General and Specific Tests for Carbohydrates

Isaac Abalos, Mark Joel Aguit, Kiel Christian Alday, Ma. Kristelle Joyce Arellano*

Department of Biological Sciences, University of Santo Tomas, Manila, Philippines

Abstract

Carbohydrates or saccharides are the most plenteous organic compounds in the

plant world. They play a role in molecular recognition, cellular protection, cell signaling,
cell adhesion, biological lubrication, control of protein trafficking, and maintenance of
biological structure. They are also known for generating and storing biological energy
(Appling, 2016). Carbohydrates are classified into three groups; the monosaccharides,
oligosaccharides, and polysaccharides. In this experiment, therehas been general tests for
carbohydrates which are the Molisch test, Anthrone test and Iodine test. Also
monosaccharides (xylose, fructose, glucose, galactose) and disaccharides (lactose,
maltose, sucrose) were subjected to spefic reactions which are the Benedict’s test,
Barfoed’s test, Mucic acid test, Seliwanoff’s test, and Bial’s Orcinol test.

Introduction

Carbohydrates are one of the three basic macronutrients needed to sustain life; it
encompasses a broad range of sugars, starches, and fibers and is widely distributed in
plants and animals. Carbohydrates are also referred to as saccharides that is derived from
the greek word sakcharon, which means sugar. Carbohydrates, represented by the general
formula Cn(H2O)n, (however, only the simple sugars or monosaccharides fit this formula
exactly) are molecular compounds made from just three elements: carbon, hydrogen, and
oxygen (Campbell and Farren, 2012). They are defined chemically as polyhydroxy
aldehydes or ketones which yield these products upon hydrolysis. They have an important
role in biological functions such as energy source and cellular communication. Aside
from that, they are also known for their structural and metabolic roles. They are
chemically defined as aldehyde or ketone derivatives of polyhydric alcohols (Chhabra,
2014). Carbohydrates are represented by the simple stoichiometric formula Cn(H2O)n
and are also referred to as saccharides. The name carbohydrate came from “hydrated
carbon”; it was first given when chemists knew only the stoichiometry of saccharides
(Mathews, 2016). Carbohydrates may be classified as monosaccharides, oligosaccharides,
or polysaccharides.

Figure 1. Different monosaccharides

Monosaccharides are simple monomeric sugar, which cannot be hydrolyzed. They are
commonly known as the building blocks of carbohydrates (Campbell, 2015). They can
either be a polyhydroxy aldehyde (aldose) or a polyhydroxy ketone (ketose).
Monosaccharides can also be classified as pentoses (5 carbons) or hexoses (6 carbons),
depending on the number of carbons they contain. Xylose is an example of pentose while
fructose, glucose, and galactose are examples of hexose (Figure 1). Monosaccharides are
water soluble due to the presence of the –OH group in their structure which interacts with
water through hydrogen bonding (Shankara, 2008). Glucose is the most abundant
monosaccharide and it is the primary energy source for living cells. Fructose is found in
fruits and honey while xylose is a sugar found in plant tissues.

Oligosaccharides on the other hand are condensation products of three to ten

monosaccharides. This consists of disaccharides, trisaccharides and so on. Examples of
disaccharides are sucrose, maltose, and lactose (figure 2). Sucrose, commonly known as
table sugar, is the most abundant disaccharide. Maltose is found in malted milk and is
produced whenever the polysaccharide starch breaks down.
 Figure 2.
Examples of
disaccharides

Polysaccharides are
long polymers of
monosaccharides
linked together. Cellulose, glycogen, and amylose are the most common example
polysaccharide.

In this experiment, the tests involved are Iodine Test, Anthrone Test, Molisch Test,
Benedict’s test, Barfoed’s test, Mucic acid test, Seliwanoff’s test, and Bial’s Orcinol test.
Benedict’s test is for reducing sugars such as glucose, galactose, xylose, and fructose.
Barfoed’s test is for reducing monosaccharides and is also positive for glucose, galactose,
xylose, and fructose. Mucic acid test is for galactose, this will detect both galactose and
lactose. Seliwanoff’s test is a test for ketohexoses; it is used in differentiating
ketohexoses and aldohexoses. Last but not the least, Bial’s Orcinol test is a test for
pentoses, it is used to differentiate pentoses from hexoses.

This experiment aims to differentiate carbohydrates by doing various specific

tests. It also aims to identify the unknowns by comparing their results with the standards
by looking at their similarities and differences.

Results and Discussion

STA (starch) CEL (cellulose) GLY (glycogen)

Anthrone Clear yellow with Clear yellow Clear yellow with
blue ring solution solution pale blue ring
solution
Molisch Violet interphase Light green and Light violet
light violet
 interphase interphase
Iodine Before heating: dark Before heating: light Before heating: light
blue yellow orange

Upon heating: Upon heating: Upon heating:

Clear pale yellow Clear light yellow Clear light yellow

solution solution solution

After heating: After heating: After heating:

Clear blue solution Clear pale yellow Clear light yellow

solution solution
Table 1: Results for General Test

The iodine test is used to test for the presence of starch. Starch turns an intense
"blue-black" colour upon addition of aqueous solutions of the triiodide anion, due to the
formation of an intermolecular charge-transfer complex. Polysaccharides can trap iodine
molecules and produce a deep blue-black product. Monosaccharides and disaccharides do
not yield deeply colored products.
Starch is a coiled structure and will turn blue-black. While Glycogen is a
branched molecule and will turn red-violet
The Anthrone test can be used for the qualitative and quantitative estimation of
polysaccharides as well as monosaccharides. The test is based on the dehydration of
monosaccharides to furfural derivatives. Furfural derivatives react with anthrone to form
a deep green color.
Molisch's test is a sensitive chemical test for the presence of carbohydrates, based
on the dehydration of the carbohydrate by sulfuric acid or hydrochloric acid to produce
an aldehyde, which condenses with two molecules of phenol resulting in a red- or purple-
colored compound.

FRU GAL GLU LAC MAL SUC XYL

 MUCIC - + - + - - -

colorless colorless colorless colorless colorless colorless

solution solution solution solution solution solution
with with
white white
crystals crystals

Table 2: Results for Mucic Acid Test

Figure 3: Mucic acid structure

Mucic acid (C6H10O8), also known as galactaric or meso-galactaric acid, is

an aldaric acid obtained by nitric acid oxidation of galactose or galactose-containing
compounds such as lactose,dulcite,quercite and most varieties of gum. It forms a
crystalline powder which melts at 230 °C and it is also insoluble in alcohol, and nearly
insoluble in cold water.

Mucic acid test will give a positive result for galactose and lactose since lactose is
a combination of glucose and galactose. Galactose, when oxidized by nitric acid, yields
soluble dicarboxylic acids. However, it will also yield an insoluble mucic acid. In the
case of lactose, mucic acid will be produced due to hydrolysis of the glycosidic linkage
between its glucose and galactose subunits. Being insoluble, galactosaccharic acid
crystals separate out.

FRU GAL GLU LAC MAL SUC XYL

 BARFOED’S + + + + + - +

2:42 55 secs 58 secs 5 mins 4 mins 5 mins 44 secs

mins

Table 3: Barfoed’s Test

Barfoed’s test is another test for reducing sugars. This test is used to differentiate
monosaccharides from disaccharides by looking at the rate of reaction.

Figure 4. Reaction of monosaccharides to Barfoed’s reagent

Figure 5. Results in Barfoed’s

test showing the negative result
(left) and positive result (right)
with a brick red precipitate

Monosaccharides are easily reduced to

Cu+ from Cu+2, which produces a brick red precipitate (Chua-Suba, 2011) (Figure 5).
Xylose, fructose, glucose, lactose galactose and xylose are the sugars positive for this
test, characterizing them as monosaccharides while maltose and sucrose appeared as a
blue solution; classifying them as disaccharides.

FRU GAL GLU LAC MAL SUC XYL

BENEDICT’S + + + + + - +
 Table 4: Results for Benedict’s Test

Figure 6. Benedict’s reagent’s reaction to sugars

In Benedict’s test, the free aldehyde or ketone groups of the reducing sugars have
the ability to reduce solutions of various metallic ions. Reducing sugars under alkaline
conditions tautomerise and form enediols, which is known to be a powerful reducing
agent that reduces cupric ions to cuprous ions. Cuprous ions then combine with –OH ions
to form yellow cuprous hydroxide, which when heated are turned into red cuprous
hydroxide (Chhabra, 2014). Ketoses can also reduce Benedict’s reagent because it
contains a ketone group on carbon 2 that can isomerize which in turn forms the aldehyde
on the first carbon. Sucrose does not give a positive result for Benedict’s test because it is
composed of an aldose and a ketose. Ketose is a five-membered ring, meaning its sixth
carbon member is hanging around therefore making it not a free anomeric carbon.
Maltose and lactose, on the other hand, are also disaccharides but they have free
anomeric carbon, which will serve as their reducing end. All the given sugar produces a
positive result except sucrose.

FRU GAL GLU LAC MAL SUC XYL

BIAL’S - - - - - - +

Table 5: Result for Bial’s Orcinol Test

Bial’s-Orcinol test is a test for pentoses—it is used to classify pentoses from

hexoses. The reagent consists of orcinol, HCl, and ferric chloride. There are two parts in
the principle of the reaction, namely: dehydration and condensation with orcinol. Furfural
is produced when pentoses are dehydrated in an acidic solution which in turn produce a
blue-green colored product when condensed with orcinol. Hexoses, however, when
dehydrated similarly yields 5-hydroxymethylfurfural and shows a brown colored product
when condensed with orcinol. Among the results, xylose appeared a positive result;
identifying it as a pentose.

FRU GAL GLU LAC MAL SUC XYL

SELIWANOFF’S + - - - - + -

Table 6: Result for Seliwanoff’s Test

Figure 7. A positive result for

Seliwanoff’s Test (on the
right), is a cherry-red solution

Seliwanoff’s test is for ketohexoses. It is

used to differentiate ketohexoses from aldohexoses. When ketohexoses reacts with
hydrochloric acid, it undergoes rapid dehydration to form 5-hydroxymethyl fualdehyde
which when condensed with resorcinol gives a cherry red complex (Chua-Suba, 2011).
This test gives a positive result for fructose and sucrose. Sucrose, consisting of glucose
and fructose, hydrolizes to form fructose which will be dehydrated and condensed to
produce a positive result.
 Figure 8. Reaction of Seliwanoff’s test to ketohexose

Experimental

There are three general test for carbohydrates, Molisch test, Anthrone test and
Iodine Test.

Molisch Test

The 10 drops of standard amylose was treated with 2 drops of Molisch’s reagent.
Down the side of the test tube, 10 drops of concentrated H2SO4 was carefully added in
the solution to form a layer.

Anthrone Test

To the spot plate, the 10 drops of Anthrone solution was placed, added to it is 1-2
drops of standard amylose solution. Repeat this using standard glycogen and standard
cellulose.

Iodine Test

A drop of iodine solution was added into 10 drops of standard amylase solution.
The mixture was then put to a water bath. Lastly, the solution was cooled for observation.

There are a total of 5 tests for specific reactions of carbohydrates namely

Benedict’s test, Barfoed’s test, Mucic acid test, Seliwanoff’s test, and Bial’s Orcinol test.

Mucic acid test

Figure 5. Positive result for Mucic acid test

 Medium-sized test tubes labeled with galactose, lactose, fructose, glucose,
maltose, sucrose and xylose were prepared. 20 drops of sugar solutions were added in
their respective test tubes. The addition of 20 drops concentrated HNO 3 in each test tube
was also done. The test tubes were plugged with cotton and were heated in a boiling
water bath for an hour. After an hour, the tubes were placed in the locker or stored in the
refrigerator. The results were observed and recorded on the next laboratory period.
Formation of crystals indicates a positive result for Mucic acid test.

Barfoed’s Test

A total of 7 test tubes were prepared labeled with glucose, galactose, maltose,
fructose, lactose and xylose.

Figure 4. Comparison of the negative (Left) and positive result for Barfoed’s test (Right)

In their respective test tubes, 10 drops of Barfoed’s reagent were placed, followed
by the addition of 5 drops of sugar solutions. The test tubes were then heated until a brick
red precipitate is observed. The test tube containing glucose should be closely observed;
once a brick red precipitate appears in it then all test tubes should be removed from the
water bath. A negative result is obtained when there is no brick red precipitate observed
after 5 minutes of heating .

Benedict’s Test

Test tubes labeled with glucose, galactose, lactose, sucrose, fructose, maltose and
xylose were prepared. After the test tubes were labeled, 5 drops of the standard sugars
were placed in their respective test tubes. The addition of 10 drops of Benedict’s reagent
was done afterwards. The test tubes were heated in the water bath until a muddy green
suspension is observed which settles as a brick red precipitate. Once the brick red
precipitate was seen, the test tubes were immediately removed from the bath and left on
the rack to cool. Results and observations were recorded.

Figure 9. Benedict’s test set-up

Bial’s Orcinol Test

Small-sized test tubes labeled with galactose, lactose, fructose, glucose, maltose,
sucrose and xylose were prepared. 5 drops of sugar solutions were placed in their
respective tubes and 10 drops of Bial’s orcinol reagent were added into each tube. The
tubes were heated in a boiling water bath until a blue-green solution is observed. Test
tubes were removed from the water bath and the results were observed and recorded.
 Figure 7. Positive result for Bial’s Orcinol test

Seliwanoff’s Test

Medium-sized test tubes labeled with galactose, lactose, fructose, glucose,

maltose, sucrose and xylose were prepared. 10 drops of Seliwanoff’s reagent was placed
in each of the labeled test tubes. The addition of 5 drops of sugar solutions in their
respective tubes was also done. The test tubes were then immersed in a boiling water bath
and heated until a cherry red solution was observed. Results and observations were
recorded.

Figure 6. Positive (right) and negative (left) result for Seliwanoff’s test

Conclusion
 Carbohydrates, being the third major class of biological molecules were subjected
to several general tests such as the Molisch test, Anthrone test and Iodine test. The
specific tests are Benedict’s test, Barfoed’s test, Mucic test, Seliwanoff’s Test, and
Bial’s-Orcinol test. In the qualitative tests, Benedict’s test is used to distinguish reducing
sugars. Barfoed’s Test also detects the presence of reducing sugars, monosaccharides and
disaccharides. On the other hand, Seliwanoff’s Test is used to detect presence of ketoses.
These are sugars containing one ketone functional group per molecule. While Bial’s-
Orcinol Test is used to detect pentoses, sugars containing five carbon atoms. In Mucic
Acid test, the concentrated HNO3 oxidizes galactose to an isomer of tetrahydroxyadipic
acid. This solution then crystallizes out from water. The resulting isomer is called mucic
acid. Galactose is a monosaccharide and an aldohexose. The disaccharide lactose can be
formed by the combination of galactose with glucose via dehydration reaction. Mucic
acid was formed from the galactose because of the oxidation of both aldehyde and
alcohol group. Several tests in the experiment yield a positive result by producing their
anticipated color change. However, some results weren’t that accurate. Nonetheless, the
objectives of the experiment were successfully carried out.

References
Shankara, S. (2008). Laboratory Manual for Practical Biochemistry. New Delhi, India:
Jaypee Brothers Medical Publishers (P) Ltd.

Appling, Anthony-Cahill, et. al. (2013). 4th edition Biochemistry. Pearson Inc: Canada.

Appling, Anthony-Cahill, et. al. (2016). Biochemistry Concepts and connections.

Pearson Inc: Canada.

Campbell, M.K. & Farrel, S.P. (2012). 7th edition Biochemistry. Cengage Learning:
International edition.

Chua-Suba, S. (2011). Laboratory Manual in Biochemistry. C&E Publishing

Incorporated: Quezon City.

Chhabra, N. (2014). Biochemistry for Medics - Lecture Notes. Retrieved March 06, 2017,
from http://www.namrata.co