Carbohydrates 2.
Disaccharides – made up of two
- are macromolecules defined as
polyhydroxyketones or polyhydroxyaldehydes.
They can be determined by a set of qualitative
chemical tests, characterized by specific
positive results, which indicate the presence of
such carbohydrate.
- is defined as an organic compound having a
general formula CnH2nOn, that is, consists only
of carbon, hydrogen and oxygen, where n
pertains to the number of carbon atom and the
hydrogen and oxygen have the same atom ratio
as that of water (H2O).
- Carbohydrates in general have either an
aldehyde group (polyhydroxyaldehyde) as in
glucose or a ketone group (polyhydroxyketone)
as in fructose.
- Those containing an aldehyde group are called
as aldoses and those containing keto groups
are called ketose,
- Carbohydrates are the most significant source
of energy, provide storage of energy (e.g.
starch and glycogen), important cellular
component (e.g. carbohydrate chain in cell
membrane), and important structural
component in organisms (e.g. chitin).
CLASSIFICATION OF CARBOHYDRATES
Carbohydrates, also known as saccharides or
glycans, are generally divided into four chemical
groups according to the number of carbons.
1. Monosaccharides – made up of one sugar unit
  Dermatan sulfate (present in skin)
o Hydroxyacetaldehyde (2-carbon sugar)
o Glyceraldehyde; Dihydroxyacetone (3-
carbon sugar)
o Erythose; Erythrulose (4-carbon sugar)
o Ribose; Ribulose; Xylose; Xylulose (5-
carbon sugar)
o Glucose; Fructose; Galactose; Mannose (6-
carbon sugar)
o Sedoheptulose (7-carbon sugar)
o Neuraminic acid (9-carbon sugar)
monosaccharide units joined by glycosidic
linkage

o Sucrose (glucose + fructose)
o Lactose (glucose + galactose)
o Maltose (glucose + glucose)
o Lactulose (fructose + galactose)
3. Oligosaccharides – short polymers of usually
three to ten monosaccharide units

o Maltotriose (3 glucose units)
o Dextrin (several glucose units)
4. Polysaccharides – long polymers of
monosaccharides
o Homoglycans (made up of only one type of
sugar unit)
 Cellulose (structural polysaccharide in
plants)
 Chitin (structural polysaccharide in
animals)
 Starch (storage polysaccharide in
plants)
 Hetastarch (water-soluble starch)
 Glycogen (storage polysaccharide in
animals)
 Inulin (polyfructan of fructofuranose)
 Dextran (homopolyglucan of α–1,6
bond)
o Heteroglycans (made up of more than one
type of sugar unit, usually a repeating
disaccharide units)
 Hyaluronic acid (present in vitreous
humor and synovial fluid)
 Chondroitin sulfate (present in cartilage,
tendons and ligaments)
 Keratan sulfate (present in nails)
 Heparan sulfate (an anticoagulant)
 Agarose (found in seaweeds)
 Peptidoglycan (present in bacterial cell
wall)
Carbohydrates and their derivatives include many
other important biomolecules that play key roles in
the immune system, fertilization, preventing
pathogenesis, blood clotting and development.

QUALITATIVE CHEMICAL TESTS FOR
CARBOHYDRATES
MOLISCH'S TEST
1. Prepare three test tubes and place 20 drops of
the following sample and label accordingly:
 precipitate. Compare the results with the control
o 1% glucose
o 1% fructose
o 1% starch
2. To all the test tubes, add 2 drops of Molisch
reagent.
3. Then pour 20 drops of concentrated sulfuric
acid down the side of the test tube (slant the
tube before adding the acid), then erect the test
tube slowly.
4. Observe the color at the junction between the
two layers. Record results.
Positive result: purple ring at the junction of the
two liquids
PRINCIPLE
 general test for carbohydrates
 carbohydrates when treated with concentrated
sulfuric acid undergo dehydration to give
furfural derivatives
 these compounds condense with α-naphthol to
form colored products
 pentoses yield furfural while hexoses yield 5-
hydroxymethylfurfural
 this is a sensitive but a non-specific test and is
given positive by all types of carbohydrates
 reagent: α-naphthol in 95% ethanol
 (+) result: an appearance of reddish violet or
purple colored ring at the junction of two liquids
Positive result for Molisch's test (glucose,
starch, fructose, galactose).
FEHLING'S TEST
PROCEDURE
1. Prepare a mixture of 2 mL Fehling’s A and 2
mL Fehling’s B in a test tube.
2. Divide the Fehling's reagent into three test
tubes with 1.0 mL each. Label one test tube as
control solution.
3. To the remaining two test tubes, place the 2.0
mL of the indicated sample and label it
individually:

o 1% sucrose
o 1% fructose
4. Shake the test tube and boil all the solutions in
a hot water bath for 2 minutes.
5. Observe for the formation of a colored
solution.
Positive result: brick-red precipitate
PRINCIPLE
 test for reducing sugars
 carbohydrates with free aldehyde or ketone
groups have the ability to reduce solutions of
various metallic ions
 the presence of aldehydes but not ketones is
detected by reduction of the deep blue solution
of copper (II) to a red precipitate of insoluble
cuprous oxide [copper (I) oxide]
 the principle involves the reduction of the cupric
ion complexed with tartrate ion to cuprous oxide
 reagent: Fehling's A [blue aqueous solution of
copper (II) sulfate pentahydrate] & Fehling's B
[clear solution of aqueous potassium sodium
tartrate (Rochelle salt) and a strong alkali
(NaOH or KOH)]
 (+) result: brick red precipitate
Positive result for Fehling's test (fructose).
Negative result with sucrose (non-reducing
sugar).
BENEDICT'S TEST
 PROCEDURE
1. Prepare three test tubes and place 20 drops of
the following sample and label accordingly:

o 1% galactose
o 1% lactose
o 1% starch
2. To all the test tubes, add 20 drops of Benedict’s
reagent.
3. Boil all the samples in a water bath for 2
minutes. Remove from heat and allow to cool.
 4. Observe for the formation of colored solution
and/or precipitate. Record results.
Positive result: green to yellow to orange to red
precipitate (intensity may vary)
 PRINCIPLE
 test for reducing sugars
 reducing sugars under alkaline conditions
tautomerise and form enediols (powerful
reducing agents)
 they reduce cupric ions to cuprous form and are
themselves converted to sugar acids
 the cuprous ions combine with OH- ions to form
yellow cuprous hydroxide which upon heating is
converted to red cuprous oxide
 PROCEDURE
 it is a semi-quantitative test; the color of the
precipitate gives a rough estimate of a reducing
sugar present in the sample
  interpretation:
o green color - up to 0.5 G% (+)
o green precipitate - 0.5-1.0 G% (++)
o yellow precipitate - 1.0-1.5 G% (+++)
o orange precipitate - 1.5-2.0 G% (++++)
o brick red precipitate - >2.0 G% (+++++)
Positive result for Benedict's test (fructose).
Negative result with
o sucrose and starch.
BARFOED'S TEST
 PROCEDURE
1. Prepare three test tubes and place 20 drops of
the following sample and label accordingly:
 PRINCIPLE

o 1% xylose
o 1% lactose
o 1% glycogen
2. To all the test tubes, add 20 drops of Barfoed’s
reagent.
3. Boil all the samples in a water bath for 3
minutes. Remove from heat and allow to cool.
4. Observe for the formation of colored precipitate.
Record results.
Positive result: scanty brick-red precipitate
   PRINCIPLE
 test for reducing monosaccharides
 aldoses and ketoses 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
 monosaccharides react very fast whereas
disaccharides react very slowly
 (+) result: scanty brick red precipitate
 Positive result for Barfoed's test
o (xylose & glucose).
 Negative result with
o glycogen.
SELIWANOFF'S TEST
1. Prepare four test tubes and place 20 drops of
the following sample and label accordingly:

o 1% xylose
o 1% fructose
o 1% sucrose
o 1% glucose
2. To all the test tubes, add 20 drops of
Seliwanoff’s reagent.
3. Boil all the samples in a water bath for 2
minutes.
4. Observe for the change in color of the solution
within 30 seconds upon heating. Record
results.
Positive result: cherry red color of the solution
(faster for ketohexoses)
 test for ketohexoses
 ketohexoses on treatment with hydrochloric
acid form 5-hydroxymethylfurfural which on
condensates with resorcinol
 this test is given positive by ketohexoses so it is
answered by fructose, sucrose and other
fructose-containing carbohydrates
 this test distinguishes between glucose and
fructose
 (+) result: cherry red color
Positive result for Seliwanoff's test (sucrose &
fructose). Negative result with xylose &
glucose.
 BIAL'S TEST 4. Add 0.1 M sodium hydroxide dropwise to both
solutions until the red litmus paper turns to
   PROCEDURE blue.
5. Perform the Fehling’s test to determine
1. Prepare four test tubes and place 20 drops of presence of reducing sugars.
the following sample and label accordingly: 6. Observe for the formation of precipitate. Record
results.

o 1% xylose Positive result: brick-red precipitate
o 1% ribose
o 1% galactose    PRINCIPLE
o 1% sucrose
 sucrose, being a disaccharide, is composed of
2. To all the test tubes, add 20 drops of Bial’s glucose and fructose units
reagent.  inverted sugar is a mixture of glucose and
3. Boil all the samples in a water bath for 2 fructose: it is obtained by splitting sucrose into
minutes. Remove from heat and set aside to these two components
cool.  the reaction is an acid hydrolysis, where these
4. Observe for the change in color of the solution. two sugar components of sucrose are split
Record results.  upon acid hydrolysis, the solution is then tested
for the presence of reducing sugars using
Positive result: blue-green color of the solution (for Fehling’s test
pentoses); brown or red color of the solution (for  (+) result of brick-red precipitate indicates
hexoses) successful hydrolysis of sucrose

 PRINCIPLE Positive result for Hydrolysis test (hydrolysis of

sucrose).
 test for pentoses
 the test reagent dehydrates pentoses to form PHENYLHYDRAZINE TEST
furfural
 furfural further reacts with orcinol and the iron    PROCEDURE
ion present in the test reagent
 reagent: ferric chloride in orcinol-HCl solution 1. Prepare four test tubes and place 40 drops of
 (+) result: formation of bluish product (all other the following sample and label accordingly:
colors indicate a negative result for pentoses)
 hexoses generally react to form green, red or 
brown products o 1% glucose
o 1% fructose
Positive result for Bial's test (xylose & ribose). o 1% galactose
Negative result with sucrose, glucose & o 1% lactose
glycogen.
2. To all the test tubes, add 0.2 g of
HYDROLYSIS TEST phenylhydrazine hydrochloride, 0.1 g of sodium
acetate, and 5 drops of glacial acetic acid.
   PROCEDURE 3. Boil all the samples in a water bath for 5
minutes. Remove from heat and set aside to
1. Prepare two test tubes and place 40 drops of cool.
1% sucrose on both test tubes. Label one test 4. Upon cooling, solid crystals will form. Observe
tube as “control” and the other one as “inverted for the shape of the crystals formed under
sucrose”. HPO. Record results.
2. On the “inverted sucrose” test tube, add 3
drops of concentrated hydrochloric acid.
3. Boil both test tubes in a water bath for 5
minutes. Remove from heat and set aside to    PRINCIPLE
cool.
  osazone crystal formation  interpretation:
 a solution of reducing sugar when heated with o Amylose - a linear chain component of
phenylhydrazine, characteristic yellow starch; gives a deep blue color
crystalline compounds called osazones are o Amylopectin - a branched chain component
formed of starch; gives a purple color
 these crystals have definite crystalline o Glycogen - gives a reddish brown color
structure, precipitation time and melting point o Dextrins - amylo, eryhthro and
for different reducing sugars achrodextrins, formed as intermediates
 reagent: phenylhydrazine-HCl during hydrolysis of starch give violet, red
 (+) results: yellow to pale orange crystals with and no color with iodine respectively
specific shape and formation
positive result for Iodine test with starch (dark
blue/violet precipitate). 

 NEEDLE
o -(glucose, mannose, fructose)
 RHOMBIC PLATES
o -(galactose)
 SUNFLOWER-SHAPED  
o (maltose)
 POWDER PUFF/HEDGEHOG-SHAPED
o (lactose)

Positive result for Phenylhydrazine test

(formation of osazone crystals specific for each
sugar).

IODINE TEST

   PROCEDURE

1. Prepare two test tubes and place 40 drops of

the following sample and label accordingly:


o 1% starch
o 1% glycogen

2. To both test tubes, add 2 drops of Iodine TS.

3. Observe for changes in the color of the
solution. Record results.

Positive result: blue-violet color for starch;

reddish-brown for glycogen

 PRINCIPLE

 test for polysaccharides

 iodine forms a coordinate complex between the
helically coiled polysaccharide chain and iodine
centrally located within the helix due to
adsorption
 the color obtained depends upon the length of
the unbranched or linear chain available for
complex formation