ORGANIC COMPONENTS: CARBOHYDRATES

Romajane Vencio Abrenio

University of the Philippines Visayas Tacloban College

Magsaysay Boulevard Tacloban City

ABSTRACT

Carbohydrates is one of the major components of organic matter due to their function
in all life forms. They act as storage of energy and sources of fuel. They are also essential in
cell-cell communication and structures of the cells. In this activity, various tests were
performed so as to detect the presence of carbohydrates and identify which specific
carbohydrates are present in the samples. The samples used were: one percent glucose, one
percent fructose, one percent lactose, one percent sucrose, one percent starch, leaf extract,
fruit extract, and distilled water. For the first half of the exercise, Molisch’s test, Seliwanoff’s
test and Benedict’s test were conducted. All of the samples gave a positive result (violet ring
formation) for the Molisch’s test, except for the control. Only sucrose and fructose gave a
positive result (red solution formation) for Seliwanoff’s test. Glucose, fructose, lactose, the leaf
extract and the fruit extract yield a positive result (brick-red precipitate formation) for Benedict’s
test. The action of acid on disaccharides and polysaccharides were observed for the second
half of the exercise. The addition of HCl to sucrose and starch hydrolysed them into glucose
and fructose (for sucrose) and into glucose units (for starch). The acid acted as a catalyst in
the reactions.

KEYWORDS

achromatic point, benedict’s test, molisch’s test, polysaccharide, seliwanoff’s test

INTRODUCTION

Carbohydrates is one of the four major classes of biomolecules along with proteins,
nucleic acids, and lipids. They can be found on both plant tissues, as well as, in animal tissues
(Jequier 1994). Most of the organic matter on Earth is made up of carbohydrates because of
their extensive roles in all forms of life. They primarily serve as energy stores, fuels, and
metabolic intermediates. Ribose and deoxyribose sugars form part of the structural framework
of RNA and DNA. Furthermore, polysaccharides are structural elements in the cell walls of
bacteria and plants. Even cellulose, the main constituent of plant cell walls, is one of the most
abundant organic compounds in the biosphere. Lastly, carbohydrates are linked to many
proteins and lipids, where they play key roles in mediating interactions among cells and
interactions between cells and other elements in the cellular environment (Berg et al. 2002).

Carbohydrates are aldehyde or ketone compounds with multiple hydroxyl groups.

Polyhydroxyaldehydes are known as aldose and polyhydroxyketones are known as ketose
(Hunt 2018). Hence, for a carbohydrate to be an aldose it needs an aldehyde group and to be
a ketose it needs a ketone group.

Carbohydrates are molecular compounds made from just three elements: carbon,
hydrogen and oxygen and they can be divided further into three general classes according to
the number of carbohydrate molecules that they have. Monosaccharides (e.g. glucose) are
termed for single sugars, disaccharides (e.g. sucrose) for sugars containing two
monosaccharide units, oligosaccharides for sugars containing three to 10 monosaccharide
units and polysaccharides (e.g. starch, glycogen cellulose) for sugars containing more than 10
monosaccharide units (RSC 2018). Both disaccharides and polysaccharides can be
hydrolyzed by heating in slightly acidic solution to yield their monosaccharide forms.
Monosaccharides are the simplest form of sugars and therefore cannot be hydrolyzed (Tatad
et al. 2014).

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 Several tests were conducted in this exercise for the students to become familiar with
some of the methods commonly used in detecting the presence of simple sugars or hydrolyzed
derivatives of polysaccharides based on their color reactions. Molisch’s test, for one, was
conducted to test the presence of carbohydrates. When simple sugars react with strong acids,
they form furfural derivatives that form colored components with a number of dyes such as α
– napthol and thymol. A positive reaction is indicated by appearance of a purple ring at the
interface between the acid and test layers (Chhabra 2014). Seliwannoff’s test was also done
to differentiate ketoses and aldoses. The reagent is a solution of resorcinol in concentrated
HCl. The acid when heated along with a sugar will produce furfural or hydroxymethylfurfural,
which further reacts to give a red color. Ketoses react more quickly than aldoses and thus the
reaction time is a means of separation or detection. Ketoses react within one minute of heating
while aldoses will require several minutes. Disaccharides containing fructose should react
intermediately between that of fructose alone and one of the aldoses (Caton 2011). In addition,

Benedict’s test was conducted to detect reducing sugars using Benedict’s reagent
which contains copper (II) ionsin alkaline solution with sodium citrate added to keep the cupric
ions in the solution. It causes isomeric transformation of ketoses to aldoses resulting in the
reduction of blue cupric ion to cuprous oxide (brick red precipitate) due to the alkaline condition
of this test (Simo et al. 2002).

MATERIALS AND METHODS

This activity was conducted on two separate meetings: the first one was on February
6, 2018 and the second was on February 8. This was done inside the Zoology Laboratory of
the Division of Natural Sciences and Mathematics (DNSM) in University of the Philippine
Visayas Tacloban College (UPVTC).

All of the solutions and reagents were freshly prepared because most of them are
unstable. In addition, all of the glassware used were properly labelled at the start of the
experiment so as to prevent human errors from happening.

Adapting the procedures by Alejar et al. (2009), the characteristic color reactions of
carbohydrates in some plant materials were divided into three parts: first is the Molisch’s test,
followed by Seliwanoff’s test and the last is Benedict’s test. Kiat kiat (Citrus reticulata) fruit and
Akapulko (Cassia alata) leaves were obtained for these parts of the experiment. Both were
washed with distilled water before the extraction process took place. The juice was extracted
from the fruit by squeezing it directly unto the beaker. The leaf extract, on the other hand, was
prepared by weighing 10 grams of it, using the analytical balance, and cutting it into smaller
pieces using a pair of scissors. The small pieces of leaves were then grinded, using an
Osterizer blender, and 10 ml distilled water was added unto the blender before grinding. The
leaf and fruit extracts were filtered with 1 layer of cheese cloth and were then placed in
separate beakers that were covered with aluminium foil.

For the Molisch’s test, eight clean test tubes were prepared. They were consecutively
labelled with the following: one percent glucose (hexose) for test tube one, one percent
fructose (hexose) for test tube 2, one percent lactose (disaccharide) for test tube three, one
percent sucrose (disaccharide) for test tube four, one percent starch (polysaccharide) for test
tube five, leaf extract for test tube six, fruit extract for test tube seven, and distilled water
(control) for test tube eight. The test tubes were then added with one ml of their labelled
solutions. To each test tube, 2 drops of five percent α – napthol was added and each test tube
were shook afterwards. The test tubes were inclined and one ml concentrated H2SO4 was
carefully added to each, allowing the acid to flow gradually on its side. The violet ring formation
was then noted at the interphase after three minutes.

The same labels were done for the Seliwanoff’s test and eight clean test tubes were
also prepared. Instead of one ml, three ml of the same solutions—the ones used in the Mol
Isch’s test, were placed to the corresponding labelled test tubes. To each test tube, three ml

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Seliwanoff’s reagent were added and all of the eight test tubes were heated simultaneously in
a boiling water bath. The exact time when the test tubes were placed in the water bath were
noted. Color development were observed and the time required for the color development in
each of the test tubes were recorded.

Same labels with Molisch’s test and Seliwanoff’s test were put on the eight test tubes
of Benedict’s test. Just like in Seliwanoff’s test, three ml of the same solutions were placed on
each of the corresponding labelled test tubes and instead of the Seliwanoff’s reagent, three
ml of the Benedict’s reagent were added to each test tube and were mixed thereafter. The test
tubes were then heated in a boiling water bath and the formation of the brick red precipitate
were observed in any of the test tubes.

For the characterization of hydrolytic products of disaccharides and polysaccharides,

the action of acid on disaccharides and polysaccharides were performed. two test tubes were
prepared for the action of acid on disaccharides. Into each of the two test tubes, five ml one
percent sucrose solution were placed using a pipette. For test tube no. one, five ml Benedict’s
reagent was added while five drops of concentrated HCl was added to test tube no. two. Test
tube no. two was warmed gently over direct flame using an alcohol lamp for two minutes, after
which, five ml Benedict’s reagent was added again. The two test tubes were then placed in a
boiling water bath and as soon as the brick red precipitate became noticeable, the test tubes
were removed. Pictures were taken for documentation.

The action of acid on polysaccharides first required the preparation of the starch-HCL
solution. In a two five ml beaker that contains 80 ml of one percent starch, 10 ml concentrated
HCl was added. The solution was mixed thoroughly via stirring using a glass rod. The solution
was then allowed to stand for 10 minutes and was set aside until it was needed.

For the actual experiment of the action of acid on polysaccharides, 10 clean test tubes
were prepared and 10 ml starch-HCl solution were transferred to test tube nos. one to eight
using a pipette. To test tube no. nine, 10 ml of one percent starch solution was transferred
while 10 ml of one percent glucose was put to test tube no. 10. Afterwards, all of the 10 test
tubes were carefully and simultaneously placed in the boiling water bath and then the time
was noted. Test tube no. one was removed after two minutes of immersion and at every two-
minute interval, one test tube was removed from the boiling water bath according to their
chronological number until test tube nos. eight, nine and 10 were the only ones left. The test
tubes that were removed were immediately placed in a cold water bath, after which, the last
three test tubes were placed into the cold water bath at the same time after the prescribed
two-minute interval. I2KI was dropped to each test tube, when all of the test tubes in the cold
water bath has sufficiently cooled down, and mixed. The color intensities of the 10 test tubes,
using test tube no. nine and 10 as a reference, were observed for their similarities and
differences. Lastly, the achromatic point, which is the absence of the starch-iodo complex
formation (blue color), was determined between the 10 test tubes.

RESULTS AND DISCUSSION

Characteristic Color Reaction of Carbohydrates

Three tests were used to determine and observe the characteristic color reaction of
carbohydrates. It includes the Molisch test, Seliwanoff’s test and Benedict’s test. The samples
used for these tests were: one percent glucose, one percent fructose, one percent lactose,
one percent sucrose, one percent starch, leaf extract, fruit extract, and distilled water.

To represent hexose carbohydrates, glucose and fructose were used. Lactose and
sucrose both represented disaccharides while starch represented polysaccharides. The leaf

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of Akapulko (Cassia alata) and fruit extracts of Kiat kiat (Citrus reticulata) were also tested to
determine the presence of carbohydrates within them. Distilled water was included in the
activity to serve as a control.

Molisch Test

Figure 1. Hexose reaction for Molisch’s test (Medical Biochemistry 2017)

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 that is either furfural or a derivative (Figure 1), which
then condenses with the phenolic structure resulting in a red or purple-colored compound
(Chhabra 2014).

Figure 2. Monosaccharides and Disaccharides

Monosaccharides give a rapid test result. Glucose (Figure 2) and fructose (Figure 2)
are monosaccharides. Disaccharides and polysaccharides, on the other hand, react more
slowly than monosaccharides. Lactose (Figure 2) and sucrose (Figure 2) are disaccharides
while starch (Figure 3) is a polysaccharide. Leaves (Figure 4) contain sufficient amounts of
starch, cellulose, hemicellulose, and pectic substances. Glucose is a product of
photosynthesis and when there is an excess of it, it will be converted to starch—its storage
form. Cellulose, hemicellulose and pectic substances are essential in the structural
composition of the cell walls in plant cells, in this case, the leaf cells (Andrews et al. 1960).
Also, starch is found in all types of orange juice (Figure five) (Prabarasi et al. 2010).

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 Figure 3. Polysaccharide and Control

All of the samples gave a positive result of a purple-colored ring, except for the distilled
water (Figure 3), since all of them are carbohydrates.

Figure 4. Leaf extract Figure five. Fruit extract

Seliwanoff’s Test

Seliwanoff’s test is a color reaction specific for ketoses and aldoses. When
concentrated HCl is added, ketoses undergo dehydration to yield furfural derivatives more
rapidly than aldoses. These derivatives (Figure 6) form complexes with resorcinol to yield deep
red color. The test reagent causes the dehydration of ketohexoses to form 5-
hydroxymethylfurfural. This 5-hydroxymethylfurfural reacts with resorcinol present in the test
reagent to produce a red product within two minutes. Aldohexoses reacts so more slowly to
form the same product (Shahidulla 1972).

Figure 6. Seliwanoff’s test reaction (Yikrazuul 2008)

Fructose (Figure 7) produced the positive result since it is the only ketose
representative among the samples. Sucrose (Figure 7) also tested positive as a ketose sugar
because it is has a fructose component in its structure, aside from glucose.

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 Figure 7. Monosaccharides and Disaccharides

Glucose (Figure 7) and lactose (Figure 7) are examples of aldoses (reducing sugar).
This was proven to be true since they reacted more slowly compared to fructose and sucrose.
Hence color light orange were observed with them. Eventually they will also form a deep red
color like the ketoses if given more time. Starch (Figure 8) is a polysaccharide made up of
glucose units so it did not form the ideal result for ketoses. The leaf extract (Figure 7), as well
as the fruit extract (Figure 8) remained with their original colors since almost all of their
components are made up of polysaccharides. Distilled water (Figure 8) also did not react with
HCl, the same way the other samples did since it does not have any carbohydrates in it.

Figure 8. Polysaccharide, Extracts and Control

Benedict’s Test

Benedict’s reagent (Figure 9) contains blue copper (II) ions (Cu2+, cupric ions) that are
reduced to copper (I) ions (Cu+, cuprous ions) by carbohydrates. These ions form precipitate
as red colored cuprous (copper (I) oxide. The negative result is a blue solution or no color
change which means that it is a non-reducing sugar (Fine 193five).

Figure 9. Benedict’s test Reaction (Aryal 201five)

Free aldehyde or keto group in the reducing sugars reduce cupric hydroxide in alkaline
medium to red colored cuprous oxide. Depending on the concentration of sugars, yellow to
green color is developed. All monosaccharides are reducing sugars as they all have a free
reactive carbonyl group. Hence, they are capable of transferring hydrogens (electrons) to
other compounds, a process called reduction. Some disaccharides, like lactose, have exposed
carbonyl groups and are also reducing sugars, but less reactive than monosaccharides
(Cammidge 1917).

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 Figure 10. Monosaccharides and Disaccharides

Glucose, fructose, lactose, the leaf extract and the fruit extract yield a positive result.
It took glucose (Figure 10) three minutes and 4five seconds to achieve the desired result on
the heating bath. Fructose (Figure 10) took a lesser time than glucose with 2 minutes and
eight seconds. Lactose (Figure 10) took three minutes and five seconds while the leaf extract
(Figure 10) took four solid minutes. The fruit extract (Figure 11) took the longest time, with five
minutes and 33 seconds for the formation time of the brick-red precipitate.

Figure 11. Polysaccharide, Extracts and Control

Sucrose (Figure 10) did not yield a positive result because it is a non-reducing sugar.
Also, complex carbohydrates, such as starches (Figure 11), do not react positive with the
Benedict’s test unless they are broken down through heating or digestion (Aryal 201five). In
the experiment, even if the starch was heated for approximately an hour, it still did not form a
brick-red precipitate. In addition, the distilled water (Figure 11) did not from any brick-red
precipitate since it does not contain carbohydrates.

Characterization of hydrolytic products of di- and polysaccharides

For the characterization of hydrolytic products of disaccharides and polysaccharides,

the action of acids were observed for sucrose and starch. Sucrose served as the
representative for disaccharides while starch represented polysaccharides. Glucose was used
in the action of acid on polysaccharides to serve as a control.

Action of acid on disaccharides

Sucrose is a disaccharide made up of glucose and fructose. The glucose and fructose
are joined by their glycosidic bond in such a way as to prevent the glucose isomerizing to
aldehyde, or the fructose to alpha-hydroxy-ketone form (Benedict 1909).

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 Sucrose, therefore, is a non-reducing sugar which does not react with Benedict's
reagent. This is the reason why no color change was observed even after the heating of the
solution (Figure 12 and Figure 13).

Upon the addition of an acid though, as seen on figure 13, the solution formed a red
solution. This is because HCl acted as a catalyst that separated glucose and fructose into
individual units. It took 2 minutes and 39 seconds before the red solution was observed.

Figure 12. Before heating Figure 13. After heating

Action of acid on polysaccharides

This part of the activity illustrated the conversion of starch (non-reducing sugar) to a
reducing sugar by the action of HCl at boiling point. HCl acted as a catalyst in the reaction.
The longer the starch was exposed to the acid, the further the hydrolysis proceeded.

Figure 14. Starch Structure (Nelson and Cox 2003)

Although starch (Figure 14) has free hemiacetal in the terminal glucose residue, it has
no reducing properties, because the percentage between the free residues is very low in
comparison to the whole molecule. Hence starch did not react or reacted very poorly with
Benedict’s reagent. Heating starch solution in acid medium, though, hydrolysed the glycosidic

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bonds that resulted many free glucose residues. These glucose molecules gave reducing
properties to the hydrolysis product.

Figure 15. Starch-HCl-I2KI solution and Glucose-I2KI solution

As a result of incorporating iodine ions (I2KI) into the starch-HCl solution, the
complexes of a characteristic colour are formed. This is called an intermolecular charge-
transfer complex. The colour depends on the chain length. Amylose gives blue colour,
almylodextrin – purple, glycogen – red. Lugol's iodine yields a blue-black colour in the
presence of starch. Heating causes the shift of absorption maximum to the ultraviolet and the
colour turns clear. After cooling the colour turns blue-black again (Ophardt 2003).

The achromatic point, which is the absence of the starch-iodo complex formation, was
achieved on the eighth test tube. The solutions turned purple, which meant that the starch is
composed mainly of amylodextrin. The starch is lighter as the number increase. This is
because it is being hydrolyzed into individual glucose units. After reaching the achromatic point
and after cooling, the solution turned to dark purple again as could be seen in b9 and b1 of
figure 1five. Test tube 10 is a glucose solution. Hence it remained clear all throughout the
activity.

CONCLUSION

Different tests were conducted in order to detect the presence of carbohydrates, as

well as to identify which specific carbohydrates are present in the samples. For the first part,

Molisch’s test, Seliwanoff’s test and Benedict’s test were conducted. All of the samples gave
a positive result (violet ring formation) for the Molisch’s test, except for the control, since all of
them were carbohydrates. Only sucrose and fructose gave a positive result (red solution
formation) since sucrose contains a ketose part and fructose is a ketose itself for Seliwanoff’s
test. Glucose, fructose, lactose, the leaf extract and the fruit extract yield a positive result
(brick-red precipitate formation) since glucose is a reducing sugar and all the others have
reducing ends or reducing sugar components for the Benedict’s test.

For the second part, the action of acid on disaccharides and polysaccharides were
observed. In the presence of the HCl, the acid used in the activity, sucrose and starch were
hydrolysed into glucose and fructose (for sucrose) and into glucose units (for starch). Both the
heating of the solution, as well as the acid, acted as catalysts in the reactions.

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 LITERATURE CITED

Alejar AA, et al. 2009. Laboratory Manual in Elementary Plant Physiology. 4th Ed. Institute
of Biological Sciences, CAS, UP Los Banos, College, Laguna, Philippines. 117 pp.

Andrews P, Hough L, Stacey B. 1960. Polysaccharide composition of leaves. Nature

18five: 166-167.

Aryal S. 201five. Benedict’s Test- Principle, Composition, Preparation, Procedure and

Result Interpretation. Retrieved on February 21 2018, from
https://microbiologyinfo. com/benedicts-test-principle-composition-preparation-
procedure-and-result-interpretation/

Benedict S. 1909. A Reagent for the Detection of Reducing Sugars. J. Biol. Chem. five
(6): 48five–487.

Berg JM, et al. 2002. Biochemistry. fiveth Ed. New York: W H Freeman. 974 pp.

Cammidge PJ. 1917. Benedict's test for sugar in the urine. Br Med J 2( 29five0): 6five-67.

Caton, KA. 2011. Seliwanoff & Benedicts Test. Retrieved on February 10 2018, from
https://www.slideshare.net/katealyssacaton/seliwanoff-benedicts-test

Chhabra N. 2014. Qualitative tests for carbohydrates: methods and significance. Retrieved
on February 10 2018, from https://www.slideshare.net/namarta28/qualitative-tests-
for-carbohydrates-3five88414five

Fine J. 193five. Benedict’s qualitative test: a further modification suitable for estimation of
urine glucose in ward or side room. Br Med J 3883: 1169-1170.

Hunt I. 2018. Aldoses and Ketoses. Retrieved on February 10 2018, from

http://www.chem.ucalgary.ca/courses/3five1/Careyfiveth/Ch2five/ch2five
-2-4.html

Jequier E. 1994. Carbohydrates as a source of energy. Am J Clin Nutr five9 :682-68five.

Medical Biochemistry. 2017. Molisch’s test. Retrieved on February 22 2018, from

http://www.medbiochemistry.com/molischs-test/

Nelson F, Cox R. 2003. Carbohydrate structure. Retrieved on February 20 2018, from

http://cbc.chem.arizona.edu/classes/bioc462/462a/NOTES/CARBO/carb_structure.ht
m

Ophardt C. 2003. Starch-Iodine. Retrieved on February 22 2018, from

http://chemistry.elmhurst.edu/vchembook/five48starchiodine.ht
ml

Prabarasi I, Pettolino F, Liao ML, Bacic A. 2010. Pectic polysaccharides from mature orange
(Citrus sinensis) fruit albedo cell walls: Sequential extraction and chemical
characterization. Carbohydrate polymers 84 (1): 46-five4.

Royal Society of Chemistry (RSC). 2018. Carbohydrates. Retrieved on February 10 2018,

From http://www.rsc.org/Education/Teachers/Resources/cfb/carbohydrates.htm

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Shahidulla M, Khorasani SS. 1972. The sensitivity and selectivity of the Seliwanoff test for
fructose. Analytica Chimica Acta 61 (2): 317-319.

Simoni RD. 2002. Benedict's Solution, a Reagent for Measuring Reducing Sugars: the
Clinical Chemistry of Stanley R. Benedict. Journal of Biological Chemistry f5:
485.

Tatad JRR, et al. 2014. Hydrolysis of polysaccharides and qualitative tests for
carbohydrates. Retrieved on February 10 2018, from
https://www.scribd.com/doc/five7744435/Hydrolysis-of-
Polysaccharides-and-Qualitative-Tests-for-Carbohydrates

Yikrazuul, R. 2008. Seliwanoff’s test reaction. Retrieved on February 22 2018, from

https://books.google.com.ph/books?id=rCU2-gvkjo0C&pg=PA71&redir_esc=y#v=one
page&q&f=false

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