Qualitative Tests for Carbohydrates

Dr. Nesrin Mwafi

Biochemistry & Molecular Biology Department
Faculty of Medicine, Mutah University
 Carbohydrates
• Our Aim in this Lab:
1. to characterize carbohydrates present in an
unknown solution

2. To distinguish between different carbohydrates

based on various chemical assays
(aldose/ketose, reducing/non-reducing sugar,
polysaccharides/simple sugars….etc. )
Classification of Carbohydrates
❑ Carbohydrates are “Sugars” or “Saccharides” consist of
the empirical formula (CH2O)n where n ≥ 3.
1. Monosaccharides: The building blocks of CHO which
cannot be hydrolyzed into smaller units like glucose,
galactose and fructose
2. Disaccharides: contain two monosaccharides covalently
linked by glycosidic bond like sucrose which consists of
glucose and fructose
3. Oligosaccharides: contain 3-10 units of
monosaccharides covalently linked by glycosidic bond like
Raffinose (a trisaccharide composed of galactose, glucose
and fructose found in cabbage and broccoli
4. Polysaccharides: are polymeric molecules composed of
long chains of monosaccharides linked together via
glycosidic bonds like starch, cellulose and glycogen
 Monosaccharides
❑ They are classified according to the number of carbon
atoms: trioses, tetroses, pentoses, hexoses …..etc.
❑ Also classified according to the chemical nature of the
carbonyl group C=O either to Aldoses (the carbonyl group
is an aldehyde) or Ketoses (the carbonyl group is a ketone)
❑ Sugar derivatives (modified monosaccharides): sugar
acids, sugar alcohols, deoxy sugars and amino sugars

Monosaccharide Sugar alcohol

Glyceraldehyde Glycerol/glycerin
Ribose Ribitol
Mannose Mannitol
Glucose Sorbitol
 Disaccharides
1. Maltose “malt sugar”: consists of two α-glucose units
Reducing
end

α-D-glucopyranosyl-(1 4)-α-D-glucopyranose

2. Lactose “milk sugar”: consists of glucose & galactose

Reducing OH Reducing
end β end
H

α- β-
β-
 Disaccharides
3. Sucrose “table sugar”: consists of glucose & fructose

• Sucrose is not a reducing sugar because the anomeric

carbon of the second residue (the reducing end) is not
free but involved in the glycosidic bond formation
 Polysaccharides
1. Starch: the storage polysaccharide in plants. It is a mixture
of amylose (20%, unbranched) and amylopectin (80%,
branched)

unbranched starch(linear) branched starch

α1 4 α1 4 α1 4 α
O
1- 4 linkage

Amylose : α (1 4) glycosidic bonds

Amylopectin: α (1 4) glycosidic bonds

with α (1 6) branch points
 Polysaccharides
2. Glycogen: the storage polysaccharide in animals and
human. It is highly branched polymer
 Polysaccharides
• Starch and glycogen have one reducing end (the molecule
end containing a free anomeric carbon C1). On the other
hand, the branches ends are all called non-reducing ends
non-reducing reducing
end (NRE) end (RE) NRE
NRE
NRE NRE
NRE
NRE NRE NRE
NRE RE RE

NRE NRE

NRE
NRE NRE
NRE NRE
NRE NRE
NRE
 Reducing Sugar
• Sugars are classified as either reducing or non-reducing
depending upon the presence of potentially free anomeric
carbon or carbonyl group
• if the reducing groups are involved in the formation of
glycosidic linkage, the sugar belongs to the non- reducing
category like sucrose
• The reducing property is mainly due to the ability of these
sugars to reduce metal ions such as copper to form
insoluble cuprous oxide
(cupric ion Cu+2 cuprous ion Cu+1)
• All monosaccharides are reducing sugars, along with
some disaccharides, oligosaccharides, and polysaccharides
 Tests for Carbohydrates
• There are a number of chemical tests for detection of
carbohydrates, determining their properties and, possibly,
their identity:

1. Molisch Test: the general test for carbohydrates

2. Iodine Test: used to differentiate simple sugars (mono/disaccharides)

and polysaccharides

3. Benedict’s Test and Barfoed’s Test: used to differentiate reducing

from non-reducing sugars.

4. Seliwanoff Test: used to distinguish ketoses from aldoses

 Molisch Test
• Principle: when carbohydrates are treated
with concentrated sulphuric acid (H2SO4),
they undergo dehydration to give furfural
derivatives. These compounds condense
with α-naphthol (Molisch reagent) to form
colored products, generally a purple/violet
ring at the interface of the two layers.
Pentoses yield furfural while Hexoses yield
5-Hydroxy methyl furfurals
 Molisch Test

• An appearance of
reddish violet or purple
colored ring at the
junction of two liquids is
observed in a positive
Molisch test
 Molisch Test
• Procedure: Take 2 ml of carbohydrate solution in a
clean and dry test tube. Add 2 drops of Molisch
reagent ( -naphthol) and mix. Tilt the test tube at a
45 degree angle and add carefully 2ml of
concentrated sulphuric acid along the side of the
test tube so as to form 2 layers.
 Molisch Test
• Interpretation: This is a sensitive but a
non-specific test and is given positive
by all types of carbohydrates. If the
oligosaccharides or polysaccharides are
present they are first hydrolyzed to
monosaccharides which are then
dehydrated to give the test positive.
 Iodine Test
• This is a test for polysaccharides such as starch
• Principle: Iodine forms a coordinate complex between the
helically coiled polysaccharide chain and iodine centrally
located within the helix due to adsorption. The color of the
complex obtained depends upon the length of the
unbranched or linear chain available for complex
formation
• Interpretation: Glycogen: gives reddish brown color
whereas starch gives deep blue color

iodine starch Starch

+ iodine
Absorption / Adsorption
 Iodine Test
• Monosaccharides and Disaccharides are two small to
trap the iodine molecules and do not form dark
colored complexes. Therefore, the iodine test can be
used to distinguish between mono/disaccharides and
polysaccharides

• Procedure: place 2 mL of each solution to be tested

in a test tube. Add 2-3 drops of the Iodine reagent
and mix. Record your results.
 Benedict’s Test
• This test is for reducing sugars which are capable
of reducing metal ions in solution
• Principle: In this test, cupric ions (Cu+2)(aqua
blue in solution) found as copper sulphate CuSo4
are reduced to cuprous ions (Cu +1) that form
Cu2O copper oxide under alkaline conditions
which is a brick-red precipitate
 Benedict’s Test
• Procedure: Add 8
drops of carbohydrate
solution to 5 ml of
Benedict’s reagent
and boil over a flame
or in a boiling water
bath for 2 minute. Let
the solution cool down
 Benedict’s Test
• Benedict’s test is a semi quantitative test (Diabetes urine
test). The color formed depends upon the amount of
reducing sugar (glucose) present in the mixture
Benedict’s Test

Urine strips for quick urine

test for diabetes
 Barfoed’s Test
• The Barfoed’s test is similar to the Benedict’s test but
differs in the specific reagent used (copper (II) acetate in
1% acetic acid solution) which is less reactive than the
Benedict’s reagent
• Principle: reducing sugars can reduce cupric ions even in
acidic conditions. This test is used to distinguish reducing
monosaccharides from disaccharides by controlling pH
and time of heating. Mono saccharides react very fast
whereas disaccharides react very slowly
• As in Benedict’s test, the primary reaction is the reduction
of Cu+2 ions to Cu2O which forms a brick red precipitate.

RCHO + 2Cu2+ + 2H2O → RCOOH + Cu2O↓ + 4H+

 Barfoed’s Test
• Procedure: add 2 ml of
carbohydrate solution to 2
ml of Barfoed‘s reagent
and keep the test tube in
the boiling water bath for
2-3 minutes only. Cool
under running water

negative positive

A scanty brick red precipitate is observed in a

positive reaction.
 Barfoed’s Test
• Interpretation:
1. The positive reaction indicates the presence of a
reducing monosaccharide

2. On prolonged heating disaccharides can also give this

test positive. Hence, the solution should be boiled for 3
minutes only.
 Seliwanoff’s Test
• Principle: Keto hexoses on treatment with hydrochloric acid
form 5-hydroxymethyl furfural which on condensation with
resorcinol (Seliwanoff reagent) gives a cherry red colored
complex
• Procedure: to 3 ml of Seliwanoff reagent (resorcinol and HCL)
add 1ml of sugar like fructose. Boil for 1- 2 min only then cool
the solution

A cherry red color is observed in a positive

reaction

negative positive
 Seliwanoff’s Test
• Interpretation:
1. This test gives positive result with ketohexoses like
fructose and fructose containing sugars (e.g. sucrose)
2. This test distinguishes between glucose and fructose
3. Overheating of the solution should be avoided as
aldoses get converted to ketoses and give a false
positive reaction with Seliwanoff reagent
Classification of Unknown Sample
U n k n o w n s a m p le

M o lis c h te st

N o c o lo r R e d -v io le t c o lo r
(n o c a rb o h y d ra te ) (n o c a rb o h y d ra te )

I o d in e te st

B lu e c o lo r C oNo
lo rreaction
le s s R e d c o lo r
(s ta rc h ) (m o n o - o r d is a c c h a rid e ) (g l y c o g e n )
B re a k d o w n e n z y m a tic a lly
a n d te s t fo r g lu c o s e B a r fo e d ’s o r B e n e d ic t’s te st

N o p r e c ip ita te M a r k e d p r e c ip ita te M a r k e d p r e c ip ita te

(s u c ro s e ) inIn5 -7
2-3mmin in in 7 -1 2 m in
B o il w ith H C L a n d (m o n o s a c c h a rid e ) (d is a c c h a rid e )
p e rfo rm S e liw a n o ff ’s te s t
fo r d e te c tio n o f fru c to s e

P e n to s e H ex ose

S e liw a n o ff’s te st

C o lo r le s s o r R e d to o r a n g e -r e d
F a in t tin t (fru c to s e )
(g lu c o s e o r g a la c to s e )
 Sugars Identification

Biochemical Glucose Fructose Maltose Lactose Sucrose

test
Molisch Positive Positive Positive Positive Positive

Benedict Positive Positive Positive Positive Negative

Barfoed Positive Positive Negative Negative Negative

Seliwanoff Negative Positive Negative Negative Positive

 Sugars Identification

Biochemical Glucose Fructose Maltose Lactose Sucrose

test
Molisch Positive Positive Positive Positive Positive

Benedict Positive Positive Positive Positive Negative

Barfoed Positive Positive Negative Negative Negative

Seliwanoff Negative Positive Negative Negative Positive