General and Specific Tests for Carbohydrates

Alcazar, Rayson M., Cebu, Katrina R., Coronel, Ana Beatrice L., Lumbo, Justine J.,

Rubiano, Brian Andrew R., Salvatera, Diana Teresa G.

College of Science, University of Santo Tomas, Espana Blvd., Manila

Abstract
Carbohydrates are aldehydes or ketones of higher polyhydric alcohols – components that
yield derivatives on hydrolysis. Unknown sugars were given to each members of the
group for identification. Different tests, general and specific reactions, are performed to
each standard sugar solutions: xylose, glucose, fructose, galactose, lactose, maltose and
sucrose; likewise to given unknowns. General tests for carbohydrates include: Molisch
test, a general test for carbohydrates; Anthrone test, a test for determining non-reducing
sugars and reducing sugars; and Iodine test, which determines the presence of starch. On
the other hand, specific reactions for carbohydrates are as follows: Mucic acid test, a test
for the the presence of galactose, forming crystals as a positive outcome; Benedict’s test,
a test for reducing sugars, positive for all standard sugars given except for sucrose;
Barfoed’s test, test to identify monosaccharide, positive for glucose, xylose, fructose and
galactose; Bial’s Orcinol test, test to distinguish pentoses, positive for xylose; and
Seliwanoff’s test, a test for the presence of ketone sugars, positive for fructose and
sucrose.

Introduction
Carbohydrates, with a general formula of CnH2nOn, are the primary source of energy
required by the human body (Asif et al, 2011). These biomolecules are built by the simple
or complex sugars and are also defined as keto or aldehyde derivatives of polyhydric
alcohols or alcohols with many hydroxyl radicals. Plants are able to produce
carbohydrates by photosynthesis while in most animals, the quickly accessible reservoir
of energy is the one regarded as carbohydrates (Benedict et al, 2010).

There are two classifications of carbohydrates based on the nature of the reactive
carbonyl group, namely: aldose and ketose. Aldoses are those that contain aldehyde
functional group while ketoses are those that have ketone functional group.
Carbohydrates could also be classified according to the number of carbon atoms (e.g. 3
carbons = triose, 5 carbons = pentose, 6 carbons = Hexose). Additionally, according to
Asif et al. (2010), it could also be classified on the basis of carbon atom present in the
carbohydrates. They could be classifies as monosaccharides, disaccharides,
oligosaccharides or polysaccharides. Monosaccharides are simple sugars that cannot be
broken down into smaller units by hydrolysis. Xylose, Glucose, Galactose and Fructose
are all classified as monosaccharides. Meanwhile, Disaccharides are the simple sugars
that are composed of two monosaccharide units and it can be hydrolyzed. Examples of
these are lactose (combination of glucose and galactose) commonly known as milk sugar,
Maltose (combination of two glucose) or malt sugar and Sucrose (combination of glucose
and fructose) also known as table sugar. Oligosaccharides on the other hand are those that
contain three to ten monosaccharides. Raffinose which is a trisaccharide consisting
glucose, fructose, and galactose and Stachyose, a tetrasaccharide, which is made up of a
glucose, fructose, and two galactose are some of the examples of oligosaccharides.
Lastly, polysaccharides are those that consist more than ten monosaccharides such as
Amylose, Cellulose and Glycogen (Manalo, 2013) and (New Age Publishers).

The objectives of the experiments performed were to characterize the group of

compounds using general tests, namely: Molisch Test, Anthrone Test and Iodine Test and
to identify the unknown sugars using specific tests, namely: Mucic acid Test, Benedict’s
Test, Barfoed’s Test, Bial’s Orcinol Test and Seliwanoff’s Test.

Methodology
Three general tests and five specific reaction tests for carbohydrates were performed
for this experiment.
A.
The first general test is the Molisch test. Ten (10) drops of the standard amylose solution
was placed in a small test tube. Two (2) drops of Molisch reagent was added to the
solution and was mixed thoroughly. The tubes were inclined and 10 drops of concentrated
H2SO4 was allowed to flow down to the side of the tube. The color of the interphase that
formed was noted. All these steps were repeated for standard glycogen and cellulose.

The second test was the Anthrone test which was performed on a spot plate. Ten (10)
drops of Anthrone reagent was placed on one of the wells of the plate. Two (2) drops of
the standard amylose solution was added and the color that was formed was noted. These
steps were repeated for standard glycogen and cellulose.

The last general test was the Iodine test. Ten (10) drops of the standard amylose solution
with a drop of Iodine solution was placed in a test tube. The tube was heated in a boiling
water bath and the changes that were produced were recorded. After cooling the test
tubes, the changes that occurred were also recorded. All steps were repeated for standard
glycogen and cellulose.
B.
For the preliminaries, each group was assigned 2 unknown sugars which were dissolved
in 10 mL of distilled water. These were used for the specific reaction tests alongside the 7
standard sugar solutions.

The first test was the Mucic Acid test. Ten (10) drops of the sugar solutions were placed
in their respective test tubes and 10 drops of concentrated HNO3 were then added. The
test tubes were plugged with cotton and were heated in a boiling water bath for 1 hour.
The tubes were placed in the lockers and results were noted the nest day.

The next test was the Benedict’s test. Ten (10) drops of Benedict’s reagent was placed to
each test tube. Five (5) drops of each sugar solution were eventually added to each tube.
The tubes were heated in the water bath until a muddy green suspension which settles as
a brick res precipitate was observed. The tubes were then cooled and the results were
recorded.
The next test is the Bial’s Orcinol test which made use of 5 drops of the sugar solutions
and 10 drops of the Bial’s Orcinol reagent. The test tubes were heated in a boiling water
bath for 10 minutes. The color changes that were observed after 5 minutes were recorded.
After another 5 minutes, the changes were also recorded.

The last two tests are Barfoed’s test and Seliwanoff’s test. These two tests were
performed by other researchers. Results for this group’s unknown sugar solutions were
obtained from the other groups.

Results and Discussion


Table 1. Molisch Test Results

Molisch Test
Sample Results
Amylose Colorless Interphase
Glycogen Colorless Interphase
Cellulose Colorless Interphase

The molisch test determines the presence of nucleic acids, glycoproteins, and
carbohydrates such as monosaccharides, disaccharides, and polysaccharides in a given
sample (Ashok, 2014). It makes use of the Molisch reagent, primarily composed of 1-
napthol dissolved in ethyl alcohol (EtOH) and a strong mineral acid such as sulfuric acid
(H2SO4) for the hydrolization of glycosidic bonds and promotes the formation of furfural
products to consolidate with the Molisch reagent causing the violet interphase observed
upon this experiment.
Monosaccharides are being dehydrated by concentrated H2SO4 corresponding to the
formation of furfural products depending on the type of monosaccharide. Afterwards, the
furfurals and hydroxymethyl will then react with the present 1-naphtol dissolved in the
Molisch reagent, yielding a violet colored interphase inside the test tube. (See Fig 1)
As for the results derived from table 1, amylose— a linear chain of D-glucose
monosaccharides attached together by 1,4-glycosidic bonds— is subjected for the
Molisch test and is expected to give a positive result for its glycosidic bonds are
hydrolyzed by the H2SO4 and yields a furfural for the Molisch reagent to interact forming
a violet interphase. Similarly, glycogen— a stored carbohydrate of mammalian cells, and
cellulose— a fibrous carbohydrate in plants, yields a colorless interphase in the Molisch
Test. Unfortunately, human error is believed to be the case. It may be caused by an
improper utilization of necessary chemical reagents or other unnecessary laboratory
technique.

Fig 1. Reaction Mechanism of the formation of furfural and its interaction with the 1-naphtol of the
Molisch Reagent (Dongre, 2015)

Table 2. Anthrone Test Results

Anthrone Test
Sample Results
Amylose Blue-green Solution
Glycogen Light Blue-green Solution
Cellulose Light Yellow with Blue Solution

The anthrone test determines the intensity or quantity of the carbohydrates present in a
given sample (Ashad, 2016). In this experiment, furfurals and hydroxymethyl furfural
was also obtained through a strong mineral acid such as H2SO4 but in this case, it will be
interacted with the anthrone dissolved in the anthrone reagent. (See Fig 2.)
Usually, a spectrophotometer is used to determine the absorbance of a given sample and
compute for the concentration using the beer-lamber law (Ashad, 2016). As for this
experiment, the carbohydrate concentration is determined through the intensity of its
color, positive to this test. Therefore amylose, being darker compared to glycogen and
cellulose, will be recognized to have a higher amount of carbohydrates. (See table 2).

Fig 2. Reaction
mechanism (Ashad,
2016)

Table 3. Iodine Test Results

Iodine Test
Sample Results
Amylose Transparent Blue Solution
Glycogen Clear Light Yellow Solution
Cellulose Clear Light Yellow Solution

The iodine test determines the presence of starch in the given storages of carbohydrates in
Table 3. Amylose, together with another polymer called amylopectin, has a helical
structure that builds up the most important source of carbohydrates in the human diet
known as starch. It occurs in the form of granules in plants and breaks down into glucose
to act as a nourishment intake. On the other hand, glycogen acts as the carbohydrate
reserve in mammalian cells in the form of granules mainly in the liver. It is highly
branched in terms of structure and is also composed of D-glucose subunits. Cellulose
exists as a fibrous carbohydrate storage in the structural component of plant cells called
cell walls. Like amylose, it is a linear polymer of glucose attached by glycosidic bonds
that allows a great deal of hydrogen bonding between OH groups on adjacent chains,
causing them to come together into fibers.
As for the results derived from Table 3, the amylose was determined to be positive for
iodine test for it contains the accommodation of an iodine molecule to undergo amylose-
iodine complex inside the helical structure of the amylose, yielding a blue-black
colorization.

Table 4. Mucic Acid Test Results

Mucic Acid Test

Sample Results
Xylose No Crystals
Fructose No Crystals
Glucose No Crystals
Galactose No Crystals
Lactose No Crystals
Maltose No Crystals
Sucrose No Crystals
Unknown A No Crystals
Unknown B No Crystals

The mucic acid test determines the presence of galactose present in a given solution
(Shadid, 2009). It makes use of a strong oxidizing agent such as concentrated nitric acid
(HNO3) in the given sample and was suspended on a boiling water bath for 1 hour. The
HNO3 will oxidize the aldehyde and primary alcohol groups of the galactose and yields a
glucaric or saccharic acid. The presence of saccharic acid could be observes as a colorless
precipitate or crystals insoluble to the whole mixture.
As for table 4, the expected results should give a positive result for galactose and if
possible, a positive result on one of our unknown samples. Unfortunately, human error
was suspected again for this outcome.
Table 5. Benedict’s Test Results

Benedict’s Test
Sample Results
Xylose Brick-red precipitate in Orange Solution
Fructose Brick-red precipitate in Orange Solution
Glucose Brick-red precipitate in Orange Solution
Galactose Brick-red precipitate in Orange Solution
Lactose Brick-red precipitate in Orange Solution
Maltose Brick-red precipitate in Orange Solution
Sucrose Blue Solution
Unknown A Brick-red precipitate in Orange Solution
Unknown B Blue Solution

The benedict’s test is used to determine the reducing sugars (aldoses) that contains a n
aldehyde functional groups (Aryal, 2015). Reducing sugars are capable of transferring
hydrogens to other compounds called reduction. The benedict’s reagent is used for the
determination of reducing sugars with the aid of potassium thiocyanate forming a white
precipitate while the Copper (II) ions of the benedict’s solution is then reduced to Copper
(I) ions that causes the color change forming an orange-red solution (See Fig 3). Upon
boiling, a red solution was monitored from xylose to maltose due to the formation of
potassium thiocyanate precipitate and the reduction of the benedict’s reagent, and are
then subjected to be reducing sugars. On the contrary, sucrose gave a negative result of
blue solution due to its disaccharide attributes. As for the given results of table 5,
unknown A is therefore a reducing sugar and unknown B is not.
Fig 3. The interaction of an aldose sample with the benedict’s solution
Table 6. Bial’s Orcinol Test

Benedict’s Test
Sample Results (5 mins) Results (10 mins)
Xylose Blue-green solution Blue-green solution
Fructose Dark brown solution Dark brown solution
Glucose Dark green solution Dark green solution
Galactose Dark green solution Dark green solution
Lactose Dark green solution Dark green solution
Maltose Dark green solution Dark green solution
Sucrose Dark brown solution Dark brown solution
Unknown 1A Yellow solution Dark brown solution
Unknown 1B Brown solution Dark brown solution
Unknown 2A Dark blue-green solution Dark brown solution
Unknown 2B Brown solution Dark brown solution
Unknown 3A Brown solution Dark brown solution
Unknown 3B Brown solution Dark brown solution w/
precipitate
Unknown 4A Brown solution w/ Dark brown solution w/
precipitate precipitate
Unknown 4B Brown solution Dark brown solution w/
precipitate
Unknown 5A Brown solution Dark brown solution w/
precipitate
Unknown 5B Brown solution Dark brown solution w/
precipitate

The bial’s orcinol test is used to determine the difference between pentoses and hexoses
or monosaccharides. They react with the bial’s orcinol reagent and are converted to
furfural, a condensed product with a color of blue-green which indicates that it is a
pentose (Shreck & Loffredo, n.a.). Xylose yielded a positive in this test, as for the
unknowns, unknown 2A could be subjected as a pentose.
Conclusion
In this experiment, different tests were subjected for the given carbohydrates to trace their
composition that serves as an essential component of human consumption. The following
are therefore concluded:
Molisch test determines the presence of nucleic acids, glycoproteins, and carbohydrates.
It promotes the hydrolization of glycosidic bonds and the formation of furfural products
yielding a violet colored interphase. Results have shown that Molisch test in amylose has
given a positive forming a violet interphase. The glycogen in the other hand yields a
colorless interphase due to unrealized human error done.
Anthrone test determines the intensity or quantity of the carbohydrates present in a given
sample. As for this experiment Amylose, results darker compared to glycogen and
cellulose, and recognized to have a higher amount of carbohydrates.
Iodine test determines the presence of starch in the given carbohydrate. Amylose,
possessing amylopectin that builds up carbohydrates known as starch then partakes a
positive results for it accommodates iodine molecule yielding a blue-black colorization.
The mucic acid test determines the presence of galactose present in a given solution. Due
to another human handling error, nonviable results were obtained hence getting into a
decision of researching which further intervened that mucid acid should give a positive
result for galactose.
The benedict’s test is used to determine the reducing sugars (aldoses) that contains an
aldehyde functional groups. Xylose to maltose were positive outcomes that resulted to a
red formation from potassium thiocyanate precipitation, and benedict’s reagent sugar
reduction, On the contrary, sucrose gave a negative result of blue solution due to
discharge solutions.
The bial’s orcinol test is used to determine the difference between pentoses and hexoses
or monosaccharides. Xylose is known to be positive for this test. unknown 2A could be
subjected as a pentose.
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