Biochemical Testing

Tests for Lipids

Test
Grease Spot
Test

Results
The filter paper
turned
translucent

Sudan III Test The margarine

(fat) turned red
and remained
separated from
the water
Emulsion
A cloudy white
Test
suspension
formed at the
top of the
solution

Explanation
The fat merges with the paper, and both have a similarly
high refractive index. The fat fills air voids, so light passes
from paper to fat, not to air (which has a very different
refractive index). This means less light is scattered and
more passes through, turning the paper translucent.
Sudan III is a red dye that stained the fat molecules,
turning the margarine red. The fat molecules are also less
dense than the water, hence they floated at the top

Lipids are soluble in ethanol but not in water. The ethanol

extracts the lipids from the sample, and when water is
added the lipids spontaneously come out of solution and
are dispersed as small droplets, forming an emulsion. As
they are less dense than water, they are suspended at the
top.

Tests for Protein

Test
Biuret Test

Results
The egg white
(protein) turned lilac

Ethanol Test

A cloudy white
suspension forms at
the top of the
solution
The egg white
solidifies

Heating Test

Explanation
The Biuret Reagant contains copper sulphate and
potassium hydroxide. When peptide bonds are
present in an alkaline solution, the copper II ions
form a complex with four nitrogen atoms in the
peptide bonds; these are purple, hence turning the
solution to lilac from blue. The more protein
present, the more intense the change.
The ethanol precipitates the protein in the egg
white, as protein is insoluble in ethanol.

When heated, the weak bonds between the amino

acids in the protein break, and the protein unfolds.
When given more energy, the amino acids form
stronger (covalent) bonds with other amino acids,
forming a network of interconnected proteins; the
water is captured and held, forming a white gel-like
substance.

Tests for Carbohydrates

Test
Benedicts
Test
(reducing
sugars test)
Non
reducing
sugars test

Results
The solution turned from
blue to green, to orange
and finally to brick red.

Iodine Test

The starch suspension

turns blue-black (inky).

The solution turned from

blue to brown, to red
and finally to brick red.

Explanation
Benedict solution contains copper sulphate. The
reducing sugars reduce the blue copper II ions to
copper I ions. These are precipitated in the form
of red-brown copper oxide
Disaccharides are hydrolysed to their constituent
monosaccharides when boiled in dilute
hydrochloric acid. The blue copper(II) ions from
copper(II) sulphate are reduced to red copper(I)
ions by the aldehyde groups in the reducing
sugars. This accounts for the colour changes
observed. The red copper(I) oxide formed is
insoluble in water and is precipitated out of
solution.
A polyiodide complex is formed with starch; a
starch-iodide complex is formed as charge is
transferred between the starch and iodide ions,
resulting in the complex absorbing light at a
different wavelength, resulting in the colour
change.