Handbook of practical Botany B.Sc.

I (CBCS)

8: Qualitative test for carbohydrates, reducing

sugar and starch
A) QUALITATIVE TEST FOR REDUCING SUGAR
Princi11le:

Carbohydrates are widely present in plants. Carbohydrates include

monosaccharide, oligosaccharide and poly-saccharide. Carbohydrate contains aldehyde
(CHO) or keto (C=O) group. Some sugars show reducing and non oxidizing properties.
Possibly all monosaccharides are reducing sugars.
Requirements: Green onion leaves (as sample), glucose (as standard) Fehling solution A,
Fehling solution B, Benedicts reagent, Mortar-pestle, test tube, burner, muslin cloth,
distilled water.
Procedure:
a) Fehling's Test:

Fehling's Solution (deep blue colored) is used to determine the presence of reducing
sugars and aldehyde. Perfonn this test with glucose. When Fehling solution is heated with
reducing sugar, gets reduced to yellow or red cuprous oxide and gets precipited. Hence
fonnation of the colored precipitate indicates the presence of reducing sugar in the test
solution.
Pre11aration of Fehling's Solution:
Fehling
Solution A

Dissolve 34.65 gms copper sulphate in distilled water and make

volume up to 500 ml

Fehling

Dissolve 125gms potassium hydroxide and l 73gms potassium

sodium tartarate in distilled water and make volume up to 500 ml.

Solution B
Plant sample:

Take 20 gms green onion leaves, cut into small pieces, crush with 50 ml
distilled water, filter through muslin cloth and save the filtrate/plant juice as plant sample.
Standard glucose Solution: Dissolve lOgms of glucose powder in lOOml of D.W. and use
as standard solution.
Test:

i) Take 1 ml of Fehling's solution A and 1 ml of Fehling solution B in the test-tube.

ii) Add 2 ml of the sugar (glucose) solution to the test tube containing Fehling solution A
and B, mix well and boil.
iii) Obsereve the brick red precipitate of cuprous oxide that fonned at the end of the
reaction.
iv) Repeat the same procedure plant sample.
v) Write your observations in the observation table.
vi) Write your results and conclusion.
b) Ben edicts test: Due to presence of potentially free aldehyde or keto group, sugar reduces
cupric hydroxide in alkaline solution to red colored cuprous oxide. Depending on
concentration of sugar solution, yellow to green color develops.

25

Handbook of practical Botany B.Sc. I (CBCS)

Pre11aration of Benedict's reagent:

A) Dissolve 173 gms sodium citrate and 100 gms sodium carbonate in 800 ml -Distilled
water. Heat to dissolve salt and filter if necessary.
B) Dissolve 17.3 gms copper sulphate in lOOml water and add it to the above solution A
with stirring and make up final volume to one liter.
Procedure:

1) Take 1 ml of Benedict reagent in a test tube.

2) Add 2 ml of the sugar solution (glucose) mix well and boil.
3) See 11reci11itation that forms at the end of the reaction.
4) Repeat above procedure for plant sample.
5) Write your observations in the observation table.
6) Write your results and conclusion.
Test

Sr.

Observation

Result

no.

Fehling's Test:

Test for glucose

Std. glucose solution +

+
Fehling solution A
Fehling solution B (boil)
upto precipitation
Plant sample + Fehling
+ Fehling
solution A
solution B (boil) upto
precipitation
B

Brick red precipitation

Reducing sugar is present

Brick red precipitation

Reducing sugar is present

Benedicts test: Test for glucose

Std. glucose solution + Solution tum green, yellow

Benedicts reagent (boil)
& finally red
2
Plant sample + Benedicts Solution tum green, yellow
& finally red
reagent (boil)
- Write your results and conclusion.
l

Reducing sugar is present

Reducing sugar is present

B) QUALITATIVE TEST FOR STARCH

Princi1llc:

Starch is polysaccharide composed of two components amylase and

amylopectin. Amylose is more soluble in water than amylopectin. Both are polymerase of
glucose. Amylose is fonned from 300-1000 molecules of glucose. Amylopectin is very
much branched chain of large number of glucose molecules. Starch is important source of
carbohydrate. It is found in cereals, in potato and in other vegetables.
Requirements: Iodine solution, Alcohol, dropper, test tube, watch glass, beaker, potato
tuber (slices) Distilled water.
Pre11aration of Starch Solution: Take 10 gms of starch powder and dissolve in 100 ml of
boiling water.

26

Handbook of practical Botany B.Sc. I (CBCS)

a) Iodine Test:

Iodine test is an indicator for the presence of starch. Iodine solution (iodine dissolved in an
aqueous solution of potassium iodide) reacts with starch producing a blue-black color.
Apply this test to all the polysaccharides provided.
Procedure:

l) To 2-3 ml of polysaccharide (Starch) solution, add 1-2 drops of iodine solution.

2) Observe the color of starch solutions.
3) Write your observations in the observation table.
4) Repeat the same procedure for the plant material.
i) Take slice of potato in a watch glass and add 2-3 drops of Iodine solution on the slice of
potato.
ii) Compare the observation with above test carried out with starch solution.
5) Write your results and conclusion.
b) Alcohol Test:

Alcohol reacts with starch and produces precipitation.

Procedure:

1)
2)
3)
4)
5)

To 2-3 ml of Starch solution, add 5 ml of alcohol.

Observe the change in colour i.e. bluish black.
Write your observations in the observation table.
Write your results and conclusion.
Repea t ti 1e above proce dure fior ti1e p lan t ma ten"al

Test

Sr.

Observation

Result

no.

1
2
3
4

5rnl starch solution + 5 ml

iodine solution
Slice of potato + 2 drops of
Iodine
5ml Starch solution + 5rnl
alcohol
Juice of Potato + 5ml alcohol

Bluish black color

Starch is present

Bluish black color

Starch is present

Precipitation

Starch is present

Precip itation

Starch is p resent

Conclusion: From the above test, carbohydrates can be detected from the given 11lant
sam11les.

27

Objective:
To characterize carbohydrates present in an unknown solution on the basis of various chemical assays.

Theory:
Carbohydrates are polyhydroxy aldehydes and ketones or substances that hydrolyze to yield
polyhydroxy aldehydes and ketones. Aldehydes (CHO) and ketones ( = CO) constitute the major
groups in carbohydrates.

Carbohydrates are mainly divided into monosaccharides,

disaccharides and polysaccharides. The

commonly occurring monosaccharides includes glucose, fructose, galactose, ribose, etc. The two
monosaccharides combine together to form disaccharides which include sucrose, lactose and
maltose. Starch and cellulose fall into the category of polysaccharides, which consist of many
monosaccharide residues.
Molischs Test:
This is a common test for all carbohydrates larger than tetroses. The test is on the basis that
pentoses and hexoses are dehydrated by conc. Sulphuric acid
to form furfural or
hydroxymethylfurfural, respectively.
condensation product.

These products condense with -naphthol to form

Furfural

-Naphthol

purple

Fehlings Test:
This forms the reduction test of carbohydrates. Fehlings solution contains blue alkaline cupric
hydroxide solution, heated with reducing sugars gets reduced to yellow or red cuprous oxide and is
precipitated. Hence, formation of the yellow or brownish-red colored precipitate helps in the detection
of reducing sugars in the test solution.
Benedicts Test:
As in Fehlings test, free aldehyde or keto group in the reducing sugars reduce cupric hydroxide in
alkaline medium to red colored cuprous oxide. Depending on the concentration of sugars, yellow to
green color is developed . All monosaccharides are reducing sugars as they all have a free reactive
carbonyl group. Some disaccharides, like maltose, have exposed carbonyl groups and are also
reducing sugars, but less reactive than monosaccharides
Barfoeds Test:
Barfoed's test is used to detect the presence of monosaccharide (reducing) sugars in solution.
Barfoed's reagent, a mixture of ethanoic (acetic) acid and copper(II) acetate, is combined with the
test solution and boiled. A red copper(II) oxide precipitate is formed will indicates the presence of
reducing sugar. The reaction will be negative in the presence of disaccharide sugars because they are
weaker reducing agents. This test is specific for monosaccharides . Due to the weakly acidic nature of
Barfoed's reagent, it is reduced only by monosaccharides.
Seliwanoffs Test:
It is a color reaction specific for ketoses. When conce: HCl is added. ketoses undergo dehydration to
yield furfural derivatives more rapidly than aldoses. These derivatives form complexes with resorcinol
to yield deep red color. The test reagent causes the dehydration of ketohexoses to form 5hydroxymethylfurfural. 5-hydroxymethylfurfural reacts with resorcinol present in the test reagent to
produce a red product within two minutes (reaction not shown). Aldohexoses reacts so more slowly to
form the same product.

Bials Test:
Bials test is used to distinguish between pentoses and hexoses. They react with Bials reagent and
are converted to furfural. Orcinol and furfural condense in the presence of ferric ion to form a colored
product. Appearance of green colour or precipitate indicates the presence of pentoses and formation of
muddy brown precipitate shows the presence of hexoses.
Iodine Test:
This test is used for the detection of starch in the solution. The blue-black colour is due to the
formation of starch-iodine complex. Starch contain polymer of -amylose and amylopectin which
forms a complex with iodine to give the blue black colour.

Osazone Test:
The ketoses and aldoses react with phenylhydrazine to produce a phenylhydrazone which further
reacts with another two molecules of phenylhydrazine to yield osazone. Needle-shaped yellow osazone
crystals are produced by glucose, fructose and mannose, whereas lactosazone produces mushroom
shaped crystals. Crystals of different shapes will be shown by different osazones. Flower-shaped
crystals are produced by maltose.

PROCEDURE

Materials Required:
1)
2)
3)
4)
5)
6)

Glassware
Test tubes
Test tube holder
Water bath
Spatula
Dropper

Reagents Required:
1)
2)

Molischs Reagent
Iodine solution

3)
4)

Fehlings reagent A
Fehlings reagent B

5)
6)

Benedicts qualitative reagent

Barfoeds reagent

7)
8)

Seliwanoff s reagent
Bials reagent

9) Phenylhydrazine hydrochloride
10) Sodium acetate
11) Glacial acetic acid
12) Glucose, fructose
13) Microscope

Procedure:
1) Molischs Test:
In a test tube, add 2 ml of the test carbohydrate solution and 2 drops of -naphthol solution.
Carefully incline the tube and pour dropwise conc. H2SO4, using a dropper,along the sides of the tube.
Observe the violet colour at the junction of the two liquids.

2) Fehlings Test:
In a test tube, add 2 ml of the test carbohydrate solution and add equal volumes of Fehling
A & Fehling B and place it in a boiling water bath for few minutes.. When the contenst of the
test tube comes to boiling, mix them together and observe any change in color or precipitate
formation. The production of yellow 'or brownish-red precipitate of cuprous oxide indicates the
presence of reducing sugars in the given sample.

3) Benedicts Test:
In the test tube with 2 ml of Benedict's reagent, add 5-6 drops of the test carbohydrate solution and
mix well. Place the test tube in a boiling water bath for 5 minutes and observe any change in color or
precipitate formation. Cool the solution. Observe the colour change from blue to green, yellow,
orange or red depending upon the amount of reducing sugar present in the test sample.

4) Barfoeds Test:
To 2 mL of the test solution add about 2-3 mL of Barfoeds reagent. Mix it well and boil it for one
minute in the water bath and allow to stand for a few minutes. Formation of a red precipitate of
cuprous oxide in the bottom and along the sides of the test tube immediately, only monosaccharides
answer this test. Since Barfoeds reagent is slightly acidic, This test is specific for monosaccharides.

5) Seliwanoffs Test:
To 2 mL of Seliwanoff s reagent, add two drops of test solution. The mixure is heated to just boiling.
A cherry red condensation product will be observed indicating the presence of ketoses in the test
sample. There will be no significant change in colour produced for aldose sugar.

6) Bials Test:

To 5 mL of Bials reagent add 23 mL of test solution and warm gently in a hot water bath for
2minutes . The formation of a bluish green product is indicative of pentoses. Hexoses generally react
to form muddy brown products.

7) Iodine Test:
Add 2 drops of iodine solution to about 2 mL of the carbohydrate containing test solution. A blueblack colour is observed which is indicative of presence of polysaccharides.

8) Osazone Test:
To 0.5 g of phenylhydrazine hydrochloride add 0.1 gram of sodium acetate and ten drops of glacial
acetic acid. Add 5 mL of test solution to this mixture and heat under boiling water bath for about half
an hour. Cool the solution slowly and examine the crystals under a microscope. Needle-shaped yellow
osazone crystals will be observed for glucose and fructose, whereas lactosazone shows mushroom
shaped and maltose produces flower-shaped crystals.

No.

Test

Observation

Inference

Molischs Test
2-3 drops of betanaphthol
solution

A deep
violet coloration is
are added to 2ml of
Presence of
produced at the
carbohydrates.
the test solution.
junction of two
Very gently add 1ml
layers.
of Conc. H2SO4 along

the side of the test

tube..

Reaction

This is due to the

formation
of
an
unstable
condensation product
of beta-naphthol with
furfural (produced by
the dehydration of
the
carbohydrate).

Iodine test
2

4-5 drops of iodine Blue

colour
solution are added to observed.
1ml of the test
solution and contents
are

mixed

gently.

is Presence
polysaccharide.

Iodine
forms
of coloured adsorption
complexes
with
polysaccharides.

Fehling's test
This is due to the
formation of cuprous
A red precipitate is Presence of reducing oxide
by
the
about
2
ml
of
reducing action of
formed
sugar
Fehlings
solution
the
sugar.
taken in a test-tube.
It is then boiled for
About 2 ml of sugar
solution is added to
3

10

min

Benedicts test
To 5 ml of Benedict's
solution, add 1ml of
the test solution and
4

If the saccharide is a
reducing sugar it will
shake
each Formation
of
a
Presence of reducing reduce Copper [Cu]
tube. Place the tube green, red, or yellow
(11) ions to Cu(1)
sugars
in a boiling water precipitate
oxide,
a
red
bath and heat for 3
precipitate
minutes.
Remove
the tubes from the
heat and allow them
to
cool.

Barfoeds test
To

ml

of

the

solution to be tested
added 2 ml of freshly A deep blue colour is
prepared
Barfoed's formed with a red
reagent. Place test ppt. settling down at
tubes into a boiling the bottom or sides
water bath and heat of the test tube.
for 3 minutes. Allow
to
cool.

Seliwanoff test

Presence of reducing
sugars. Appearance of
a red ppt as a thin
film at the bottom of
the test tube within
3-5 min. is indicative
of reducing monosaccharide. If the ppt
formation takes more
time, then it is a
reducing disaccharide.

If the saccharide is
a reducing sugar it
will reduce Cu (11)
ions to Cu(1) oxide

A
cherry
red Presence of ketoses When reacted with
colored
precipitate [Sucrose
gives
a Seliwanoff reagent,

of within 5 minutes is positive

test
Seliwanoffs reagent, obtained.
add 1ml of the test
To

3ml

of

solution.
Boil
in
water bath for 2
minutes.

Presence
A
faint
red
colour
produced

Bial's test
Add 3ml of Bials
reagent to 0.2ml of
7

A
product

blue-green

ketohexose ketoses react within

] 2 minutes forming a
cherry
red
condensation product

of

Aldopentoses
react
aldoses slowly, forming the
coloured
condensation
product.

Presence of pentoses.

Presence
the test solution.
A muddy brown to
hexoses.
Heat the solution in
gray
product
a boiling water bath
for
2
minutes.

The furfurals formed

produces
of
condensation
products with specific
colour.

Osazone Test

of
Two two ml of the Formation
yellow
test solution, add beautiful
crystals
of
osazone
3ml
of
phenyl
Glucose/fructose
Needle
shaped
hydrazine
crystals
hydrochloride
Presence of lactose
Hedgehog
crystals
Presence of maltose
solution and mix.
Sunflower
shaped
Keep in a boiling crystals
water
bath
for

Reducing
sugars
forms ozazone on
treating
with
phenylhydrazine

30mts.
Cool
the
solution and observe
the crystals
microscope.

under

Differences Encountered in a Real Laboratory:

In an actual laboratory setting, there are certain important steps that are not necessarily applicable in
a virtual lab:

1.

Always wear lab coat and gloves when you are in the lab. When you enter the lab,switch on the
exhaust fan and make sure that all the reagents required for the experiment are available. If it is not

available, prepare the reagents using the components for reagent preparation.
2. Care should be taken while handling caustic acids like Conc. Sulphuric acid [H2SO4], nitric acid [HNO3],
Hydrochloric acid [HCl]. These acids should be opened and used in FUMEHOOD only. Accidental spill of
these acids will cause severe burns and itching. Wash the spilled area with cold water and inform the
lab assistant immediately.
3. When Sodium hydroxide is prepared, make sure that it is handled with care as the sodium hydroxide
solution is caustic in nature.
4. Always check the water level in the water bath and if it is up to the level [nearly half the volume],
switch on the water bath and adjust to the required temperature. Take care while using the water
bath for the boiling step in the experiment. Hold the test tube using a test tube holder.
5. There should be a proportion between the reagents added and the test solution to obtain good result
within the time mentioned. The droppers used should not be mixed between the reagents, always use
individual droppers for each reagent.
6. The color formed will depend upon the quality of the reagents. So care should be taken while
preparing the reagents. If commercially available reagents are used assure that it is not kept open for
long time.
7. Clean the test tubes and glass wares with soap and distilled water. Recap the reagent bottles once the
experiment is completed. The water bath and the exhaust fan should be switched off .

OSAZONE FORMATION
Carbohydrates react with phenylhydrazine to fonn crystalline derivatives called

osazones.
CHO

HC=NNHPh

CHOH

CHOH

C=NNHPh

CHOR

HC=NNHPh

PhNHNll2

CHOH

2PhNHNH2

CHOH

CHOH

CHOH

CHOH

CHOH

CHOH

CHOH

CH20H

CH20H

CH20H

I
I

I
I

+ NH3 + PhNH2

I
I

An osazone

An osazone can be isolated as a derivative and its melting point determined. However, some
of the 1nonosaccharides give identical osazones (glucose, fructose, and mannose). Also, the
1nelting points of different osazones are often in the sa1ne range. This litnits the usefulness
of an isolation of the osazone derivative.
A good experilnental use for the osazone is to observe its rate of formation. The rates
of reaction vary greatly even though the same osazone 1nay be produced from different sug
ars. For example, fructose forms a precipitate in about 2 minutes, whereas glucose forms a
precipitate about 5 minutes later. The osazone is the same in each case. The crystal struc
ture of the osazone is often distinctive. Arabinose, for example, produces a fine precipitate;
glucose produces a coarse precipitate.

Dissolve 66.6 g of copper(IJ) acetate in I L of distilled water. FiJter the solution, if necessary, and add 9 mL of glacial ace

tic acid.

Caution: Phenylhydrazine 1s a suspected carcinogen. Handle with

gloves.

Procedure for Osazone Formation.

A boiling water bath is needed for this ex

periment. Place 0.5 ml of each of the following 10% carbohydrate solutions in sepa
rate test tubes: xylose, arabinose, glucose, galactose, fructose, lactose, sucrose, starch
(shake it), and glycogen. Add 2 ml of phenylhydrazine reagent to each tube.6 Place
the tubes in a boiling water bath simultaneously. Watch for a precipitate or, in some
cases, cloudiness. Note the time at which the precipitate begins to form. After 30 min
utes, cool the tubes and record the crystalline form of the precipitates. Reducing di
saccharides will not precipitate until the tubes are cooled. Nonreducing disaccharides
will hydrolyze first, and then the osazones will precipitate.