Discussion
1. Molisch’s Test
Molisch’s Test is used to test the presence of carbohydrates. The reaction is based on the
fact that concentrated Sulphuric acid catalyzes 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 coloured product (furfuryl-diphenyl-
methane-dyes). Polysaccharides and glycoproteins also give a positive reaction. In the event of
the carbohydrate being a poly- or disaccharide, the acid first hydrolyses it into component
monosaccharides, which then get dehydrated to form furfural or its derivatives. The carbohydrate
used such 1% Glucose, 1% Sucrose, 1% Fructose and 1% Starch showed presence of
carbohydrate which can be observed purple in colour, indicating a positive result.
2. Benedict’s Test for reducing sugars
Benedict's Test is used to detect the presence of reducing sugars. The Benedict method
describes the elimination of sugars (monosaccharides and certain disaccharides) that have
functional groups of free ketones or aldehyde 1. The formula from Benedict can be used to
monitor for sugar in urine1. Some sugars like glucose are called reducing sugar because their
capability to transfer hydrogen (electrons) to other compounds, a process called reduction 1. A
reduction reaction allows the Benedicts reagent to change color as reducing sugars are combined
with the Benedicts reagent and warmed. Depending on the amount and form of sugar, the colour
ranges from green to dark red (brick) or rusty-brown 1. The result showed that the carbohydrates
including 0.5% Glucose, 1% Glucose, 2% Glucose, 1% Lactose and 1% Fructose are positive
reaction while 1% Sucrose and 1 % Starch showed negative reaction, no changes in colour.
Sucrose showed negative reaction because these two carbohydrates are non-reducing sugar due
its chemical structure does not allow certain organic compounds to form a hemiacetal2. On the
other hand, starch is not a reducing sugar. The first ring can’t break down because there's no
hydrogen on the circled oxygen to allow for ring opening 3. Similarly, the next ring, and the next
ring, et cetera, cannot be break down as well3. So, polysaccharides, such as starch, are not
reducing sugars3. They need to be hydrolyzed and broken down into smaller units,
such as glucose or perhaps maltose, before the rings can open up3.
3. Barfoed’s Test for reducing monosaccharides
Barfoed’s Test is used to differentiate between reducing monosaccharides and reducing
disaccharides. The presence of reduced sugar is indicated by the formation of a red copper (II)
oxide precipitate4. In the case of disaccharide sugars, the reaction will be unfavorable because
they are slower reducing agents4. This test is for monosaccharides specific 4. Because of the
Barfoed reagent's weakly acidic nature, it is reduced only by monosaccharides 4. The result
showed that 1% Glucose, 1% Fructose and 1% Galactose form brick-red precipitate while 1%
Sucrose and 1% Lactose showed no changes in colour at all. Therefore, 1% Glucose, 1%
Fructose and 1% Galactose is monosaccharide sugars whereby 1% Sucrose and 1% Starch are
considered disaccharide sugars.
4. Seliwanoff’s Test
Seliwanoff’s Test is a specific test used for ketoses. When adding HCl concentration,
ketoses undergo dehydration to yield furfural derivatives faster than aldoses. This derivatives
form complexes that create deep red color with resorcinol 4. The study reagent induces 5-
hydroxymethylfurfural oxidation of ketohexoses. In the test reagent, 5-hydroxymethylfurfural
reactions with resorcinol to produce a red product within two minutes (reaction not shown). To
form the same material, aldohexoses reacts more gradually4. The result showed that only 1%
Fructose showed positive reaction by turning slighting orange. Fructose is a ketose, non-reducing
sugar which has 5 atom rings rather than six atom rings5; carbohydrates like 1% glucose, 1%
Ribose, 1% Sucrose and 1% Starch showed negative reaction, no changes in colour. Glucose and
galactose are hexoses but both of these carbohydrates are non-reducing sugars5.
5. Bial’s Test
Bial’s test is used to detect the presence of pentose sugar in the samples. This test can be
used to distinguish pentoses from hexoses. Pentoses react to furfural in the presence of
concentrated HCl, while hexoses give hydroxymethyfurfural. Orcinol and furfural condensation
to form a colored product in the presence of ferric ion. Green or precipitate presentation indicates
the presence of pentoses. Hexoses, which on dehydration give 5-hydroxyfurfural, react to a
brownish color with Bial's reagent. Di-and polysaccharides yield the same effects, but much
more slowly. Based on the result above, 1% Glucose and 1% Fructose give a positive result by
change in brownish color; while 1% ribose gives a negative result, which form a dark green
color. From this result, 1% Glucose and 1% Fructose are pentoses and 1% Ribose is Hexoses.
6. Osazone Test
Osazone test is used to identify reducing sugars. In this experiment, a monosaccharide
reacts with a crystalline compound called phenyl hydrazine6. The sugars that will reduce as a
result of this reaction will contribute to osazone formation 6. This will only arise when there is an
excess of phenyl hydrazine and they must be at a boiling temperature for this reason because the
temperature plays an important role in these experiments. All the crystal morphology is observed
under the microscope at 400x magnification. The test result showed that 1% Glucose and 1%
Fructose form needle-shaped crystals because Glucose and Fructose produce osazone because of
the similarities in their molecule structure. Next, 1% Galactose form Rhombic plate-shaped
crystals, 1% Lactose form powder puff-shaped crystals and 1% sucrose show no formation of
crystals.
7. Action of alkali on sugars
The principle is that sugar reduction exhibits instability and behavior that resembles the
changes that accompany the use of sugars by living cells in an alkaline solution in some
important respects7. These develop strong reduction strength and become self-oxidizable, are to a
considerable extent interconvertible, become polymerized and depolymerized, and in the absence
of oxidizing agents, with the intermediate formulation of methylglyoxal, these are partly
converted into lactic acid7. In addition to the fascination of reactions on purely chemical grounds,
the fact that each of these phenomena has its counterpart in carbohydrate metabolism gives the
obvious activation of sugars by alkali a special biochemical interest7. In this test result both
samples showed brick-red precipitate which indicate these non-reducing sugars undergo strong
reduction and become self-oxidizable. The brick red precipitate is formed due to the presence of
monosaccharides when it is reacted with benedict’s reagent.
8. The inversion of Sucrose
Sucrose is dextrorotatory with a specific rotation +66.5 ⁰. On hydrolysis in presence of
HCl or enzyme invertase, it gives and equimolar mixture of glucose with a specific rotation
+52.7⁰ and fructose with a specific rotation - 92.4⁰. Since the specific rotation of D- (+) glucose
is less than that of D- (-) fructose, the resulting solution after hydrolysis becomes levorotatory.
Since the hydrolysis of sucrose to an equimolar mixture D-(+) glucose and D-(-)fructose is
accompanied by the change in sign of optical rotation from dextrorotatory to levorotatory,
hydrolysis of sucrose is called inversion of sugar and the equimolar mixture of D-(+) glucose and
D-(-)fructose obtained as a result of hydrolysis is called invert sugar. The test results showed that
1% Sucrose with 1mL of 40% NaOH formed brick-red precipitate while 1% Sucrose without
1mL of 40% NaOH has no precipitate formed.
9. Iodine Test
The Iodine Test for Starch is used to determine the presence of starch in biological materials 8.
The test result showed that starch turn blue black when reacted with iodine reagent. This is
because Amylose in starch in the presence of iodine is responsible for the creation of a deep blue
pigment9. Inside the amylose coil, the iodine molecule slips9. Iodine-KI Reagent: Iodine is not
very water-soluble; the iodine reagent is therefore produced by dissolving iodine in water in the
presence of potassium iodide9. This makes a linear triiodide ion complex with solubility that slips
into the starch's coil resulting in an intense blue-black color9.
Question
7. Action of alkali on sugars
1. Explain the reaction of glucose in the alkali medium by giving your reasons and related
structures.
Answer:
glucose is unstable in the presence of base and undergoes the Lobry de Bruyn–Alberda Ekenstein
transformation whereby it is partially converted to a mixture of fructose and mannose.
2. Explain the difference between glucose solution and alkali treated glucose solution when a test
for reducing sugar is applied.
Answer:
In alkaline solutions, all mono and disaccharides with a free aldehyde or keto group act as
reduction agents10. The reduction of sugar products relies on the presence of actual or potential
groups of aldehydes or ketone10. An important consideration in reduction analysis is the
enolization of sugars in alkaline conditions 10. A sugar's ability to reduce alkaline test reagents
depends on an aldehyde or keto group being present to reduce reactions 10. Monosaccharides vary
between an open ring shape and a closed ring shape10. Using these studies, it is possible to reduce
the ketone (-C= O) group for Fructose and the aldehyde group (-CHO) for Glucose in the ring
open forms10. Many disaccharide sugar units often fluctuate between an open ring shape and a
closed ring shape10. These disaccharides also reduce sugars as they have a ketone or aldehyde to
react to the ring open form10. Sucrose is one of the few disaccharides that do not have an open
ring shape, so it is a sugar that is not reduced10.
9. Iodine Test
Explain the reaction between iodine solution and polysaccharides by giving the structures of
related compounds.
Answer:
The basis for this test is that starch is a coiled polymer of glucose. Iodine interacts with these
coiled molecules and becomes bluish black. Other non-coiled carbohydrates do not react with
iodine. Therefore, a bluish black color is a positive test for starch, and a yellow-ish brown color
(i.e., no color change) is a negative test for starch. Glycogen, the common polysaccharide in
animals, has a slight difference in structure and produces only an intermediate color reaction.
Figure: Coiled polysaccharide, amylose and amylopectin
Each polysaccharide tested gives different colour results with the iodine test. Explain the reason
briefly.
Answer:
When following the changes in some inorganic oxidation reduction reactions, iodine may be
used as an indicator to follow the changes of iodide ion and iodine element. Soluble starch
solution is added. Only iodine element in the presence of iodide ion will give the characteristic
blue black color. Neither iodine element alone nor iodide ions alone will give the color result.
Therefore, other polysaccharide showed no changes in colour, yellowish color.
Reference
1. Aryal S, Love B, barnabas A, Khare N, train w. Benedict's Test- Principle, Preparation,
Procedure and Result Interpretation [Internet]. Microbiology Info.com. 2019 [cited 30 November
2019]. Available from: https://microbiologyinfo.com/benedicts-test-principle-composition-
preparation-procedure-and-result-interpretation/
2. [Internet]. Sciencing.com. 2019 [cited 30 November 2019]. Available from:
https://sciencing.com/sucrose-nonreducing-sugar-5882980.html
3. Disaccharides and Polysaccharides [Internet]. Dl.clackamas.edu. 2019 [cited 30 November
2019]. Available from: http://dl.clackamas.edu/ch106-07/disaccha.htm
4. Qualitative Analysis of Carbohydrates (Theory) : Biochemistry Virtual Lab I : Biotechnology
and Biomedical Engineering : Amrita Vishwa Vidyapeetham Virtual Lab [Internet].
Vlab.amrita.edu. 2019 [cited 30 November 2019]. Available from: http://vlab.amrita.edu/?
sub=3&brch=63&sim=631&cnt=1
5. Chemistry for Biologists: Carbohydrates [Internet]. Rsc.org. 2019 [cited 30 November 2019].
Available from: https://www.rsc.org/Education/Teachers/Resources/cfb/carbohydrates.htm
6. Osazone Test: Principle, Procedure, and Results - All Medical Tests [Internet]. All Medical
Tests. 2019 [cited 1 December 2019]. Available from: http://allmedtests.com/osazone-test-
principle-procedure/
7. [Internet]. Jbc.org. 2019 [cited 1 December 2019]. Available from:
http://www.jbc.org/content/86/1/345.full.pdf
8. Iodine Test for Starch [Internet]. Brilliant Biology Student. 2019 [cited 1 December 2019].
Available from: http://brilliantbiologystudent.weebly.com/iodine-test-for-starch.html
9. Starch and Iodine [Internet]. Chemistry LibreTexts. 2019 [cited 1 December 2019]. Available
from:
https://chem.libretexts.org/Bookshelves/Biological_Chemistry/Supplemental_Modules_(Biologi
cal_Chemistry)/Carbohydrates/Case_Studies/Starch_and_Iodine
10. Qualitative Tests Used For Carbohydrates Biology Essay [Internet]. UKEssays.com. 2019
[cited 1 December 2019]. Available from: https://www.ukessays.com/essays/biology/qualitative-
tests-used-for-carbohydrates-biology-essay.php
