Detoxicol

SDLS 2008
Medicine for the intoxicated

File Created on 10/04/2004 08:08 PM Topic: Biochemistry Laboratory Carbohydrates

Transcriber(s): JC Tayco Lecturer: Prof. Patricia Generoso, Dr. Esperanza Uy
Editors: JC Tayco, Mark Lomboy, Pre Ausan No. of pages: 4

 Carbohydrates → starch and glycogen will give a positive result

→ are ployhydric alcohols having an aldehyde
(aldoses) or a ketone (ketoses) group
→ glucose is the most common monosaccharide
in mammalian metabolism
→ glucose is the major source of energy for
various cells
→ oxidation of carbohydrates is the central
energy yielding pathway in most non-
photosynthetic cells
→ in the diet, carbohydrates are taken up as
starch
 ANTHRONE TEST
 Melliturias
→ condition in which abnormal amounts of
specific sugars are excreted in the urine
→ the most common type is glucosuria in which
D-glucose is excreted in large amounts in the
urine
* in Diabetes mellitus → glucosuria is
evident
→ other sugars excreted include:
 Fructose
 Lactose
 Galactose
 Arabinose (a pentose)
because the sulfuric acid is still capable of
breaking down the glycosidic bonds
→ Procedure:
1 mL test solution + 2 drops molisch’s
reagent + 1 mL concentrated H2SO4
→ nice to know:
 Sulfuric acid removes 3 molecules of
water
 Sulfuric acid breaks glycosidic bonds
and acts as dehydrating agent
→ another general test for carbohydrates
→ very sensitive test
→ concentrated sulfuric acid hydrolyzes bound
sugars (breaking of glycosidic bonds)
→ dehydrates the monosaccharides to form
furfural (if a pentose) or hydroxymethyl
furfural (if a hexose)
→ the furfural derivatives reacts with anthrone
→ reactions:

 Glycosidic bonds
→ links a monosaccharide to another
monosaccharide

 GENERAL TESTS FOR CARBOHYDRATES

 MOLISCH’S TEST (Alpha – Naphthol
Reaction)
→ a general test for carbohydrates in free or
combined forms
→ concentrated sulfuric acid hydrolyzes bound
sugars (breaking of glycosidic bonds)
→ dehydrates the monosaccharides to form → used in quantitative colorimetric determination
furfural (if a pentose) or hydroxymethyl of glycogen, insulin and blood sugar
furfural (if a hexose) → 1 mole of anthrone is needed to react with 1
→ the furfural derivatives reacts with α- naphthol mole of sugar in the furfural form to give the
→ reactions: color compound
→ remember that to form a furfural ring, at least
5 carbons are needed
→ Procedure:
1 mL anthrone solution + 3 drops test solution

 TEST FOR REDUCING SUGARS

→ dependent on the presence of aldehyde or
ketone groups
→ sugars have reducing properties due to the
presence of an aldehyde group or a ketone
group
→ only free aldehyde or ketone group is
capable of reducing cupric ions into cuprous
oxide

 BENEDICT’S TEST
→ 1 mole of sugar : 1 mole of α- naphthol
→ under alkaline medium where sugars are
more reactive
→ Benedict’s reagent is a solution of Cu(OH) 2, is
stabilized by forming a soluble complex with
the alcohol group of sodium citrate in an
alkaline medium of sodium carbonate
→ this test is not specific for sugar
→ active component is cupric ion (Cu++)
→ for qualitative and quantitative analysis of
sugar in blood and urine
→ all monosaccharides are reducing sugars
→ disaccharides are reducing sugars except
for sucrose
→ remember:
 Reactive part of aldoses like glucose
is carbon number 1
 Reactive part of ketoses like fructose
is carbon number 2
→ Procedure:
1 mL Benedict’s solution + 8 drops of test
solution then boil in water bath
→ cupric ion is reduced and the resulting cuprous
ion is less soluble and cuprous oxide
precipitates as a brick red solid
→ reaction:
→ sucrose is a non-reducing disaccharide because
the reducing groups in both glucose and
fructose are involved in the linkage between
the two sugar units
 BARFOED’S TEST
→ also a copper reduction test but it is carried out
in acidic medium where sugar is less reactive
→ Barfoed’s solution consists of: copper acetate
and glacial acetic acid
→ this test differentiates monosaccharides from
disaccharides
→ in acidic medium, monosaccharides are
much reactive than disaccharides
→ prolonged heating of disaccharides gives a
positive result because they are already
broken down into their monosaccharide
components
→ Procedure:
1 mL Barfoed’s reagent + 6 drops of test
solution + boil in water for 3 minutes then
cool
→ reaction:
 TEST FOR PENTOSES
→ positive result only for pentose thus eliminates
the possibility that the sugar is hexose
→ pentoses are monosaccharides containing 5
carbon atoms
→ prolonged heating with mineral acids will yield
furfural
→ complex carbohydrates in the form of
pentosamine, gums, xylans and arabans,
wchich on hydrolysis yield pantoses
 TAUBER’S BENZIDINE TEST
→ more specific for pentoses
→ can detect up to 0.01 mg of the pentose either
in its free of combined form
→ Procedure:
0.5 mL Tauber’s reagent + 5 drops test
solution, boil over open flame for 1-2 minutes
 BIAL’S ORCINOL TEST
→ based on the reaction with strong acid and
phenol
→ hexuronic acid is positive because they are
decarboxylated and forms pentose
→ pentose should not be bound in order to react
with Bial’s reagent
→ pentose should form a furfural ring to bind with
orcinol to produce a blue colored product at
C1
→ Bial’s reagent dehydrates the sugar
→ complex carbohydrates in the form of
pentosans, gums, xylans and arabans, wchich
on hydrolysis yield pantoses
→ involves the decomposition o the sugars when
heated with concentrated HCl to a furfural
which condenses with orcinol (3,5 dihydroxy
toluene)
→ Procedure:
1 mL Bial’s reagent + 1 mL test solution, boil
until bubles come then dilute to 10 mL with
water
→ reaction:
 TOLLEN’S PHLOROGLUCINOL
TEST
→ based on the formation of similar intermediate
furfurals which condenses with phloroglucinol
→ in the presence of resorcinol and an acid,
ketohexoses form a red color in a much faster
rate than aldohexoses
→ sucrose, when heated longer also shows a red
color since the prolonged heating hydrolyzes
sucrose into glucose and fructose
→ fructose is a ketohexose
2
 → galactose and glucoronic acid give positive
Tollen’s test
→ Procedure:
1 mL test solution + 1 mL concentrated HCl +
2-3 drops Tollen’s reagent
→ reaction:
 TEST FOR KETOSES
 SELIWANOFF’S TEST
→ no acid was used instead heat was applied
→ the formation of the furfural ring is due to heat
→ 2 moles of resorcinol : 1 mole of 5-
hydroxymethyl furfural
→ sucrose will give a positive result only if heated
longer
→ the ketone group of the ketohexoses is the
active part in the reaction (dehydration)
→ ketoses are dehydrated much faster than  COMBISTIX
aldohexoses
→ Procedure:
1 mL of Seliwanoff’s reagent + 7 drops of test
solution, then heat in water bath for 3
minutes
→ reaction:
 Answers to Questions:
 Give the reason/rationale for each of the
 ENZYME TEST FOR GLUCOSE
→ quantitative and qualitative test
→ most specific test because of the use of an
enzyme reagent (glucose oxidase)
→ glucose oxidase oxidizes glucose to gluconic
acid and hydrogen peroxide
→ Determines the amount of glucose in urine
→ DOUBLE SEQUENTIAL ENZYME REACTION
→ Glucose oxidase catalyzes the reaction
between glucose and room air to produce
gluconic acid and peroxide
→ Peroxide catalyzes the reaction between
peroxide and chromogen to form an oxidized
colored compound representing the presence
of glucose
→ Positive result (if the reagent strip is):
→ Potassium iodide: Green → brown
→ Tetramethylbenzidine: yellow → green
→ Reactions involved:
 Differentiate Benedict’s Test from Barfoed’s
→ Interferences:
→ False positive:
 Contamination by oxidizing agents and
detergents
→ False negative
 High levels of ascorbic acid
 High levels of ketones
 High specific gravity
 Low temperature
 Improperly preserved specimens
 Tes Tape
→ paper that contains glucose oxidase
→ with additional enzyme peroxidase to catalyze
reaction between peroxide and potassium
iodide chromogen
→ Oxidizing the chromogen to give a green to
brown color
→ ascorbic acid and bilirubin gives false positive
result because they will compete with
chromogen
following:
a. sucrose gives a negative result to
Benedict’s test
→ Benedict’s test is used to detect reducing
sugars, sucrose is a non-reducing
disaccharide because the reducing groups
in both glucose and fructose are involved
in the linkage between the two sugar
units.
b. arabinose yields positive result to
Orcinol Test
→ Orcinol Test is used to detect the presence
of pentoses, arabinose is a pentose thus
yields a positive Orcinol Test
c. no brick red color is formed by lactose
when heated with Barfoed’s Test
→ Barfoed’s test is used to detect the
presence of reducing monosaccharides,
lactose is a disaccharide thus will give a
negative Barfoed’d test
d. Seliwanoff’s Test is positive with the
sugar fructose
→ Seliwanoff’s test is used to detect the
presence of ketoses, fructose is a ketose
thus will give a positive Seliwanoff’s test
Test in terms of: reaction medium, and
reactivity of the monosaccharides and
disaccharides.
→ Benedict’s test is done in an alkaline medium
thus both monosaccharides and disaccharides
3
 are reactive. Barfoed’s test on the other hand,
is done in an acidic medium. In acidic
medium, sugars are less reactive but between
monosaccharides and disaccharides,
monosaccharides are more reactive.

 Differentiate fructose and sucrose

using Seliwanoff’s test.
→ Seliwanoff’s test is used to detect the presence
of ketoses, fructose is a ketose thus will give a
positive Seliwanoff’s test. Sucrose on the
other hand would not give a positive
Seliwanoff’s test but if sucrose is heated
longer, HCl in Seliwanoff’s reagent will break
down sucrose to glucose and fructose only
then will it give a positive Seliwanoff’s test.

 Why is Glucose Oxidase Test (Tes-

tape) a more specific test for
urinary glucose than Benedict’s
Test? Explain.
→ Benedict’s test is used to detect the presence
of reducing sugars, glucose will give a positive
Benedict’s test but the test is not specific for
glucose since glucose is not the only reducing
sugar. Glucose Oxidase Test is more specific
for glucose because the enzyme oxidizes
selectively glucose.

 Summary
Schematic diagram for the determination of unknown sugars:

4
Tests Reagents Used Principle Visible Results Detected

5
 Involved
Molisch’s Test Molisch’s reagent Dehydration with Purple ring at the All Carbohydrates
(α – naphthol (5% α – naphthol in subsequent junction
reaction) 95 % Alcohol) condensation

Anthrone Test Anthrone solution Dehydration with Green or blue All Carbohydrates
(0.2% Anthrone in subsequent green color
concentrated condensation
H2SO4)
Benedict’s Test Benedict’s solution Reduction of  Greenish Reducing sugars
(cupric carbonate, sugars precipitate (+) (both
sodium carbonate, (Oxidation  Yellowish brown monosaccharides
citrate) Reduction precipitate (++) and disaccharides)
Reactions)  Brownish red
precipitate (++ *sucrose is not a
+) reducing sugar
 Brick red
precipitate (+++
+)
Barfoed’s Test Barfoed’s reagent Reduction of Brick red Reducing sugars
(copper acetate sugars precipitate (monosaccharides
and glacial acetic (Oxidation ONLY!)
acid) Reduction
Reactions)
Tauber’s Benzidine Tauber’s reagent Cherry red color Pentoses
Test (4% benzidine
dihydrochloride in
glacial acetic acid)

Bial’s Orcinol Test Bial’s reagent Dehydration with Blue green Pentoses
(0.3% orcinol and subsequent
0.02%FeCl3 in 10N condensation * brown to green-
HCl) brown for hexoses
* hexuronic acid
gives positive
result due to
conditions of the
test
Tollen’s Concentrated HCl Dehydration with Red color Pentoses
Phloroglucinol Test Tollen’s reagent subsequent (ketohexoses has
(2% Phloroglucinol condensation faster reaction than
in 95% Alcohol) aldohexoses)
Galactose
Glucoronic acid

Seliwanoff’s Test Seliwanoff’s Dehydration with Red color Ketoses

reagent subsequent (sometimes there
(0.05% resorcinol condensation may be brown red
in 12% HCl) precipitate)
Tes Tape Tes tape Oxidation Changes into green Glucose
after 30 seconds

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