Exercise 5 and 6

TEST FOR CARBOHYDRATES AND IDENTIFICATION OF THE UNKNOWN

CARBOHYDRATE
Bate AC, Enriquez IJ, Loberiano AK, Ochea C

ABSTRACT

Different qualitative tests for carbohydrates determine the different properties that distinguish
carbohydrates from one another. Two unknown carbohydrates, Unknowns G and H, were
identified by performing different tests for carbohydrates. The first test performed was the Iodine
Test, which identified the unknown samples as both a saccharide. The Barfoed Test was
performed next, verifying that both sugars are monosaccharides. The next test was Bial’s
Orcinol Test, which confirmed that Unknown G was a hexose and Unknown H was a pentose
sugar. The Seliwanoff’s Test was performed after the Bial’s Orcinol Test, which identified
Unknown G as fructose. Unknown H tested negative for Seliwanoff’s Test so the Fermentation
Test was performed, which identified Unknown H as galactose. The results derived from the
experiment differed from the expected identities of sugars, which are fructose and arabinose.
The error in identifying correctly Unknown H arose during the Seliwanoff’s Test, where the
“colorless” result and the “faint red” result were confused, hence mistaking arabinose for
galactose.

INTRODUCTION

A carbohydrate is an organic compound that made up the majority of organic substances found
on earth. It performs countless roles in the living things; Some of which is being an energy
source. A carbohydrate consisting of carbon, hydrogen and oxygen with a general formula of
C​n​(H​2​O)​n​. 1​ ​Carbohydrates can either be a mono-, oligo- or polysaccharide. A monosaccharide is
the building blocks of carbohydrates. An oligosaccharide is a combination of a few chains of
monosaccharides. While a polysaccharide is a combination of many chains of monosaccharides
that are bonded together. 2​

A certain enzyme may only break down a specific carbohydrate or there are certain methods for
a certain carbohydrate to be absorbed by the body.​3 ​There are also certain medical conditions
that the absence of certain enzymes does not allow breaking down of a certain sugar. It is
important to identify certain sugars present to make sure that the body has enough source of
carbohydrates.

In this experiment, two unknown sugar samples were identified. In the process of identifying, a
diagram on the set qualitative tests for the identification of common sugar was used.

OBJECTIVES
The objective(s) of the experiment is/are as follows:
 ● To correctly identify the unknown carbohydrates by performing the different qualitative
tests for carbohydrates
● To determine the appropriate tests required in identifying the provided unknown samples

METHODOLOGY

In this experiment, two unknown samples were examined. In the process of correctly naming its
identity, a diagram on the set of qualitative tests was followed.

The Molisch Test is a test that detects the presence of carbohydrates, in whether a free or
combined form. A positive test is characterized by a red or a purple-colored ring compound.
Carbohydrates that exhibit this kind of results are polysaccharides, monosaccharides,
disaccharides and glycoproteins.​4 ​To perform the Molisch Test, 3 drops of a 15% of
alpha-naphthol in alcohol or Molisch’s reagent was added to 5 drops of the samples. The
solution was mixed well and inclined to slowly pour down 15 drops of sulfuric acid. Observations
were noted.

In the determination of whether the sugar is a starch, monosaccharide, disaccharide, glycogen

or erythrodextrin, an Iodine Test was performed. This test is also called the Starch Test because
it detects the presence of starch in a solution. The result is positive when the solution forms a
dark blue solution, negative if it forms a colorless solution for mono- and disaccharide, and red if
it is a glycogen or erythrodextrin. 5​ ​To perform the Iodine Test, 1 drop of KI solution was added
to 5 mL of the diluted unknown sample solution. The solution was heated to boiling and was
cool down to room temperature. All the appearance of the color in each procedure was noted.

The Barfoed’s Test is used to differentiate monosaccharides from disaccharides. It is also a

copper reduction reaction where the reagent is made in an acidic medium (copper (II) acetate
and acetic acid). A positive test is characterized by the formation of brick red precipitate after
heating. The time it took for a sugar to form the precipitate is what determines whether the
sugar is a mono- or a disaccharide. If the precipitate is formed within 7 minutes, the sugar is a
monosaccharide. If it is formed after 7 minutes, then the sugar is a disaccharide.​6 To perform
the Barfoed’s Test, 1.5 mL of the Barfoed’s reagent was added to 5 drops of unknown sugar
solution. It is then heated for 5 minutes and cooled for 15 minutes. Observations were also
noted.

The Bial’s Orcinol Test is used to differentiate pentoses from hexoses. For this test, the bial
reagent, which contains orcinol, is dissolved in concentrated HCl and a small amount of FeCl​3​.
When mixed with the reagent, pentoses are converted to furfural.​7
 Figure 1. ​Formation of furfural in pentose and hexose sugars.

A positive test is characterized by the formation of a blue or blue-green compound. Thus, the
solution is a pentose. 7​ ​To perform the Bial’s Orcinol Test, 4.5 mL of the Orcinol Reagent was
added to 0.5 mL of the unknown sugar solution. It is then heated for 15 minutes. The
appearance of the color was noted.

The Seliwanoff’s Test is used to distinguish between aldose and ketose sugars.​8 To perform
Seliwanoff’s Test, 4 drops of Seliwanoff’s Reagent was added to 5 drops of the unknown sugar
solution. The mixture was heated for 15 minutes and the color changes were observed. Ketoses
yield furfurals when reacting to the Seliwanoff reagent which is characterized by a cherry-red
complex. Aldoses yield a negative, or a colorless result.​13

The Fermentation Test is a test that examines whether a bacteria can utilize a certain
carbohydrate or not. A positive result is observed if bubbles of CO​2 were formed.​9 To perform
the Fermentation Test, 10 drops of 20 % suspension of ordinary Baker’s yeast was added to 5
drops of the sugar solution. It kept standing for 30 minutes and bubbling formation was noted.

The Benedict’s Test helps in detecting simple sugars that are reducing sugars.​10 A positive
result indicates that the sugar is a mono- or disaccharide. A negative result means the sugar is
a sucrose. To perform Benedict’s Test, 2 mL of Benedict’s solution was added to 5 drops of the
unknown solution. The mixture was heated for 2 minutes and observations were noted.
RESULTS AND DISCUSSION

In finding the unknown sugars, various tests for carbohydrates were performed in the order
indicated in the diagram below:

Figure 2.​ Qualitative Tests for the Identification of Carbohydrates

This diagram is based on the provided reference from this exercise. The highlighted boxes are
the tests that were performed during the experiment, which are based on the reactions exhibited
on each of the processes. The marks of the unknown samples G and H are placed according to
the observed results from the conducted tests.

The first test performed was the Iodine Test. The process of heating has resulted in both of the
samples, initially appearing yellow, to become colorless. It exhibited a negative result to the test
which means that both samples are saccharides.
 Figure 3. ​Iodine Test for Unknowns G and H.

After confirming that the unknown sugars are saccharides, the Barfoed’s test was performed. It
was found that both unknown sugars G and H are monosaccharides since a brick-red
precipitate was formed within 7 minutes into heating the solutions. This observation has further
suggested that neither of the samples is a sucrose.

Figure 4. ​Barfoed’s Test Result of Unknowns G and H.

The Bial’s Orcinol test was conducted consequently after both samples obtained positive for
Barfoed’s Test. The samples were found to have different reactions to the test, suggesting that
the two samples are different types of monosaccharide. Unknown H changed its color to green,
hence it is a pentose. Unknown G changed its color to dark brown, indicating that it is a hexose.
 Figure 5 ​Results obtained from performing Bial’s Orcinol Test in Experiment 5.

Figure 6. ​Results of Unknowns G and H for Bial’s Orcinol Test.

The Unknown H had a green solution and blue precipitate which would indicate a no reaction
since a blue solution should be the positive result. The result observed from Unknown H is
similar to the reaction of sucrose and fructose observed in Experiment 5.

The Seliwanoff’s Test was immediately performed, and a color change to a dark red was
observed in Unknown G determining its identity, which is fructose. Unknown H remained
colorless so the Fermentation Test was performed.

Figure 7. ​Results of Unknowns G and H for Seliwanoff’s Test.

The Fermentation Test was performed to verify the identity of Unknown G and determine
Unknown H. It was observed that Unknown G formed a thin layer of bubbles while Unknown H
formed no bubbles. According to the diagram, if no bubbles were formed, then the sugar is
galactose.

Figure 8. ​Results of Unknowns G and H for Fermentation Test.

The experiment was performed twice to confirm the identities of Unknowns G and H, but the
experimental results did not coincide with the expected identities. Unknown G is fructose but
Unknown H is supposed to be arabinose. This error may have arisen due to the
misinterpretation of the results obtained from the Seliwanoff’s Test, where a faint red result
would mean the sugar is arabinose. Although aldoses, like arabinose, yield a colorless or faint
solution in Seliwanoff’s Test. The results obtained from Experiment 5 shows that arabinose
should result in a colorless solution.

Figure 9. ​Result of arabinose in Seliwanoff’s Test from Experiment 5.

Fructose is found in many fruits, vegetables, and honey, and is sweeter than glucose. It is
absorbed more slowly in the intestine, and is converted to glucose and glycogen in the liver.
Glucose levels are reduced when fructose replaces sucrose or starch in the diet of diabetic
people.​14
Like fructose, arabinose is also significant especially to diabetic people. It can reduce glucose
levels and suppress insulin levels. It can also reduce the appearance of heart disease, lower
blood cholesterol, and manage body weight and composition.​15

CONCLUSION

Different tests were done to determine the identity of the unknown carbohydrates. The iodine
test identified the unknown as saccharides. The Barfoed’s test determined that both samples
are monosaccharides. The Bial’s Orcinol test differentiated the two samples; Unknown G is a
hexose while Unknown H is a pentose. The Seliwanoff’s test confirmed that Unknown G is
fructose. Fermentation test was performed and it was concluded that Unknown H was
galactose, which did not coincide with the expected identity of the unknown sample. A mistake
in determining the correct result for Seliwanoff’s Test caused the error, and Unknown H was
identified as arabinose.

REFERENCES

1. Qualitative Analysis of Carbohydrates. Stud Fier-Blogspot ; 2012.

http://stud-fier.blogspot.com/2012/01/carbohydrates.html?m=1​ (Accessed
February 26, 2020)
2. Campbell, M. K.; Farrell, S. O. In ​Biochemistry;​ Mary Finch, 2012; p 451.
3. (1) 3.3: Digestion and Absorption of Carbohydrates - Medicine LibreTexts
https://med.libretexts.org/Courses/American_Public_University/APUS%3A_An_In
troduction_to_Nutrition_(Byerley)/Text/04%3A_Carbohydrates/3.3%3A_Digestion
_and_Absorption_of_Carbohydrates (Accessed Mar 4, 2020).
4. Gautam, R. ​What is the principle of the Molisch Test for carbohydrates?​ Quora;
2018.​https://www.quora.com/What-is-the-principle-of-the-Molisch-test-for-carboh
ydrates​ (Accessed February 28, 2020)
5. Goedecke, C. ​Why does Iodine turn starch Blue?​, 2016.
https://www.chemistryviews.org/details/education/10128441/Why_Does_Iodine_
Turn_Starch_Blue.html​ (Accessed Mar 4, 2020)
6. Chatterjea, M. N.; Shinde, R. In ​Textbook of Medical Biochemistry.​
7. Hein, M.; Best, L. R.; Miner, R. L.; Ritchey, J. M. In ​Introduction to General,
Organic, and Biochemistry in the Laboratory​; Brooks/Cole Publishing Company;
pp 257–261.
8. Seliwanoff's Test. Socratic Q & A: 2017.
https://socratic.org/questions/a-positive-seliwanoff-s-test-is-obtained-with-a-gluco
se-b-fructose-c-galactose-d​ (Accessed Mar 4, 2020)
9. Carbohydrate Fermentation Test. Iowa State University Microbiology.
https://www.micro.iastate.edu/video/microbiology-007-carbohydrate-fermentation-
test​ (Accessed Mar 4, 2020)
10. Ashenhurst, J. Reducing Sugars, 2019.
https://www.masterorganicchemistry.com/2017/09/12/reducing-sugars
11. Carbohydrates - Barfoed’s Test
http://dept.harpercollege.edu/chemistry/chm/100/dgodambe/thedisk/carbo/barf/ba
rfoed.htm​ (accessed Mar 5, 2020).
12. Course Book of Chemistry 2 (Practical) Proteins [Online] p. 18 Mohamed, A.
http://bu.edu.eg/portal/uploads/Agriculture/Agricultural%20Biochemistry/1117/crs-
13490/Practical%20Biochemistry.doc.pdf​ (Accessed January 27, 2020).
13. Tenks; Yash; Bleluv; Bleluv; Anthony; Dee; Dee; Madhuparna; Madhuparna;
Shahid; Shahid; Sule, B.; Sule, B.; Courtney; Courtney; Naseer; Naseer;
Rabee'u, A.; Rabee'u, A.; Imtiaz, M.; Imtiaz, M.; Deen; Deen; Nasir; Nasir;
Himanshu; Himanshu. Seliwanoffs Test Principle and Procedure.
https://allmedtests.com/seliwanoffs-test-principle-procedure/ (accessed Mar 5,
2020).
14. Wolfsdorf, J. I.; Garvey, K. C. Management of Diabetes in Children .
Endocrinology: Adult and Pediatric (Seventh Edition)​ 2016, 854–882.
15. Noelles, R.; Benschop, A. L-Arabinose: A Novel Food Ingredient For A Healthier
Living.