CARBOHYDRATES

QUALITATIVE TESTS
LABORATORY
EXERCISE 5:
CARBOHYDRATES
General Qualitative Tests
LESSON OUTLINE

• General Tests for Carbohydrates

– Molisch Test
– Anthrone Test
– Action of Alkali Test
– Iodine Test
GENERAL TESTS FOR
CARBOHYDRARES
• Molisch’s Test
– This test is used to detect the presence of carbohydrates in a given
sample.
– A mixture of dissolved α-naphthol in ethanol is used to make a
Molisch’s reagent. Additional concentrated sulfuric acid is also
required in this test.
– The formation of the purple colored ring occurs at the interface
between the sulfuric acid and the test solution. The sulfuric acid
remains above the test solution as the acid is denser than the test
solution.
– The reaction is based on the fact that the concentrated acid
catalyzes the dehydration of sugars to form furfural (from
GENERAL TESTS FOR
CARBOHYDRARES
• Molisch’s Test
GENERAL TESTS FOR
CARBOHYDRARES
• Anthrone Test
– This test is used to detect the presence of carbohydrates in a
given solution.
– Anthrone reagent is made by dissolving anthrone in
concentrated sulfuric acid.
– The presence of a blue-green complex indicates the presence
of carbohydrates in the given solution.
– If carbohydrate is present in the form of free carbohydrate as
poly- or monosaccharide or bound as in a glycoprotein or a
glycolipid, the concentrated acid in the Anthrone reagent first
hydrolyses it into component monosaccharide.
GENERAL TESTS FOR
CARBOHYDRARES
• Anthrone Test
GENERAL TESTS FOR
CARBOHYDRARES
• Action of Alkali
– This test is done for identification of reducing sugars.
– Sodium hydroxide solution is used in this test.
– A yellowish brown color and caramel odor is observed to
detect the presence of carbohydrates in a sample.
– The sugars give enols by reacting with NaOH and the
medium becomes basic. The colorless solution turns to
yellow color during heating with NaOH and upon further
heating solution turns into brown color. This reaction is
reversible.
GENERAL TESTS FOR
CARBOHYDRARES
• Action of Alkali
GENERAL TESTS FOR
CARBOHYDRARES
• Iodine Test
– This test is detect the presence of polysaccharides, primarily
starch.
– Lugol’s iodine solution is used in this test.
– The appearance of blue-black or purple color represents a positive
test, indicating the presence of starch.
– The iodine test is based on the fact that polyiodide ions form
colored adsorption complex with helical chains of glucose residue
of amylase (blue-black), dextrin (black), or glycogen (reddish-
brown). Iodine on its own is insoluble in water. Addition of
potassium iodine results in a reversible reaction of the iodine ion
with iodine to form a triiodide ion, which further reacts with an
GENERAL TESTS FOR
CARBOHYDRARES
• Iodine Test
EXERCISE 6: TEST
FOR
CARBOHYDRATES
BASED ON THEIR ABILITY TO FORM FURFURAL AND
ITS DERIVATIVES
LESSON OUTLINE

• Tests Based on Their Formation of Furfural

and Its Derivatives
– Seliwanoff’s Test
– Bial’s Orcinol Test
– Mucic Acid Test
TESTS BASED ON THEIR
FORMATION OF FURFURAL AND ITS
DERIVATIVES
•
Seliwanoff’s Test
– This test is used to detect the presence of ketohexoses in a given
sample and distinguish ketoses from aldoses.
– Resorcinol in HCl is used to make a Seliwanoff’s reagent.
– The formation of the cherry red-colored complex indicates a positive
result which means that the given sample contains ketoses. The
absence of such color or the appearance of the color after a
prolonged period of time indicates a negative result which means that
the test sample doesn’t have ketoses.
– The acid hydrolysis of polysaccharides and oligosaccharides yields
simpler sugars. Ketoses are more rapidly dehydrated than aldoses.
Ketoses undergo dehydration in the presence of concentrated acid to
yield 5-hydroxymethyl furfural.
TESTS BASED ON THEIR
FORMATION OF FURFURAL AND ITS
DERIVATIVES
• Seliwanoff’s Test
TESTS BASED ON THEIR
FORMATION OF FURFURAL AND ITS
DERIVATIVES
• Bial’s Orcinol Test
– This test is used to distinguish the pentoses and pentosans from
other derivatives of carbohydrates like the hexoses.
– Mixture of orcinol, ethanol and FeCl3•6H2O is used to make a Bial’s
reagent.
– The presence of a blue-green complex indicates the presence of
pentoses in the sample.
– This test is based on the principle that under hydrolysis pentosans
are hydrolyzed into pentoses. Further, pentoses are dehydrated to
yield furfural, which in turn condense with orcinol to form a blue-
green precipitate.
TESTS BASED ON THEIR
FORMATION OF FURFURAL AND ITS
DERIVATIVES
• Bial’s Orcinol Test
TESTS BASED ON THEIR
FORMATION OF FURFURAL AND ITS
DERIVATIVES
• Mucic Acid Test
– This test is used to detect the presence of galactose and lactose in a
given sample.
– Mucic acid reagent is composed of concentrated nitric acid.
– The formation of crystal at the bottom of the tube indicates a positive
result which means that the sample solution has galactose or its
derivatives.
– Monosaccharides upon treating with potent oxidizing agents like nitric
acid yield saccharic acids (dicarboxylic acids). Nitric acid has the capacity
to oxidize both aldehyde and primary alcoholic groups present at C1 and
C6 respectively of galactose to yield an insoluble precipitate (rod-shaped
crystals) of mucic acid under higher temperature. Lactose also yields a
mucic acid, due to the hydrolysis of the glycosidic bond between the
TESTS BASED ON THEIR
FORMATION OF FURFURAL AND ITS
DERIVATIVES
• Mucic Acid Test
EXERCISE 7: TEST
FOR
CARBOHYDRATES
BASED ON THE REDUCING PROPERTY OF SUGARS
LESSON OUTLINE

• Redox Reactions of Sugars

• Tests Based on The Reducing Property of Sugars
– Fehling’s Test
– Benedict’s Test
– Barfoed’s Test
– Picric Acid Test
– Nylander’s Test
– Tollen’s Test
REDOX REACTIONS OF SUGARS
• Oxidation to Produce Acidic Sugars
– The redox chemistry of monosaccharides is closely linked
to the alcohol and aldehyde functional groups
– Oxidation can yield three different types of acidic sugars
depending on the type of oxidizing agent used
• Aldonic acid - formed when weak oxidizing agents such as
Tollen’s and Benedict’s solutions oxidize the aldehyde end
• Reducing sugar – carbohydrate that gives a positive test with
Tollen’s and Benedict’s solutions
REDOX REACTIONS OF SUGARS
• Oxidation to Produce Acidic Sugars
REDOX REACTIONS OF SUGARS
• Oxidation to Produce Acidic Sugars

Mild oxidizing agent(s):

• +
Tollens’ reagent (Ag ), Fehling’s
2+
reagent (Cu in aqueous sodium

tartrate), and Benedict’s

2+
reagent (Cu in aqueous sodium citrate)
REDOX REACTIONS OF SUGARS
• Oxidation to Produce Acidic Sugars
– Strong oxidizing agents can oxidize both ends of a
monosaccharide at the same time (the carbonyl group
and the terminal primary alcohol group) to produce a
dicarboxylic acid
• Such polyhydroxy dicarboxylic acids are known as aldaric acids
– In biochemical systems, enzymes can oxidize the primary
alcohol end of an aldose such as glucose, without
oxidation of the aldehyde group, to produce an alduronic
acid
REDOX REACTIONS OF SUGARS
• Oxidation to Produce Acidic Sugars
– Monosaccharide oxidation can yield three different types
of acidic sugars. The oxidizing agent used determines the
product.
REDOX REACTIONS OF SUGARS
• Reduction to Produce Alcohol Sugars
– The carbonyl group in a monosaccharide (either an
aldose or a ketose) is reduced to a hydroxyl group using
hydrogen as the reducing agent
• The product is the corresponding polyhydroxy alcohol called
sugar alcohol or alditol
• Sorbitol - Used as a moisturizing agent in foods and cosmetics
and as a sweetening agent in chewing gum
REDOX REACTIONS OF SUGARS
• Reduction to Produce Alcohol Sugars

Common reagents: H2/Pd; NaBH4/H2O

TESTS BASED ON THE REDUCING
PROPERTY OF SUGARS
• Fehling’s Test
– This test is used to differentiate between reducing and non-
reducing sugars.
– Fehling’s reagent is composed of two parts: Fehling’s reagent A
(copper sulfate heptahydrate solution) and Fehling’s reagent B
(mixture of KOH and potassium tartrate).
– The appearance of a reddish-brown precipitate indicates a positive
result and the presence of reducing sugars.
TESTS BASED ON THE REDUCING
PROPERTY OF SUGARS
• Fehling’s Test
– The Fehling’s solution appears deep blue in color and consists of
copper sulfate mixed with potassium sodium tartrate and strong
alkali, which is usually sodium hydroxide. On heating, the sample
with the Fehling’s solution, bistartarocuprate (II) complex oxidizes
the aldoses to corresponding aldonic acids.
– In the process, the copper (II) ions of the complex are reduced to
insoluble yellow or red-colored precipitate or cuprous (I) oxide
(Cu2O) ions.
– The tartrate ions prevent the formation of insoluble Cu(OH)2 from
the reaction of copper sulfate and sodium hydroxide present in the
solution by forming a bistartarocuprate (II) complex. This complex
TESTS BASED ON THE REDUCING
PROPERTY OF SUGARS
• Fehling’s Test
TESTS BASED ON THE REDUCING
PROPERTY OF SUGARS
• Fehling’s Test
TESTS BASED ON THE REDUCING
PROPERTY OF SUGARS
• Benedict’s Test
– This test is used detect the presence of reducing sugar in the
sample solution.
– A mixture of copper sulfate, sodium citrate and anhydrous sodium
carbonate is used to prepare a Benedict’s reagent.
– Any change in color from blue to green or yellow or orange or brick
red within 3 minutes indicates a positive Benedict test i.e.
presence of reducing sugar in the sample.
TESTS BASED ON THE REDUCING
PROPERTY OF SUGARS
• Benedict’s Test
– Sodium carbonate in the Benedict reagent increases the pH of the sample-
reagent solution mixture. Under warm alkaline conditions reducing sugars
are tautomerism to strong reducing agents, enediols. These enediols reduce
the cupric ions (Cu2+) (present as Copper Sulfate (CuSO4)) of Benedict
reagent into cuprous ions (Cu+). The cuprous particles are present in form of
insoluble Copper (I) oxide or cuprous oxide (Cu2O) which is of red color.
These red-colored copper oxides get precipitated.
– The concentration of reducing sugar in the sample differs from the intensity
and shade of the color of the reaction mixture. This shade of color can be
used to estimate the concentration of reducing sugar in the sample. Color
may vary from greenish to yellow to orange-red to brick-red. As the
concentration of reducing sugar increases color gradually changes from
greenish to yellowish to orange to brick-red.
TESTS BASED ON THE REDUCING
PROPERTY OF SUGARS
• Benedict’s Test
TESTS BASED ON THE REDUCING
PROPERTY OF SUGARS
• Barfoed’s Test
– This test is used distinguish reducing monosaccharides from
disaccharides.
– Copper acetate in acetic acid is used to prepare a Barfoed’s reagent.
– The presence of red precipitate detects the presence of reducing
monosaccharides in the sample. If the color appears within the first few
minutes, the sample contains reducing monosaccharides. However, if the
color appears later than the first 3 minutes, the sample is of reducing
disaccharides.
– The Barfoed’s reagent is made up of copper acetate in a dilute solution of
acetic acid. Since acidic pH is unfavorable for reduction,
monosaccharides, which are strong reducing agents, react in about 1-2
min. However, the reducing disaccharides take a longer time of about 7-8
minutes, having first to get hydrolyzed in the acidic solution and then
TESTS BASED ON THE REDUCING
PROPERTY OF SUGARS
• Barfoed’s Test
TESTS BASED ON THE REDUCING
PROPERTY OF SUGARS
• Picric Acid Test
– This test is used detect the presence of reducing sugar in the
sample solution.
– Saturated picric acid is used to conduct this test.
– Appearance of mahogany red color indicates the presence of
reducing sugar in the solution.
– The picric acid test for carbohydrates is a very sensitive chemical
test for the presence of reducing sugars. The reducing sugars react
with picric Acid (toxic yellow crystalline solid) also chemically
known as 2,4,6-trinitrophenol (TNP) to form a red colored picramic
acid.
TESTS BASED ON THE REDUCING
PROPERTY OF SUGARS
• Picric Acid Test
TESTS BASED ON THE REDUCING
PROPERTY OF SUGARS
• Nylander’s Test
– This test is used detect the presence of reducing sugar in the
sample solution.
– Mixture of Rochelle salt and bismuth in potassium hydroxide is
used to prepare Nylander’s reagent.
– The black-colored precipitates form within a short time (bismuth
subnitrate is reduced to bismuth black), indicates the positive test.
– Aldehyde group or free ketone of carbohydrate reduces and forms
black precipitates bismuth subnitrate reduced to black bismuth. A
very tiny amounts of glucose (0.08%) can be identified through
this test.
TESTS BASED ON THE REDUCING
PROPERTY OF SUGARS
• Nylander’s Test
TESTS BASED ON THE REDUCING
PROPERTY OF SUGARS
• Tollen’s Test
– This test is used to detect the presence of aldehyde containing
carbohydrates and distinguish reducing sugars from non-reducing
sugars in the sample solution.
– Mixture of silver nitrate, potassium hydroxide and ammonia is used
to prepare Tollen’s reagent.
– The formation of a dark grey precipitate or silver mirror on the
bottom and sides of the test tube indicates a positive result, which
means that the given sample contains reducing sugars/ aldoses.
TESTS BASED ON THE REDUCING
PROPERTY OF SUGARS
• Tollen’s Test
– The Tollen’s reagent is the alkaline solution of silver nitrate (AgNO3)
mixed with liquid ammonia (NH3), which results in the formation of
a complex. The aqueous solution of silver nitrate forms a silver
aqua complex where the water acts as a ligand. The aqua
complexes are then converted into silver oxides (Ag2O) by the
action of hydroxide ions.
– Silver oxide forms a brown precipitate, which is then dissolved by
aqueous ammonia resulting in the formation of the [Ag(NH3)2]+
complex. This complex is the primary component of the Tollen’s
reagent and is a strong oxidizing agent. The complex then oxidizes
the aldehyde group present in some sugars to form a carboxylic
TESTS BASED ON THE REDUCING
PROPERTY OF SUGARS
• Tollen’s Test