LAB - 4

Fospholipids, steroids and fat-soluble vitamins

Aim of the class: preparation of phospholipids, study on phospholipid composition, detection of some
steroids and vitamins.

Phospholipids

Phospholipids exist mainly in biological membranes. Glycerol or more composed alcohol –

sphingosine - constitutes a skeleton of phospholipid molecules. Derivatives of glycerol, phospholipids,
are named glycerophospholipids. One of them is phosphatydylcholine (lecithin), which contains glycerol,
two chains of fatty acids, phosphate and choline. Large amounts of lecithin exist in the yolk of hen egg. It
may be extracted with a mixture of chloroform with methanol (ratio 2:1). Lecithin is hardly soluble in
anhydrated acetone. The presence of hydrophilic phosphocholine group makes possible to form a stable
suspension (emulsion) of lecithin in water.

Heating lecithin with hydrosulphate (VI) results in dehydration of glycerol molecule and
formation of unsaturated aldehyde – acrolein – a substance of unpleasant, irritating smell. It may be
detected with a reductive test, e.g. with potassium chromate (VI). Acrolein reduces an orange potassium
chromate (VI) – to green chromium (III) sulphate.

Steroids

Steroids are derivatives of cyclopentanoperhydrophenantrene. They may be classified into several

classes, dependently on functional groups, attached to cyclopentanoperhydrophenantrene rings. The
mains of them are alcohols (e.g. cholesterol), alcoholoacids (e.g. bile acids), phenols and ketones (e.g.
steroid hormones).

♠ Cholesterol
Cholesterol is a representative of animal sterols. It is a component of cell membranes and plasma
lipoproteins. Furthermore, cholesterol is a precursor of bile acids, cholecalciferol (a precursor of vitamin
D3) and steroid hormones. The presence of a double bond in one of cholesterol rings is responsible for its
ability to form colour products in the presence of concentrated inorganic acids. The action of concentrated
sulphuric acid results in dehydration of cholesterol molecule (Salkowski test) with a formation of a red
bicholestadien disulphonate, which in the presence of acetate anhydride forms a green colour
bicholestadien monosulphonate (Lieberman-Burchard test). Traces of water make this reaction
impossible.

♠ Bile acids
Bile acids are steroid compounds, which are synthesised from cholesterol in the liver. Human bile
contains mainly cholic and deoxycholoic acids. A part of these acids interact with glycine or taurine
forming glicocholic and taurocholic acids. The mechanism of this interaction is similar to a peptide bond
formation. The carboxyl groups of bile acids interact with amino group of glycine or taurine. The
hydrocarbon character of both the ring structures and the side chains, as well as the presence of polar –
OH groups and electrically charged groups, -COO-, -SO3H-, cause that the bile acids demonstrate
character of strong detergents. They reduce the surface tension of alimentary lipids contained in the
intestine. The decrease in surface tension may be detected by a Hay test. If a powdered sulphur (in
laboratory dialect it is referred to as a „sulphur flower”) is applied to a test tube (containing emulsion of
olive oil in water) the sulphur floats on the surface of this suspension. If bile acids are present in such a

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suspension, the emulsion of olive oil in water becomes more stable and the sulphur flower falls down to
the bottom of test tube. The chemical structure of bile acids resembles some other compounds, which
contain both ring components and several –OH groups, like resorcin or α-naphtol. For this reason the bile
acids are able to condense with hydroxymethylenfurfural, which is produced by the action of concentrated
sulphuric acid on saccharose. An appearance of red colour product of condensation process indicates the
presence of bile acids.

Fat-soluble vitamins

Main forms of vitamin D are ergocalciferol (vitamin D2) and cholecalciferol (vitamin D3). They
are synthesised in human body from precursors, which are unsaturated sterols. Vitamin D2 is produced in
human skin from ergosterol under the action of UV-light (which is a component of sun light). Vitamin D3
exists in cod-liver oil. It is synthesised in human liver from 7-dehydrocholesterol.

Vitamins A demonstrate isoprene structure. They exist exclusively in animal tissues in two main
forms, A1 (retinol-1) and A2 (retinol-2). Both of them are alcohols containing 6-carbon ring, with a long
side chain – composed of two isoprene units. Vitamin A alcohol form is oxidised in human body to
aldehyde. Many plants contain a group of substances, refereed to as carotene, which are polyunsaturated
derivatives of isoprene. The high number of conjugated double bonds in carotene structure makes them
colour. In human body the carotenes: α, β and γ play a role of provitamins A. One molecule of β-carotene
is converted into 2 molecules of vitamin A, whereas α and γ are transformed with a release of one
molecule of vitamin A.

The presence of conjugated double bonds, both in vitamin A and D, allows them to form
characteristic colour complexes with antimony chloride (SbCl3). Vitamin A1 gives a product of a blue
colour, whereas vitamin D3 – gives a product of a red-violet colour.

Laboratory procedures

1. Extraction of lecithin from the yolk of hen egg

Grind about 2-3 g of dried yolk of hen egg in a mortar in 10 ml of solvent containing chloroform and
methanol (mixed in a ratio 2:1). Filter the extract into a dry test tube through a paper moistened with the
same solvent. Evaporate the solvent in a boiling water bath. Observe an appearance of a dark-brown, oil-
looking substance, which appears vaseline-like consistency after cooling. This is a lecithin contaminated
with other constituents of yolk compounds.

2. The solubility of lecithin

Transfer about 3 drops of lecithin into 2 test tubes. Supplement one of the samples with 2 ml of H2O
and heat it for a few seconds in a boiling water bath. Lecithin does not dissolve in water but forms a
stable turbid suspension. Supplement the second sample with 1 ml of chloroform and heat it in a boiling
water bath for a few seconds. Notice that lecithin dissolves in chloroform. An addition of 2 ml of acetone
results in precipitation of lecithin from the solution.

3. The chemical composition of lecithin

a. detection of glycerol - acrolein test

Transfer about 3 drops of lecithin into a dry test tube. Add 1.5 ml of orange colour potassium
dichromate (K2Cr2O7) and acidify it with 3 drops of concentrated sulphuric acid. Put the test tube into
boiling water bath until green colour of solution appears. Notice a change of an orange potassium
dichromate (K2Cr2O7) into a green chromium sulphate (Cr2 (SO4)3) .
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 b. detection of fatty acids – saponification reaction

Transfer about 3 drops of lecithin into a test tube and supplement it with 3 ml of 10% KOH
alcoholic solution. Heat the mixture in a boiling water bath for a few minutes. This procedure results in
hydrolytic cleavage of lecithin. The released fatty acids are converted into potassium salts (soaps). Add 5
ml of distilled water and shake the tube. Because the presence of soap the solution covers with foam. This
indicates the presence of fatty acids in lecithin.

c. detection of choline

Prepare a test tube containing about 3 drops of lecithin and add 2 ml of 20% NaOH. Heat the
sample in a boiling water bath for 5 minutes. The action of strongly alkaline medium and high
temperature results in hydrolysis of ester bond between choline and phosphate residue, contained in
lecithin, with a release of free choline. The released choline decomposes with a release of ethylene glycol
and trimethylamine, which demonstrates a characteristic bad smell.

d. detection of phosphorus

Prepare a test tube containing about 3 drops of lecithin and add 0.5 ml of 20% NaOH. Heat the
sample in a boiling water bath for 2 minutes and then add 2 ml of ammonium molybdate solution.
Observe an appearance of yellow colour ammonium phosphomolybdate solution.

4. Detection of cholesterol

a. Salkowski reaction

Transfer 0.5 ml of cholesterol (dissolved in chloroform) into a dry test tube and then introduce
gently on the test tube wall about 0.5 ml of concentrated sulphuric acid (H2SO4). Two-phase system is
formed. The upper (chloroform-containing) layer stains red, whereas the lower (H2SO4- containing) layer
fluoresces green.

b. Lieberman-Burchard reaction

Transfer 1 ml of cholesterol solution (in chloroform) into a dry test tube, add 3 drops of acetate
anhydride and introduce gently 2 drops of concentrated H2SO4. The solution stains red and then changes
its colour into blue and finally into green.

5. Detection of bile acids

a. Hay test

Prepare 2 test tubes containing 3 ml of distilled water. Supplement one of them with a drop of bile.
Introduce a few grains of powdered sulphur to each sample. The sulphur grains float on the surface of
water, whereas in a sample containing the bile they fall down to the bottom of the test tube.

b. emulsifying action of bile acids

Prepare 2 test tubes containing 3 ml of distilled water and add 3 drops of olive oil to each of them.
Supplement one of the samples with a few drops of bile. Shake intensively both tubes. The oil does not
dissolve in water. The water-oil mixture separates into two phases. The oil locates on the top and water
drops down to the bottom. A phase border is visible. In contrast to that in the sample containing water,
olive oil and bile (bile acids) forms a stable emulsion.
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6. Detection of fat-soluble vitamins

Prepare 1 ml of saturated solution of antimony chloride (SbCl3) in chloroform and add 2 drops of
pharmacological preparation of vitamins A+D3. The blue colour, which appears immediately after
mixing, indicates the presence of vitamin A. The change of colour into a red-violet indicates the presence
of vitamin D3.

Laboratory task

Check, if the tested solution contains cholesterol or bile acids?