Bio2 Activity 3

ST.
ALEXIUS COLLEGE
Gen. San. Drive, City of Koronadal, South Cotabato, Philippines 09506, Tel.: (083) 228-2019, Fax: (083) 228-4015, Email: st.alexiuscollege@yahoo.com
Name: JENIFEL ARISTOSA Score: Course, Year & Section: Date performed:
Activity 3: Test for Carbohydrates
Objectives
At the end of the experiment, student should be able to:
● Identify different the test for carbohydrates
● Differentiate the different classes of carbohydrates
Procedures:
A. Iodine Test for Polysaccharides
1. Perform this procedure using starch, glucose, sucrose, galactose, fructose and lactose .
Run a blank test using distilled water.
2. Place 3mL of starch solution in each of two test tubes. To one, add several drops of 40%
NaOH. Add a drop of very dilute iodine solution to each test tube. Note the color. Heat
the tube in which the color developed. Cool and note any change. Compare with the
blank tube.
3. Perform the iodine test on starch powder. Note the result. Compare the result with that of
the starch solution.
4. Using other polysaccharides, perform the test by adding one drop of iodine solution to
5mL of 1% solution of carbohydrate. Observe and record your results.
B. Molisch Test
1. To three separate test tubes, place 0.5mL of 1.0% solutions of glucose, sucrose, and
starch respectively.
2. Add 2mL water and 2 drops of Molish Reagent.
3. Mix each well. Incline each tube and slowly add 3mL concentrated sulfuric acid on the
side of the tube to form a layer below the sugar solution.
4. A purple ring at the junction is indicative of a carbohydrate.
C. Osazone/ Phenylhydrazine Test

1. In seven separate test tubes, add 1 mL of 1.0% solutions of glucose, fructose,
galactose, lactose, and sucrose and label properly.
2. Add 1mL of phenylhydrazine reagent. Mix the contents well and stopper with cotton
loosely.
3. Put all the tubes at the same time in a large beaker of boiling water. Note the time of
immersion and the time of formation of each osazone.
4. Shake the tube occasionally to avoid supersaturation.
5. Remove the tubes after 30 minutes and cool. Note the sugars that form osazones only
after cooling.
1 |Biochemistry Manual
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6. Examine the crystals under low power microscope. The crystals may be washed several
times with distilled water before examination. Better crystals are obtained when allowed
to cool in a water bath.
7. Draw the crystals you obtained.
D. Benedict’s Test
1. Prepare 0.5% and 2.0% glucose, fructose, sucrose and starch.
2. To 1mL reagent, add exactly 2 drops of the sugar solution. Mix well.
3. Boil vigorously for two minutes (or place in boiling water bath for 3 minutes).
4. Allow the solution to cool spontaneously. Do not hasten cooling by immersion in cold
water.
E. Barfoed’s Test
1. Use 1.0% solution of the following sugars for this test: glucose, sucrose, and
fructose. To 1mL of the reagent, add two drops of the sugar solution.
2. Heat to boiling for 30 seconds. If no precipitate form, allow the tubes to stand for
about 15 minutes and examine.
3. To compare the reactions of monosaccharides and disaccharides, place the tubes at
the same time in a boiling water bath. Note the time when signs of reduction appear
in each tube.
4. Continue boiling for 15 minutes and remove the tubes from the bath. Note the
amounts of precipitate in each tube after standing for 15 minutes.
5. Record all observations.
F. Seliwanoff’s Test
1. Use 0.1% and 1.0% solutions of glucose, fructose and sucrose for this test.
2. To 5mL of the reagent, place 1mL of the sugar solution.
3. Place them in a boiling water bath for exactly 1 minute. Note the results.
4. Continue heating and observe the color change at one minute intervals for 4
minutes. Run a blank test.
G. Bial’s Test
1. Use 1.0% solutions of glucose, fructose and galactose for this test.
2. To 3mL of Bial’s reagent, add 2mL of the sugar solution.
3. Heat until it just begins to boil.
4. A blu egreen color forming at once or on cooling indicates a pentose. Ketohexoses and
methyl pentoses produce orange or olive-green colored solutions.
H. Tollen’s Pholoroglucinol Test

1. Use 1.0% solutions of glucose, fructose and galactose for this test.
2. To 1 ml of each of the sugar solution, add 1 mL of concentrated hydrochloric acid and 2-
3 drops of Tollen’s Phloroglucinol reagent.
3. Heat it in a boiling water bath. A positive test is indicated by a red color.
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I. Mucic Acid Test

1. Use 1.0% solutions of galactose, glucose and lactose for this test.
2. Place 10mL of sugar solution in a small beaker.
3. Add 5mL concentrated nitric acid and evaporate to approximately 5mL on the steam
bath. Do not evaporate to dryness.
4. Allow the solution to cool, or preferably to stand in your locker until the next laboratory
period.
5. Note and draw the appearance of any crystal formed under the microscope.
ACTIVITY:
A. Classification of Carbohydrates
1. Identify five samples of food found in your home.
2. List down the carbohydrate present in the ingredients.
3. Classify on what class of carbohydrate does it belong and identify its use.
Sample Carbohydrate Present Classification of Use

Carbohydrate
Yakult Glucose Monosaccharide Used to treat

hyperkalemia
Rice Starch Polysaccharide Rice's high-fiber

properties aid in
digestion and lowering
blood pressure.
Honey Fructose and Glucose Monosaccharide orally to treat coughs

and topically to treat
burns and promote
wound healing.
Potato Starch Polysaccharide a good source of fiber,

which can help you
lose weight by keeping
you full longer.
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Milk Lactose Disaccharide Adding milk and dairy

products to your diet
may prevent bone
diseases like
osteoporosis.
Bread Starch Polysaccharide helps to keep our

digestive system
healthy, helps control
blood sugar and
cholesterol levels and
makes us feel fuller for
longer.
B. Test for Carbohydrates

1. Write down the significance or purpose for each given test.
2. Identify the principles involved for each test.
3. Indicate the positive result for each test.
Test Significance/Purpose Principle Involved Positive Result
Iodine Test This test is used to The iodine test is based on Violet to Black
differentiate the absorptive properties
polysaccharides from the possessed by large
rest of carbohydrates. It polysaccharide molecules.
is given positive by The glucose chains in most
starch and glycogen. It of polysaccharides are
can also be used to organized to form helices.
differentiate between The space between the
glycogen, starch, and turns of the helix can hold
cellulose. small iodine molecules. This
is seen with amylase chains
found in starch. Glycogen
and amylopectin can also
absorb these iodine
molecules on their surface.
The absorptive property of
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polysaccharides decreases
upon heating.
Molisch Test It is a screening test for This test is based on the Purple Ring
confirming the presence reaction of the alpha-
or absence of naphthol with carbohydrate
carbohydrates in a given in the presence of sulfuric
solution. It is a highly acid. The sugars react with
sensitive test for alpha-naphthol in an acidic
carbohydrates. environment to form purple-
Monosaccharides, colored furfural or
oligosaccharides, and hydroxymethylfurfural
polysaccharides all give derivatives. The intensity of
positive Molisch’s test. the color is directly
proportional to the amount
of carbohydrate present in
the solution.
Osazone Test It is a confirmatory test Phenyl hydrazine reacts Yellow

for carbohydrates. It with reducing sugar in an
gives you the final acidic environment at high
inference about the type temperature to form
of carbohydrate present phenylhydrazone. Phenyl
in the solution. Osazone hydrazine further reacts with
derivative of a the sugar on heating and
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carbohydrate form forms crystals of osazone

specific crystals that are specific to that
characteristic to it. The carbohydrate.
shape of the crystal tells
us about the nature of
carbohydrate present.
Benedict’s It is a test for reducing This test is based on the Bright Orange
Test sugars. Carbohydrates ability of reducing sugars to
having free functional undergo oxidation in alkaline
solutions. In the presence of
group, that is not
an alkali, reducing sugars
involved in a glycosidic undergo tautomerization to
bond, give this test form enediols. These
positive. All enediols reduce the cupric
monosaccharides and ions (Cu+2) to form cuprous
disaccharides except ions (Cu+). The cuprous ions
sucrose give positive from cuprous hydroxide.
Upon heating, it is converted
Benedict’s test. This test
to cuprous oxide that forms
is negative for precipitates.
polysaccharides. The citrate ions present in
the reagent release the
cuprous ions slowly for
reduction and prevent the
formation of Cu(OH)2 until
the oxidation-reduction
process is completed.
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Barfoed’s It is a differentiating test Reducing sugar undergo Brick Red

Test to distinguish between tautomerization in mildly
monosaccharides and acidic medium to form
enediols. These enediols
disaccharides. Barfoed’s
reduce cupric ions to
test is also based on the cuprous ions that form
reducing ability of sugar. cuprous hydroxide. This
However, sucrose also cuprous hydroxide us
gives this test positive as converted to cuprous oxide
it undergoes hydrolysis on heating and precipitates
in the presence of an are formed.
acid. Monosaccharides
Its principle is similar to
give early positive test
Benedict’s test except the
while the disaccharides acidic environment.
give late positive. Monosaccharides being
strong reducing agents give
this test much early.
Seliwanoff’s This test is used to This test involves the Red Color
Test detect monosaccharides formation of furfural
with a ketonic functional derivatives by
group. It is widely used monosaccharides with
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to differentiate fructose, hydrochloric acid. The

a keto sugar, from furfural derivatives formed
glucose and galactose by a sugar with ketonic
functional group condense
with resorcinol to form a
chromogen having cherry-
red color.
Bial’s Test It is a general test for Pentoses form furfural Blue Color
carbohydrates and is compounds in the presence
sensitive only for of concentrated acid. The
pentoses. Any furfural compounds formed
compound that contains by pentoses condense with
a pentose sugar will give orcinol to form blue colored
a positive Bial’s test. compounds.
Tollen’s It is a type of Dark Grey

Phloroglucinol precipitate or
biochemical test Tollen’s reagent is the
Test Silver Mirror on
which is used to mixture of silver nitrate the bottom and
(AgNO3) and liquid
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distinguish reducing sides of the test

tube
sugars from non- ammonia (NH3), which
reducing sugars. This results in the formation
of a complex. The
biochemical test is
aqueous solution of
also known as the silver nitrate helps in the
silver mirror test formation of a silver
based on the end aqua complex while the
product of this test. water functions as a
This test was also ligand.
used to differentiate
Now, the hydroxide ions
between aldehydes
convert the aqua
and ketones through complexes into silver
routine qualitative oxides (Ag2O) which
organic analysis. appear as a brown
precipitate. This brown
precipitate dissolved by
aqueous ammonia
resulting in the formation
of the [Ag(NH3)2]+
complex. This complex
is considered as the
primary component of
the Tollen’s reagent and
is a strong oxidizing
agent.
Now, the aldehyde

groups present in the
sugars are oxidized by
this complex and form a
carboxylic acid. At the
same time, the silver
ions present in the
reagent are reduced to
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metallic silver, as a
result of this reduction
leads to the formation of
a silver mirror on the
bottom and sides of the
test tube. However, an
α-hydroxy ketone gives
a positive Tollen’s test
as the Tollen’s reagent
oxidizes the α-hydroxy
ketone into an aldehyde.
Mucic Acid This is a specific test for Oxidation of most The

Test galactose and is given by monosaccharides by nitric acid development of
galactose as well as yields soluble dicarboxylic crystals on the
lactose, which is made up of acids. However, oxidation of bottom of the
galactose and glucose. galactose yields an insoluble tube is an
mucic acid. Lactose will also increase in the
yield a mucic acid, due to amount of
hydrolysis of the glycosidic crystal, which
indicates that
linkage between its glucose and
the sample
galactose subunits. Being
solution
insoluble, galactosaccharic acid
contains
crystals separate out.
galactose , or
derivatives of it.
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ST.

Activity 3: Test for Carbohydrates

C. Osazone/ Phenylhydrazine Test

H. Tollen’s Pholoroglucinol Test

I. Mucic Acid Test

Sample Carbohydrate Present Classification of Use

Yakult Glucose Monosaccharide Used to treat

Rice Starch Polysaccharide Rice's high-fiber

Honey Fructose and Glucose Monosaccharide orally to treat coughs

Potato Starch Polysaccharide a good source of fiber,

Milk Lactose Disaccharide Adding milk and dairy

Bread Starch Polysaccharide helps to keep our

B. Test for Carbohydrates

Test Significance/Purpose Principle Involved Positive Result

Osazone Test It is a confirmatory test Phenyl hydrazine reacts Yellow

carbohydrate form forms crystals of osazone

Barfoed’s It is a differentiating test Reducing sugar undergo Brick Red

to differentiate fructose, hydrochloric acid. The

Tollen’s It is a type of Dark Grey

distinguish reducing sides of the test

Now, the aldehyde

Mucic Acid This is a specific test for Oxidation of most The

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