Characterization of Some Monosaccharides, Disaccharides, and

Polysaccharides by Colorimetric Tests and Tests based on Furfural

Formation, Oxidation, and Reducing Property of Sugars

Martinez, Githea Philline C., Obmasca, Ma. Cristine Joy A., Palalay, John Alvin D.

*Paraguison, Arthur Lorenz D.

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

Abstract
Three common polysaccharides (amylose, glycogen, and cellulose) were subjected into colorimetric tests
such as the Molisch, Anthrone, and Iodine tests which characterized them. Standard sugar solutions of
some monosaccharides and disaccharides were done several tests such as the Mucic Acid, Barfoeds,
Benedicts, Bials Orcinol, and Seliwanoffs tests which yielded varying results per sugar solution that led
to the comparison and in turn solving the problem of identification of an unknown sugar solution.
____________________________________________________________________________________

Introduction
There are four essential macromolecules that supports life and its processes, and one of

these which are also the most abundant are called carbohydrates. Carbohydrates serve

as a vital source of energy for all living organisms. They are characterized by having the

general formula CnH2nOn (with some exceptions), and are able to be classified into many

types. As the term carbohydrate is synonymous with saccharide (Gk. sakkharon =

sugar), it can then be categorized into four groups based on the number of carbohydrate

molecules: monosaccharide (one), disaccharide (two), oligosaccharides (2-10), and

polysaccharides (>10).

Monosaccharides are further classified by either: (1) the number of carbon atoms they

contain. Trioses have three carbon atoms, tetroses have four, pentoses have five, and

hexoses have six; and (2) on whether the carboxyl group (C=O) is attached to the first, or
second carbon in the structure, in which the carboxyl group attached to the first carbon in

the structure are called aldoses, since it has the presence of an aldehyde (-CHO) group,

and when attached to the second carbon, they are known as ketoses, because it contains

a keto (-C=O) group. Examples of monosaccharides are glucose (blood sugar), fructose

(fruit sugar), and galactose (brain sugar). In turn, a five-carbon aldose is known as

aldopentose (glucose and galactose), and fructose is a ketohexose, meaning it contains

six carbon atoms and the carboxyl group is attached to the second carbon in the structure.

Disaccharides are also defined by the combination of two monosaccharides, wherein

some examples are maltose (malt sugar, from the combination of two glucose

compounds), sucrose (table sugar, from the combination of glucose and fructose), and

lactose (milk sugar, from the combination of glucose and galactose). Oligosaccharides

encompasses the disaccharides, and also may contain two to ten monosaccharide units,

which are all linked by glycosidic bonds, a type of covalent bond.

Finally, polysaccharides are the most complex form of carbohydrates, where each play

an essential role in biological activities. It is synthesized by plants and animals to be

utilized either as food, for support, or for metabolization of energy. An example of which

is glycogen, a major glucose storage polymer found in animals, and its function in the

release of glucose from storage can be found in muscle cells during exercise, and in the

liver, during digestion where it aids, with the pancreas, in facilitation of the blood sugar

levels. Glycogen is a compact structure which resulted from coiling of polymer chains, in

which this permits large amounts of energy to be stored within a small volume. In the case

of plants, they produce starch, a polysaccharide synthesized from photosynthesis, which

serves as its food supply. Starch can be further dissected into amylose, which makes up
10-30% of starch and is water-soluble, and amylopectin which is 70-90% composition of

starch and is water-insoluble, making the former more desirable in the storage of plant

energy. And lastly, Cellulose, is a carbohydrate which function is found in the cell wall of

plants, giving it its rigidity. In the industry it is made to produce paper, cotton, etc.

The objectives to be accomplished by the end of this experiment are: (1) to subject

standard amylose, glycogen, and cellulose to several color reactions which are based on

the dehydrating action of concentrated acids on sugars producing furfural and its

derivatives, which in turn, condenses with phenols or aromatic amines forming dyes.; and

(2) to perform characterization and specific tests on the following group of compounds

called carbohydrates to be able to identify an unknown monosaccharide or disaccharide

Methodology

A. General Tests for Carbohydrates

A.1 Molisch test

Five drops of Molisch reagent was added to a small test tube which contained

ten drops of standard amylose. It was then thoroughly mixed. The tube was

inclined and ten drops of concentrated H2SO4 (sulfuric acid) was then carefully

allowed to flow down the side of the tube. Finally, the color formed at the

interphase of the two liquids was noted and then recorded. The steps above

were also done this time using standard glycogen, and then standard cellulose.
 A.2 Anthrone test

Ten drops of Anthrone solution was added to the well of a spot plate which

contained five drops of the standard amylose solution. The contents of the well

was thoroughly mixed. Then at last, the color formed was noted and recorded.

The steps above were also done this time using standard glycogen, and then

standard cellulose.

A.3 Iodine test

Three drops of iodine solution was added to the smallest size of test tube which

contained ten drops of the standard amylose solution. A first record of the

observation was made for the result. After which, the tube was heated in a

water bath, and changes produced was also noted as a second record. The

tube was then removed from the bath and the solution cooled. The third record

of observation was made for this. The steps above were also done this time

using standard glycogen, and then standard cellulose.

B. Specific Reactions of Carbohydrates

B.1 Preliminaries

A big, dry, clean, and stoppered test tube was prepared and then labeled with

the seat number, course, year and section and was submitted to the instructors

for the identification of the unknown. It was then acquired after it was made

available. In a large test tube, half of the solid unknown sugar was dissolved in

10 mL of distilled H2O, which served as the unknown solution to be used for

the specific chemical tests. The other half was stored for future use, and finally
 a water bath was prepared on a hot plate, where all tubes required to be heated

was placed.

B.2 Mucic acid test

Eleven medium-sized test tubes with the standard sugars and the unknown

were labeled. Ten drops of the sugar solution was then placed in their

respective tubes. The solutions were mixed well after the addition of ten drops

of concentrated HNO3 (nitric acid) to each tube. The tubes were plugged with

cotton and was then heated in a boiling water bath for one hour, and was left

to stand until the next laboratory period and was stored in a refrigerator. The

standard sugars which formed crystals were noted.

B.3 Benedicts test

Eleven medium-sized test tubes with the standard sugars and the unknown

were labeled. Ten drops of Benedicts reagent was placed to each of the test

tubes, followed by adding ten drops of the sugar solutions in their respective

tubes. The tubes were then heated in the water bath until a muddy green

solution was observed which settled as a brick red precipitate. The tubes were

then removed after five minutes or even at the instant it formed the color

change and was cooled on the rack. Results were then recorded.
B.4 Barfoeds test

Eleven medium-sized test tubes with the standard sugars and the unknown

were labeled, where ten drops of Barfoeds reagent were placed, and was then

mixed with 10 drops of the sugar solutions. The tubes were heated in the water

bath until a brick red precipitate was observed, and the time it took for it to

appear was noted. The tubes were finally removed and observations were

recorded.

B.5 Bials Orcinol test

Eleven small-sized test tubes with the standard sugars and the unknown were

labeled, where ten drops of the sugar solutions were placed, and was then

mixed with 10 drops of Bials orcinol reagent. The tubes were heated in the

water bath until a blue-green solution was observed, and the time it took for it

to appear was noted. The colors formed during 5 minutes of heating were

noted, the tubes were removed from the water bath, and observations were

recorded.

B.6 Seliwanoffs test

Eleven medium-sized test tubes with the standard sugars and the unknown

were labeled, where ten drops of Seliwanoffs reagent were placed, and was
 then mixed with 10 drops of the sugar solutions. The tubes were immersed in

the water bath until a cherry red solution was observed, and the time it took for

it to appear was noted. The tubes were finally removed and observations were

recorded.

Results and Discussion

A. General Tests for Carbohydrates

I. Molisch Test

Table 1: Results for Molisch Test

Result
Amylose purple ring at interphase
Glycogen purple ring at interphase
Cellulose purple ring at interphase

Molisch test, also known as -naphthol reaction, is a test for the presence of

carbohydrates, meaning all sugar solutions are expected to show a positive result, which

is the formation of a purple ring between the two layers of the solution. The Molisch test

is useful for identifying any compound which can be dehydrated to furfural of

hydroxymethylfurfural in the presence of sulfuric acid. The reagent used, -naphthol in

95% ethanol, reacts with cyclic aldehydes, forming a purple product at the interphase.

The tests principles include the hydrolysis of the glycosidic bonds forming the reduced

sugar, dehydration of the monosaccharide into hydroxymethyl furfurals, and lastly, the

condensation of the furfural with -naphthol, shown in the figure below, wherein the
furfurals are shown to react with -naphthol present in the test reagent, producing the

purple product at the interphase.

Figure 1: Condensation of furfural with -naphthol

(courtesy of Google images)

II. Anthrone Test

Table 2: Results for Anthrone Test
Result
Amylose Dark blue-green solution
Glycogen Blue-green solution
Cellulose Light blue-green solution

The Anthrone test is a known as a colorimetic test that yields a blue-green solution to

appear in the presence of sugar in a sample, again meaning that all samples must exhibit

a positive result in this test. It also determines how much sugar concentration is in a

sample of any substance. The reagent used is Anthrone reagent (0.3 g anthrone added

into 100 mL conc. H2SO4). The principle of this test is shown in the figure below: in the

hydrolysis of carbohydrates, dehydration forming either a furfural or a 5-

hydroxymethylfurfural, and the condensation of anthrone via enthranol intermediate.

 III. Iodine Test

Table 3: Results for Iodine Test

before heating during heating After cooling
Amylose Dark blue Clear blue Dark blue
solution solution solution
Glycogen Clear light Clear light yellow Clear light
yellow solution solution yellow solution
Cellulose Clear light Clear light yellow Clear light
yellow solution solution yellow solution

The Iodine test is a test which is for helical carbohydrates, such as amylose. It is the

polysaccharides which trap iodine molecules and produce a dark black-blue product

which is the indicator of a positive result. The reagent used, IKI, or also known as Lugols

iodine reagent, is responsible for the black-blue color in the presence of starch. The

reaction is due to the formation of polyiodide chains from the reaction of amylose and

iodine. The principle of this test in the case of amylose as a component of starch, where

it forms helices where iodine molecules (I2) assemble, which forms a dark blue or black

color tapped in the helical structure. When a color change does not occur, as in the case

of the cellulose, it indicates that there was a completion in hydrolysis of the

polysaccharide. Theoretically, cellulose should have also yielded a positive result but a

different color (red) instead, but a possible error could be contamination.

B. Specific Reactions of Carbohydrates

I. Mucic acid test (for galactose and lactose)

Mucic acid, also known as galactaric acid, undergoes a reaction in which there is an

oxidation of most monosaccharides by nitric acid and yields soluble dicarboxylic acid.
However, oxidation of galactose yields an insoluble mucic acid, which will also be the

result in lactose due to hydrolysis of the glyccosidic linkage between glucose and

galactose, and the indicator of this positive result is the formation of crystals in the solution

after being left for ~2 days. One reagent uses is concentrated HNO3. The principle

involved is the 1,6-oxidation of sugars where galactose-containing carbohydrates forming

a meso compound which upon standing will yield crystals. In the identification of unknown,

in unknown sample #28 it seems that this test will determine whether the unknown is

actually glucose (negative result), or galactose (positive result).

II. Tests based on the reducing property of the carbohydrate

A. Benedicts test

Sugar solution Result

Xylose (XYL) Dark brown ppt in brown solution

Fructose (FRU) dark brown ppt in brown solution

Glucose (GLU) Brick red ppt in yellow solution

Galactose (GAL) Brick red ppt in yellow solution

Lactose (LAC) Brick red ppt in yellow solution

Maltose (MAL) Brick red ppt in yellow solution

Sucrose (SUC) green solution

#25 Greenish-brown solution

#26 Brick red ppt in brownish-orange solution

#27 Yellow green solution

#28 Brick red ppt in muddy green solution

Table 4: Results for Benedicts test

The Benedicts test is a test which is specifically for reducing sugars, and was performed

in mildly basic conditions. The reagents are CuSO4, Na2CO3, Na3C6H5O7, and sodium

citrate in H2O. Sodium carbonate is required to turn the solution basic, and the Benedicts

reagent, which incorporates sodium citrate to keep the cupric salts in the solution by

forming complex ions with them, preventing precipitation of copper (II) carbonate. The

positive result will yield a muddy green suspension which settles as a brick red precipitate

(which is Copper (I) oxide), which indicates the reducing power of the sugar. The principle

involves the oxidation of carbohydrates by copper ions to form a carboxylate ion group.

Sodium gluconate (Na3C6H5O7) is the sodium salt of gluconic acid. All except sucrose will

yield a positive result in this test, and the reason for sucrose being a nonreducing sugar

is that in contrast to maltose and lactoses reducing ends from a free anomeric carbon, in

which is not free in sucrose, and since it is a disaccharide containing an aldose and a

ketose, there is no free carbon in this sugar which makes it nonreducting. The unknowns

#25 and #27 yielded a negative result in the test, having a high possibility that sucrose is

the identity of their unknown sugar solution, although since the standard solution of

sucrose in the laboratory was contaminated, further tests and analyzation should be done.
 B. Barfoeds Test

Table 5: Results for Barfoeds Test

Sugar solution Result Reaction Time (min)

Xylose (XYL) Brick red ppt 1.68

Fructose (FRU) Brick red ppt 1.60

Glucose (GLU) Brick red ppt 1.85

Galactose (GAL) Brick red ppt 1.30

Lactose (LAC) Brick red ppt 4.85

Maltose (MAL) Brick red ppt 3.76

Sucrose (SUC) Brick red ppt 4.50

#25 Brick red ppt -

#26 Brick red ppt 0.77

#27 Brick red ppt 3.75

#28 Brick red ppt 1.83

The Barfoeds test will distinguish reducing monosaccharides and disaccharides by a

difference in the rate of reaction, where the former will always tend to have a faster

reaction rate as compared to the latter. The reagents used are copper (II) acetate, and

diluted acetic acid, which is the component in the positive result, a brick-red precipitate

which is copper (I) oxide. The principle is the oxidation of a reducing monosaccharide is

faster than that of the disaccharide, where the monosaccharides are oxidized by the

copper ion in the solution, forming a carboxylic acid and the brick red precipitate normally

within three minutes. From this, it can be inferred that unknowns #26 and #28 are actually
reducing monosaccharides and #27 is more or less a disaccharide, for it exceeded three

minutes, indicating a slow rate of reaction.

Figure 2: the oxidation reaction in the Barfoeds test

III. Tests for production of furfural or its derivative

Aldo- and ketopentoses rapidly undergo dehydration to give furfural under acidic

conditions, while ketohexoses also rapidly undergo dehydration to yield 5-

hydroxymethylfurfural, and aldohexoses are slowly dehydrated to 5-

hydroxymethylfurfural under acidic conditions as well.

A. Bials Orcinol Test

Table 6: Results for Bials Orcinol test

Sugar solution Result Reaction time (min:sec)

Xylose (XYL) Blue-green solution 1:09

Fructose (FRU) Black-brown solution 5:00

Glucose (GLU) Yellowish-brown solution 5:00

Galactose (GAL) Yellowish-brown solution 5:00

Lactose (LAC) Yellowish-brown solution 5:00

Maltose (MAL) Yellowish-brown solution 5:00

Sucrose (SUC) Black-brown solution 5:00

 #25 Yellowish-brown solution 5:00

#26 Dark brown solution 5:00

#27 - -

#28 Yellowish-brown solution 5:00

The Bials Orcinol test is a specific test for pentoses (a carbohydrate which contains five

carbon atoms), to differentiate it from hexoses (a carbohydrate which contains six carbon

atoms). The reagents used are Orcinol, also known as 5-methylresorcinol, as the

condensation reagent, 10% ferric chloride (FeCl3) as the catalyst, and concentrated

hydrochloric acid (HCl). The visible positive result for this test is a blue-green solution,

which is only evident in the only standard pentose sugar, which is xylose. The principle

involved is dehydration forming a furfural and condensation with orcinol. The reaction that

occurred was when the pentose sugar yielded furfural on dehydration in an acidic

solution, the furfural in turn reacted with orcinol and ferric chloride which gave the blue-

green condensation product. The other sugars yielded 5-hydroxymethylfurfural which was

responsible for the brown solutions. Since all of the unknown did not yield a blue-green

solution, xylose was crossed out of the possible identity of the unknown of the members.
 A. Seliwanoffs Test

Table 7: Results for Seliwanoffs Test

Sugar solution Result Reaction Time (min:sec)

Xylose (XYL) Green solution 5:00

Fructose (FRU) Cherry red solution 3:00

Glucose (GLU) Cherry red solution 3:29

Galactose (GAL) Cherry red solution 5:23

Lactose (LAC) Light red solution 8:23

Maltose (MAL) Cherry red solution 2:12

Sucrose (SUC) Cherry red solution 2:51

#25 Cherry red solution -

#26 Cherry red solution 1:41

#27 Cherry red solution -

#28 Dark green solution 5:00

Seliwanoffs test is a specific test for ketohexoses, a ketose sugar with six carbon atoms,

which are fructose, and sucrose (from glucose + fructose). The test is dependent on the

relative rates of dehydration of carbohydrates, in distinguishing aldohexoses from

ketohexoses. The reagents used were resorcinol, as the condensation reagent, and

hydrochloric acid (HCl), as the dehydrating acid. The positive result is the formation of a

cherry red condensation product. The principle is the rapid dehydration forming 5-

hydroxymethyfurfural and condensation reaction with resorcinol to yield a cherry red

solution. In the reaction, the ketohexose reacts quickly to yield 5-hydroxymethylfurfural,

while the aldohexose will have a slower rate of reaction. 5-hydroxymethylfurfurals

reaction with resorcinol will then yield a cherry red condensation product, a light-colored

red or pink product is an indicator that it took a longer time to react with Seliwanoffs

reagent. An error might have occurred but nonetheless a negative result in observing a

green solution in unknown #28. The fastest time, for unknown #26, may indicate that it is

a ketohexose.

So, as a recap:
Table 8: Results of the unknown in each specific test
Test (time of Unknown #25 #26 #27 #28
appearance)
Mucic Acid - - - -
Benedicts Greenish- Brownish- Yellow green Brick red ppt in
brown solution orange solution solution muddy green
with red ppt solution
Barfoeds Brick red ppt in Brick red ppt in Brick red ppt in Brick red ppt in
blue solution blue solution blue green green solution
(3:15) (0:38) solution (3:45) (1:49)
Bials Orcinol Yellowish- Dark brown - Yellowish-
brown solution solution (5:00) brown solution
(5:00) (5:00)
Seliwanoffs Cherry red Cherry red Cherry red Dark green
solution solution (1:41) solution solution (5
mins)

The results above therefore indicate or give clues as to what is the identity of their

unknown. Unknown #25 exhibited a negative result for Benedicts test, which makes the

probability of the identity of the unknown is actually the disaccharide sucrose, as

supported by the negative result for Bials Orcinol test, crossing out xylose for even all of

the unknowns (except for unknown #27, where the test was not able to be performed),

also the negative result for the Barfoeds test, implying that most likely it is a disaccharide,

and its positive reaction for the Seliwanoffs test, inferring that it is a ketohexose.
For unknown #26, as exhibited by its positive reactions to Benedicts and its quick reaction

to Barfoeds test, most likely implying that it is a reducing sugar and a monosaccharide.

Finally, it reacted rapidly as well in Seliwanoffs test, meaning that it is a ketohexose as

well. If the pieces were put together, it can be suggested that fructose is the identity of

unknown #26.

For unknown #27, it did not yield a brick red precipitate or a muddy green suspension in

the Benedicts test, once more increasing the probability that the unknown is also actually

sucrose, as it is a nonreducing sugar. It also showed a negative result for the Barfoeds

test, implying that it can be a disaccharide because of its slow rate of reaction. There was

no record of Bials Orcinol test which was unable to be done, and a record of the time for

Seliwanoffs test, thus there are still doubts but most likely the identity of unknown #27 is

sucrose as well.

For unknown #28, it showed a positive result for both Benedicts and Barfoeds test,

meaning that it is a reducing sugar and its fast rate of reaction will tell that it is a

monosaccharide. As it also yielded a negative result for Bials Orcinol as well as

Seliwanoffs test, xylose and fructose were taken out of the picture and the possibilities

lie between glucose and galactose. However, since the mucic acid test is yet to be seen

whether it will form crystals (galactose) or not (glucose).

Conclusion
Standard amylose, glycogen, and cellulose are able to be subjected into colorimetric

general tests which characterized their properties in the production of furfural and/or its

derivatives based on the dehydrating action of concentrated acids on the

polysaccharides. All of them are carbohydrates and contain sugars as assured by the
Molisch and Anthrone tests, respectively, and the Iodine test displayed which

polysaccharides have helical structure (amylose).

For the specific tests, it were all utilized to be able to narrow down the identity of ones

unknown sugar solution. A positive result exhibited in the Mucic acid test would narrow it

down to galactose and lactose, in Benedictss test, only sucrose would display a negative

result, monosaccharides and disaccharides are able to be distinguished by Barfoeds test,

pentoses and hexoses by Bials Orcinol test, and ketohexoses and aldohexoses by

Seliwanoffs test.

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