LAB 11 - EXPERIMENTAL PART

Physical and chemical properties of lipids

1. Solubility of lipids
Prepare four test tubes and place about 3-4 drops of oil in all of them. Then to the 1 st tube add 2
ml of distilled water, to the 2nd tube - 2 ml of dishwasher detergent, to the 3 rd tube - 2 ml of ethanol,
and to the 4th tube – 2 ml of chloroform. Mix test tubes well and place them in test tube rack. Observe
the solubility of lipids.
Interpretation:
Lipids are water insoluble (probe no 1), they are emulsified by detergents (probe no 2), they are
soluble in non-polar solvents such as chloroform (probe no 4) and they are little soluble in ethanol
(probe no 3).

2. Acrolein’s test for glycerol identify

Place 2-3 drops of oil (or glycerol) into dry the test tube and add a little of KHSO4 (potassium
hydrosulphide). In the outlet of the test tube, place the blotting-paper wetted (moisten) earlier in
Tollens’ reagent. Place the test tube in the boiling water bath for ten minutes. Observe the colour
change of blotting-paper.
Interpretation:
Glycerol as a component of lipid, on the strength of heating with KHSO 4, loses 2 water –
molecules and creates an acrolein (acrylate aldehyde) – an irritant the mucosal membrane compound.
Acrolein, as an aldehyde, has reducing properties. Steams of acrolein reduce Ag+ ions onto Ag0. Ag
ions are in the blotting paper wetted in Tollens’ reagent.

3. Identification of fatty acid.

Place 0.5 ml of oil into the test tube and add 5 ml of 10% alcohol solvent of NaOH. Mix it well
and place the test tube in boiling water bath for ten minutes until ethanol’s evaporation (KEEP THE
CAUTION!!!). A new opalescent solution is arising, which makes foam during shaking. Divide this
arisen solvent into equal parts – into three test tubes. To the 1st tube - add few drops of 1 M H2SO4
(sulphuric acid) until acidic reaction gaining (check it by a litmus paper). To the 2 nd tube - add few
drops of CaCl2 (calcium chloride) or BaCl2 (barium chloride). To the 3 rd tube - add NaCl (sodium
chloride) in substantia and place it in boiling water bath. Record any changes.
Interpretation:
Hydrolysis of lipids takes place in alkaline environment. The products of hydrolysis are glycerol
and soaps – the salts of higher fatty acid. In probe no 1 - free fatty acids are arisen. In probes no 2 and
no 3 – soaps. Sodium and potassium soaps (probe no 3) are water soluble, calcium, barium or
magnesium soaps (probe no 2) are water insoluble.

4. Reduction of potassium permanganate

Place 5 ml of 1% solvent of Na2CO3 (sodium carbonate) into the test tube and add 5 drops of oil.
Place it in boiling water bath for 2 minutes and later add 1-2 drops of 1% solvent of KMnO 4
(potassium permanganate). Observe the reaction.
Interpretation:
Oils in their composition have more unsaturated fatty acids than saturated fatty acids. Unsaturated
fatty acids have one or more double bonds, which by the strength of KMnO 4 undergo the oxidation.
Potassium permanganate decolour itself, because Mn7+ ions are reduced onto Mn4+ ions, and violet
colour of KMnO4 disappears.

5. Acidic Value Determination

Place 1 ml of oil into Erlenmeyer, add 5 ml of ethanol (96%) and 2 drops of phenolphthalein.
Titrate the content of the flask by 0,1 M KOH until pink colour obtaining, which will maintain for 2
minutes. Make a control sample (blank) with no oil-adding.

AV = 5,611 × (a – b)/c

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where:
a – ml of KOH used for sample
b – ml of KOH used for blank
c – weight of used fat

Acid Value - number of milligrams of KOH required to neutralize the free fatty acids in 1 g of fat.
The acid number is a measure of the amount of carboxylic acid groups in a chemical compound, such
as a fatty acid, or in a mixture of compounds.

Detection of cholesterol

1. Salkowski’s test
Prepare one dry test tube. Place 1 ml of chloroform solvent of cholesterol and add 0.5 ml of
concentrated sulphuric acid (H2SO4). The test tube is red-coloured.
Interpretation:
By the strength of concentrated sulphuric acid, dehydration and formation of disulphide
bicholestadien acid take place. Disulphide bicholestadien acid is red-coloured.

2. Libermann-Burchard’s
Prepare one dry test tube. Place 1 ml of chloroform solvent of cholesterol and add 10 drops of
acetic anhydride ((CH3CO)2O) and 1-2 drops of concentrated sulphuric acid (H 2SO4). The test tube is
green-coloured.
Interpretation:
As a result of acetic anhydride and concentrated sulphuric acid fusion, arise monosulphide
bicholestadien acid, which causes green-coloured probe.

Detection of bile acids

1. Hay’s test
Prepare two test tubes and to each of them add 2 ml of water. To the first one add a few drops of
bile. Next, to both of them add a little of flower of sulphur. Leave test tubes for 10 minutes and
observe any changes.
Interpretation:
In the first one tube, flower of sulphur falls to the bottom of the tube and in the second one
remains on the surface. This happens, because bile acids contained in the bile, reduce the surface
tension, and therefore in the tube where bile was added, sulphur falls to the bottom.

2. Pettenkofer’s test
To the test tube pour 2 ml of water and add 2-3 drops of bile. Shake it. Next add 0,5 ml of 0.5%
sucrose, then incline the test tube and slowly add concentrated H2SO4. Coloured rings should be
observed.
Interpretation:
Sucrose, in the reaction with concentrated sulphuric acid, forms furfural derivatives. These
derivatives react with bile acids and give coloured compounds.

Detection of bilirubin

1.Gmelin's test
Put 2 ml of bile into a clean test tube, incline the test tube and add 1ml of concentrated HNO3.
Green rings of bilirubin oxidation products appear between two liquid phases.

2. Rosin's test
Put 2 ml of bile into a clean test tube and add 1% iodine alcohol solution on it. Green rings appear
between two liquid phases. The colour comes from bilirubin oxidation products.

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 Enzymatic fats digestion by lipase

Performing:
Place 2 ml of lipase into two test tubes. The first test tube place in boiling water bath for 2
minutes. Then into both test tubes add 4 ml of milk and 1-2 drops of phenolphthalein. Next into each
one, add drop by drop 1% solvent of Na2CO3 (sodium carbonate) until obtaining the pink colour of the
test tube. Both test tubes place in water bath at 37°C for 1 hour. Observe any colour changes.

Interpretation;
In this reaction milk acts as a substrate for lipase, because it contains emulsified fat. Sodium
carbonate alkalizes the environment to the optimum of lipase acting (pH 8-9). During incubation
(lipase acting), free fatty acids (which acidize the environment) are cleaved off, what is observe as
decolourization of indicator (phenolphthalein).