Group Members: Group No.

YB2G
1. Caryll Apostol Section: CHEM 133L YB2
2. Riah Santos Date Submitted: October 2, 2020

Expt. 2 Isolation of Polysaccharides and

Analysis of Carbohydrates

I. Objectives Member 1 Member 2

• To isolate the polysaccharides of chicken liver Pre-lab Data
and predict the name and its reactivity during Data and Observations
the tests.
• To test the presence of carbohydrates Lab Report
in the given test samples using the Sub-total
molisch test and anthrone test Online Act Participation
• To test the presence of starch in the
given tests samples using iodine test. Total Score
• To test the presence of sugars with the
given test samples using the Benedict’s,
Barfoed’s, Seliwanoff’s and Orcinol’s tests
• To describe the basis and principles of the
general tests, iodine tests and qualitative tests

II. Apparatus:
• Filter paper
• Graduated cylinder
• Filter funnel
• Beaker
• Cutting board and knife
IV. Procedure: (Note: Write schematic diagram on the sheet provided.)
III. Symbols of Chemicals:
• Micro test tube • CH3COOH (10% acetic acid)
• Spot plate • C2H5OH (95% ethanol)
• H2SO4 (50% sulfuric acid)
• Droppers
• Test tube • C14H10O (Anthrone reagent)
• Test tube rack • C6H12O6 (glucose, fructose)
• C5H10O5 (arabinose)
• Water bath
• C12H22O11 (maltose,
sucrose)
• C6H12O6 (galactose)
• C12H22O11 (lactose)
• C6H10O5 (starch)
• C18H32O16 (dextrin)
• C15H20NNaO4(gum Arabic)
• H2O (Water)

V. Data and Observations

Part 1. Isolation of Polysaccharides

Sample used
Description of filtrate After filtering the chicken liver solution, the liquid filtrate
shows a faint transparent yellow color.

Description of precipitate After adding 95% ethyl alcohol to the filtrate and mixing it, a
cloudy solution is observed. White precipitate at the bottom
of the beaker is present.

1
 Part 2. Analysis of Carbohydrates

A. GENERAL TESTS FOR CARBOHYDRATES

Observations
Test Sample
MOLISCH TEST ANTHRONE TEST
A dark purple colored ring After mixing glucose with the
Glucose separates the cloudy light purple anthrone reagent, a dark blue color
liquid(glucose) from the green (close to black) mixture is observed.
colored liquid (sulfuric acid) found
in the bottom of the test tube.
A dark purple liquid covers more A dark blue color (close to black) is
Sucrose than half of the test tube and the observed after mixing surcrose and
light purple liquid (sucrose) on the the anthrone reagent.
top forms a gradient with the dark
purple. The sulfuric acid found in
the bottom of the test tube turned
yellowish green.
The dark purple ring is twice After mixing arabinose and the
Arabinose bigger than the glucose molisch anthrone reagent, a dark blue color
test. The analyte which is that is close to black is observed.
Arabinose is cloudy light purple
while the sulfuric acid at the
bottom of the test tube becomes
color green and it mixes with the
dark purple (molisch reagent)
Compared to the other test tubes, After mixing the starch and anthrone
Starch the sulfuric acid which turned reagent, the color shifted from green
green is not visible. The test tube to a dark blue color after 30 minutes.
is divided in two colors, the starch
which is on top that shows a
cloudy light purple color and the
molisch reagent which is found on
the bottom half of the test tube.
A dark purple ring forms in A transparent yellow liquid is formed
Cotton or Filter Paper between the cotton suspension from mixing the cotton suspension
Suspension (light purple) and sulfuric acid and anthrone reagent.
(light green).
Out from all the test tubes, only There is a slight color difference from
Water water does not have the dark the cotton suspension analyte and
purple color. The analyte, water water analyte solution. After mixing
changes to a cloudy orange on water and the anthrone reagent, the
top of the test tube and a yellow liquid possesses a darker yellow
ring separates it with the sulfuric compared to the cotton suspension.
acid which became a transparent
light green color.
A light purple ring will be When mixing glycogen with anthrone
Glycogen observed since glycogen is reagent, a dark blue color (close to
multibranched polysaccharide of black) shall be observed.
glucose.
2
 *Predict reactivity for filtrate from sample

B. IODINE TEST FOR POLYSACCHARIDES

Test Sample Color Response to Iodine

A cloudy black color develops.
Starch
A dark black purple develops
Dextrin
After adding the yellowish iodine solution, the gum-arabic analyte
Gum-arabic changes to a color yellow.
The yellow iodine solution mixes with the water and develops a
Water yellowish color.
After adding the yellowish iodine solution, the colorless
Glycogen filtrate/glycogen changes to a blue-black color.
*Predict reactivity for filtrate from sample

3
 C. QUALITATIVE TESTS FOR SUGARS (Indicate color of solution/precipitate & write + or -)

Benedict’s Test Barfoed's Test Seliwanoff's Test Orcinol’s Test

Test Solution
color +/ color +/- color +/- color +/-
-
orange + blue with + - -
Glucose minimal red Light yellow Light yellow
precipitate
&
suspension
+ blue with + - -
Galactose brown minimal red Light yellow Light yellow
precipitate
&
suspension
+ blue with + Bright red + +
Fructose Brown minimal red Dirty green
precipitate
+ blue with + -
Arabinose orange minimal red Light yellow Dirty green +
precipitate
+ - - -
Maltose orange transparent Light yellow Light yellow
blue
+ - - -
Lactose orange transparent Light yellow Light yellow
blue
- - + -
Sucrose Transparent transparent Bright red
blue blue Light yellow
- transparent - - -
Water Transparent blue Light yellow Light yellow
blue

4
 VI. Introduction

This laboratory report considers the isolation of polysaccharides from chicken liver and the
different tests in determining the presence of specific saccharides (carbohydrates). The first
experiment performed is the isolation of polysaccharides. Chicken liver is used because of the
profound presence of the storage polysaccharide, glycogen. Glycogen is a soluble glucose polymer
found in animal tissue. Glycogen is also found in animal muscle, however for this experiment,
chicken liver is used. The following tests that were performed are Molisch, Anthrone, Iodine,
Benedict’s, Barfoed’s, Seliwanoff’s and Orcinol’s. Each and every test follow an array of different
procedures and are based of contrasting principles. The procedures or schematic diagrams for each
test are attached in page 9-11. The basis and principles of each test will be discussed thoroughly
in part VII analysis of this lab report.
The analytes used for the general tests of carbohydrates (Molisch & Anthrone) are glucose,
arabinose (monosaccharides), sucrose(disaccharide), starch, cotton or filter paper suspension,
glycogen (polysaccharides) and lastly the control variable, water. An expected purple ring will form
when adding the Molisch reagent to the test sample. Sulfuric acid is added carefully added to
facilitate the formation of a layer and to avoid mixing. For the anthrone test, a bluish green color is
to be expected in addition of the anthrone reagent to the analyte. In conditions when the solution
appears to be milky, add sulfuric acid to dilute.
The addition of iodine solution in the Iodine test is expected to yield a blue-black color when
reacting with a polysaccharide. The tests samples are starch, dextrin, gum-arabic and glycogen all
of which are polysaccharides. A control variable, water is also a test sample for this experiment.
The last four experiments are qualitative tests for sugars. The analytes used are glucose,
galactose, fructose, arabinose(monosaccharides), maltose, lactose, sucrose(disaccharides) and
lastly water (control). Each test follows the same procedure, however different reagents are used in
every experiment. For the benedict’s test a green to dim red or brown is an expected observation.
The addition of the Barfoed’s reagent in the Barfoed’s test should possess red precipitate in the
solution. In the Seliwanoff’s test, the color is dependent on the presence of ketones and aldoses. A
bright red is expected when ketones is present while a light pink color is observed in aldoses. Lastly,
for orcinols test, the expected color is green. A plus sign is then indicated when a change of color
is observed.
This experiment is performed for students to understand and differentiate the different
carbohydrates through the use of different tests. At the end of this lab report, students will have a
deeper comprehension of the objectives and principles of each tests.

VII. Analysis

Carbohydrates, also known as saccharides, are carbon compounds that contain large
quantities of hydroxyl groups and are also the most important sources of energy. They have the
basic general formula Cn(H2O)n and they are the most commonly found organic compounds
in living organisms. They are classified into several groups, namely monosaccharides,
disaccharides, and polysaccharides, depending on the number of their monosaccharide units.
(Genuino et.al, 2018)
Glycogen, the major glucose storage polymer in animals, has a highly branched structure
which permits rapid release of glucose from glycogen stores, for example in muscle cells during
exercise. Glycogen is a very compact structure that results from the coiling of the polymer chains.
This compactness allows large amounts of carbon energy to be stored in a small volume, with little
effect on cellular osmolarity. In this experiment, glycogen was isolated from the chicken liver with
the use of precipitation. Chicken liver is used in this experiment because it is a good source to

5
 isolate glycogen from. The first steps for isolating polysaccharides from an animal source such as
the chicken liver is by rinsing, drying, and cutting it into small pieces. Through cutting the sample
into small pieces, it can be easier to isolate and extract such substances that will be used and
observed. While the sample is still being heated in the water bath, 10% of acetic acid is being added
in order to coagulate the proteins. The next step in isolation of polysaccharides from the sample is
by filtration using filter paper and a funnel. Through filtration, it is important to immediately filter the
solution while it is still hot so that it can filter the impurities present especially during isolation. Once
the solution is finished being filtered, the filtrate is added with two volumes of 95% ethyl alcohol.
Ethanol can help break-down or extract more of the filtrate. After mixing the solution with ethyl
alcohol a cloudy solution is observed. White precipitate at the bottom of the beaker is present. This
precipitate is glycogen. The glycogen elicited a positive result upon the addition of Molisch’s
Reagent and sulfuric acid because the Molisch’s Test is a test for carbohydrates. Same happens
with the Anthrone test, a dark blue color is observed. It also produced a positive result for Iodine
Reaction because it will react with glycogen found in a polysaccharide or in the solution.

General tests for Carbohydrates

Molisch’s test is a chemical test which is used to check for the presence of carbohydrates in a
given analyte. Molisch’s test involves the addition of Molisch’s reagent to the analyte and the
subsequent addition of a few drops of concentrated H2SO4 (sulphuric acid) to the mixture. The
formation of a purple or a purplish-red ring at the point of contact between the H2SO4 and the analyte
and Molisch’s reagent mixture confirms the presence of carbohydrates in the analyte. The Molisch’s
Test shows positive results (purple) for all carbohydrates, with monosaccharides (glucose,
arabinose) reacting much faster than disaccharides (sucrose) and polysaccharides (starch,
cotton or filter paper suspension/cellulose, glycogen). The addition of Molisch reagent to the water
test tube yielded a negative result, therefore concluding that there are no carbohydrates present in
water.
In Molisch’s test, the carbohydrate undergoes dehydration upon the introduction of concentrated
hydrochloric or sulphuric acid, resulting in the formation of an aldehyde. This aldehyde undergoes
condensation along with two phenol-type molecules (such as ∝-naphthol, resorcinol, and thymol),
resulting in the formation of a purple or reddish-purple colored complex.

Anthrone test is also another general test for all carbohydrates. The anthrone reaction is the basis
of a rapid and convenient method for determination of carbohydrates, either free or present in
polysaccharides. It can be used as qualitative test, since different sugars dehydrate at different rates
and produce a variety of colors. After adding the anthrone reagent to the test analytes, glucose,
sucrose, arabinose, starch and glycogen are the only test samples that yielded a positive result. A
dark blue color was observed for the five test samples. Therefore, indicating that there are
carbohydrates present. The water test sample in the other hard yielded an expected color yellow,
indicating the absence of carbohydrates. For the cotton or filter paper suspension/cellulose, a rather
unexpected result was obtained. Upon mixing with the anthrone reagent, a transparent yellow color
is observed. Although, the Molisch test produced a positive reaction from the cellulose, the anthrone
test did not. The possible reason that it may have not worked is because the reagent added must’ve
been too little. Another reason is because there are limitations for the anthrone test for carbohydrates.
This test requires the initial formation of furfural or a derivative of it. In this test carbohydrate should
first get dehydrated when reacting with conc. H2SO4 to form furfural. This furfural reacts with
anthrone to give bluish green colored complex.

Iodine Test for Polysaccharides

The Iodine Test, on the other hand, is used to identify polysaccharides. Polysaccharides

6
 combine with iodine to form a positive result (a blue-black.) The Iodine Test for Starch is utilized
to decide the presence of starch in natural materials. The test can be subjective or quantitative.
At the point when treated with Iodine solution, iodine broke up in a watery arrangement of
potassium iodide the tri-iodide-anion edifices with starch, creating a blue/purple coloring. On the
other hand, when you are observing, and no color change appears in the solution then it means
that starch is not present in the solution. Starch, Dextrin and Glycogen developed a dark color
indicating the presence of starch. Gum-arabic a polysaccharide yielded an unexpected negative
result; the reason may be the lack of iodine solution added on the gum Arabic. The difference in
color is apparently related to the extent of coiling in the different samples of glycogen. As different
samples of glycogen give the iodine solution varying colors from wine red, red, red brown to
brown. Gum-arabic a linear structure is not coiled therefore giving no color to the iodine.

Qualitative Tests for Sugars

The Benedict's test allows us to detect the presence of reducing sugars or sugars with a
free aldehyde or ketone group. At the point when reducing sugars are blended with Benedict’s
reagent and warmed, a decrease response causes Benedict’s reagent to change its coloring. The
color fluctuates from green to dim red or brown, depending upon the measure of and sort of
sugar. The concentration of the sugar affects the color; blue- sugar is absent; green-0.5% sugar;
yellow- 1% sugar; orange 1.5% sugar; brick-red 2% or more sugar.
Glucose, Maltose, Arabinose, and Lactose yields an orange color after boiling indicating
the presence of reducing sugars. A brown color is observed with the test samples, Galactose and
Fructose, indicating the presence of reducing sugars. Although fructose a ketone is not strictly a
reducing sugar, it is an alpha-hydroxy-ketone and gives a positive test because it is converted to
the aldose’s glucose and mannose by the base in the reagent. The control variable, water yields
a negative result indicating the absence of reducing sugars.

Barfoed’s Test also detects the presence of reducing sugars. By the use of the
Barfoed's reagent we can distinguish monosaccharides from disaccharides. This happens by
controlling such conditions as: PH, time of heating and sugar concentration. At the point, when
barfoed’s reagent mixes with series of monosaccharide or disaccharide and warmed in bubbling
water bath, they react, and a precipitate solution is formed. Copper acetic acid derivation which
is available in barfoed’s reagent of copper oxide gives block red solution when it reacts with
monosaccharide or disaccharides. Monosaccharide responds quickly while disaccharide
responds gradually. Red Precipitate = positive. Only the monosaccharides Glucose, Galactose,
Fructose and Arabinose show minimal red precipitate in the solution. Although it is also expected
for maltose, lactose and sucrose to yield a positive result, these disaccharides need to be boiled
longer than the monosaccharides. Water, the last test sample is observed to have a negative
result therefore indicating the absence of reducing sugars.

Seliwanoff’s Test is used to detect ketoses, sugars containing one ketone group
per molecule. Ketoses are differentiated from aldose by means of their ketone/aldehyde
usefulness. If the sugar contains a ketone bunch, it’s a ketose. If the sugar contains an aldehyde
bunch, it is an aldose. This test depends on the rule that, when heated, ketones are more quickly
dried out than aldoses. At the point when it is added to a solution containing ketones, a red
shading is framed quickly giving a positive test. At the point when added to an element containing
aldoses, a slower framing pink light is seen. A cherry-red shading shows the nearness of
ketohexoses. Ketopentoses yield blue-green series while aldoses and disaccharides give no
shading change. Sucrose and Fructose are the only analytes that show a positive result. Both
sucrose and fructose are two common sugars which give a positive test. Sucrose gives a positive
test as it is a disaccharide consisting of fructose and glucose. Ketose get dehydrated faster and

7
 hence they give the test faster. Aldoses react very slowly and give faint colors. The aldoses
present in the experiment are glucose and galactose, both did not give positive reaction. The
control test sample water yields a negative result indicating the absence of ketoses in the solution.

Orcinol Test is used to detect pentoses with a test reagent consisting of orcinol, HCl,
and ferric chloride. In the presence of pentoses, the test reagent dehydrates pentoses to
form furfural. Furfural, then, reacts with orcinol. A pentose, if present, will be dehydrated to form
furfural which then reacts with the orcinol to generate a colored substance. The solution will turn
bluish and a precipitate may form. However, if a hexose is present a brown complex will form. In
the results, fructose and arabinose developed a dirty green color after boiling the solution
indicating the presence of hexoses. The control variable, water yields a negative result indicating
the absence of pentoses.

VIII. Conclusion

The Molisch’s Test and Anthrone test, is the addition of Molisch’s reagent and Anthrone
reagent to the test samples such as glucose, sucrose, arabinose, starch, cotton suspension,
water, and filtrate from sample that were used as analyte with the subsequent addition of a few
drops of H2SO4 (Sulfuric acid) to the solution. In this way it can identify the presence of
carbohydrates. The purpose of H2SO4 (Sulfuric acid) dehydrates carbohydrates when reacted
with the concentration. Identifying the presence of carbohydrates from in the Molisch tests is the
formation of a purple to dark blue in color solution, which indicates a positive result for the
presence of carbohydrates. In identifying a positive result in the anthrone test a dark blue complex
is observed for identification of carbohydrates.

The Iodine test, with the addition of drops of iodine solution to the given test samples such
as starch, dextrin, gum Arabic, and water - it can detect the starch or polysaccharides present.
To identify if a starch is present, the iodine interacts with the coiled molecules which results to
dark blue to black in color solution. Through this experiment, this is due to formation of polyiodide
chains from the reaction of starch and iodine. When a dark blue to black color solution occurred,
this results from the amylose in starch that forms helices where iodine molecules assemble. If a
yellowish color solution occurs, this is due to the starch that is hydrolyzed into smaller
carbohydrate units that indicates the completion of hydrolysis which in other words, indicates the
absence of starch.

Same procedures with the same test solutions are followed for the qualitative test for
sugars. When the color of solution is red, brown, orange, yellow, green, this indicates a positive
result – which means to say that there are traces of sugars that is reduced. If the color of solution
is blue, this indicates a negative result or tells the absence of reduced sugar. In Benedict’s test,
this identifies the reducing sugars in which functional groups such as ketone and aldehyde are
free. For Barfoed’s test, this indicates monosaccharide sugars in solution. The Seliwanoff’s test
causes dehydration of ketohexoses. From the Orcinol’s test, the appearance of green color of
solution this indicates presence of minimal traces of pentoses.

At the end of the experiment, students were able to differentiate the different saccharides
from the qualitative tests. The students were also able to analyze the presence of carbohydrates
with the use of Molisch, Anthrone and iodine test with the use of reagents.

8
 For the exclusive use of Chem 133L class in Xavier-Ateneo EXPERIMENT 2

Schematic Diagram of Procedure

Isolation of Polysaccharides using chicken liver MOLISCH TEST

chicken liver Place sample

solutions in six
wash, dry & cut to small pieces separate test
place sample to 30mL tubes
boiling water
Mix 1mL of sample
boil for 20 mins until 20mL sol’n + 1 drop of
note opalescence of solution
Molisch reagent
Put few drops of 10%
acetic acid
Pur 1mL sulfuric
acid
proteins start to
coagulate
filter mixture using filter paper & funnel -Slowly, down the side of test
note the appearance of filtrate tube to form layer under
-Repeat to other samples
add 2 volumes of 95%
ethanol to the filtrate
Stir well Record
observations
record observations

THIS MATERIAL IS OWNED BY THE CHEMISTRY DEPT OF XAVIER-ATENEO

 For the exclusive use of Chem 133L class in Xavier-Ateneo EXPERIMENT 2

ANTHRONE TEST

Place sample
solutions in six
separate test tubes
add 3 drops of sample solution
add 1/2 mL anthrone reagent
mix well

if results are not if results are

milky milky

record observations dilute 50% H2S04

observe several to 30 minutes

record observations

IODINE TEST FOR POLYSACCHARIDES

Place sample solutions in

separate wells of spot plate
Add 2 drops of sample solution
Add 1 drop iodine solution in each well

Heat in boiling ater bath

Remove once visible results are
observed

Record observations

THIS MATERIAL IS OWNED BY THE CHEMISTRY DEPT OF XAVIER-ATENEO

 For the exclusive use of Chem 133L class in Xavier-Ateneo EXPERIMENT 2

BENEDICTS TEST BARFOEDS TEST

Place sample solutions in Place sample solutions in

separate micro test tubes separate micro test tubes
Add 1 drops of sample solution + Mix 1 drop sample solution + Barfoed’s
1 drop benedicts reagent reagent

Heat in boiling water Heat in boiling water

bath bath
Remove once visible results are Remove once visible results are
observed observed

Record observations Record observations

SELIWANOFF’S TEST ORCINOL’S TEST

Place sample solutions in Place sample solutions in

separate micro test tubes separate micro test tubes
Mix 1 drop sample solution + 1 drop Mix 1 drop sample solution + 1 drop
seliwanoff’s reagent orcinol’s reagent

Heat in boiling water Heat in boiling water

bath bath
Remove once visible results are Remove once visible results are
observed observed

Record observations Record observations

THIS MATERIAL IS OWNED BY THE CHEMISTRY DEPT OF XAVIER-ATENEO

 References:

Admin. (2020, September 25). Molisch's Test - Principle, Procedure, Reaction, & Reagent Preparation.
Retrieved October 02, 2020, from https://byjus.com/chemistry/molischs-test/

Admin. (n.d.). Laboratory Tests. Retrieved October 02, 2020, from https://allmedtests.com/

ISOLATION AND CHARACTERIZATION OF CARBOHYDRATES. (n.d.). Retrieved October 02,

2020, from https://pdfslide.net/documents/isolation-and-characterization-of-carbohydrates.html

Admin_masterflow. (2019, November 06). The Significance of Filtration. Retrieved September 28, 2020,
from https://www.masterflow.net.au/the-significance-of-filtration/
Guo, M., Hu, X., Wang, C., & Ai, L. (2017, November 29). Polysaccharides: Structure and Solubility.
Retrieved September 28, 2020, from https://www.intechopen.com/books/solubility-of-
polysaccharides/polysaccharides-structure-and-solubility

MegaIceRaccoon5267. (n.d.). Ring at the junction of the two liquids upon addition of concentrated H 2 SO
4: Course Hero. Retrieved September 28, 2020, from https://www.coursehero.com/file/p58h9tg/ring-at-the-
junction-of-the-two-liquids-upon-addition-of-concentrated-H-2-SO-4/
Nilay GhoshNilay Ghosh 17.1k1717 gold badges6565 silver badges137137 bronze badges, & Faded
Giant♦Faded Giant 39.7k1010 gold badges100100 silver badges216216 bronze badges. (1965, February
01). Why does fructose reduce Tollen's reagent and Fehling's solution? Retrieved September 28, 2020, from
https://chemistry.stackexchange.com/questions/40127/why-does-fructose-reduce-tollens-reagent-and-
fehlings-solution

Admin. (2020, September 25). Molisch's Test - Principle, Procedure, Reaction, & Reagent Preparation.
Retrieved October 01, 2020, from https://byjus.com/chemistry/molischs-test/

Aryal, S., Prajjwal, Love, B., Barnabas, A., Jean, Khare, N., . . . Nikhil. (2019, August 15). Benedict's
Test- Principle, Preparation, Procedure and Result Interpretation. Retrieved October 01, 2020, from
https://microbiologyinfo.com/benedicts-test-principle-composition-preparation-procedure-and-result-
interpretation/

Iodine test Definition and Examples - Biology Online Dictionary. (n.d.). Retrieved October 01, 2020,
from https://www.biologyonline.com/dictionary/iodine-test

Karki, G., & Satapathy, S. (2018, December 18). Anthrone Test: Objective, Principle, Reagents,
Procedure and Result. Retrieved October 01, 2020, from https://www.onlinebiologynotes.com/anthrone-
test-objective-principle-reagents-procedure-and-result/

Qualitative Analysis of Carbohydrates. (n.d.). Retrieved October 01, 2020, from

http://vlab.amrita.edu/?sub=3