POST-LAB ACTIVITY NO.

12
TEST FOR CARBOHYDRATES

I. DISCUSSION
Classification and Structure of Carbohydrates
Simple Sugars (Aldoses and Ketoses, Pentoses and Hexoses)
Reducing and Non-Reducing Sugars
Name of Test, purpose of test, composition of reagent result

II. PROCEDURE

A. GENERAL COLOR TESTS

1. Molisch Test
To a test tube containing 2 ml. of carbohydrate solution (e.g. glucose, sucrose and starch) add
1-2 drops of alpha-naphthol solution and mix. Incline the test tube and slowly and carefully add about
1 ml. of concentrated sulfuric acid (CAUTION!) down the side of the tube to form a layer below the
sugar solution. Note the color formed at the junction of the two liquids.

Results: A violet ring is formed at the junction of two layers

The Molisch Test

Shows positive test for:
All carbohydrates. Monosaccharides give a rapid positive test. Disaccharides and polysaccharides react slower.

Reactions:
The test reagent dehydrates pentoses to form furfural (top reaction) and dehydrates hexoses to form 5-hydroxymethyl
furfural (bottom reaction). The furfurals further react with -naphthol present in the test reagent to produce a purple
product (reaction not shown).

A positive test is indicated by:

The formation of a purple product at the interface of the two layers.

a negative test (left) and a positive test (right)

2. Anthrone Test
To 5 drops of sugar solution in a test tube, Add 1
ml. each of anthrone reagent. Mix thoroughly by swirling.
Record observe the color formed.
Results: Blue-green color complex is formed which infers presence of carbohydrate
Anthrone Test:
Objective, Principle, Reagents, Procedure and Result
Objective:
To detect carbohydrate in the given solution
Principle:
Anthrone test is also another general test for all carbohydrates. In this test also, carbohydrate gets dehydrated
when react with conc. H2SO4 to form furfural. This furfural reacts with anthrone to give bluish green colored
complex.

Result interpretation:
Positive test: all carbohydrate give test positive
B. TESTS FOR REDUCING SUGARS
1. Benedict’s Test
Add 2 ml. of Benedict’s reagent to 2 ml. of
glucose, sucrose and starch solutionsin separate test tubes. Shake each tube thoroughly
and place each tube in a boilingwater bath. Heat for 5 minutes and observe what happens.
Record your results in a table.
Substance Result Obtained Tested

glucose It is a reducing sugar that reduces the

CU(II) ions in the test reagent to red
precipitate of copper (I) oxide

sucrose Color remains the same (Blue) after

placing it in boiling water. Test is
negative, indicating that the sucrose is
non-reducing sugar.
 starch Color remains the same (Blue) after
placing it in boiling water. Test is
negative, indicating that the sucrose is
non-reducing sugar.

2. Nylander’s Test
Nylander's test is a chemical test used for
detecting the presence of reducing sugars.
Glucose or fructose reduces bismuth
oxynitrate to bismuth under alkaline
conditions. When Nylander's reagent, which
consists of bismuth nitrate, potassium
sodium tartrate and potassium hydroxide, is
added to a solution with reducing sugars, a
__black_____ precipitate of metallic bismuth is formed. Fill in the blank what color
is formed.
3. Barfoed’s Test

Add 1 ml. of barfoed’s reagent to 1 ml. of glucose, sucrose and lactose solutions placed in
separate test tubes. Heat in boiling water, take out after 10 mins and see the record the
result in the table.
Substance Result Obtained Tested

glucose The test is positive. There’s a formation

of reddish precipitate.

sucrose Color remains the same (Blue) after

placing it in boiling water. Test is
negative.

lactose Color remains the same (Blue) after

placing it in boiling water. Test is
negative.

C. MUSIC AICD TEST FOR GALACTOSE AND LACTOSE

Mucic acid test is a test that is highly specific and is used for the detection of the presence of galactose
and lactose. It is also termed galactaric acid that is named after the product of the reaction.

Objectives of Mucic acid test

To detect the presence of galactose and lactose in a given sample.
To distinguish between the galactose containing saccharides and other sugars.
PROCEDURE:

1. To 1 ml. of galactose and lactose solution in separate evaporating dishes, add 5 ml. of
conc. nitric acid. Evaporate on a steam bath under the hood to a volume of 2 ml.

2. Allow to cool cover the evaporating dishes with watch glasses and set aside in your
locker until the next laboratory period.

3. Add 5 ml. of distilled water to

each dish, mix
well a stirring rod, and pour the
contentsof the
dishes into clean test tubes.

4. Examine the crystals under the

microscope.
Questions:
1. What does the formation of crystals
of the tube
indicates? 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.
2. What does the absence of such crystals
indicate?
The absence of such crystals
indicates a
negative result and represents that the sample
doesn’t have galactose or its derivative. The solution
might still have other carbohydrates.

D. SELIWANOFF’S TEST FOR KETOSES

Place 1 ml. each of solutions of maltose, fructose and glucose in separate test tubes. Add 5 ml. of
Seliwanoff’s reagent to each tube, mix and heat for exactly 30 sec in the boiling bath, observe and
record results at this time.

Results: Fructose turns cherry-red colored (positive Seliwanoff’s test) while maltose (aldose) and
 glucose (aldose) did not undergo such color change.

Seliwanoff’s test definition

Seliwanoff’s test is used to differentiate between sugars that have a ketone group (ketose) and sugars that
have an aldehyde group (aldoses). This test is a timed
color reaction specific to ketohexoses.
• 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.

Uses of Seliwanoff’s test

• Seliwanoff’s color reaction is used in the method for the
colorimetric determination of fructose in fermentation media.
• A modified version of this test can be used for the determination
of the concentration of ketoses in a given sample.

Limitations of Seliwanoff’s test

• The high concentration of glucose or other sugar may interfere by producing similar colored compounds with
Seliwanoff’s reagent.
• Prolonged boiling can transform glucose to fructose by the catalytic action of acid and form cherry red-complex giving a
false-positive result.
• This test is a generalized test and doesn’t
distinguish between specific ketoses, and a
separate test is required for the particular
ketose sugar identification.

E. TESTS FOR PENTOSES

A. Aniline Acetate Test
Place 3 ml. of arabinose in a
test tube. Add 2 ml.
of conc. HCI and carefully boil the
resulting solution for
about 1 minute. Discontinue heating
and hold a small
piece of filter paper moistened with
aniline acetate over
the mouth of the tube. Do not allow the
paper to rest on
the mouth of the tube. Hold the paper,
otherwise false
results may be obtained.
Result: ____(+) pink color___
The aniline acetate test
It is a chemical test to identify the presence of certain carbohydrates. When any of these
carbohydrates reacts with aniline acetate, furfural is produced, causing a bright pink color.
Hydrochloric acid interacts with pentoses (sugars containing five carbon atoms) to produce
furfural. This causes a bright pink color. This distinguishes pentoses (sugars containing five
carbons) and hexoses (sugars containing six carbons). 3-Furanaldehyde responds to the usual
aldehyde tests, but unlike 2-furanaldehyde it gives no color test with aniline acetate.

B. Bial’s Test
To 1 ml. of Bial’s reagent in a test tube, add 1 ml. of arabinose and heat in boiling water
bath for two mins. Observe and record the changes.
Result: There is a formation of bluish product which indicates a (+) result of the Bial’s Test
What is Bial's test?
Bial’s test is a chemical test for the presence of pentoses. It is named after Manfred Bial, a German
physician. The components include orcinol, hydrochloric acid, and ferric chloride. A pentose, if present,
will be dehydrated to form furfural which then reacts with the orcinol to generate a coloured substance
Indication of the presence of pentoses
The test gives us the result by the following colour changes
A blue-green colour indicates pentoses or nucleotides containing pentoses.
A yellow-green colour indicates hexoses, and disaccharides are yellow.
Inference
Bial’s Test will distinguish between mono- and disaccharides, lactose will not react. It can be noted that
Bial’s test is commonly used to distinguish between pentoses and hexoses and is, therefore, often
conducted after these groups have been identified with the help of Molisch’s test (which is a test which
checks for the presence of pentoses and hexoses).

References:

Amiritacreate. (2017). Qualitative Analysis of Carbohydrates. Www.Youtube.com.

https://www.youtube.com/watch?v=Ewe7i1D9lSQ&t=433s

Amit. (2020). Molisch Test for Carbohydrates || Biochemistry Practical. Www.Youtube.com.

https://www.youtube.com/watch?v=zHcmxBi_wG0

Aniline acetate test. (n.d.). Academic Dictionaries and Encyclopedias.

 https://enacademic.com/dic.nsf/enwiki/3718875?

fbclid=IwAR381crJ4QahECGptlhzMQo b-td1vmhnzvh60H3IE2SxO4fJQHJHd0SFDc4

Anthrone’s Test. (2014). Www.Youtube.com.

https://www.youtube.com/watch?v=Wq5uh0bVJzM&feature=youtu.be

Carbohydrates: The Molisch Test. (2019). Harpercollege.Edu.

http://dept.harpercollege.edu/chemistry/chm/100/dgodambe/thedisk/carbo/molisch/molis

c h.htm

Chemistry: Nylander’s test. (2020). Handwiki.org.

https://handwiki.org/wiki/Chemistry:Nylander%27s_test

Ramos, V. (2020). Qualitative tests for carbohydrates (Discussion). Www.Youtube.com.

https://www.youtube.com/watch?v=0aWSHlK7Xtk&t=843s

What is Bial’s test? (n.d.). BYJUS. https://byjus.com/questions/what-is-bials-test/