NSC1115- LABORATORY PRACTICAL ONE

Tests for Biological Molecules (Part One)

In this laboratory practical, food samples will be tested for biological molecules present. The
tests will include Iodine test for Starch, Ethanol Emulsion test for Fats and oils and
Biuret Test for proteins.

Starch is a polysaccharide that consists of glucose molecules joined together by glycosidic

bonds. Linear molecules of starch are called amylose and the branches molecules are called
amylopectin.

The iodine test for starch is used to determine the presence of starch in biological materials.
Iodine on its own is insoluble in water. Therefore, Potassium tri-iodide solution, Iodine
dissolved in potassium iodide solution, is used as a reagent in the test. The potassium iodide
dissociates and the iodide ion reacts reversibly with the iodine to yield the tri-iodide ion. A
further reaction between a tri-iodide ion and an iodine molecule yields the penta-iodide ion.
The tri-iodide and penta-iodide ions slip inside the amylose (a form of starch) forming a
starch-iodide complex which appears as an intense purple or blue-black colour.

The ethanol emulsion test for fats and oils exploits the solubility of lipids in ethanol. Lipids
are insoluble in water, but soluble in organic solvents. Ethanol is one such organic substance
that can dissolve lipids. However, ethanol is also soluble in water due to the presence of the
hydroxyl (-OH) functional groups which participates in hydrogen bonding with water. Thus,
ethanol can extract lipids from crushed solid samples and form a solution. When water is
added, lipids are spontaneously removed from solution and dispersed as micelles (small
droplets) throughout the solution of ethanol and water. A layer is formed at the top as lipids
are less dense than water, giving a cloudy white appearance.

Biuret test is a general test for compounds (proteins and peptides) having two or more peptide
(CO-NH) bonds. When biuret is treated with dilute copper sulfate in alkaline medium, a
purple colored compound is formed. It is believed that the color is due to the formation of a
copper co-ordinated complex or chelate complex. Cupric ions or Cu (II) ions form a violet-
colored chelate complex with unshared electron pairs of peptide nitrogen and oxygen of
water. The chelate complex absorbs light at 540 nm and hence appears violet. The color
change from blue to violet indicates the presence of proteins.

The greater the number of peptide bonds in a protein, the greater the color intensity. Hence,
the color change is from blue to pink if the concentration of peptide bonds is low like
in short-chain peptides. The principle of biuret test is conveniently used to detect the
presence of proteins in biological fluids.

Materials

Samples A, B, C and D Beakers

Droppers Test tubes

Reagents: Iodine, Biuret, Ethanol White tile

Procedure

Iodine Test for Starch

- Put 2cm3 of sample solution into a test tube and add 2 to 3 drops of iodine
- Observe colour changes

Biuret Test for Proteins

- Put 2cm3 of sample solution in a test tube and add 2cm3 of Biuret reagent.
- Shake well and leave to stand for about a minute.
- Observe any colour changes

Ethanol Emulsion test for Fats and oils

- Put 2cm3 of sample solution in a test tube and add 2cm3 ethanol and shake thoroughly
- Add 2cm3 of distilled water
- Make observations

Results

Copy and complete the table below:

Biuret Test for Proteins Iodine Test for Starch

Ethanol Emulsion Test for
fats and oils
Observations Conclusion Observations Conclusion Observations Conclusion

Sample

Sample

Sample

Sample

Write a full laboratory report using the attached guidelines.