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Biochem Expt. 5

Biochem Expt. 5

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General and Specific Reactions for Qualitative Analysis of Carbohydrates Yurika Kinoshita, Franz

Liangco, John Bosco Magsuci and Princess Maristela Department of Chemistry, Univeristy of Santo
Tomas, Manila, Philippines

Abstract
Carbohydrates, which are polyhydroxy aldehydes or ketones, are considered the most abundant
biomolecules on Earth. Amylose and cellulose, which are polysaccharides, serve as storage and structural
molecules, respectively, by most organisms. Both of these molecules gave positive results for Molisch and
Anthrone test, general tests that indicate the presence of carbohydrates, and only amylose yield a positive
result for Iodine test, a general test for starches. Mono- and disaccharides are characterized by specific
tests, where sucrose, yielded a negative result in Benedict’s test, a test for reducing sugars; xylose, a
pentose, gave a positive result for Bial’s orcinol test; galactose and lactose were both positive in Mucic acid
test; disaccharides sucrose, maltose and lactose gave negative results in Barfoed’s test; and fructose and
sucrose gave quick positive results in Seliwanoff’s test.

Introduction
Carbohydrates are the most abundant molecules on earth which are predominantly polyhydroxy aldehydes
or ketones, or compounds that yield polyhydroxy aldehydes or ketones on hydrolysis. Certain
carbohydrates are dietary staples in most parts of the world and the oxidation of carbohydrates is the central
energy yielding pathway in most non-photosynthetic cells. Insoluble carbohydrates provide structural and
protective elements in cell walls of bacteria so as in plants and in the connective tissue of plants. More
complex carbohydrate polymers present which covalently attached to proteins or lipids that acts as signals
to determine the intracellular location or metabolic fate of the hybrid molecules called glycoconjugates. [2]

When the word “carbohydrate” was coined, it originally referred to compounds of the general formula
Cn(H2O)n. However, not all carbohydrates have this empirical formula, only the simple sugars or
monosaccharides fit thisformula exactly. The other existing types of carbohydrates are oligosaccharides and
polysaccharides based on the monosaccharide units and have slightly different formulas. [1]
Monosaccharides are simple sugars referred to as the basic of carbohydrates. [5] It can be a polyhydroxy
aldehyde or polyhydroxy ketone. The simplest monosaccharides contain three carbon atoms atoms and are
called the trioses. Glyceraldedhyde is the aldose with three carbon atoms, and the dihydroxyacetyone is the
ketose with three carbon atoms (a ketotriose). By far, the most important monosaccharide is D-glucose. [1]
Furthermore, monosaccharides with an aldehyde functional group are called aldoses whereas those with a
ketone group are ketoses.[2] All aldoses are reducing sugars, for their terminal carbonyl group may reduce
certain metals such as Ag and Cu, and in turn, be oxidized to form carboxylic acid. Disaccharides that have
a free anomeric hydroxyl group are also reducing sugars.[3] Oligosaccharides consist of short chain of
monosaccharide units (three to ten units) joined by glycosidic bonds. Three of the most important examples
of oligosaccharides are disaccharides namely sucrose, lactose and maltose. Sucrose is the common table
sugar extracted from sugarcane and sugar beets. It is made up of alpha-D glucose and beta-D-fructose.
Sucrose is not a reducing sugar because both anomeric groups are involved in glycosidic linkage. Lactose is
a disaccharide made up of beta-D-galactose and D-glucose. On the other hand, maltose is a disaccharide
obtained from the hydrolysis of starch. It does consist of two residues of D-glucose in an alpha (1→4)
linkage. Polysaccharides are many monosacharides linked together. Polysaccharides that occur in
organisms are often composed of a very few type of monosaccharide components. [1]

Fig. 1 Structures of the Seven Sugars used as Standards in the Specific tests Taken from
http://course1.winona.edu/sberg/241f00/Lec-note/Metabol.htm Glycosidic linkages occur between
monosaccharides by bonding of the anomeric (carbon 1) hydroxyl with another atom from the other
monosaccharide. The α-1→4 glycosidic bond in starch, for example, happens between the α-hydroxyl of
carbon 1 in a glucose subunit and carbon 4 in the other glucose subunit. The glycosidic bond, unlike the
peptide bond, does not have a single definite form, allowing carbohydrates to bond with a wide array of
atoms and molecules, many of which are called glycoconjugates. Examples are theglycoproteins that play a
role in membrane transport. Nonetheless, both types of covalent linkages produce as many variations of
macromolecules.[3]

This experiment aims to characterize the three polysaccharides, amylose, glycogen and cellulose, by
subjecting them to general reactions and to identify the unknown sugars with the virtue of specific tests
done along with standard sugar solutions.

Methodology
Materials
The solutions and reagents used for the general reactions were the following: standard amylose solution,
standard cellulose solution, Molisch reagent, concentrated H2SO4, anthrone solution, iodine solution.

The materials used for the specific reactions that determine the identity of the given unknown sugar solids
for the four group members were twenty percent standard solutions of xylose, fructose, glucose, galactose,
lactose, maltose and sucrose, conc. HNO3, Benedict’s reagent, Barfoed’s reagent, Bial’s Orcinol reagent,
Seliwanoff’s reagent. Procedure

In Molisch test, 10 drops of standard amylase solution was put into a small test and two drops of molisch
reagent was added to it. The solution was mix thoroughly. The tube was then inclined and 10 drops of
concentrated H2SO4 were allowed to flow down the side of the tube. The color formed at interphase of the
solution was noted and result was recorded. Steps 1-4 were repeated with the use of standard glycogen and
standard cellulose. In Anthrone test, 10 drops of Anthrone solution was placed to one of the well of a spot
plate and 1-2 drops of standard amylase solution was added to it. The color formed was noted and the result
was recorded. The former procedures was repeated but with the use of standard glycogen and standard
cellulose instead of standard amylase. In Iodine test, a drop of iodine solution was added to three test tubes
containing standard amylase, standard glycogen and standard cellulose. The observations were recorded.
The three test tubes were heated in a boiling water bath and the changes in each test tubes were noted.
Lastly, the tubes were removed from the bath and cooled.The observations were recorded. For the
preparation of the solution of the unknown samples, about half of the given amount of sugar was dissolved
in 5 ml of distilled water. At the same time, the water bath used from the general tests was refilled with
water and was used for all of the specific reactions. In Mucic acid test, 7 medium sized test tubes were
labelled with the standard sugars and another 4 test tubes were labeled as the unknowns for each member.
10 drops of sugar solutions were placed in their respective tubes and 10 drops of concentrated HNO3 to
each tube. The tubes were plugged with cotton and were heated in boiling bath for 1 hour. The tubes was
placed inside the locker until tne next laboratory period. The standard sugars and unknowns which
produced crystals were noted. In Benedict’s test, 7 test tubes were labelled with the standard sugars and
another four test tubes were labelled as the unknowns for each group member. 10 drops of Benedict’s
reagent were placed to each test tube. 5 drops of sugar solutions were added in their respective test tubes.
The tubes were heated in the water bath until a muddy green suspension was observed and settled as brick
red precipitate. The tubes were immediately remove from the bath and cooled in the rack. The results were
recorded. In Barfoed’s test, 7 test tubes were labelled with the standard sugars and another four tests tubes
for the unknowns. 10 drops of Barfoed’s reagent were placed to each of the labelled. 5 drops of the sugar
solutions and the unknowns were placed in their respective tubes. The tubes were heated in the water bath
until a brick red precipitate was observed. The time when the brick red precipitate was observed was noted.
The tubes were immediately removed from the bath. The results were recorded. In Bial’s Orcinol test, 7
small-sized test tubes with the standard sugars and another 4 test tubes for the unknowns of each member. 5
drops of sugar solutions and the unknowns were placed in their respective tubes. 10 drops of Bial’s orcinol
reagent was placed into each tube. The tubes were heated in a boiling water bath until a blue gree solution
was observed. The time when the blue green solution appeared was noted. The colors formed during 5
minutes of heating were noted. The tubes were removed from the bath and the results were recorded. In
Seliwanoff’s test, 7 medium-sized test tubes were labelled with the standard sugars and another 4 test tubes
for the unknowns. 10 drops of Seliwanoff’s reagent were placed to each labelled test tubes. 5 drops of the
sugar solutions wereadded to their respective test tubes. The tubes were immersed in a boiling water bath.
The tubes were heated until a cherry red solution was observed and were removed from the water bath. The
time when the cherry red solution was produced was noted. The results were recorded.

Results and Discussion
Several carbohydrates were subjected to tests which yielded different colors based on the reaction. The
chemical test wherein a color will be generated or the color of the reagent’s color changes upon the reaction
with an analyte of interest is referred to as colorimetric test. There are many existing test for carbohydrates
wherein in the general reactions are used to identify the presence of carbohydrates and the specific
reactions are for determining aldoses from ketoses. In table 1 is the summary of the three general reactions:
Molisch test, Anthrone test and Iodine te General Tests| | Polysaccharides|

| Standard Amylose Solution| Standard Glycogen Solution| Standard Cellulose Solution| 1. Molisch | The
color of the interphase produced was purple.| The color of the interphase produced was pink.| The color of
the interphase produced was dark violet.| 2. Anthrone| The solution immediately turned blue-green.| The
color of the solution produced was yellow green.| The color of the solution produced was yellow green
solution.| 3. Iodine| Light violet solution was produced after adding 1 drop of iodine. From a light violet
solution, it turned into clear colorless solution during heating and after cooling. | Clear colorless solution
was produced after adding 1 drop of iodine. It remained as clear colorless solution during heating and after
cooling.| White and cloudy solution with precipitate was produced after adding 1 drop of iodine. The
solution was white and cloudy during heating and after cooling.| Table 1 Results of generals tests for
amylose and cellulose

Molisch’s Test is a sensitive chemical test for all carbohydrates, and some compounds containing
carbohydrates in a combined form, based on the dehydration of the carbohydrate by sulfuric acid to
produce an aldehyde (either furfural or a derivative), which then condenses with the phenolicstructure
resulting in a red or purple-colored compound. [6] Amylose, glycogen and cellulose yielded positives
results in this test therefore they all contain carbohydrates. Anthrone test is also a general test for all
carbohydrates. Anthrone solution contains 10H-Anthracen-9-one (anthrone) and sulfuric acid. Sulfuric acid
dehydrates the sugar as in Molisch test, as well as converts anthone to anthronol, and condensation with
anthronol yields a blue-green colored precipitate. [7] Iodine test is used to indicate the presence of starch.
The test uses Lugol’s iodine or iodine in aqueous potassium iodide. The solution produces the triiodide ion
(I3-) which forms the complex with starch molecules. Amylose, the starch used in this test, is a helical
polysaccharide of glucose residues bonded by α-1→4 glycosidic linkages. As a result of the helical
configuration, the center of the molecule is hollow or empty, which may be occupied by iodine atoms and
thereby, forming a dark blue or purple complex. Since cellulose is a linear polysaccharide, it cannot form a
complex with triiodide. Thus, the yellow solution is considered a negative result. As a result, Iodine test is a
general test only for starches. When the solutions are heated however, the glycosidic linkages in amylose
were hydrolyzed, thus destroying the structure and liberating the iodine. Consequently, the helical structure
of amylose is destroyed, leaving a colorless solution. Only the amylase gave a positive result.[7] After the
general reactions which determined the presence of carbohydrates, the specific tests were done in order to
characterize a monosaccharide from a polysaccharide. Below is table 2 containing the summarized results
of the specific test reactions done with the 7 standard sugar solutions: xylose, fructose, glucose, galactose,
lactose, maltose and sucrose.

Carbohydrates| Specific Tests|
| Barfoed’s| Benedict’s| Bial’s Orcinol| Seliwanoff’s| Mucic acid| 1. Xylose| Clear light blue solution with
brick red precipitate in 2minutes and 36 seconds| Brick red precipitate| Blue green solution in 1minute and
30 seconds| Dark green solution| No formation of white crystals| 2. Fructose| Clear blue solution with brick
red precipitate in 2minutes and 7 secs.| Orange solution with orange precipitate| Greenish solution with
brown precipitate| Cherry red solution| No formation of white crystals|3. Glucose| Clear light blue solution
with brick red precipitate in 2minutes and 54 seconds| Orange solution with brick red precipitate| Blue
green solution in 3minutes and 40 seconds| Light red solution| No formation of white crystals| 4. Galactose|
Clear blue solution with brick red precipitate in 3minutes and 7 seconds| Orange solution with brick red
precipitate| Greenish solution with brown precipitate| Light red solution| Formation of white crystals| 5.
Lactose| Clear light blue solution| Orange solution with brick red precipitate| Muddy green solution in
2minutes and 30 seconds| Light red solution| Formation of white crystals| 6. Maltose| Clear light blue
solution| Orange solution with brick red precipitate| Blue green solution in 3minutes and 7 seconds| Peach
solution| No formation of white crystals| 7. Sucrose| Clear light blue solution| Light blue solution| Greenish
solution with brown precipitate| Cherry red solution| No formation of white crystals| Table 2 Results of
Specific Tests on Standard Sugar Solutions The first test that was performed was the mucic acid test which
needed an hour of heating. This test is one in which concentrated HNO3 is heated along with an aldose
sugar to give a dicarboxylic acid. Nitric acid is able to oxidize the terminal groups of aldoses, but leaves the
secondary hydroxyl groups unchanged. The dicarboxylic acid formed from galactose is called mucic
acid and is insoluble in cold aqueous solution. Those acids formed from the other common sugars are
soluble in H20. Thus the formation of the insoluble precipitate is an indication of the presence of galactose.
[7] Benedict’s test distinguishes the reducing from the non-reducing sugars. Benedict’s reagent contains a
mixture of copper(II) sulfate, sodium citrate and sodium carbonate. The initial color of the reagent is blue.
Sodium carbonate provides a weakly basic environment whereas citrate ion from sodium citrate, acts as a
buffer. The presence of a reducing sugar reduces the Cu+2 to Cu-, which is visible as a brick red precipitate
as copper(I) oxide. All of the aldoses are reducing sugars because of their terminal carbonyl group.
Fructose, despite not having any terminal carbonyl groups, is also a reducing sugar due to the fact that it
can undergo a series of keto-enol tautameric shifts to a mixture of glucose and mannose in basic
conditions.[4] All yielded positive results except for the sucrose which has no reducing end considering
that no anomeric hydroxyl groups may be located.Barfoed’s reagent, cupric acetate in acetic acid is slightly
acidic and is balanced so that is can only be reduced by monosaccharides but not less powerful reducing
sugars. Disaccharides (maltose, lactose, sucrose) may also react with this reagent, but the reaction is much
slower when compared to monosaccharides (glucose, galactose, xylose and fructose).[6] Bial’s Test is to
determine the presence of pentoses (5C sugars). The components of this reagent are resorcinol, HCl, and
ferric chloride. In this test, the pentose is dehydrated to form furfural and the solution turns bluish and a
precipitate may form.[6] Seliwanoff’s Test distinguishes between aldose and ketose sugars. Ketoses are
distinguished from aldoses via their ketone/aldehyde functionality. If the sugar contains a ketone group, it
is a ketose and if it contains an aldehyde group, it is an aldose. This test is based on the fact that, when
heated, ketoses are more rapidly dehydrated than aldoses.[6] Conclusion

The positive results in both Molisch and Anthrone tests indicated that amylose, glycogen and cellulose are
carbohydrates. Amylose yielded a positive result in Iodine test therefore it is definitely a starch; on the
other hand, cellulose and glycogen are not. References

[1] Campbell, M. K., & Farrell, S. (2003). Biochemistry. USA: Thompson Learning, Inc. [2] McKee, T., &
Mckee, J. (2003). Biochemistry: Molecular Basis of Life. New York: McGraw Hill Companies, Inc. [3]
Pratt, C. W., & Cornely, K. (2011). Essential Biochemistry. USA: John Wiley & Sons, Inc. [4] McMurry, J.
(2011). Organic Chemistry with Biological Applications. Canada: Brooks/Cole, Cengage Learning. [5]
Bettelheim, F. A., & March, J. (1990). Biochemistry. USA: Saunders College Publishing, Winstons, Inc.
[6] Retrieved January 10, 2013 from
http://www.chem.boun.edu.tr/webpages/courses/Chem415/Chem%20415%20Experiment%201.pdf [7]
Schrek, J. O., & Loffredo, W. M. (1994). Qualitative Testing for Carbohydrates. Retrieved July 30, 2012,
from Modular Laboratory Program in Chemistry:
http://kurdchemists.org/files/carbohydrates%20test%20new.pdf

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