Analysis of Carbohydrates

A. MOLISCH TEST
 When carbohydrates are exposed to concentrated H2SO4 in the presence of heat of
dilution, they undergo dehydration to form furaldehyde derivatives. Hexoses form
hydroxymethyl furaldehyde. It condenses with α-naphthol to form a colored
chromogen.

Result Interpretation of Molisch’s test:

 Positive Molisch’s test: purple color complex

 Negative Molisch’s test: no purple color

B. HYDROLYSIS TEST( for Sucrose)

*Sucrose on hydrolysis with HCl is converted to glucose and fructose.
* The presence of these two monosaccharides can be confirmed by Benedict’s and
Seliwanoff tests.
 Sucrose is non-reducing sugar, since it does not
have free aldehyde or ketone group to cause
reduction, hence it gives a negative reaction with
Benedict’s reagent.
 But upon boiling with HCl, sucrose is hydrolyzed
to yield glucose and fructose, which gives
positive reactions with Benedict’s and Seliwanoff
reagents.

 C. IODINE TEST
 Starch is made up of two polysaccharides fraction amylase and amylopectin.
Iodine is trapped inside the helix and the complex is responsible for the blue
color. On heating, the helical coil unwinds, which is equivalent to
denaturation. Iodine is released and the color disappears. On cooling,
renaturation takes place and iodine is again bound to the reformed helix.

D. BIAL’S TEST
-Bial’s Test is to determine the presence of pentoses (5C sugars).
This test is specific for pentoses and the compounds containing pentoses and thus
useful for the determination of pentose sugars. When pentoses are heated with
concentrated HCl, furfural is formed which condenses with orcinol, in the presence of
ferric ion, to give a blue-green color. The reaction is not absolutely specific for pentoses
since prolonged of some hexoses yields hydroxyl methyl furfural, which also reacts with
orcinol, to give colored complexes.
Positive Result: Formation of bluish green solution and precipitate indicates the presence
of a pentose sugar.
Negative Result: All other colors indicate a negative result for pentoses.
E. BENEDICT’S TEST AND FEHLING’S TEST
 Under alkaline conditions, reducing sugars tautomerize with
enediol. These reducing sugars reduce cupric ions to cuprous ions. The
cuprous hydroxide formed is then converted to brick-red cuprous oxide
upon heating.
 Results:
- Positive Benedict’s Test: Formation of a reddish precipitate within three
minutes. Reducing sugars present.
Example:.Glucose
- Negative Benedict’s Test: No color change (Remains Blue). Reducing sugars
absent.
Example: Sucrose.

Result:

- Positive Fehling’s test: reddish brown ppt (

glucose, fructose, lactose)
- Negative Fehling’s test: No red ppt (sucrose,
starch)

F. SELIWANOFF’S TEST
 The principle of the test is the formation of hydroxymethyl furaldehyde and
its condensation with resorcinol, which forms a colored chromogen
 Results:

- After the test is being performed, you will

observe the color changes in the solution. If
the color turns to Red, then this means that
your results are positive and keto sugar is
present inside the solution.
G. TOLLEN’S TEST (Silver Mirror)
-is used to test for the presence of the aldehydes. In this reaction, an aldehyde is oxidized
to a carboxylic acid while the Ag+ is reduced to silver metal, which deposits as a thin
film on the inner surface of the glass.

A positive test with Tollens' reagent is indicated by the precipitation of elemental silver,
often producing a characteristic "silver mirror" on the inner surface of the reaction vessel.

H. TOLLEN’S PHLOROGLUCINOL

–> differentiates pentose from hexose

Phloroglucin –> behaves as ketone
*water- very light yellow
*glucose – red wine (hexose)
*fructose- greenish brown (hexose)
*arabinose- reddish brown
– betadine-like color (pentose)
Positive test if the color is betadine like.

I. BARFOED’S TEST (modified)

 Under slightly acidic conditions, monosaccharides easily reduce Cu+2 to Cu.+
On treatment with phosphomolybdic acid, Cu + reduces colorless
phosphomlybdic acid to blue phosphomlyduos acid.
 Results:
- Positive Barfoed’s test: development of brick red color ppt within 3-5
minutes
- Negative Barfoed’s test: absence of red color
J. OSAZONE TEST
 When reducing sugars are treated with phenylhydrazone first,
phenylhydrazone is formed. On heating, the hydrazone further reacts with
phenylhydrazone to form osazones.
 The formation of the Osazones as a result of performing this test tells us that
sugars are present and thus, we identify them. However, listed below are
some other formations in this test.
 In this test, you will see Maltose forming different petal shaped crystals.
 As for Lactose, you will see it forming puff shaped crystals.
 Another substance Galactose will form phombi-plate shaped crystals.