WEEK 3: BIOCHEMICAL TESTS ON ORGANIC MOLECULES

Organic compounds contain carbon atoms linked together to form chains or rings.
Four classes of organic compounds—carbohydrates, lipids, proteins and nucleic
acids—are found in large amounts in living organisms. The chemical properties of
the different classes depend on the presence of specific functional groups. In
general, the larger molecules in each class are formed by joining one or more
building block molecules together in a dehydration synthesis reaction during which a
molecule of water is formed for each building block added. Large molecules are
broken down into the smaller building block molecules by a reverse reaction called
hydrolysis during which water is added. In this exercise, you will learn how to test for
the presence of these organic molecules in a solution.

Carbohydrates are the main energy-storage molecules in most organisms. They are
also important structural components for many organisms. The building blocks of
carbohydrates are small molecules called sugars, composed of carbon, hydrogen
and oxygen. Carbohydrates are classified according to the number of sugar
molecules they contain. Monosaccharides, such as glucose, fructose, ribose, and
galactose, contain only one sugar molecule. Disaccharides, such as sucrose,
maltose and lactose, contain two sugar molecules linked together. Polysaccharides,
such as starch, glycogen, cellulose and chitin, contain many sugar molecules linked
together.

Lipids are organic molecules that are insoluble in water and other polar solvents.
Lipids are readily soluble in nonpolar solvents, such as chloroform, benzene, and
ether. Lipids include fats and oils (important as energy storage compounds),
phospholipids and glycolipids (part of the structure of cell membranes), waxes
(protective surface coatings on many plants and animals), and steroids (found in
some cell membranes and many hormones).

Proteins are complex, specialized molecules composed of carbon, hydrogen,

oxygen, and nitrogen. Many proteins also contain sulfur. The building blocks of
proteins are the amino acids. There are twenty different amino acids commonly
found in proteins. All of these amino acids have a similar structure. At the center of
the molecule is the alpha carbon which is bonded to four different groups: (1) an
amino group (NH2), (2) a carboxyl group (COOH), (3) a hydrogen atom and (4) the R
group (also called the side chain). The different amino acids have different R groups
otherwise; the twenty amino acids have identical structures.

Biuret test for proteins

Biuret reagent is a light blue solution, which turns purple when mixed with a solution
containing protein. The purple colour is formed when copper ions in the Biuret
reagent react with the peptide bonds of the polypeptide chains to form a complex.
The reagent used in the Biuret Test is a solution of copper sulphate (CuSO4) and
sodium hydroxide (NaOH). The NaOH is there to raise the pH of the solution to
alkaline levels; the crucial component is the copper II ion (Cu2+) from the
CuSO4.When peptide bonds are present in this alkaline solution, the Cu2+ ions will
form a coordination complex with 4 nitrogen atoms from peptide bonds (see figure 2
below).

Fig. 9 Fig. 10

The complex of Cu2+ ions and nitrogen atoms change the colour of CuSO4 solution
from blue to violet. This colour change is dependent on the number of peptide bonds
in the solution, so the more protein, the more intense the change (see figure 3
above).
Table 1: Biuret Test

Sample Biuret reaction

tested Observation Conclusion

Water    

Albumin    

Starch    

Lugol’s test for starch – Using a spot plate

Lugol’s reagent (I2KI) changes from a yellowish-brown to blue-black in the presence
of starch. Monosaccharides and disaccharides do not react with Lugol’s reagent.
Iodine dissolved in an aqueous solution of potassium iodide - reacts with starch
producing a deep blue-black colour. This reaction is the result of the formation of
polyiodide chains from the reaction of starch and iodine. The amylose, or straight
chain portion of starch, causes the dark blue/black colour. The amylopectin, or
branched portion of starch, causes the formation of an orange/yellow hue. When
starch is broken down or hydrolysed into smaller carbohydrate units, the blue-black
colour is not produced. The iodine solution will also react with glycogen and
cellulose, although the colour produced is browner and much less intense.
Table 2: Reducing sugars and Carbohydrates test (starch test)

Sample tested Benedict’s Test Lugol’s Test

Colour before Colour Colour before Colour

boiling after boiling Lugol solution after Lugol solution
Starch        

Glucose        

Sucrose

Water

Benedict’s test for carbohydrates (reducing sugars) – Using test tubes

Benedict’s reagent (a blue coloured solution containing copper ions) is used to test
for the presence of reducing sugars. When a solution containing Benedict’s reagent
and a reducing sugar is heated, the copper (II) ions in the Benedict’s reagent are
reduced to copper (I) ions and the solution changes from blue to green to orange to
red-orange to brick-red. A brick-red precipitate (solid), copper (II) oxide (Cu 2O), and
may appear in the bottom of the tube. The more reducing sugar present in the
mixture, the more precipitate will form in the bottom of the tube. A positive Benedict’s
test requires a free aldehyde or ketone group. All monosaccharides are reducing
sugars. Some disaccharides are reducing sugars, and some are not.
Polysaccharides do not test positive for reducing sugars unless they undergo a
hydrolysis reaction (by heating or digestion) during which the polysaccharides are
broken down to form monosaccharides.

Benedict’s test for carbohydrates (non-reducing sugars) – Using test tubes

If the reducing sugar test comes out as negative (no colour change), the non-
reducing sugar test can be done
The sugar sucrose is a non-reducing sugar. It is formed in a condensation reaction
making a glycosidic bond between a glucose molecule and a fructose molecule.
Fructose and glucose are both monosaccharides, and sucrose a disaccharide. The
glycosidic bond formed in sucrose is different from that one found in, for example,
maltose (maltose being a reducing sugar). It is this difference which prevents the
sucrose from reacting with the Benedict’s solution.

The non-reducing sugar test works because if there is any sucrose present (which is
a non-reducing sugar), it is broken down into those monosaccharides, which can be
tested for reducing sugars using the ordinary reducing sugar test. A positive result
therefore means non-reducing sugars are present on the original sample. The same
scale applies:

(nothing) blue → green → yellow → orange → red (lots)

If a substance does not react with Benedict’s solution, the following

procedure is performed.

 Boil the sample with hydrochloric acid – this hydrolyses any sucrose present,
splitting sucrose molecules to give glucose and fructose (see below)
 Cool the solution and neutralise it by adding sodium carbonate solution (an
alkali solution)
 Carry out the reducing sugar test (Benedict’s test) again: if there were non-
reducing sugars present in the original sample, the test will now come out as
positive (as they have been broken down into reducing sugars glucose and
fructose)

Test for lipids using Ethanol

Ethanol is a volatile, flammable, colourless liquid with a slight chemical odour. It is

used for a fat test. The molecule is a simple one, being an ethyl group linked to a
hydroxyl group. Its structural formula, CH3CH2OH, is often abbreviated as C2H5OH,
C2H6O or EtOH.
The emulsion test is a method to determine the presence of lipids using wet
chemistry. The procedure is for the sample to be suspended in ethanol, allowing
lipids present to dissolve (lipids are soluble in alcohols). The liquid (alcohol with
dissolved fat) is then decanted into water.

Sudan red (Sudan V) is also used in identifying lipids. Sudan red is a lipid soluble
dye that will move into the lipid layer coloring it red when added to a mixture of lipids
and water:

Table 3: Lipids test

Sample tested Lipid test using EtOH

Sunflower oil

Water