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Enzymatic Hydrolysis, Analysis of Mucic Acid Crystals and Osazones, and Thin-
Layer Chromatography of Carbohydrates from Cassava
Mendoza, P.*, Mesina, K.*, Nodora, C.*, Nombre., M., Pineda, E.
Group 6, 2A Pharmacy
Department of Pharmacy, Faculty of Pharmacy
University of Santo Tomas

Abstract
Carbohydrates contain carbon, hydrogen, and oxygen. They are sugars, starches, or fibers found
in fruits, grains, and milk and these are essential in human life. Manihot esculenta, known as
cassava, manioc, or yuca, is composed of two types of molecules namely: amylose and
amylopectin. Amylase helps in digesting carbohydrates and found in the saliva and made in the
pancreas. Different tests are conducted in this experiment namely enzymatic hydrolysis, mucic
acid test, phenylhydrazone test, and thin layer chromatography. This experiment aims to
determine the role of amylase on the hydrolysis of isolated polysaccharide, to examine
microscopically the different mucic and osazone crystals, and to perform thin-layer
chromatography on the carbohydrate hydrolysates. Enzymatic hydrolysate obtained a positive
result in Benedict’s test, which is a brick red precipitate of Cu O. The resulting observation from
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the carbohydrate solution in Mucic Acid test are yellow, clustered masses indicative of a
negative result and absence of galactose. Osazone crystals from Phenylhydrazone test possess
yellow color. Glucose and Fructose give alike osazones named glucosazones and fructosazones.
Chromatography is a procedure that divides compounds into their individual components.
Several compounds move in different distances and with different speed on the plate by
capillary action. The Rf value of the enzymatic hydrolysate, being close to the value of glucose
would suggest that it mostly contains glucose molecules. Some of the results acquired were
accurate with the principles involved in each experiment and their expected results; however,
some were otherwise due to the inevitable errors during the execution of the experiments.

Introduction Manihot esculenta, known as
cassava, manioc, or yuca, belongs to the
Carbohydrates are spurge family of plants called
polyhydroxyaldehydes (aldoses) and Euphobiaceae. It is most likely originated in
polyhydroxyketones (ketoses) with the South American forest regions and it is
general formula (CH2O)n where n equals 3 or commonly consumed in tropical countries
more. At the chemical level, they are called in parts of Asia, Africa, and South America.
carbohydrates because they contain It is known to contain starch. [2] It is
carbon, hydrogen, and oxygen. composed of two types of molecules
Carbohydrates are sugars, starches, or namely: the linear and helical amylose and
fibers found in fruits, grains, and milk. the branched amylopectin.[8]
These are the most important sources of
energy and these are essential in human
life. Carbohydrates are classified into
different classes depending on the number
of monosaccharide units they contain
namely monosaccharides, disaccharides,
oligosaccharides, and polysaccharides.[9]

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Thin layer chromatography (TLC) is

a procedure done to separate and identify
compounds of interest and makes use of
the polarity of the compounds. This is also
done for the qualitative analysis of the
carbohydrate constituents from the
enzymatic hydrolysate and is usually
Figure 5.1: The structure of amylose (top) visualized using various visualizing agents.[2]
and amylopectin (bottom).
This experiment aims to determine
Different tests are conducted in this the role of amylase on the hydrolysis of
experiment namely enzymatic hydrolysis, isolated polysaccharide, to examine
mucic acid test, phenylhydrazone test, and microscopically the different mucic and
thin layer chromatography. osazone crystals, and to perform thin-layer
chromatography on the carbohydrate
Amylase is an enzyme that helps in hydrolysates.
digesting carbohydrates. It is found in the
saliva and is made in the pancreas. Methodology
Enzymatic hydrolysis is a method in which
enzymes promote the separation of bonds Enzymatic Hydrolysis
in molecules with the addition of the
components of water and presents a crucial A. Preparation of Dialyzing Bag
function in the digestion of food.[7]
The collodion solution was poured
The oxidation of most into a clean and dry hard glass (ignition)
monosaccharides by nitric acid produce tube. The inside of the tube was
dissolvable dicarboxylic acids, however, completely and carefully coated by slowly
oxidation of galactose generates an rotating it in a horizontal position while
insoluble mucic acid. Lactose also produces the excess collodion solution was poured
a mucic acid, owing to hydrolysis of the off back into its container. The tube was
glycosidic bonds linking its glucose and suspended allowing the inner coating of
galactose subunits.[6] collodion solution to dry. The film around
the tube was loosened and cold water was
Phenylhydrazone test comprises allowed to run in between it and the
the reaction of monosaccharides with membrane. The dialyzing bag was rinsed
phenyl hydrazine, which appears as a with distilled water.
crystalline compound.[4]
B. Enzymatic Hydrolysis proper

Ten milliliters of isolated
carbohydrate was placed into a beaker then
2.3 mL of saliva was added and allowed to
stand at room temperature for a total of
thirty minutes and observed for any
Figure 5.2: The representation of the changes in the hydrolysate’s viscosity. The
reaction involved in the Phenylhydrazone solution was poured into a dialyzing bag
test. and suspended overnight in a small flask or
beaker with 50 mL distilled water. The
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dialyzing bag was then removed and cm from the bottom, was lightly lined
discarded. The solution inside the flask was across with a pencil. Equidistant points
concentrated using an open flame to the were marked along the origin for the
volume of 10 mL. The presence of reducing standards and the enzymatic hydrolysates.
sugar in the hydrolysate was tested by The standards were applied five times and
performing Benedict’s Test. the enzymatic hydrolysate ten times using
capillary tubes, drying the spots after every
Mucic Acid Test application. The spotted TLC plate was
placed inside the developing chamber
Three drops each of galactose and ensuring that the solvent system is below
lactose were each mixed with 3 drops of the the line of origin. The developing chamber
concentrated HNO3 on a glass slide. The was covered and the TLC plate was allowed
glass slides containing the mixtures were to be developed until the solvent is about 1
passed over a small flame from an alcohol cm from the top of the TLC plate. The
lamp until they were almost dry. The chromatoplate was removed from the
mixtures were cooled at room temperature. chamber after development and the solvent
The crystals were examined under the front was marked with a pencil. It was then
microscope and then drawn. The glass slide air-dried and sprayed with the visualizing
was allowed to stand until the next period if agent of 5 mL p-anisaldehyde, 9.0 mL 95%
no crystals appeared. CH3CH2OH, 0.5 mL H2SO4, and 0.1 mL
CH3COOH. The plate was then placed in the
Phenylhydrazone Test oven and heated at 100°C to 150°C for a
total of 10 minutes. Colored spots are
Two grams of the phenylhydrazine indicators of sugars present. The spots
hydrochloride and 3 g of the CH3COONa identified are then encircled and their Rf
were mixed with 10 mL of distilled water. values calculated. The Rf values of the
The mixture was placed in a warm water standards are compared with that of the
bath until clear. Two drops each of glucose, enzymatic hydrolysate to identify the
fructose, xylose, lactose, sucrose, and components of the hydrolysate.
starch were mixed with 4 drops of freshly
prepared phenylhydrazine reagent in Results and Discussion
separate test tubes. The test tubes were
covered with cotton then heated in a The following figures and tables
boiling water bath for 30 minutes. The time below show the observations acquired from
recorded was when the crystals first the results of the Benedict’s test performed
appear. The tubes are then cooled and the on the enzymatic hydrolysate, and the
crystals were observed under the analysis of the crystals formed by the mucic
microscope and drawn. and phenylhydrazone tests, followed by the
tables showing the measurements or values
Thin-Layer Chromatography that were computed from the thin- layer
chromatography.
Forty milliliters of the solvent
system, acetonitrile:ethyl acetate:n-propyl
alcohol:water (85:20:50:90 v/v), was placed
into a beaker as a developing chamber and
then covered with inverted watch glass. The
chamber was equilibriated for a total of 10
minutes. The end of the TLC plate, about 2
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hydrolysate is a green or yellow-green

precipitate indicative of traces of reducing
sugars. This was due to the fact that
enzymatic hydrolysis is incomplete
hydrolysis utilizing the enzyme found in our
saliva called amylase.

Mucic acid is also known as
galactaric acid and is particularly valuable in
the identification of galactose. Galactose, a
Figure 5.3: The result observed from the C-4 epimer of glucose, is a kind of sugar that
Benedict’s test done on the enzymatic is less sweet than glucose and is recognized
hydrolysate. as a nutritive sweetener because it has food
energy. Galactose, a monosaccharide,
Benedict’s test is a test used for combined together with glucose via a
identifying reducing sugars dehydration reaction, results into the
(monosaccharides and most disaccharides) disaccharide known as lactose.[3]
wherein cupric ions in alkaline medium
shows a positive result by appearing as Galactose is transformed to
brick red precipitate of Cu2O. Benedict’s saccharic acid upon heating with HNO3,
test originates from the alteration of which is a strong oxidizing agent. Saccharic
Fehling’s test. The problems encountered acid is insoluble in cold water and assists in
by Fehling’s test are, consequently, not the classification of galactose.[5]
encountered in Benedict's test.[6] The
principle behind this is the isomerization of
ketoses to aldoses. This is found to be
reliable in the testing of urine for
pathological measures of sugars.[1]

Figure 5.5: The resulting crystal observed
from the Mucic acid test viewed under the
microscope.

Figure 5.4: Side-by-side comparison of the
result (right) obtained from the
hydrolysate from the color interpretation
standards (left).
Figure 5.6: Side-by-side comparison of the
As seen in the figure above we can result (left) obtained from the
compare the obtained result of the carbohydrate solution from the expected
Benedict’s test from the enzymatic mucic acid crystals formed by galactose
hydrolysate to the expected positive result, (right).
which is a brick red precipitate of Cu2O. The
resulting product observed from the
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Concentrated nitric acid heated shaped crystals, whereas the glucose and
together with galactose results into a fructose form alike needle-shaped crystals.
dicarboxylic acid and appears as a white
precipitate, indicative of a positive result.[5] Based on the duration of time for
As seen in the figure above we can compare the formation of the crystals, Fructosazone
the obtained result of the Mucic acid test takes 2 minutes to form, Glucosazone takes
from the carbohydrate solution to the 5 minutes, Galactosazone takes 20 minutes,
expected positive result, which is a white Maltosazone takes 30-45 minutes, and
precipitate of mucic acid crystals. The Lactosazone takes 30-45 minutes to form.[4]
resulting observation from the
carbohydrate solution are yellow, clustered Chromatography is a procedure
masses indicative of a negative result and that divides compounds into their individual
absence of galactose. components. Several compounds move in
different distances and with different speed
Phenylhydrazine, an organic on the plate by capillary action. To take this
compound, reacts with carbonyl carbon of variation into account, the ratio of the two
sugar to form the osazones. These osazone distances (distance by the compound and
crystals possess yellow color, characteristics distance by the solvent front) is calculated
shapes, melting point, time of formation, and reported. This ratio is called the
and solubility. retention factor (Rf).

Table 5.1: Data obtained for the Thin-Layer
Chromatography of standards and
hydrolysate.

Figure 5.7: Microscopic Observations of
the Osazones of fructose, glucose, starch,
xylose, lactose, and sucrose respectively.

Glucose and Fructose give alike
osazones named glucosazones and
fructosazones because disregarding the first
two carbons, which are used in the
formation of osazone, the remaining four
carbon atoms then have an identical
arrangement in both of them. Sucrose
doesn't form any osazone because it has no Figure 5.8: The TLC Plate Result.
reducing group ready for reaction with
phenyl hydrazine. But upon hydrolysis, In the thin-layer chromatography,
however, it yields osazone. The lactose the stationary phase is a highly uniform
produces powder puff shape crystals, absorbent paper. Cellulose (nonpolar and a
maltose makes sunflower-shaped or star- polymer of the simple sugar, glucose) in the
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form of paper sheets creates a fitting execution of the experiments or the lack of
medium where various components are time given.
absorbed by the fibers thus the
components will visibly separate. Cellulose Reference
typically applies for partition of
carbohydrates, sugars, alcohols, amino [1] Aryal, S. (2015). Benedict’s test principle,
acids, carboxylic acids, fatty acids in paper composition, preparation,
chromatography. The mobile phase, procedure and result
acetonitrile:ethyl acetate:n-propyl interpretation. Retrieved from
alcohol:water (85:20:50:90 v/v), would then http://www.microbiologyinfo.com/
be polar. The compound with the larger Rf benedicts-test-principle-
value is less polar because it does not stick composition-preparation-
to the stationary phase. From the table procedure-and-result-
above we can infer that although the Rf interpretation/
values are close to each other, still maltose
from the standards is shown to be the least [2] Bathan, G., Bayquen, A., Cruz, C.,
polar due to its high Rf value. The Rf value Crisostomo, A., De Guia, R., Farrow,
of the enzymatic hydrolysate, being close to F., Peña, G., ... Torres, P. (2014).
the value of glucose would suggest that it Laboratory manual in Organic
mostly contains glucose molecules. Chemistry (Revised ed.). Quezon
City, Philippines: C & E Publishing,
Conclusion Inc.

Different tests were used to [3] Caton, K. (2011). Mucic and Barfoed's
determine the identity of carbohydrates tests. Retrived from
such as Benedict's test, which exhibits a http://www.microbiologyinfo.com/
principle of isomerization of ketoses and katealyssacaton/mucic-and-
aldoses. Others include Mucic acid tests, barfoeds-test
which is used to identify galactose and
Phenylhydrazone test to identify osazones [4] Chhabra, N. (2015). Osazone test.
(fructose and galactose). For quantitative Retrieved from
analysis, Thin-Layer Chromatography was http://usmle.biochemistryformedic
used for the identification of the s.com/osazone-test/
components of carbohydrates and their
respective polarity. These tests are useful [5] Hernandez, G. (2017). Mucic acid.
and essential to human life because it helps Retrieved from
us to determine carbohydrates present in http://www.academia.edu/228322
the food we eat. 64/Mucic_Acid_Test

These experiments were conducted [6] Kumar, P. (2016). Qualitative and
as an application of the knowledge on the quantitative tests for
principles involved in each of tests and carbohydrates. Retrieved from
procedures done. Some of the results http://www.biologydiscussion.com/
gathered were coherent and accurate with carbohydrates/test/qualitative-and-
the said principles and their expected quantitative-tests-for-
results although some were otherwise due carbohydrates/13042
to the inevitable errors during the
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[7] Lal, S. (2015). Amylase – blood.

Retrieved from
https://medlineplus.gov/ency/articl
e/003464.htm

[8] Mercola, J. (2016). What Is Cassava
Good For? Retrieved from
http://articles.mercola.com/sites/ar
ticles/archive/2016/07/25/cassava-
benefits.aspx

[9] Szalay, J. (2015). What are
Carbohydrates? Retrieved from
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-carbohydrates.ht
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