QUALITATIVE TESTS FOR CARBOHYDRATES
1) MOLISCH TEST
Ø general test for carbohydrates
Ø Principle: Carbohydrates when treated with
concentrated sulphuric acid undergo
dehydration to give furfural derivatives.
These compounds condense with Alpha
naphthol to form colored products.
Ø Pentoses yield furfural while Hexoses yield 5-
Hydroxy methyl furfurals.
Chemical Equation:
Reducing sugars
If
the
oxygen
on
the
anomeric
carbon
of
a
sugar
is
not
a4ached
to
any
other
structure,
that
sugar
can
act
as
a
reducing
agent
and
is
termed
a
reducing
sugar.
v Anomeric Reducing end
carbon
Redox Reactions of Monosaccharides
Ø Under prescribed conditions, some sugars reduce silver
ions to free silver and copper(II) ions to copper(I) ions.
Such sugars are called reducing sugars.
A reducing sugar will have one of the following
groups:
§ an aldehyde group (as in glyceraldehyde)
§ a hydroxyketone (as in fructose)
§ a cyclic hemiacetal group (as in glucose and
maltose)
Reactions of Monosaccharides
Reducing sugars
Ø Sugars with the hemiacetal
structure can be reducing
sugars under alkaline
conditions because the ring
opens forming an aldehyde
group.
Reactions of Monosaccharides
Reducing sugars
Ø Many clinical tests that monitor color change are
based on the oxidation reaction shown here.
Redox Reactions of Monosaccharides
Ø The Benedict, Barfoed, and Fehling tests are based
on the formation of a brick red copper(I) oxide
precipitate as a positive result while the Tollens test
is based on the formation of a silver mirror.
Ø The Barfoed test is more sensitive in that it can
distinguish a reducing monosaccharide from a
reducing disaccharide.
Ø The sugars are oxidized to carboxylic acids and the
metal ions are reduced
Reducing Tests for Sugars
2) BENEDICT’S TEST
BENEDICT’S TEST Principle:
Ø is used to test for simple carbohydrates.
Ø The Benedict’s test identifies reducing sugars
(monosaccharide’s and some disaccharides), which have
free ketone or aldehyde functional groups.
Ø Some sugars such as glucose are called reducing sugars
because they are capable of transferring hydrogens
(electrons) to other compounds, a process called
reduction.
Result Interpretation of Benedict’s Test
Ø When reducing sugars are mixed with Benedicts reagent and heated, a
reduction reaction causes the Benedicts reagent to change color. The color
varies from green to dark red (brick) or rusty-brown, depending on the
amount of and type of sugar.
• If the color upon boiling is changed into green, 0.1 to 0.5 % sugar in
solution.
• If it changes color to yellow, 0.5 to 1 % sugar is present.
• If it changes to orange, 1 to 1.5 % sugar is present.
• If color changes to red, 1.5 to 2.0 % sugar is present.
• if color changes to brick red,it means that more than 2 % sugar is present
in solution.
Composition of Benedict’s Reagent:
q CuSO4, Sodium citrate, Na2CO3
Brick red ppt.
3. FEHLING’S TEST
Composition of Fehling’s Reagent:
q Fehling’s A: CuSO4
q Fehling’s B: NaOH, Sodium Potassium tartrate
(Rochelle’s salt)
4. BARFOED’S TEST
Ø Barfoed's test is a chemical test used for detecting
the presence of monosaccharides. It is based on the
reduction of copper(II) acetate to copper(I) oxide
(Cu2O), which forms a brick-red precipitate.
RCHO + 2Cu2+ + 2H2O → RCOOH + Cu2O↓ + 4H+
Ø Disaccharides may also react, but the reaction is
much slower. The aldehyde group of the
monosaccharide which normally forms a cyclic
hemiacetal is oxidized to the carboxylate.
Barfoed’s Reagent: Cu(C2H3O2)2 in HAc
5. Seliwanoff’s TEST
Ø Seliwanoff’s test is a chemical test which
distinguishes between aldose and ketose
sugars.
Ø Ketoses are distinguished from aldoses via
their ketone/aldehyde functionality.
Ø This test relies on the principle that, when
heated, ketoses are more rapidly
dehydrated than aldoses.
Seliwanoff’s Reagent: resorcinol and concentrated HCl
Ø When Seliwanoff reagent is added to a
solution containing ketoses, a cherry
red color is formed rapidly indicating a
positive test. When added to a solution
containing aldoses, a slower forming
light pink is observed instead.
Seliwanoff-Reaction
6. BIAL’S TEST
Ø Bial's test is a chemical test for the presence of
pentoses.
Ø It is used to distinguish pentoses from hexoses
Ø Bial’s Reagent: orcinol, hydrochloric acid, and
ferric chloride.
Ø A pentose, if present, will be dehydrated to
form furfural which then reacts with the orcinol
to generate a colored substance.
BIAL’S REACTION
Ø Bial's test is used to distinguish pentoses from hexoses;
this distinction is based on the color that develops in the
presence of orcinol and iron (III) chloride.
Ø Furfural from pentoses gives a blue or green color. The
related hydroxymethylfurfural from hexoses may give a
muddy-brown or gray solution, but this is easily
distinguishable from the green color of pentoses.
Mucic Acid (Galactaric acid) Test
Ø Oxidation of galactose resulted in the formation of
Galactaric acid. It is a meso compound insoluble in
water and have zero optical rotation.
Hydrolysis of sucrose:
TT1: C12H22O11 H2O, Δ Mixture A
sucrose
Mixture A + Fehling’s Reagent Blue solution
Δ
Mixture A + Seliwanoff’s Reagent light red orange
Δ
TT2: C12H22O11 H+, Δ Mixture B NaOH
(C6H12O6)+ (C6H12O6) Mixture C
sucrose H2O
Glucose + fructose
Mixture C + Fehling’s Reagent Brick red ppt.
Mixture C + Seliwanoff’s Reagent Cherry red
Iodine Test for starch:
Observed Result:
Blue solution turns
Δ Colorless
Dark Blue solution
Hydrolysis of starch:
H +, Δ, H O
H+,Δ, H2O (C H O ) 2
(C6H10O5)y
(C6H10O5)n or 6 10 5 x or
enzyme amylodextrin enzyme erythrodextrin
starch
Dark blue complex Purple Reddish brown
(Rx’n with I2 in KI)
- A polyiodide complex H+, Δ, H2O or
enzyme
H+, Δ H+, Δ, H2O (C H O )
n C6H12O6 n/2 C12H22O11 6 10 5 z
H2O or achroodextrin
glucose maltose enzyme
Yellow Yellow brown
Decreasing Molar mass: x >y> z
