EXPERIMENT 2 CARBOHYDRATES A solution of iodine (I2) and potassium iodide (KI) in

water has a light orange-brown color. If it is added to

GENERAL TEST FOR CARBOHYDRATES
a sample that contains starch, it gives a color-change
Molisch Test to a deep blue coloration.

PRINCIPLE EXPLAIN THE COLOR CHANGES

A general test for all carbohydrates. All

carbohydrates (monosaccharide, disaccharide,
polysaccharide) give positive reaction for Molisch
Test . It is based on the dehydration of the
carbohydrates by Sulfuric Acid to produce an
aldehyde, which condenses with two molecule of
alpha-naphthol, resulting in the appearance of a
purple ring at the interface.
PURPOSE OF THE TEST
Molisch Test is a general test for Carbohydrates
identification
IMPORTANCE AND INDENTIFIED
CARBOHYDRATES
Iodine test is important in detecting starch, given that
the test uses iodine solution which gives a dark
blue color result if starch is present.
Only iodine element in the presence of iodide ion will
give the characteristic dark blue color. Neither
iodine element alone nor iodide ions alone will give
the color result.
STARCH. After the experiment one test tube turned

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Carbohydrates when reacted with concentrated

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into a dark blue coloration, which means that starch
sulfuric acid get dehydrated to form furfural and its
is present.

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derivatives.

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DIFFERENCE OF AMYLOSE, AMYLOPECTIN,
Positive Molisch Test give the formation of a

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GLYCOGEN AND CELLOLUSE
PURPLE RING at the interface
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PROVIDE THE TYPE EQUATION USED IN THE
TEST
SOLUBILITY
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The solubility of polysaccharides is determined by

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their molecular structures. Any structural feature

REAGENTS AND PURPOSE
that hinders the intermolecular association leads
SULFURIC ACID to higher solubility, such as branching structure;
on the opposite, structural characters that
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Strong dehydrating agent promote the intermolecular association result in a

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Dehydrate sugar into hydroxy methyl furfural w/c poor solubility, such as linear chain.
condense with alpha naphthol
MOLISCH REAGENT
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COMPATIVE TEST REACTION FOR

Solution of 10% a- naphthol (C10H8OH) dissolved in CARBOHYDRATES
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ethanol Fehling’s Test

Use for the detection of carbohydrates PRINCIPLE
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PROVIDE A POSSIBLE EXPMANATION FOR THE The presence of aldehydes are detected. Cupric ion
POSITIVE AND NEGATIVE RESULT is reduced to Cuprous ion by the aldehyde and
precipitates Cuprous Oxide. Reduction of the deep
blue solution of copper(II) to a red precipitate of
insoluble copper oxide.
Iodine Test PURPOSE
PURPOSE The test is commonly used to determine reducing
sugars
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aldehydes. Brick- red precipitate indicates presence
of aldehyde group.
COMPONENTS OF FEHLING’S REAGENT AND
FUNCTIONS
Soln. A Contains 7% CuSO4 dissolved in distilled
water containing 2 drops of dilute sulfuric acid.
Soln. B Contains 25% KOH and 35% NaOH and 35g
of potassium tartarate
Fehling’s solution A is aqueous copper sulphate and
Fehling’s solution B is alkaline sodium potassium
tartarate ( Rochelle salt). Rochelle salts (sodium
potassium tartarate) present in the reagent acts as
the chelating agent in this reaction.
PROVIDE THE TYPE EQUATION USED IN THE
TEST
WHY IS THE FEHLING’S TEST NOT USED IN
ANALYZING CARBOHYDRATES IN CLINICAL
SAMPLES?
Fehling test is an alternative to Benedict’s test. It
differs from Benedict’s test in that it contains sodium
potassium Tartrate in place of Sodium citrate and
potassium hydroxide as an alkali in place of sodium
carbonate in Benedict’s reagent. It is not a preferred
test over Benedict’s test since the strong alkali
present causes caramelization of the sugars; hence it
is less sensitive than Benedict’s reagent
STATE THE ADVANTAGE OF BENEDICT’S
REAGENT INSTEAD OF FEHLING’S REAGENT
Benedict’s solution is available as a ready-to-use
reagent. On the other hand, Fehling’s solution has to
be prepared by mixing two solutions: Fehling’s A and
Fehling’s B.

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Fehling's solution is made up out of two separate
solutions, is caustic, and doesn't keep well.

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SHOW THE SAMPLE EQUATION INVOLVED IN Benedict's solution is more stable, is a single
THE REACTION BETWEEN FEHLING’S REAGENT solution, and has no caustic properties, making it

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AND THE SAMPLE WHICH GAVE A POSITIVE easier to handle.
RESULT rs e
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Benedict’s solution is stable and does not deteriorate
quickly. Fehling’s Solution: Fehling’s solution
deteriorates quickly. Therefore, it is prepared only
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when needed.
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PROVIDE A POSSIBLE EXPMANATION FOR THE Benedict’s Test

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POSITIVE AND NEGATIVE RESULT

WHAT IS THE PRINCIPLE INVOLVED IN
The POSITIVE results obtained with your sample/s. BENEDICT’S TEST?
GLUCOSE: Has positive result because it has SPECIFIC TEST FOR CARBOHYDRATES
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
aldehyde group in its open chain
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Barfoed’s Test
 LACTOSE: The ring of the glucose able to
open even though the glycosidic bond is PRINCIPLE
present
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Barfoed’s test reaction is based on the reduction of

 FRUCTOSE: Cupric acetate [Cu (CH3COO) 2] by reducing
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monosaccharides and reducing disaccharides.

The NEGATIVE results obtained with your sample/s. Reduction of cupric acetate produces cuprous oxide
[Cu2O] which gives a brick red precipitate.
 SUCROSE: It did not react because the ring
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of glucose cannot open due to the glycosidic Reducing monosaccharides

bond. It is not a reducing sugar.
• Glucose
WHY IS THERE A NEED TO FRESHLY PREPARE
THE FEHLING’S REAGENT • Xylose

Fehling's reagent must be freshly prepared because • Fructose

it may undergo oxidation if it is mixed beforehand • Galactose
thus causing the effectivity of the reagent to lessen to
compare samples. PURPOSE
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Barfoed's test is a chemical test used for detecting
the presence of monosaccharides. Disaccharides
may also react, but the reaction is much slower. Also,
Barfoed’s test can be used for distinguishing
monosaccharides from reducing disaccharides.
COMPOSITION OF BARFOED'S REAGENT AND
ITS ROLE
A solution of Copper acetate which can be reduced
by a Ketone or Aldehyde to form Copper oxide which
presents as Red or Brown precipitate and Acetic acid
CH3COOH which provides acidic conditions or
decrease pH less than 7.
PROVIDE THE TYPE OF EQUATION USED IN THE
TEST
Aldehydes and ketone have the property to reduce
copper ions to copper oxide Cu2O which is red or
brown in color; monosaccharides and disaccharides
WHICH IS MORE ACCURATE?
Fehling’s and Benedict’s
Fehling’s: In this test 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
copper oxide. The test is commonly used for
reducing sugars but is known to be NOT specific for
aldehydes. Benedict’s: Benedict's test determines
whether a monosaccharide or disaccharide is a
reducing sugar, and is hence similar in purpose to
the Fehling's test.
Fehling’s: A positive test is indicated by a green
suspension and a red precipitate. Benedict’s: The
color ranges from green to yellow to orange to brick-
red depending on the amount of reducing sugar in
the sample.
Barfoed’s Test

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can have a free aldehyde and ketone group.
This another variation of tests for reducing sugars,

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RCHO + 2Cu2+ + 2H2O → RCOOH + Cu2O↓ + 4H+ but determines if a carbohydrate is amonosaccharide

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or a disaccharide. Barfoed's reagent reacts with
EXPLANATION FOR POSITIVE AND NEGATIVE

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monosaccharides to producecuprous oxide at a
RESULT
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POSITIVE
The reaction will be negative in the presence of
Both the test sugars fructose and glucose came out reducing disaccharide sugars as they areweaker
positive in this experiment. This is simply because reducing agents and react at a slower rate. A copious
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they are monosaccharides. Monosaccharides are all amount of brick-red precipitate indicates a reducing
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considered to be reducing sugars with free aldehyde monosaccharide

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or ketone groups. In this experiment, the aldehyde

groups of the sugars would react with the reagent of
this test creating the brick red precipitate.
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Result Interpretation of Benedict’s Test:
If the color upon boiling is changed into green, then
there would be 0.1 to 0.5 percent sugar in solution.

NEGATIVE
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If it changes color to yellow, then 0.5 to 1 percent

The test sugars sucrose and lactose are both
sugar is present.
dissaccharides, a notable difference between them in
relation to this experiment would be the fact that If it changes to orange, then it means that 1 to 1.5
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sucrose is a non-reducing sugar while lactose is a percent sugar is present.

reducing one.
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If color changes to red, then 1.5 to 2.0 percent sugar

WHY IS HEATING THE MIXTURE LIMITED TO is present.
ONLY 3 MINUTES?
And if color changes to brick red, it means that more
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Heating the mixture is limited to 3 minutes only than 2 percent sugar is present in solution.
because if it goes more than that the dissaccharide,
specifically lactose, will also start forming the brick
red precipitate. If this were to happen, it will make the Result Interpretation of Benedict's Test
this test useless since there will no longer be any
kind of clear distinction between monosaccharides
and dissaccharides.
Positive Benedict’s Test: Formation of a reddish
FEHLING'S AND BENEDICT'S TEST VS. precipitate within three minutes. Reducing sugars
BARFOED'S TEST
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Negative Benedict’s Test: No color change (Remains
Blue). Reducing sugars absent. Example: Sucrose.
Seliwanoff’s Test
PRINCIPLE
When heated, ketoses are more rapidly dehydrated
than aldoses. When Seliwanoff's reagent is added to
a solution containing ketoses, a cherry red color is
formed rapidly indicating a positive result. When
added to a solution containing aldoses, if heated for
a longer period of time , a slower forming light pink or
faint red is observed instead.
PURPOSE
To distinguish if the carbohydrate present in the
solution is an aldose or ketose sugar.
COMPOSITION OF THE REAGENT AND ITS ROLE
dehydrated into smaller saccharide units. Molisch
test is a sensitive but non-specific chemical test for
the presence of all carbohydrates while Seliwanoff’s
Test is a specific test on detecting ketose sugars.
Bial’s Orcinol Test
WHAT IS THE PRINCIPLE INVOLVED IN THE
BIAL’S ORCINOL TEST?
Bial's test is useful in distinguishing pentoses sugar
from hexoses sugars. Pentosses (such as ribose
sugar) form furfural in acidic medium which
condense with orcinol in presence of ferric ion to give
blue green colored complex which is soluble in butyl
alcohol.
WHAT IS THE PURPOSE OF THE TEST?
Bial's test is a chemical test for the presence of
pentoses. A pentose, if present, will be dehydrated to

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HYDROCHLORIC ACID – It is a dehydrating acid. form furfural which then reacts with the orcinol to

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The acid hydrolyzes polysaccharide and generate a colored substance.

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oligosaccharide ketoses to simpler sugars, followed

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WHAT COMPONENTS MAKE UP THE BIAL’S
by furfural
ORCINOL REAGENT? STATE THE FUNCTION OF

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RESORCINOL- It is a condensation reagent. The EACH COMPONENT.
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dehydrated ketose then reacts with two equivalents
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Bial's reagent consists of 0.4 g orcinol, 200 ml of
of resorcinol in a series of condensation reactions to
concentrated hydrochloric acid and 0.5 ml of a 10%
produce a molecule with a deep cherry red color.
solution of ferric chloride. Bial's test is used to
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PROVIDE A POSSIBLE EXPMANATION FOR THE distinguish pentoses from hexoses; this distinction is
based on the color that develops in the presence of
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POSITIVE AND NEGATIVE RESULT

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orcinol and iron (III) chloride.

POSITIVE
Provide a possible explanation for:
Ketose sugars get dried out in the nearness of
concentrated acids to yield furfurals or their The positive results obtained with your sample/s –
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subsidiaries which react with resorcinol in Seliwanoff the substance will yield a Blue colour formation that
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reagent to yield a cherry-red hue complex as the detects the presence of pentose.
positive result.
The negative results obtained with your sample/s –
NEGATIVE the substance’s color will stay the same. However, a
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brown color reaction detects the presence of Hexose.

With this test, fructose will bring about cherry red
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shading, while the aldose sugars like glucose will Green: Pentose Brown: Hexose
give a negative result with no red shading showing
COMPARE THIS TEST WITH MOLISCH’S TEST.
up in the series. In any case, if the warming is not as
it is expected (which is more than 5 minutes), aldose Molisch Test
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sugar will create a pink shading. While the sucrose (a

A. Not specific to carbohydrates.
blend of fructose and glucose) will create a cherry
red shading as a result of the fructose in it. B. Generalized test that cannot distinguished
carbohydrates.
COMPOSITION
C. Negative result will rule out the presence of
Both tests are used to identify presence of
carbohydrates.
carbohydrates using condensation reagents which
reacts with the carbohydrates to produce highly
colored products.
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 D. The positive result will show a purple-coloured
compound that appears as a ring layer at the
interface
Bial’s orcinol Test
A. Specific in detecting Pentose sugar.
B. Positive result ranges from green to yellow
(Pentose) or red-brown (hexose) depending on the
derivative present.
Mucic Test
WHAT IS THE PRINCIPLE INVOLVED IN THE
MUCIC ACID TEST?
This test is highly specific for galactose. Oxidation of
most monosaccharides, such as glucose in this case,
by nitric acid yields soluble dicarboxylic acids.
However, oxidation of galactose yields an insoluble
mucic acid. Lactose, a dissacharide will also yield a
CH2OH-(CHOH)4-CHO + HNO3 -> CH2OH-(CHOH)4-
CHO-NO3 + H+
PROVIDE A POSSIBLE EXPLANATION FOR:
The positive results obtained with your sample/s:
When concentrated nitric acid is heated together with
the galactose sample, a dicarboxylic acid called
mucic acid forms as a white precipitate, which counts
as a positive result.
Lactose is a dimer of glucose and galactose. The
concentrated nitric acid will hydrolyze the dimer and
so lactose will also give a positive result.
B. The negative results obtained with your sample/s:
Glucose sample give similar acids but they are water
soluble under conditions of the test.
EXPLAIN WHY ONLY GALACTOSE FORMS THE

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WHITE SANDY CRYSTALS.

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mucic acid, due to hydrolysis of the glycosidic linkage
between its glucose and galactose subunits.

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When the sugar galactose is heated together or

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oxidized with the concentrated nitric acid HNO 3, it
WHAT IS THE PURPOSE OF THE TEST?
forms a mucic acid – an isomer of a saccharic acid.

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The purpose of this mucic acid test is to identify the Mucic is insoluble and forms characteristics of sandy
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presence of the sugar galactose in food or in crystals which serve to identify galactose.
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synthetic manufacture.
LACTOSE HAS ALSO BEEN KNOWN TO FORM
WHAT COMPONENTS MAKE UP THE MUCIC CRYSTALS IN THE MUCIC ACID TEST.
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ACID TEST? STATE THE PURPOSE OF EACH RATIONALIZE WHY THIS IS POSSIBLE.
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COMPONENT.
Lactose also form crystals due to hydrolysis of the
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Sugar Solutions (Glucose, Galactose, Lactose) - glycosidic linkage between its glucose and galactose
these serve as the base for the experiment subunits.
Concentrated Nitric Acid - since mucic acid test is
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a test made for galactose, conc. nitric acid will bond

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forming mucic acid; able to oxidize the terminal

groups of aldoses, but leaves the secondary hydroxyl
groups unchanged
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Dicarboxylic acid - it is the mucic acid formed from

galactose; it is insoluble.
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PROVIDE THE TYPE OF EQUATION USED IN THE

TEST.
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HC=O-(CHOH)4-COOH (aq.) + 2 HNO3 (aq.) ->

HOC=O-(CHOH)4-COOH (s) + H2O (l) + 2 NO2 (g)
SHOW THE SAMPLE EQUATION INVOLVED IN
THE REACTION BETWEEN THE MUCIC ACID
REAGENT AND YOUR SAMPLE/S WHICH GAVE A
POSITIVE RESULT.
Galactose reacts with HNO3:
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