General and Specific Test of Carbohydrates

Raylene Audrey A. Diolata*, Kathleen May C. Gordola, and Maria Carmela C. Guerra
College of Science, University of Santo Tomas, Manila, Philippines

Abstract
Carbohydrates are one of the four biomolecules that make up life and commonly
called as sugars. They are divided into monosaccharides, disaccharides,
oligosaccharides and polysaccharides. These sugars were identified using a set of
biochemical tests that is categorized into general and specific reaction tests. General
tests such as Molisch, Anthrone and Iodine were done on polysaccharides. While
Specific reaction tests such as Mucic, Benedict’s, Barfoed’s, Bial’s Orcinol and
Seliwanoff were done on monosaccharides and disaccharides. These tests were able to
set standards in identifying the unknown sugar, which was concluded to be Xylose.

Introduction
One of the most important sources of energy is Carbohydrates, a polyhydroxyl
alcohol consisting of aldehyde and ketone. They are commonly called as sugars and
can be divided into four groups: monosaccharides, disaccharides, oligosaccharides and
polysaccharides. These groups can be differentiated by chemical tests formulated
based on their degree of polymerization. These tests can be categorized into General
and Specific Reaction. These General tests included Molisch, Anthrone and Iodine
tests. While the Specific Reaction tests included Mucic, Benedict’s, Barfoed’s, Bial’s
Orcinol and Seliwanoff tests.
There were three standards used for the General tests: Amylose, Glycogen and
Cellulose. These sugars are polysaccharides hence they are used for the general tests.
For the Specific Reaction tests, there were Glucose, Galactose, Fructose, Xylose,
Maltose, Lactose, and Sucrose. The first four of the sugars are monosaccharides and
the rest are disaccharides. These is a distinction between the two categories and
between all of the sugars, hence the use of a specific reaction test.
This experiment aims to understand the principles and procedures of these
chemical tests used to identify carbohydrates. An unknown sugar, along with standard
sugars, will be tested and identified based on the standard’s reactions.

Results and Discussion

Amylose and Glycogen are storage polysaccharides found in plants and animals
respectively. Amylose is a linear polymer chain containing glucose and make up 20-
25% of starch, while Glycogen is a branching polymer chain – similar to Amylopectin –
and serves as primary energy storage for animals and fungi. Lastly, Cellulose is a linear
β-sheet polyer chain and the main component of plant cell wall.
 General tests were conducted to qualitatively analyze the given samples. The
tests conducted included Molisch test, Anthrone test, and Iodine test. The samples were
three kinds of carbohydrates found in three different groups of organisms.

Tests/Sugars Amylose Glycogen Cellulose

Light pink Lighter pink
Molisch Clear pink
w/ suspension w/ suspension
Anthrone Yellow green Yellow green Yellow green
Cloudy light yellow
Blue solution Light yellow solution
solution
Iodine Cloudy lighter yellow
Light Yellow solution Lighter yellow solution
(Before, During, After) solution
Cloudy lighter yellow
Blue solution Lighter yellow solution
solution
Table 1 Results for General Tests

The table shows the reactions of amylose, glycogen and cellulose to the general
tests. The sugars didn’t show the ideal results of the test, which a purple ring that
indicates for positive reaction. This should he be all positive due to the presence of
carbohydrates, which are essentially the samples themselves. This should also hold
true for Anthrone test, which tests for the carbohydrates as well. They should have a
blue-green complex however, there were only yellow solutions. Lastly, the iodine test
should only show a positive result to the amylose due to the presence of it’s α-D
gluxose units and form a deep-blue color. The rest only showed a yellow solution.

Monosaccharides and Disaccharides were also used in the test.

Monosaccharides are the simplest unit of carbohydrates and serve as the building
blocks for other complex saccharides. They are soluble in water and usually colorless.
These sugars can be classified based on the functional group present. If there is a
ketone group present in the inner atom, this is called ketoses. Meanwhile, if an
aldehyde group can be found at the end of the molecule, the monosaccharide is an
aldose. These differences in their structure are helpful in identifying the sugars from
each other. Examples of these – which were used in the experiment – are glucose,
fructose, galactose and xylose.

Meanwhile, Disaccharides are when two monosaccharides are bound together

by a glycosidic bond. They are joined by dehydration or also known as condensation
reaction. Common examples of these sugars – that were used on the experiment – are
Maltose, Lactose and Sucrose. Maltose is made up of two glucose, Lactose is a
galactose and a glucose. And then Sucrose is made up of glucose and fructose.

Specific reaction tests were done to differentiate or to pin point the type sugar the
unknown is. The tests included Benedict’s test, Barfoed’s test, Bial’s Orcinol test and
Seliwanoff’s test. Mucic was only used for comparison but was not done due to
limitations of the laboratory facility. The results are as follows.
Test/Sugar Glucose Maltose Fructose Sucrose Xylose Galactose Lactose

Clear Clear Clear Clear Rhombic Rhombic Clear

Mucic
solution solution solution solution Crystals Crystals solution
Brick red Brick red Brick red Blue Brick red Brick red Brick red
Benedict’s
precipitate precipitate precipitate solution precipitate precipitate precipitate
Brick red Blue Brick red Blue Brick red Brick red Blue
Barfoed’s precipitate solution precipitate solution precipitate precipitate solution
fast slow fast slow fast fast slow
Blue-
Bial’s Clear Clear Clear Clear Clear Clear
green
Orcinol solution solution solution solution solution solution
solution
Cherry Cherry Blackish- Red-ish Red-ish
Red Clear
Seliwanoff’s red red yellow orange orange
solution solution
solution solution solution solution solution
Table 2 Results of Standards for Specific Tests

The table shows the distinct reactions of sugars to each specific test. For the
Mucic test, we can see that only xylose and galactose produced rhombic crystals, while
the rest has no color reaction. For the Benedict’s test, all standards produced a brick
red precipitate except sucrose. In the Barfoed’s test, blue solutions were observed in
maltose, lactose and sucrose after 5 minutes. They were categorized as slow
(disaccharide) due to having needed more time to produce a brick red precipitate.
Meanwhile the rest were labeled fast (monosaccharides), having produced brick red
precipitates for less than 5 minutes. However in the Bial’s Orcinol’s test, only Xylose
produce a blue solution while the rest stayed clear after 5 minutes of heating. Lastly, in
the Seliwanoff’s test, the fructose and sucrose standards produce a cherry red solution.
The galactose and lactose similarly produced red-orange solutions, while glucose and
xylose produced red and blackish-yellow solutions respectively.

The specific reaction tests were done on the unknown sugar as to characterize
the sugar. The results were compared as follows.

Tests Unknown Sugar Xylose

Mucic N/A Rhombic Crystals
Benedict’s Brick red precipitate Brick red precipitate
Brick red precipitate Brick red precipitate
Barfoed’s
(fast) (fast)
Blue-green solution
Bial’s Orcinol Blue-green solution
34 secs
Seliwanoff’s Blackish-yellow solution Blackish-yellow solution
Conclusion: Xylose
Table 3 Comparison of Unknown Sugar for Specific Tests Results

The table shows that the unknown sugar had a similar result from the xylose
standard with the specific reaction test. The results of both the standard and the
unknown sugar we’re similar for all the tests done. Only Mucic test was not done due to
the limitations of the laboratory. And so, it is concluded that Xylose is Unknown sugar.
Experimental
There were two parts to the experiment: General test on Polysaccharides and
Specific Reaction test on various standard sugars. For the General tests, the following
procedures were done accordingly to the test.
Molisch Test
In three medium sized test tubes, ten drops of standard amylose,
glycogen and cellulose solutions were added respectively. Then, two drops of
Molisch reagent – which is α-naphthol dissolved in ethanol – per test tube were
added and mixed thoroughly. After so, ten drops of concentrated H2SO4 were slid
down the side of the inclined test tube. In principle, this concentrated acid would
dehydrate the carbohydrates, therefore should only show a reaction if sample
was carbohydrates. The test tubes were observed for a red violet or purple ring
formed at the interphase of two liquids. Data was recorded accordingly per sugar
standard.

Anthrone test
In a spot plate, ten drops of Anthrone solution – which consists of
anthrone reagent and concentrated H2SO4 – was placed in each well assigned for
the three standards. Then, one to two drops of the standards were added. The
concentrated H2SO4 would dehydrate the carbohydrates and the anthrone would
then condense to form a blue green color complex. These reactions were
observed among all the wells with the standards - a color change indicating the
presence of carbohydrates.

Iodine test
In separate test tubes, ten drops of the standards were added followed by
a drop of iodine solution. This iodine solution should form a deep blue color
 complex linear and helical saccharides, a red violet color with branched
saccharides and no complex with beta sheet saccharides – which will give a
yellow color only. Observations of the mixture were recorded. Then, the tubes
were heated in a boiling water bath and observed again. Once reaction was
recorded, remove the tube and cool the solution. Observations on the color
reactions were once again recorded.

For Specific Reaction tests, an unknown solid was given and half of it was
dissolved in 10mL distilled water. This sugar solution was tested alongside with seven
sugar standards. The following procedures were done accordingly.
Mucic test
In 8 medium sized test tubes, ten drops of the sugar solutions – the 7
standards including the unknown – should be placed in respective tubes. Ten
drops of concentrated HNO3 will be added to each tube and plugged with cotton
before heating in boiling water bath for 1 hour. The tubes will be removed from
heat and put inside locker until next meeting. Crystals would be observed due to
oxidation of both aldehyde and primary alcohol group at C1 and C6.
Benedict’s test
In 8 medium sized test tubes, ten drops of Benedict’s reagent were placed
and then five drops of the corresponding sugar solutions were added for each
tube. The tubes were heated in a water bath until muddy green suspension was
observed. This will settle as a precipitate and will give the rough estimate of
reducing sugar present.

Barfoed’s test
In 8 medium sized test tubes, ten drops of Benedict’s reagent were placed
and then five drops of the corresponding sugar solutions were added for each
tube. The tubes were heated in a water bath until brick red precipitate was
observed. Time during reaction must be recorded. Remove from water bath
immediately after the color reaction and cool. This reaction is due to Aldoses and
Ketoses reducing cupric ions in acidic conditions. Monosaccharides will reduce
much faster than disaccharides. Therefore, if precipitate is observed in less than
5 minutes, the standard is a monosaccharide. The test would be negative result if
brick red precipitate is not observed after 10 minutes.
Bial’s Orcinol test
In 8 medium sized test tubes, five drops of the sugar solutions was placed
in their respective tubes and then was added with ten drops of Bial’s Orcinol
reagent each. The tubes were heated until a blue-green solution was observed –
the time was noted. This color was due to pentoses dehydrating to form furfural
that will react with orcinol and iron ion that form the blue complex. The color
reactions in the duration of the 5 minute heating were recorded before the tubes
were removed from the water bath.

Seliwanoff’s test
In 8 medium sized test tubes, ten drops of Seliwanoff’s reagent were
placed and then five drops of the corresponding sugar solutions were added for
each tube. The tubes were heated in a boiling water bath until a cherry red
solution is produced – time was noted. This cherry red color is due to the 5-
hydroxy methyl furfural (which had come from hydrochloric acid treatment of keto
hexoses) condensed with resorcinol. Observe the color reactions and record.
Conclusion
In conclusion, carbohydrates are categorized into monosaccharides, disaccharides,
oligosaccharides and polysaccharides. These sugars can be put under tests that can
identify them using the results as their characteristics. These tests are general and
specific reaction. In the experiement, we were able to identify the unknown sugar was
xylose due to its similar results from the specific reaction tests.

References:
http://study.com/academy/lesson/amylose-structure-formula-function.html
https://byjus.com/chemistry/glycogen/
http://study.com/academy/lesson/cellulose-in-plants-function-structure-quiz.html
https://msu.edu/course/lbs/145/luckie/Lab1.html
http://study.com/academy/lesson/monosaccharides-definition-structure-examples.html\
http://study.com/academy/lesson/disaccharides-definition-structure-types-
examples.html