General Test for Carbohydrates

Biochemistry Laboratory #3
A. Furfural reaction for Carbohydrates

1. Molisch test (Alpha-naphthol reaction) - general test for carbohydrates. Purple ring indicates presence of
carbohydrates except triose and Tetrose.
Components of the test:
1ml water,
1 mL 0.02 glucose
1 mL 0.02m sucrose
1 mL 0.5% starch
5% alpha-naphthol
Con. Sulphuric acid
Molisch's Test Procedure
The development of a purple ring at the layer formed by the concentrated acid is a positive indicator for Molisch's test. If
no purple or reddish-purple colour arises, the given analyte does not contain any carbohydrate.
Molisch’s test is based on the dehydration of sulphuric acid into furfural. One hydroxyl group is removed from a sugar
molecule when a sample containing carbohydrate molecules is treated with sulphuric acid. Water is used to eliminate the
hydroxyl group. After the hydroxyl group is removed from the sugar molecule, furfural is formed. The resulting furfural
reacts with Molisch’s reagent (sulphonated – naphthol) to produce a purplish red product.

 Monosaccharide (like glucose) give a more rapid positive reaction as compared to disaccharide (sucrose) and
polysaccharide (starch) this is because of the additional time required by the latter 3 complex sugars to break down
through acid hydrolysis.
 When the carbohydrates (glucose, sucrose, starch) are treated with concentrated mineral acid, they lose 3 molecules of
water and forms furfural derivatives which combines with alpha naphthol to give purple coloured ring at the junction
of two solutions.
Control DW: Negative
Glucose, Sucrose, Starch: Positive, has Purple Ring colour.

 Furfural is formed by
dehydrating five-carbon
sugars. Triose and Tetrose
show negative result because
they lack the 5 carbon
backbone.
2. Seliwanoff’s reaction (resorcinol-HCI test) – will turn red indicating the presence of ketoses
Components:
5mL Seliwanoff’s reagent (HCI – Resorcinol)
1ml water
1 mL 0.01M glucose
1 mL 0.02m fructose
1 mL 0.01M arabinose.
Seliwanoff's test is a chemical test which distinguishes between aldose and ketose sugars. If the sugar contains a ketone
group, it is a ketose. If a sugar contains an aldehyde group, it is an aldose. This test relies on the principle that, when
heated, ketoses are more rapidly dehydrated than aldoses. When added to a solution containing aldoses, a slower forming
light pink is observed instead.
When added to a solution containing ketoses, a red colour is formed rapidly indicating a positive test.
Fructose and sucrose are two common sugars which give a positive test. Sucrose gives a positive test as it is a
disaccharide consisting of fructose and glucose.
Generally, 6M HCl is used to run this test. Ketoses are dehydrated faster and give stronger colors. Aldoses react very
slowly and give faint colors.

 This test can be used to identify ketose and aldose sugars. This test utilizes hydrochloric acid as a dehydrating agent,
and the agent resorcinol to condense. When treated with HCL ketoses are dehydrated more quickly, giving a the
furfural variant (5-hydroxymethyl fufural) that is then condensed by the chemical resorcinol, resulting in a cherry-red
colour compound. Aldoses react to create the identical product but with a slower rate producing a faint pink to yellow
colour complex.
A. Fructose – is a Ketose. Positive result, turned to cherry red after heating it.
B. Glucose – Aldose. Negative result, no changes after water bath.
C. Arabinose – Aldose. Negative result, no changes after water bath.
D. Water - Negative result, no changes.

3. Tollen’s Phloroglucin Reaction –used for identification of pentoses indicated by brown colour result.

Components:
5 mL Phloroglucin
1ml water
1 mL 0.01M glucose
1 mL 0.01m arabinose

Phloroglucinol is a reagent of the Tollens' test for pentoses. This test relies on reaction of the furfural with Phloroglucinol
to produce a coloured compound with high molar absorptivity.
The furfural derivatives formed by galactose then condense with the phloroglucinol to form a red-coloured compound.
Lactose also gives this test positive as it is hydrolysed by acid to yield glucose and galactose. The furfural compounds
formed by pentoses having a keto group also form a similar red-coloured compound with phloroglucinol.
Water: Negative
Glucose: Hexose and aldose, turned to reddish brown. Gives off reddish colour just like galactose.
Arabinose: Pentose, Positive, turned to dark brown.

 This test also involves the formation of furfural derivates in the presence of concentrated HCL.
 Pentose (like arabinose) is dehydrated by HCl then it is converted in the form of furfural (C 5H4O2). Furfural will
combine with Phloroglucinol (C6H6O3) which will result to Phloroglucide (C11H10O5). This gives its brown coloured
result.

B. Reactions of Starch
1. Reaction with Iodine –
Components:
1 drop of iodine,
5 mL starch
Potassium iodide

This test is specific for polysaccharides (like starch). This test is used to differentiate polysaccharides from the rest of
carbohydrates. It is given positive by starch and glycogen. It can also be used to differentiate between glycogen, starch,
and cellulose.
A positive result for the iodine test (starch is present) is a colour change ranging from violet to black; a negative result (no
starch) is a yellow colour of the iodine solution.
Iodine forms a blue, black, or grey complex with starch and is used as an experimental test for the presence of starch. The
colour of the complex formed depends on the structure of the polysaccharide and the strength and age of the iodine
solution. Iodine does not form a complex with simpler carbohydrates (monosaccharides and disaccharides)

 Iodine is used to test if starch is present. More specifically, a solution of I2 in KI is used. In this solution, the tri-
iodide ion forms. When this is added to something containing starch, the tri-iodide ion slips inside helical starch
molecules, and this complex absorbs most of the light, turning dark blue-black. So a positive test will be seen by a
dark colour appearing.
 This test can be reversed, if you need to detect iodine. Starch is used to detect iodine, giving the same dark colour.

2. Fehling’s test – test involved in differentiating between reducing and non-reducing sugars. The appearance of a
reddish-brown precipitate indicates a positive result and the presence of reducing sugars.
The absence of the reddish precipitate or the appearance of deep blue colour indicates a negative result and lack of
reducing sugars.
It should be remembered here that starch is a non-reducing sugar as it does not have any reducing group present.
Components:
1 mL Fehling’s A
1 mL Fehling’s B
1 mL Starch Solution
  Starch is a polysaccharide and it does not feature a free aldehyde or keto group. So, it gives negative test with
Fehling's reagent.
 Positive results in the Fehling test indicate presence of glucose, fructose and lactose or presence of reducing sugar
in the sample. The negative result of the Fehling test indicates the presence of non-reducing sugars such as
sucrose, starch.

3. Hydrolysis of Starch –
Components:  Starches are carbohydrates contain a large number of glucose
molecules that are bound together. These simple glucose sugars can
1 mL Starch Solution be separated from one another using an acid like hydrochloric acid.
 Starch is a condensation polymer made from glucose monomer units.
5 mL con. HCI When these large starch molecules react with water they break down
into smaller sugar molecules. The starch is said to be hydrolysed.
Iodine

Hydrolysis of Starch:
Neutralize the acid in the second test tube using sodium bicarbonate. Slowly add chunks of solid sodium bicarbonate to
the test tube until the solution ceases fizzing.
Allow the test tubes to cool off until they can be handled easily.

 Observe the colour of each test tube, which corresponds to the amount of sugar present in the solution. The second
test tube will be more dark red or brown, indicating that the hydrochloric acid hydrolyzed the starch in the solution
and produced greater quantities of simple sugars.

Hydrolysis of Starch: Bacteria-related

 This test is used to identify bacteria that can hydrolyze starch (amylose and amylopectin) using the enzymes a-
amylase and oligo-1,6-glucosidase. Often used to differentiate species from the genera Clostridium and Bacillus.
Because of the large size of amylose and amylopectin molecules, these organisms can not pass through the bacterial
cell wall. In order to use these starches as a carbon source, bacteria must secrete a-amylase and oligo-1,6-glucosidase
into the extracellular space. These enzymes break the starch molecules into smaller glucose subunits which can then
enter directly into the glycolytic pathway. In order to interpret the results of the starch hydrolysis test, iodine must be
added to the agar. The iodine reacts with the starch to form a dark brown colour. Thus, hydrolysis of the starch will
create a clear zone around the bacterial growth. Bacillus subtilis is positive for starch hydrolysis (pictured below on
the left). The organism shown on the right is negative for starch hydrolysis.