QUALITATIVE TESTS FOR CARBOHYDRATES

Zoe Angela M. Espinosa, Ma. Pamela Rica M. Fajardo, Emmanuel Joshua M. Garcia, Liana Celina D.
Geluz, Jillian Pamela D. Go, Jennifer Mae C. Heredia
Group 4 2A Medical Technology CHEM600 Laboratory

ABSTRACT
Carbohydrates are generally hydrates of carbon with the common formula (CH 2O)x, ranging from simple sugars such
as glucose and fructose to complex molecules such as starch and cellulose. In the experiment, the solutions of
carbohydrates that were used were glucose, fructose, lactose, xylose, sucrose, and starch. Different qualitative tests
were performed on the different samples namely Benedicts Test, Barfoeds Test, Seliwanoffs Test, Bials Test, Mucic
Acid Test, and Phenylhydrazone Test. Glucose, fructose, lactose, xylose, and sucrose produced a brick-red pptfor
Benedicts Test, indicating a positive result. For Barfoeds Test, only fructose and xylose showed a brick-red pptbut
glucose was also expected to produce a positive result. Seliwanoffs Test generated a cherry red pptfor fructose and
sucrose while Bials Orcinol Test gave a blue green coloration to glucose, xylose, lactose, and starch even when xylose
is the only one expected to give the positive result. Crystals from galactose were observed from the mucic acid test.
Lastly, phenylhydrazone test gave glucose, fructose, and xylose needle-shaped yellow osazone crystals. Lactose also
formed needle shaped crystals. Sucrose did not form osazone crystals. Starch produced some needle shaped crystals.

INTRODUCTION
Qualitative analysis shows whether a particular
substance is present, but does not give an
indication of how much is present [2]. In this
experiment, several qualitative tests were used
to analyze the distinguishing features of standard
carbohydrate solutions.
Benedict's Test detects reducing sugars through
the use of Benedict's reagent [1]. Benedict's
reagent is a chemical reagent named after an
American chemist, Stanley Rossiter Benedict.
When
Benedicts
solution
and
simple
carbohydrates are heated, the solution changes
to orange red/ brick red. This reaction is caused
by
the
reducing
property
of
simple
carbohydrates. Barfoed's test is a chemical test
used
for
detecting
the
presence
of
monosaccharides. It is based on the reduction of
copper(II) acetate to copper(I) oxide (Cu2O),
which forms a brick-red ppt[7].Seliwanoffs test is
a chemical test which distinguishes between
aldose and ketose sugars. It is named after
Theodor Seliwanoff, the chemist that devised the
test. The test reagent, resorcinol in 6M HCl,
dehydrates
ketohexoses
to
form
5hydroxymethylfurfural. Bial's-Orcinol Test or Bial's
Test is a qualitative test which involves a reagent
containing 5-methylorcinolin concentrated HCl
with a small amount of FeCl3. Pentoses undergo
dehydration to furfural that condenses with
orcinol to create a blue-green solution [6]. Mucic
Acid Test employ the principle that aldohexoses
are converted to their corresponding dicarboxylic
acids in the presence of strong oxidizing agents
like concentrated HNO3. The dicarboxylic acidwhich is mucic acid produced from the oxidation
of galactose is relatively insoluble and separates
out as colorless crystals [6]. Osazone or
Phenylhydrazone Test involves a carbohydrate
specific reaction with phenylhydrazine to form
osazone crystals of specific shape which confirms

the presence of a particular sugar [10]. Different

reducing sugars form distinct crystals with
phenylhydrazine
reagent.
Microscopic
examination and comparison of formed osazones
with
reference
standards
may
lead
to
identification of the reducing sugar [6].
The objectives of the experiment are, 1) to
perform five qualitative tests on the carbohydrate
standards namely, Benedict's Test, Barfoed's Test,
Seliwanoff's Test, Bial-Orcinol's Test, Mucic Acid
Test and Phenylhydrazone Test, 2) to examine
microscopically the different osazone and mucic
acid crystals, and 3) to describe the different
crystals formed.
EXPERIMENTAL
A. Samples Used
0.1 M Glucose, 0.1 M Lactose, 0.1 M Xylose,
0.1 M Lactose, 0.1 M Sucrose, 0.1 M Starch
B. Procedure
1. Benedict's, Barfoed's, Seliwanoff's, and
Bial's-Orcinol Tests
In six separate test tubes, five drops of
carbohydrate solution (each of glucose, fructose,
xylose, lactose, sucrose and starch) were mixed
with 1 mL of the respective reagent for each test.
It was noted that all carbohydrates for a given
test were given the reagents at the same time.
The test tubes were placed at the same time into
a boiling water bath. Once positive results were
seen for a test, the test tubes were removed
from the water bath.
2. Mucic Acid Test
On two separate glass slides, three drops of
each of the standard carbohydrate solutions (only
galactose and lactose) were mixed with three
drops of concentrated HNO3. The mixtures were
left on top of a hot plate until they were almost
dry. The glass slides were left to cool to room
temperature. The crystals were examined under

a compound binocular microscope. The shape

and color of the crystals were noted.
3. Phenylhydrazone Test
The phenylhydrazine reagent was prepared by
mixing 2 g phenylhydrazine hydrochloride, 3 g
NaCH3COOH, and 10 ml distilled water. The
reagent was placed in a warm water bath until it
was clear. In six different test tubes, two drops of
the carbohydrate standard (glucose, fructose,
xylose, lactose, sucrose, and starch) were mixed

with
4
drops
of
the
freshly
prepared
phenylhydrazine reagent. The test tubes were
mixed well and were covered with a cotton
stopper. The test tubes were subject to a boiling
water bath for 30 minutes and the time when
crystals first appear was noted. The test tubes
were cooled and the crystals were observed
under the microscope. The shape and color of
each of the crystals were noted.

RESULTS AND DISCUSSION

Carbohydrate
Solution
Glucose
Fructose
Xylose
Lactose
Sucrose
Starch

Benedicts Test
Brick Red ppt (+)
Brick Red ppt (+)
Brick Red ppt (+)
Brick Red ppt (+)
Brick Red ppt (+)
Blue soln (-)

Barfoeds Test
Blue soln (*)
Brick Red ppt (+)
Brick Red ppt (+)
Blue soln (-)
Blue soln (-)
Blue soln (-)

Visible Results
Seliwanoffs Test
Peach soln (-)
Cherry Red ppt (+)
Peach soln (-)
Peach soln (-)
Cherry Red ppt(+)
Peach soln (-)

Bials Orcinol Test

Blue Green Coloration(*)
Green Ppt(-)
Blue Green Coloration(+)
Blue Green Coloration(*)
Dark Green Ppt(-)
Blue Green Coloration(*)

Table 1. Results of the Qualitative Test for Carbohydrates

In the table below, the results from the
performed qualitative tests were shown. The
positive sign (+) indicates that a positive result is
shown on the performed experiment while the
negative sign (-) indicates a negative result. The
asterisk (*) indicates questionable or inaccurate
result.
1. Benedicts test
Benedicts test is used to test for simple
carbohydrates. The principle behind Benedicts
test is that carbohydrates with free aldehyde or
ketone groups have the ability to reduce
solutions of various metallic ions. Reducing
sugars under alkaline conditions tautomerise
forming enediols. These enediols are powerful
reducing agents that has the ability to reduce
cupric ions to cuprous and are themselves
converted to sugar acids. The formed cuprous
ions combine with hydroxyl (-OH) ions to form
yellow cuprous hydroxide which upon heating
turns to red cuprous oxide, giving the brick red
pptfor a positive result. Five of the carbohydrate
samples namely glucose, fructose, xylose,
lactose, and sucrose produced a positive result
which
confirms
that
they
are
reducing
carbohydrates. The equation below shows one
mole of a reducing carbohydrate reacting with
two moles of copper (II) to yield a carboxylate
product and cuprous oxide, responsible for the
brick-red ppt[1].

Figure 1. Reaction Mechanism for Benedicts Test

Figure 2. Results of Benedicts Test for Glucose,

Fructose, Xylose, Lactose, Sucrose, and Starch
(L-R)
2. Barfoeds test
Barfoeds test is a qualitative test used to
confirm the presence of monosaccharide and
distinguish them from disaccharides. The
principle behind Barfoeds test is that aldoses and
ketoses are known to reduce cupric ions even in
acidic conditions. Monosaccharides react very fast
whereas disaccharides react very slowly. Copper
(II) acetate, [Cu(CH3COO)2], which is present in
Barfoeds reagent is converted to copper (II)
oxide, CuO, and therefore gives a brick red
pptwhen reacted with monosaccharides. A
positive result indicates the presence of a
reducing monosaccharide but on prolonged
heating, disaccharides can also produce a
positive result. Fructose and Xylose generated a
positive result that confirmed they are reducing
monosaccharides. Glucose showed a negative
result in the test but it is also a reducing
monosaccharide. We can infer that there may be
contaminants present in the solutions used or
human error caused the inaccuracy in the result.
The equation below shows one mole of a reducing
carbohydrate reacting with two moles of copper

(II) to yield a carboxylate product and cuprous

oxide, responsible for the brick-red ppt [7].

Figure 3. Reaction Equation for Barfoeds Test

Figure 4. Results of Barfoeds Test for Glucose,

Fructose, Xylose, Lactose, Sucrose, and Starch
(L-R)
3. Seliwanoffs test
Seliwanoffs test is a chemical test that can
distinguish between aldose and ketose sugars
through
the
present
functional
group.
Ketohexoses on treatment with hydrochloric acid
form
5-hydroxymethyl
furfural
which
on
condensation with resorcinol gives a cherry red
colored complex [2]. Ketohexoses gives a
positive result; therefore, fructose and sucrose
produced the cherry red pptthat confirmed they
are ketohexoses. Ketoses are dehydrated at a
faster rate than aldoses. Aldoses are capable of
reacting and forming the same product but at a
slower rate. Fructose and sucrose generated a
positive result which confirmed that they are
ketohexoses. The reaction below shows how the
ketose is dehydrated by a strong acid, and once
dehydrated, will react with two moles of
resorcinol to form the product.

Figure 5. Reaction Equation for Seliwanoffs Test

Figure 6. Results of Seliwanoffs Test for

Glucose, Fructose, Xylose, Lactose, Sucrose, and
Starch (L-R)
4. Bials Orcinol Test

Bials Orcinol test is a chemical test used to

confirm the presence of a pentose. The test
reagent dehydrates pentoses to form furfural.
Furfural then reacts further with orcinol and the
iron ion present in the test reagent to produce a
bluish green coloration in the product. Bials
reagent
contains
5-methylresorcinol
in
concentrated HCl with minimal amount of FeCl3.
This test is specific for pentises. Hexoses
generally react to form green, red, or brown
products. Xylose is supposed to be the only
carbohydrate positive for this test in the given
samples because it is the only pentose.
Innacuracy in the result of glucose, lactose, and
starch may be due to contaminants or human
error. The equation below shows how a pentose,
under acidic conditions will react with the Bial's
reagent to form furfural, then condensing with
orcinol to form a blue-green solution.

Figure 7. Reaction Equation for Bials Orcinol

Test

Figure 8. Results of Bials Orcinol Test for

Glucose, Fructose, Xylose, Lactose, Sucrose, and
Starch (L-R)
5. Mucic Acid Test
The mucic acid test is a test specifically useful
in the identification of galactose as it is oxidized
to form a dicarboxylic acid (mucic acid) in the
presence
of
concentraned
HNO3,
forming
insoluble crystals as it separates [6].

Figure 9. Oxidation by Nitric Acid

The aldehyde and primary alcohol groups
present in aldohexoses such as galactose are
oxidized to carboxyl groups in its reaction with
HNO3 forming saccharic acid. The saccharic acid

formed from the free or combined glucose is

insoluble and separates out as colorless crystals
in the cold [3].

Figure 10. Mucic Acid Crystals

In the experiment performed, mucic acid
crystals were observed in mixing the galactose
solution with concentrated HNO3. The crystals
observed here are not colorless as the solution
was not completely dried when viewed under the
microscope. Nonetheless, it is still considered a
positive result.
6. Phenylhydrazone Test
This test makes use of the chemical reagent
known as phenylhydrazine with the formula
C6H5NHNH2. The technique was developed by Emil
Fischer, a German chemist, to identify different
sugars. Fischer was able to differentiate the types
of sugar by studying the crystals that formed
from his procedure [4].

Figure 11. Formation of Osazones

In using excess amounts of the phenylhydrazine
reagent at a kept temperature, reducing sugars
with a free aldehyde or a free ketone group
reacts to form insoluble crystals [4]. Three
molecules of phenylhydrazine are required for
reaction to take place at the first two carbon
atoms. The upper equation shows the general
form of the osazone reaction, which affects
alpha-carbon oxidation with formation of a bisphenylhydrazone, known as an osazone [5].
Osazone crystals have definite crystalline
structure, precipitation time, and melting point
for different formed crystals [3]. Comparison of
the formed osazones with a reference standard
will help in the identification of the reducing
sugar [6].
Sugars known as disaccharides include sucrose
which forms sunflower-like crystals. Lactose
osazone crystals are more akin to tight balls of
needles. Monosaccharides like glucose, fructose,
and xylose form needle-like or broomstick-

shaped crystals, showing that the positions of

their first and second carbons are not affected in
the crystal formation [9].
Carbohydrate
Solution

Crystalline
Structure

Glucose

Needle
shape

Fructose

Needle
shape

Xylose

Needle
shape

Lactose

Needle
shape

Sucrose

No crystals

Starch

Needle
shape

Microscopic
Observation

Figure 12. Phenylhydrazine Test Crystals

Glucose, fructose, and xylose produce needleshaped or broom-shaped yellow osazone crystals
due to the similarities in their molecular
structures as monosaccharides. The difference in
these sugars present on the first and second
carbon atoms are masked when osazone crystals
are formed. Hence these sugars form similar
needle-shaped crystals arranged like sheaves of
corn or a broom [5].
Lactose crystals were also needle shaped in
appearance. Reference guides for lactose
described formations of powder puff or cotton
ball or hedge hog shaped crystals as positive
results. Problems on focusing and contaminants
may have caused inconsistencies with the results
obtained.
Sucrose did not form osazone crystals since its
a non-reducing sugar [9]. It has no free reactive
group because the anomeric carbons of both
monosaccharide units are involved in the
glycosidic bond, where the configuration of this

glycosidic linkage is alpha- for glucose and betafor fructose. Hence, sucrose neither shows
reducing nor mutarotation characters [5].

Figure 13. Structure of Sucrose

Starch which is hydrolyzed to form
monosaccharide glucose showed needle-like
crystals under observance in low power objective
using light microscope.
Times for the formation of yellow crystals for
each sugar were not recorded for they were kept
inside lockers before precipitation occurred, and
so were unobserved throughout the duration of
their precipitation.
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Biochemistry. Quezon City, Philippines. C & E
Publishing, Inc.
From the internet (on-line)
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Composition, Preparation, Procedure and Result
Interpretation. 5/1/16
BBC (2010). Qualitative Tests.
http://www.bbc.co.uk/schools/gcsebitesize/scienc
e/triple_edexcel/qualitative_analysis/qualitative_
analysis/revision/1/ 4/30/16
Caton, Kate (2016).
http://www.slideshare.net/katealyssacaton/mucic
-and-barfoeds-test 5/3/16
Caton, Kate (2016).
http://www.slideshare.net/katealyssacaton/osazo
ne-test-8609621 5/3/16
Chhabra, Namrata (2015).
http://usmle.biochemistryformedics.com/osazone
-test/ 5/3/16
Harper College (2010). Barfoed's Test.
http://www.harpercollege.edu/tmps/chm/100/dgodambe/thedisk/carbo/barf/barfo
ed.htm 5/1/16
Harper College (2010). Seliwanoff Test.
http://www.harpercollege.edu/tmps/chm/100/dgodambe/thedisk/carbo/seli/seli.ht
m 5/1/16
Laing, Martin (2016)
http://www.ehow.com/info_8392949_differentosazone-crystals.html 5/3/16
Pubchem (2009). Phenylhydrazine and formation
of crystals.
https://pubchem.ncbi.nlm.nih.gov/compound/65
37404 5/1/16