Molisch Test

Molisch's test is a sensitive chemical test, nafor the presence of carbohydrates, based on the
dehydration of the carbohydrate by sulfuric acid or hydrochloric acid to produce an aldehyde,
which condenses with two molecules of a phenol (usually α-naphthol, though other phenols
such as resorcinol and thymol also give colored products), resulting in a violet ring.

All carbohydrates – monosaccharides, disaccharides, and polysaccharides (except trioses and

tetroses)– should give a positive reaction, and nucleic acids and glycoproteins also give a
positive reaction, as all these compounds are eventually hydrolyzed to monosaccharides by
strong mineral acids. Pentoses are then dehydrated to furfural, while hexoses are dehydrated
to 5-hydroxymethylfurfural. Either of these aldehydes, if present, will condense with two
molecules of α-naphthol to form a purple-colored product, as illustrated below by the example
of glucose:

Anthrone Test

The anthrone test can be used for the qualitative and quantitative estimation of
polysaccharides as well as monosaccharides. The test is based on the dehydration of
monosaccharides to furfural derivatives.
Anthrone can be prepared from anthraquinone by reduction with tin, glacial acetic acid and
hydrochloric acid, or by cyclization of o-benzylbenzoic acid with liquid hydrogen fluoride.

Anthrone syntheses
Anthrone test is also another general test for all carbohydrates. In this test also, carbohydrate
gets dehydrated when react with conc. H2SO4 to form furfural. This furfural reacts with
anthrone to give bluish green colored complex.

Iodine Test
The iodine test is used to test for the presence of starch. Starch turns into an intense
"blue-black" colour upon addition of aqueous solutions of the triiodide anion, due to the
formation of an intermolecular charge-transfer complex. In the absence of starch, the brown
colour of the aqueous solution remains.

To produce the triiodide anion (I3−), elemental iodine is dissolved in an aqueous solution of
potassium iodide. The resulting complex with starch produces an intense "blue-black" colour.
The intensity of the colour decreases with increasing temperature and with the presence of
water-miscible organic solvents such as ethanol. The test cannot be performed at very low pH
due to the hydrolysis of the starch under these conditions.
 Schematic view of I3− ions embedded in amylose helix

Benedict’s Reagent
Benedict's reagent (often called Benedict's qualitative solution or Benedict's solution) is a
chemical reagent and complex mixture of sodium carbonate, sodium citrate and copper(II)
sulfate pentahydrate., often used in place of Fehling's solution to detect the presence of
reducing sugars. The presence of other reducing substances also gives a positive reaction.
Such tests that use this reagent are called the Benedict's tests. A positive test with Benedict's
reagent is shown by a color change from clear blue to a brick-red precipitate.

Generally, Benedict's test detects the presence of aldehydes and alpha-hydroxy-ketones, also
by hemiacetal, including those that occur in certain ketoses. Thus, although the ketose
fructose is not strictly a reducing sugar, it is an alpha-hydroxy-ketone, and gives a positive
test because it is converted to the aldoses glucose and mannose by the base in the reagent.

The principle of Benedict's test is that when reducing sugars are heated in the presence of an
alkali they are converted to powerful reducing species known as enediols. Enediols reduce the
cupric compounds (Cu2+) present in the Benedict's reagent to cuprous compounds (Cu+)
which are precipitated as insoluble red copper(I) oxide(Cu2O).

The color of the obtained precipitate gives an idea about the quantity of sugar present in the
solution, hence the test is semi-quantitative. A greenish precipitate indicates about 0.5 g%
concentration; yellow precipitate indicates 1 g% concentration; orange indicates 1.5 g% and
red indicates 2 g% or higher concentration.
Barfoed’s Test

Barfoed’s test used copper (II) ions in a slightly acidic medium. Reducing monosaccharides
are oxidized by the copper ion in solution to form a carboxylic acid and a reddishprecipitate
of copper (I) oxide within three minutes. Reducing disaccharidesundergo the same reaction,
but do so at a slower rate. The nonreducing sugars give negative result.

Seliwanoff’s Test

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 ketoses, a red color is formed rapidly indicating a positive test. When
added to a solution containing aldoses, a slower forming light pink is observed instead.

The hydrochloric acid reacts with ketose to form

5-hydroxy methyl furfural,C6H6O3 which on condensation forms dehydrated ketose and reaction with
resorcinol forms a red colored complex. Fructose is a ketose sugar among all the sugars in the options
so it will give a positive test.