IDENTIFICATION OF CARBOHYDRATES

Lab partner; Breinburg Selena

Abstract
De aim of this experiment was to detect biologically significant compounds in various solutions.
Doing multiple tests by using various test reagents, carbohydrates can be detected in the solutions.
These tests conducted were based on the detection of corresponding functional groups of different
carbohydrates and on specific functional groups of the different carbohydrates. Based on the
functional groups detected by the test reagents, it was possible to make statements about which
carbohydrate was present in the known and unknown solutions obtained. The results were that the
identification was successful for all but one monster which is monster 1. Because of lack for more
information of the unknowns, these were difficult to identify.

Introduction
Carbohydrates are compounds are extremely important in biological processes. They provide
energy through oxidation, supply carbon for the synthesis of cell components, serve as a form of
stored chemical energy and form part of the structures of some cells and tissues. Carbohydrates
are known as biomolecules because of the closely association with living organisms. Sugars also
called carbohydrates, are carbon compounds that contain substantial amounts of hydroxyl groups.
Polyhydroxy aldehydes are carbohydrates that contain an aldehyde moiety. Polyhydroxy ketones
are carbohydrates that contain a ketone moiety. Carbohydrates can be classified as either
monosaccharides, oligosaccharides or polysaccharides. Carbohydrates containing single
polyhydroxy aldehyde or ketone unit are classified as monosaccharides. Disaccharides contain two
monosaccharide units linked together by a covalent bond. Oligosaccharides contain from 3 to 10
monosaccharide units. Carbohydrates with very long branched or straight chains of hundreds or
thousands of monosaccharide units are called Polysaccharides.
There are some tests that can be used for just these kinds of qualitative analysis. The tests used for
this experiment are: The Molisch test for Carbohydrates, Benedict Test for Reducing Sugars
,Barfoed’s Test for Monosaccharides ,Lasker and Enkelwitz Test for Ketoses, Mucic Acid Test for
Galactose and the Iodine Test for Starch and Glycogen

Experimental procedure
Test1. Molisch test for carbohydrates
Eight test tubes were labeled from 1-8. In each test tube 2 mL of 0.2M sugar solution was added,
Afterwards Two drops of Molisch reagent (solution of alpha-naphthol in 95% ethanol) were
added to each of the eight test tubes. The tubes were inclined and gently 5 mL of concentrated
sulfuric acid was added to each of the eight test tubes. The results were then observed
Test 2. Benedict’s test for reducing sugars
Nine test tubes were labeled from 1-9. In each test tube 1 mL of 0.2M sugar solution was added.
5 mL of Benedict’s solution (solution of CuSO 4 , Na2CO3 and sodium citrate with a pH of 10.5)
was added to each of the nine test tubes. Each test tube was then placed in a boiling water bath
for three minutes. After three minutes the test tubes were cooled for 15 minutes and the results
were observed

Test 3. Barfoed’s test for monosaccharides

Eight test tubes were labeled from 1-8. In each test tube 1 mL of the different sugar solutions
was added. 3 mL of the freshly prepared Barfoed’s reagent (solution of Cu(CH3COO) 2 in dilute
acetic acid with a pH of 4.6) was added to each of the eight test tubes. Each test tube was then
placed in a boiling water bath for 3 minutes. After 3 minutes the test tubes were allowed to cool
for 10 minutes and the results were observed.

Test 4. Lasker and Enkelwitz test for ketoses

Eight test tubes were labeled from 1-8. In each test tube 1 mL of 0.02M of the different sugar
solutions was added. 5 mL Benedict’s solution was added to each of the eight test tubes and
mixed well. The test tubes were heated in a 55 °C water bath for 20 minutes. Notes were taken
on what happened at 10 minutes and 20 minutes.

Test 6. Mucic acid test for galactose

Nine test tubes were labeled from 1-9. In each test tube 5 mL of a sugar solution was added. 1
mL of concentrated nitric acid was added to each of the nine test tubes. The test tubes were
placed in a boiling water bath for approximately three hours. After withdrawing the test tubes
from the water bath, they were left to cool down overnight. The following day the results were
observed

Test 7. Iodine test for starch and glycogen

10 test tubes were labeled from 1-9. In each test tube 5 mL of a sugar solution was added and a
self-prepared potato juice were added. 2-3 drops of Lugol’s iodine solution (aqueous KI and I 2
solution) were added to each of the nine test tubes. 3 mL of this potato juice was added to a test
tube and to the test tube 2-3 drops of Lugol’s iodine solution was added.
Results and Discussion

Table 1 Test1 Molisch test for Carbohydrates

Color Conclusion
Fructose Purple Carbohydrate
Glucose Purple Carbohydrate
Galactose Purple Carbohydrate
Ribose Purple Carbohydrate
Sucrose Purple Carbohydrate
Starch Purple Carbohydrate
Lactose Purple Carbohydrate
Glucose Purple Carbohydrate
Mixture Purple Carbohydrate

In this part of the experiment the solutions were tested with the molisch reagent. Molisch test is
very important because it is a general test for the presence of carbohydrates. All the solutions
containing starch, sucrose, glucose, lactose, galactose, ribose and fructose turned dark purple. We
can conclude that all these solutions contained carbohydrate. starch, sucrose, glucose, lactose,
galactose, ribose and fructose are carbohydrates. All of these solutions turned purple because the
reaction is based on the fact that concentrated sulfuric acid catalyses the dehydration of sugars to
form furfural (from pentoses) or hydroxymethyl furfural (from hexoses). These furfurals then
condense with sulfonated alpha-naphthol to give a purple or violet colored product (furfuryl-
diphenyl-methane-dyes).[5]
Table 2 Benedict's test

Color Precipitate Reducing sugars

Fructose Red precipitate Reducing sugar
Glucose Red precipitate Reducing sugar
Lactose No precipitate Not a reducing sugar
Galactose Red precipitate Reducing sugar
Ribose No precipitate Not a reducing sugar
Sucrose No precipitate Not a reducing sugar
Starch No precipitate Not a reducing sugar
Mixture No precipitate Not a reducing sugar
Monster 3 Red precipitate Reducing sugar

The principle of Benedict’s test is based on forming a red precipitate Cu2O. Reducing sugars under
alkaline condition tautomerize and form powerful reducing agents called enediols. They can
reduce cupric ions (Cu2+) (in the benedict solution) to cuprous form (Cu+), under carefully
controlled conditions the precipitate Cu2O is formed, which is responsible for the change in color
of the reaction mixture. The functional groups tested here are a free aldehyde or free ketone a
reducing carbohydrate possesses.

Figure 1 Formation of cuprous oxide

The results show that fructose, glucose, galactose and the carbohydrate in monster 3 are reducing
carbohydrates. Theoretically ribose is also a reducing sugar, because it is a monosaccharide with
an aldehyde group,[3] Which means that the ribose sample should have also given precipitate, but
due to inaccurate timing the ribose wasn’t 3 minutes in the water bath so the ribose didn’t have the
chance to reduce the alkaline solution of copper. Many disaccharides including lactose [4] are also
identified with the benedict solutions. In this experiment the lactose solution should have given
precipitate but due to inaccurate timing the lactose wasn’t 3 minutes in the water bath so the lactose
didn’t have the chance to reduce the alkaline solution of copper.
The results show that monster 3 was a reducing agent, which means that it could either be fructose,
glucose or galactose. This test also showed us that the mixture did not contain a reducing agent,
so to identify the mixture it already rules out the 3 found reducing agents.
Table 3 Barfoed's test for monosaccharides

Color precipitate Type:

Fructose Red precipitate Monosaccharides

Glucose Red precipitate with a bit of Monosaccharides

yellow
Lactose - -

Galactose Red precipitate Monosaccharides

Ribose Red precipitate Monosaccharides
Sucrose - -

Starch - -
Mixture Red precipitate Monosaccharides

This test is used to distinguish monosaccharide from disaccharide and polysaccharides. The
Barfoed’s test is a reduction test carried out in acidic medium. This test is based on the reduction
of copper(II)acetate to copper(I) oxide (Cu2O), which forms a brick-red precipitate.
Monosaccharides react very fast whereas disaccharides and react very slowly.

Figure 2 Barfoed's reaction

Disaccharides will also respond to the test with extreme heating and under proper conditions of
acidity. [6]
This experiment was not done under heating condition which only gave us the results for
monosaccharides.
This test yielded in a positive result for fructose, glucose, galactose and ribose, because these
sugars are monosaccharides. Lactose, sucrose and starch yielded a negative result because they
aren’t monosaccharides. Lactose and sucrose are disaccharides and starch is a polysaccharide.
From this result it also came to light that the mixture contained a monosaccharide, but from test 2,
fructose, galactose and glucose were rules out, so it leaves ribose.
Table 4 Lasker and Enkelwitz test for ketoses

Color at 10 min Color at 20 min Type:

Fructose Blue Blue solution ketohexones
Red precipitate
Glucose 2 layers: 2 layers: ketopentoses
Top = red Top = orange
Bottom = blue Bottom = blue
Red precipitate
Lactose 2 layers: 2 layers ketopentoses
Top = red Top = red
Bottom = blue Bottom = blue
Galactose 2 layers: 2 layers = Ketopentoses
Top layer = light Top layer = light
orange/red orange
Bottom = blue Bottom = blue
Little bit of red
precipitate
Ribose Blue Mostly Blue with very Aldoses
thin orange layer on
top
Sucrose Blue Blue Aldoses
Starch Blue Blue Aldoses
Mixture 2 layers: 2 layers: Ketopentoses
Top = red Top =light orange
Bottom = blue Bottom = blue
The Lasker and Enkelwitz test utilizes Benedict's solution. The Lasker and Enkelwitz test is carried
out at a much lower temperature. The color changes that are seen during this test are the same as
with Benedict's solution. For the Lasker and Enkelwitz test it is favored to use dilute sugar
solutions. [7]
A ketohexose is a ketone-containing hexose (a six-carbon monosaccharide). A ketohexose would
react around 20 minutes. In the obtained results in this experiment it shows that the sugar that is a
ketohexose is fructose. Ribose started to form a thin orange layer, but this can be explained with
the temperature of the water bath; at some point around the 15 minutes the temperate was almost
80℃, while it should have been 55℃.
A pentose is a monosaccharide with five carbon atoms. From the results in table 4 the sugars that
are ketopentoses are glucose, lactose, galactose. The mixture must be a sample or solvent
containing a ketepentoses too, because that is what test showed.
The sugars that didn’t react in the 20-minute time period are aldoses. These sugars must have been
ribose, sucrose and starch, because they remained blue throughout the whole 20 minutes.
From the result of test 4 we can see that the mixture contained a ketopentoses, which means that
it could be lactose.
Table 5 Micic acid test for galactose

Color after 30 mins of boiling Overnight Results

Fructose Clear No crystals

Glucose Clear No crystals

Lactose Clear with light yellow tint Crystals
Galactose Clear with light yellow tint Crystals
Ribose Clear light yellow No crystals

Sucrose Clear with brown stain on the No crystals

side of the tube
Starch Clear with light yellow tint No crystals
Mixture Clear with light yellow tint No crystals
Monster 2 Clear Crystals

Nitric acid oxidizes most monosaccharides and this reaction yields soluble dicarboxylic acids.
When galactose is oxidized an insoluble mucic acid is yielded. Lactose will also yield a mucic
acid. The functional group detected by this test is the glycosidic linkage between its glucose and
galactose subunits. [8]
Mucic acid are white crystals. The obtained results show us that Lactose produces crystals, because
of hydrolysis of the glyosidic linkage between its glucose and galactose subunits. [8] The other 2
sugars in which crystals were formed are galactose (theoretical confirmed) and the monster. The
monster2 for mucic test contained lactose or galactose.
Table 6 iodine test for starch and glycogen

Color
Fructose Brown-yellow
Glucose Brown-yellow

Lactose Brown-yellow
Galactose Brown-yellow
Ribose Brown-yellow
Sucrose Brown-yellow

Starch Blue-black
Mixture Brown-yellow
Monster 1 Brown-yellow
Potato Juice Blue-black

This test is used for the detection of starch in the solution. The blue-black color is due to the
formation of starch-iodine complex. Starch contain polymer of α-amylose and amylopectin which
forms a complex with iodine to give the blue-black color. Glycogen reacts with the iodine reagent
to give a brown-blue color. Other polysaccharides and monosaccharides yield no color change; the
test solution remains brown-yellow (color of the reagent). None of the solutions gave a brown-
blue color this indicates that none of the solutions contained/were glycogen. The monster and the
mixture in this experiment did not contain glycogen nor starch because it remained the color of the
reagent (brown-yellow).
References
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%209_Sacharidy/Doplnkove%20studijne%20materialy/Carbohydrates_Boudreaux.pdf
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th
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