that the brown colour is changed to
blue. 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.
Result:
The chromic ions oxidize the glycerol and in this process they are reduced to chromous ions which
give the blue colour.

Determination of Iodine Number

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.

Procedure: 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) Na2S2O3 (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.
Result:

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.

Salkowski’s Test (H2SO4 Test)

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.
Procedure:
Dissolve cholesterol in 2 ml of chloroform in dry test tube. Add equal amount of con. H 2SO4. Shake
gently. The upper layer turns red and the sulphuric acid layer shows a yellow colour with a green
fluorescence.

Result:
If cholesterol is present, the solution becomes bluish red and slowlychanges to a violet red, and the s
ulfuric acid becomes red with a green fluorescence. (for indole) to the solutionto be tested, a little nitri
c acid is added and then slowly a solution of 2 per cent potassium nitrite; a red color showsthat indole
is present.

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.

Result:

A cherry colour is developed in the chloroform. Then a blue colour is developed when the chloroform
is added by 2-3 drops of acetic anhydride.
References:

General, Organic and Biochemistry Book

http://www.harpercollege.edu/tm-ps/chm/100/dgodambe/thedisk/food/grease/grease.htm

https://www.coursehero.com/file/p5m0ls/9-Sudan-Red-Test-Test-for-the-presence-of-lipids-Sudan-
red-is-a-lipid-soluble/

https://biochemistryisagoodthing.wordpress.com/2013/02/17/lab-review-2/

http://biology-igcse.weebly.com/-food-test-3---emulsion-ethanol-test-for-fats.html

http://brilliantbiologystudent.weebly.com/ethanol-emulsion-test-for-lipids.html

http://www.seplessons.org/node/362

http://fac.ksu.edu.sa/sites/default/files/Qualitative%20test%20of%20Lipids%20II.pdf

http://www.biologydiscussion.com/lipids/tests/qualitative-and-quantitative-tests-for-lipids/13050

https://en.wikipedia.org/wiki/Liebermann%E2%80%93Burchard_test

https://www.scribd.com/doc/94115612/Reaction-of-Lipids