ISOLATION AND HYDROLYSIS OF GLYCOGEN, CHARACTERIZATION OF DIFFERENT

POLYSACCHARIDES, SEPARATION OF MONOSACCHARIDES, AND QUANTITATIVE

DETERMINATION OF DIFFERENT POLYSACCHARIDES

Nogoy, Rodrigo John1 Pineda, Alexandra2 Rotulo, Jhon Justin3 Villegas, 

Psyche Jane Mar T.4 Yow, Mark Nicholas5
1Department of Biochemistry

Abstract

Carbohydrates are the most abundant class of organic compounds. It is also a vital source of
energy storage in biological life. The current study for this experiment is to isolate polysaccharide
samples from the chicken liver; to perform the general test for carbohydrates; to compare the hydrolysate
products; to perform dialysis separation of products from enzymatic hydrolysis; to illustrate the specificity
of 𝛼-amylase on hydrolysis; to perform thin-layer chromatography on carbohydrates and identify its
monosaccharide; to classify the unknown carbohydrate; to determine the amount of reducing sugars on
the sample; and lastly to explain all of the test in its principle regarding the reaction. In this experiment,
isolation of Glycogen was performed via hydrolysis and precipitation. Glycogen produced a violet color on
Molich’s test and produced a red color on I test. Each of the sugars were then put on a qualitative test.
2

Glucose, fructose, xylose, lactose formed a brick-red precipitate in Benedict's test. Glucose, fructose,
xylose produced a brick-red precipitate in Barfoed’s test. Xylose, lactose, sucrose and starch formed
blue-green coloration in Bial’s orcinol test. Fructose and sucrose produced a light-red orange solution in
Seliwanoff’s test. Lactose formed broken glass crystals, observed via microscope in Mucic acid test. The
acid hydrolysate consists of slightly glucose whereas the base hydrolysate contains dextrin via Thin-layer
chromatography. Lastly, Quantitative tests were then performed via Nelson’s reagent on different
concentrations of standard glucose. The results showed that glucose, fructose, xylose, and lactose are
reducing sugars and monosaccharides excluding lactose; sucrose, starch, acid, and enzyme hydrolysate
are non-reducing sugars; xylose, lactose, sucrose and starch are pentoses; fructose and sucrose are
ketoses; and lactose is an aldohexose.

Introduction

Carbohydrates are the most abundant class of organic compounds with a variety of
functions. They serve as metabolic precursors for virtually all other biomolecules and mainly as
a major form of energy storage in plants and animals. Monomers, consisting of simple sugars
called monosaccharides, contain either an aldehyde group (aldoses) or a ketone group
(ketoses). The number of carbon units a sugar contains also determines whether
monosaccharides are labelled as pentoses (a sugar with five carbons) or hexoses (a sugar with
six carbons). A type of covalent bond, termed as a glycosidic linkage, enables monosaccharides
to bond with other monosaccharide units and form oligosaccharides or polysaccharides.
Oligosaccharides, originating from the word “oligo” meaning few, contain two to ten
monosaccharide units. Polysaccharides contain more than ten subunits.

The main source of energy for all organisms comes from a homopolysaccharide with
repeating linkages of the monomer glucose. In animals, this polymer takes on a branched form
called glycogen. In plants, it takes on the form of starch which exists in both linear and branched
forms called amylose and amylopectin, respectively. A number of qualitative tests and
quantitative tests have been developed over the years to determine the components of
carbohydrates in isolated samples of the aforementioned polysaccharide. Molich’s test and the
 Iodine test are examples of tests that are able to confirm the presence of a carbohydrate. Other
tests are able to determine more specific components such as whether the sugar contains an
aldehyde group or a ketone group through the use of the Seliwanoff’s test, or if the
monosaccharide is a pentose using the Bial’s Orcinol test. Barfoed’s test specifies the presence
of a monosaccharide or polysaccharide, and whether the sugar is reducing or not is detected by
the use of Benedict’s test. Reducing sugars may be quantified by the use of Nelson’s test.
Mucic acid is a specific test for galactose. Thin-layer chromatography is also utilised to ascertain
components of acid and enzymatic hydrolysate.

This experiment aims to isolate the polysaccharide glycogen from animal sources.
General tests will likewise be conducted to confirm the presence of carbohydrates. Hydrolysate
products yielded from enzymatic and acid hydrolysis will be compared, and dialysis separation
of products from the latter will be illustrated. Thin-Layer chromatography as well as qualitative
tests will be performed and correlated to identify the monosaccharide present in the isolate.
Reducing sugars are to be determined using the Nelson’s Test. All principles involved will be
explained.

Methodology

I. Extraction of Glycogen from Chicken Liver

3g of chicken liver was weighed approximately and transferred in a petri dish. It was then
minced into smaller pieces and transferred in a beaker. Then, 12 mL of boiling water was added
to the liver and stirred using a glass rod. The mixture was boiled for 2 minutes in order to
precipitate the proteins. The mixture was transferred into a mortar and the sample was grinded
until lumps were no longer visible. 3 mL of distilled water was added again to the mixture. Then,
it was heated in boiling water for 30 minutes and to avoid the mixture from drying, water was
consecutively added. After heating, the glycogen became visible. 1 mL of 0.1% of acetic acid
was added in order to further precipitate the proteins. The mixture was filtered and the isolated
glycogen solution was divided into four separate test tubes labeled from 1 to 4. The glycogen
solution was then used on different tests.

II. Glycogen Precipitation by Ethanol

From the glycogen solution that was collected from the filtration, test tube 1 was used. 5
to 10 drops of ethanol were added on 1 mL glycogen solution. As the water shell of glycogen
molecules loses, precipitation was formed.

III. General Test for Polysaccharides

A. Molisch’s Test

A few drops of Molisch’s reagent (5% α-naphthol in 95% ethanol) was added into 1 mL
of glycogen solution. Then 2 mL of concentrated H SO was added on the side of the test tube.
2 4

A layer was formed, and the color was then observed at the junction of the two liquids.

B. Iodine Test

0.01 M I solution was added in 1 mL of glycogen solution. Then the mixture was heated
2

in a water bath to observe if there was a change in color. The mixture was then cooled and
recorded the result. 
IV. Hydrolysis of Polysaccharides

A. Acid Hydrolysis

5 drops of concentrated HCl was added into the 5 mL solution of the isolate. The test
tube was then covered with marble and transferred into a water bath for 30 minutes. The
hydrolysate was stored in the refrigerator for Benedict’s test.

B. Enzymatic Hydrolysis

10 mL of the isolated glycogen was transferred on a beaker. 2.3 mL of saliva was

extracted from the mouth rinsed with warm water for 1 minute and transferred the washings on
a beaker. The mixture was set aside for 30 minutes, and a change in viscosity was observed.
The solution was then transferred into a dialyzing bag and was suspended in a small flask filled
with 50 mL distilled water. Then the solution from the dialyzing bag was removed and discarded
the dialyzing bag. The solution was concentrated inside the flask from the hot plate into a
volume of 10 mL. The presence of reducing sugars in the hydrolysate was observed after
performing Benedict’s test.

V. Qualitative test for Carbohydrates

A. Benedict’s, Barfoed’s, Seliwanoff’s, and Bial’s Orcinol Tests

5 drops of standard carbohydrate solution such as glucose, fructose, xylose, lactose,

sucrose and starch were added into 1 mL of each reagent required for each test. The test was
performed individually on different carbohydrate solutions at the same time. Then all the test
tubes containing the sample with specific reagent were heated on a boiling water at the same
time. The test tubes were removed from the water bath after the tubes formed a visible positive
result. The procedure was repeated for other tests.

B.  Mucic Acid Test

3 drops of standard lactose solution and 3 drops of concentrated HNO were mixed on a
3

glass slide. The glass slide containing the solution was then placed over a small flame until the
mixture was almost dry. Then the glass slide was cooled at room temperature. Microscope was
used to examine the crystals formed. The mucic acid crystals were then drawn to record the
data.

VI. Thin-Layer Chromatography

20mL of n-butyl alcohol: acetic acid: ether: water (9:6:3:1 v/v) solvent system was placed
in a developing chamber. The developing chamber was covered with an inverted watch glass
and was equilibrated for 10 minutes A 1cm of line was drawn from the bottom using a pencil
across one end of the TLC plate. The equidistant points were marked along the origin line for
the standards, acid hydrolyzed sample, and enzymatic hydrolyzed sample. Then the standards
were applied 5 times and the hydrolysate samples 10 times using the capillary tube, it was dried
every application of the standard or sample on the TLC. The TLC was then placed inside the
developing chamber and made sure the solvent was below the origin line. The developing
chamber was covered again after placing the TLC and let the solvent develop in the TLC until
the solvent was about 1 cm distant from the top of the TLC plate. Then the chromatoplate was
removed from the developing chamber. The chromatoplate was dried and sprayed with a
visualizing agent. Then, it was carefully placed on top of the hot plate avoiding it to be totally
burned. The spots were then appeared. Rf values of each spot on the chromatoplate was
computed and was compared to the Rf values of standards with the products of the acid and
enzymatic hydrolysates. The components of the acid and enzymatic hydrolysates were then
identified.

VII. Quantitative Analysis of Carbohydrates

12.5 mL of Nelson’s A and 0.5 mL of Nelson’s B were mixed to prepare the Nelson’s reagent.
Then, 7 test tubes having standard glucose solution with distilled water  were labeled. 1.0 mL of
the prepared Nelson’s reagent were put and mixed into the test tubes. After that, all test tubes
were put into a boiling water bath for about 20 minutes. After heating, all test tubes were
allowed to cool and 1 mL of arsenomolybdenate reagent were put into the test tubes until the
Cu2O precipitate dissolved. Small amounts of the mixture were transferred into 96-well plate
and absorbances were read at 480 nm. A standard curve was constructed using all the
absorbances and concentration of the unknown solution was determined.

Results and Discussion

I. General Test for Polysaccharides

The precipitation of the proteins was done by boiling the solution. During heating, glycogen was
left soluble in the solution while proteins were precipitated. The precipitation process of
glycogen was catalyzed by the addition of 0.1% acetic acid, the impurities or precipitate was
separated from the solution using filtration.

(insert extract picture) 

Precipitation using ethanol 

Glycogen is a polymer which is used to trap the nucleic acids. In ethanol, glycogen is insoluble
so it forms polymer structure which shows as white precipitate. In this reaction, glycogen does
not react with ethanol that is why it forms a precipitate because if it did react, it will not for a
precipitate and it will be dissolved in alcohol.

Table 1. Isolation of Polysaccharide

Description Molisch’s Test KI/I2
Isolate Precipitate Violet pale yellow
Figure 1. Molisch’s Test of Glycogen

Molisch’s Test is a general test specifically to all types of carbohydrates. This test
determines whether the isolate is a carbohydrate or not. Upon addition of a strong acid, sugars
undergo dehydration and formation of furfural for pentoses occur while on the other hand,
hexoses form 5-hydroxymethylfurfural. This results in purple coloration at the interface of two
liquids. The glycogen, which was the isolate, gave a positive result as seen on Figure 1.

Figure 2. Iodine Test of Glycogen upon Heating

Complexation is the principle of iodine test for polysaccharides. For starch, when iodine
solution is subjected to it, it forms a violet color while for
glycogen, it gives a red complex due to branching. When heating, both colors disappear but
reappear when cold. As seen on Figure 2, upon heating the solution, the red complex
disappeared.

II. Hydrolysis of Isolated Polysaccharide

Table 2. Hydrolysis of Isolated Polysaccharide

Hydrolysate Description (Viscosity) Benedict’s test

 
 Acid Hydrolysate No change in viscosity Blue solution (-)

Enzyme More viscous Blue solution (-)

Hydrolysate

Acid Hydrolysis

Glycogen is a polymer of glucose. This is easily demonstrated by acid-catalyzed hydrolysis to

the monosaccharide. The acid hydrolysis is addition of acid to a covalent bond. In the case of
glycogen, the glycosidic covalent bonds are the target of acid hydrolysis. Heating of glycogen in
the presence of conc. HCl causes its hydrolysis into glucose because of the free aldehyde
group, making glycogen a strongly reducing monosaccharide.Many oligosaccharides form
eventually result as glucose. The reaction is shown as: 

C H O +H /H O-------->2(C H O )
12 22 11
+
2 6 12 6

Acid hydrolysis of acetals regenerates the carbonyl and alcohol components, in the case of the
glucose derivative, the result will be a tetramethyl ether of the pyranose hemiacetal. This
compound will, of course, undergo typical aldehyde reactions. 

Figure ?. Acid Hydrolysate from the Glycogen Extract

The acid hydrolysis resulted negative on Benedict’s test but this should not be the case as
glucose is a reducing sugar. The possible cause of the result is that glycogen was not fully
hydrolyzed by the acid which still leads to a non free aldehyde group. Thus it will still not react to
Benedict's test.
Figure ?. Benedict’s test of Acid Hydrolysis

  Enzymatic Hydrolysis

Enzyme-catalyzed hydrolysis are more specific with respect to bonds cleaved, for example, α-
amylase of human saliva. The α-amylase catalyzes the rapid, random hydrolysis of internal α-
1,4 bonds. They do not hydrolyze α-1,6 linkages regardless of the size nor do they hydrolyze
maltose. So, glycogen is initially split by α-amylase action into branched dextrin of medium
molecular weight and little amounts if maltose is formed. The final degradation products of the
action of α-amylase on glycogen are glucose, maltose and isomaltose. The glucose is formed
by the relatively slow end cleavages of the oligosaccharides.

Enzymatic hydrolysis was done by the process of dialysis, which includes a semi-
permeable membrane that allows molecules to pass through via diffusion into the surrounding
medium. In this experiment, the dialyzing bag which is a colloid solution composed of pyroxylin
film, ether, and alcohol, served as the membrane that allows monosaccharides and
disaccharides to pass through into the distilled water medium. 

Figure ?. Enzyme Hydrolysate from the Glycogen Extract

III. Specific Reactions of Carbohydrates

(bials, seliwanoff etc.)

Mucic Acid Test

 Mucic Acid Test is specifically for galactose and lactose only. The carbon 1 and carbon 6
of lactose undergo oxidation that leads to formation of aldaric acids. Positive result of this test
gives broken glass-like crystals under a microscope. Lactose as seen on Figure ? gave a
positive result.

Figure 3. Mucic Acid Test of Lactose Under a Microscope

V. Qualitative Tests for Carbohydrates

Most carbohydrates that are present can be identified using different tests like
benedict’s, barfoed’s, seliwanoff’s, and bial’s orcinol. 
Benedict's test is used to detect the presence of a reducing sugar that has either an
open-chain form with a free aldehyde, a free hemiacetal, or a free ketone group. It is a semi-
quantitative test where the color gives an estimate of reducing sugar is present. All
monosaccharides have free reactive carbonyl groups and some disaccharides have exposed
carbonyl groups and also a reducing sugar, but other disaccharides are non-reducing sugar.
Based on figure 3, glucose had a result of orange precipitate that showed a moderate level (1.5-
2.0 gm%) of reducing sugar (Pandya, 2013). While fructose and xylose had a result of green
solution with orange precipitate meaning the level of reducing sugar is traceable (0.5-1.0 gm%).
And the remaining carbohydrate solution such as lactose, sucrose, and starch showed a
negative result which is just a blue solution because it contains non-reducing sugars. The
precipitates happens because the CuSO4in Benedict’s solution reacts with the group of the
reducing sugar and it is then oxidized by the Cu ions in the solution that forms a carboxylic acid
and a brick-red precipitate of CuO2.

Figure 4. Result for Benedict’s Test of different carbohydrate solutions where positive
result has red-orange precipitate and negative result for a color blue solution (left to
right: glucose, fructose, xylose, lactose, sucrose, and starch).

In Barfoed’s test, a reducing monosaccharide and reducing disaccharide can be

distinguished. In figure 4  all of the carbohydrate solutions showed a negative result which is
blue solution. For the positive result, brick-red precipitate forms where Cu ions in a slightly
acidic medium oxidize the carbohydrate and within 3 minutes, positive result will be visible for
reducing monosaccharides and reducing disaccharides undergoes in the same reaction but the
result appears after approximately 10 minutes (Schreck, 1994).

Figure 5. Result for Barfoed’s Test of different carbohydrate solutions where all solutions
showed negative results  with a visible color blue solution (left to right: glucose,
fructose, xylose, lactose, sucrose, and starch).

In Seliwanoff’s test, it is used in determining between aldoses and ketoses. It has HCl as
a dehydrating agent and resorcinol as condensation reagent. The HCl dehydrates the ketoses
to form 5-hydroxymethylfurfural then it condenses with resorcinol that produces a cherry-red
solution within 2 minutes (Jahangirpuria, et. al, 2017).  The aldohexoses also reacts to form the
same product but the result appears more slowly and gives a pale-yellow to light-pink color
solution. Like in figure ---, fructose and sucrose showed a positive result where the solution has
a light red-orange solution while the rest of the carbohydrate showed a negative result where
the solution if pale-yellow.

Figure 6. Result for Seliwanoff’s Test of different carbohydrate solutions where positive
result has a cherry red solution on the second and fifth test tube while negative result for
the rest that have had a pale-yellow solution (left to right: glucose, fructose, xylose,
lactose, sucrose, and starch).

In Bial’s Orcinol test, pentose monosaccharide and hexose monosaccharide is being

distinguished. Concentrated HCl is being used as a dehydrating acid and orcinol +  FeCl3as a
condensation agent. Pentoses is being dehydrated to form furfural then further reacts with
orcinol and the Fe ion present in the reagent that produces a moss green or blue-green solution
and hexoses produces muddy-brown to grey solution (Figueira, 2013).

Figure 7. Result for Bial’s Orcinol Test of different carbohydrate solutions where second,
third, and fifth test tube showed positive results with a visible blue-green and green
solution (left to right: glucose, fructose, xylose, lactose, sucrose, and starch).

Thin Layer Chromatography

In order to determine the components of acid and enzyme hydrolysate, thin layer
chromatography was performed and the hydrolysate was compared to the different
carbohydrate solutions (glucose, maltose and dextrin). 

Table 3. Thin Layer Chromatography of Hydrolysates and Glucose Standards

 Standards Hydrolysates
 

Glucos Maltose Dextrin Acid Enzymatic

e

Distance traveled by 6.3 cm 6.3 cm 6.3 cm 6.3 cm 6.3 cm

solvent

Distance traveled by solute 0 cm 1.6 cm 6.2 cm 3.9 cm 0 cm

Rf value 0 0.25 0.98 0.62 0

Identity of components _ _ _ Glucose Dextrin

Figure ?. Thin Layer Chromatography of Acid and Enzyme Hydrolysate with the Glucose
Standards

Based on the data obtained for the retention factor, it was inferred that the identity of the
enzyme hydrolysate is dextrin while the identity of the acid hydrolysate is glucose. The enzyme
hydrolysate tested negative in all classification tests pertaining to a polysaccharide. The
obtained result from the acid hydrolysate tested negative in Benedict’s test which delineates into
glycogen which is a non-reducing sugar. The two hydrolysates tested negative in Benedict’s test
since both of it are non-reducing sugar and no other sugar can bond with them.  

The results that were gathered show that the acid hydrolysate’s identity is dextrin. According to
Salway, Dextrin is a carbohydrate produced by hydrolysis of glycogen or starch and is made up
of polymers of D-glucose linked by α-(1→4) or α-(1→6) glycosidic bonds. During acid
hydrolysis, it undergoes complete hydrolysis which means that it completely breaks the
glycosidic bonds of dextrin to get the D-glucose components. In conclusion, the retention factor
of dextrin should be lower than glucose because dextrin is more polar than glucose.

The obtained component for the enzymatic hydrolysis resulted as glucose which means that the
hydrolysis undergoes complete hydrolysis because the glycogen was hydrolyzed into glucose.
According to Richens, it is completely plausible for the hydrolysates to be dextrin and glucose in
acid and enzymatic hydrolysis respectively, due to the fact that there are factors which limits or
exceeds the expected results which can only be seen or determined experimentally.

However, if the obtained data from the TLC experiment would be compared to the results of the
qualitative tests, the hydrolysates would be both dextrin because both hydrolysates tested
negative on Benedict’s test but in the theoretical aspect of acid hydrolysis, acid hydrolysate
should have a positive result in Benedict’s test because it should have a reducing sugar due to
complete hydrolysis.

Some possible errors occurred could be the lack of distilled water in the flask was not exactly 50
mL. Also, maybe the hydrochloric acid was not sufficient to undergo complete hydrolysis since it
should cleave all the glycosidic bonds in the glycogen.

Table 4. Visible Results of Qualitative Tests for Different Carbohydrate Solutions.  

Carbohydrate Visible Results
Solution

Benedict’s Test Barfoed’s Seliwanoff’s Test Bial’s Orcinol Test

Test

Glucose Orange precipitate  Blue solution Pale-yellow solution Yellow solution

Fructose Green solution with orange precipitate Blue solution Light red-orange Green solution
solution

Xylose Green solution with orange precipitate Blue solution Pale-yellow solution Blue-green solution

Lactose Blue solution Blue solution Pale-yellow solution Yellow solution

Sucrose Blue solution Blue solution Light red-orange Yellow-green solution

solution

Starch Blue solution Blue solution Pale-yellow solution Yellow solution

VII. Quantitative Analysis of Carbohydrates

Reducing sugars are sugars that contain a free formyl or carbonyl functional group and
therefore are able to function as reducing agents. All monomers are classified as reducing
sugars. Several disaccharides and oligosaccharides are likewise able to undergo reduction-
oxidation reactions depending on whether the aldehyde or ketone group is free and non-
bonding. The amount of reducing sugars generally present in a carbohydrate sample can be
quantitatively determined through the Nelson-Somoyogi method of quantification or simply
Nelson’s Assay.
The Nelson-Somoyogi method of quantification is used to create a standard curve for glucose.
While it may be utilized for quantifying reducing sugars, it does not distinguish between reducing
monosaccharides, disaccharides, or polysaccharides. 
Nelson’s reagent is composed of two separate reagents mixed together, namely Nelson’s A
reagent and Nelson’s B reagent. Nelson’s A reagent is slightly basic and is a mixture of 
Na2CO3, NaKtartrate, NaHCO3, and Na2SO. Nelson’s B reagent serves as the oxidizing agent
and is comprised of CuSO4 • 5H2O and H2SO4. 12.5mL of Nelson’s A and 0.5mL of Nelson’s B
is integrated into samples containing known volumes of standard glucose solution, and the
mixture’s absorbances are individually read after the solution has been heated for an ample
amount of time.

Figure ?: Samples Depicting the Formation of Molybdenum Blue

When reducing sugars are heated with copper tartrate in an alkaline environment, copper is
reduced from the +2 or cupric state to the +1 or cuprous state. This effectively forms cuprous
oxide which is visibly denoted by a brick-red precipitate. Thereafter, arsenomolybdate reagent
(–(NH4)2MoO4, H2SO4, Na2HasO4 • 7H2O) is infused into the reduced solution and the
reduction of this substance results in the formation of the compound molybdenum blue after
dissolution of cuprous oxide. The consequential blue color developed per test tube is transferred
to a microwell plate and the absorbances read against a reagent blank at 480nm. 

Table 5: Concentrations and Absorbances of Glucose Standard Solution and Unknown

1 2 3 4 5 6 7 8

Glucose Standard (M) 0 0.001 0.002 0.004 0.006 0.008 0.01 -

Glucose Standard (mg/mL) 0 0.180156 0.36012 0.720624 1.080936 1.44124 1.80156 -

8

Corrected Absorbance (nm) 0 0.292 0.202 0.562 0.649 0.878 1.077 0.077

Concentration of Unknown (M) - - - - - - - 0

Concentration of Unknown - - - - - - - 0
(mg/mL)
The determination of reducing sugars present in a carbohydrate sample is measured
colorimetrically. Such is possible due to the direct correlation of the blue color formation of
molybdenum blue and the amount of reducing sugars. Figure __ below suggests that tube
number 7 has the most amount of reducing sugars. This is further supported by the data found
in table ____. Tube 7 purely contains 1.0 mL of the 0.01M glucose standard solution. Tube 1
serves as the blank, comprising of only water and 1.0 mL of Nelson’s reagent. This explains the
lack of blue coloration. Tubes 2 to 6 contain diluted samples of the glucose solution. 

Test tube 8, containing the unknown sample, has a slightly darker green coloration as compared
to the blank but a lighter hue than tube 2. This is indicative of availability of reducing sugars,
however not in abundance. Its corrected absorbance of 0.077 also hints that a small amount of
reducing sugars is present despite the computed 0 M or 0 mg/mL concentration from the linear
equation generated from the standard glucose curve in figure ___. An error that may have lead
to the conflicting data could be attributed to the fact that 200 microliters were not uniformly used
when individual solutions from the 8 test tubes were transferred to the 96-microwell plate. This
error is highlighted by the sudden drop in absorbance in tube 3 despite it having a higher
concentration than tube 2.

Figure ?: Glucose Standard Curve

Conclusion

In this experiment, a lot of principles were involved in isolating and characterizing the
carbohydrates. 
The general test for glycogen from chicken liver is Molisch’s Test where the
polysaccharides undergo hydrolysis, dehydration that forms furfural or 5-hydroxymethylfurfural,
and condensation with 𝞪-naphthol. And Iodine test that forms a red complex due to branching. 
Hydrolysis of the isolated polysaccharides consists of two components, acid and
enzymatic hydrolysis. The acid hydrolysis is the imparts of complete hydrolysis and the product
of it is monosaccharides. While the enzymatic hydrolysis is where the salivary amylase was
placed in a dialyzing bag to separate the molecules.
For the specific reactions of carbohydrates, there are several tests based on– (a) Production of
Furfural Derivatives: Bial’s Orcinol Test for pentoses that undergoes dehydration to form furfural
and condensation with orcinol then complexation with iron ions and Seliwanoff’s Test for
ketohexoses that undergoes dehydration that forms 5-hydroxymethylfurfural and condensation
with resorcinol; (b) Based on Oxidation: Benedict’s Test for the reducing sugars, Barfoed’s Test
for reducing monosaccharides from disaccharides, and Mucic Acid Test for galactose and
lactose; and (c) Property of Carbonyl Carbon: Osazone Test for all sugars with free carbonyl
group at C-1 or C-2.
 In separating the components of monosaccharides, Thin-layer chromatography was
used where normal phase chromatography and the mobile phase of N-butyl alcohol, acetic acid,
ether, and water was used.
Lastly, the quantitative determination of reducing sugars was used through Nelson’s
Test where the intensity of blue coloration is dependent on the amount of copper oxide formed
that is related to the amount of reducing sugar oxidized.
By all counts and with proven results, this experiment is successful and precise. There
were few errors encountered in the experiment but it didn’t affect the results.

References

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