BC 102: Biochemistry Activity 02: Qualitative Analysis of Carbohydrates

Qualitative Analysis of Carbohydrates

ACTIVITY 02

INTRODUCTION

Carbohydrates are the most abundant organic compounds found in living organisms and
are mainly composed of carbon, hydrogen and oxygen. Carbohydrates acts as the primary source
of energy for living organisms. Such compounds are called carbohydrates because they can be
considered as hydrates of carbon. Most of them have the general formula of Cx(H2O)y.

They are defined as polyhydroxy aldehydes or polyhydroxy ketones—the compounds

which produces such products upon hydrolysis. Carbohydrates are also called saccharides, some
of which have a sweet taste and are called sugars. They can also be classified through their
reactivity.

The following are some of the tests that you can use to detect the presence of
carbohydrates:
A. Molisch’s Test
Molisch’s reagent is an alcoholic solution of α-naphthol that is used for conducting
Molisch’s test on carbohydrates. This is a common chemical test used to detect the presence
of carbohydrates, where they will undergo dehydration by sulfuric acid to produce an
aldehyde. The aldehyde then condenses with two molecules of phenol—usually α-naphthol,
though other phenols (resorcinol or thymol) also give coloured products.

B. Iodine Test
The Iodine test is used to test for the presence of starch. The iodine forms a coloured
adsorption complex with polysaccharides. This makes it a useful and convenient method for
the detection of amylase, amylopectin, & glycogen.
The models below show four views (left panel) in turning from the side to an end view.
Here, the iodine molecule slips inside of the coil (right panel).

C. Benedict’s Test
Benedict’s test distinguishes a reducing sugar from a non-reducing sugar. Benedict’s
reagent contains blue copper (II) ions (Cu2+, cupric ions) that are reduced to copper (I) ions
(Cu+, cuprous ions) by carbohydrates. These ions form a coloured precipitate as a cuprous
(copper (I) oxide. This reaction can be illustrated below:

CuSO4 → Cu2+ + SO42−

Cu2+ + Reducing Sugar → Cu+

Cu+ → Cu2O

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BC 102: Biochemistry Activity 02: Qualitative Analysis of Carbohydrates

D. Fehling’s Test
The test is used to distinguish aldehyde from ketone functional groups in a saccharide.
Carbohydrates with free aldehyde or ketone groups reduce copper sulfate to cuprous oxide
forming a coloured precipitate.

OBJECTIVE:

In this activity, you will conduct some simple tests for the available carbohydrates.

SAFETY PRECAUTIONS!

1. When dealing with acid and bases avoid direct contact with your skin and eyes. If any
gets on your skin, wash thoroughly with water.
2. Wear eye protection! If any gets in your eye, rinse immediately and thoroughly with
water.
3. Never add water to acid. Always add acid to water.

MATERIALS CHEMICALS
3 x Beaker (250 mL) Molisch’s Reagent
1 x Bunsen burner Concentrated H2SO4
1 x Test tube rack Lugol’s Reagent
1 x Test tube holder Benedict’s Reagent A
25x Test tubes (10.0 mL) Benedict’s Reagent B
1 x Tripod 1% Glucose
1 x Wash bottle 1% Fructose
1 x Wire gauze 1% Maltose
1% Sucrose
1% Starch
Distilled Water

PROCEDURE

I. Physical Test
A. Solubility
1. Test the solubilities of the following solid sugars:
arabinose, fructose, glucose, sucrose, and starch in 3 solvents (water, 10% NaCl and ethanol).
2. Use only a pinch of solid to be tested in 5 mL of solvent.
3. Describe your results as soluble, slightly soluble, and insoluble.
4. Take a photo of your results and attach the image on your Lab Sheet.

II. Reactions of Carbohydrates

A. Molisch’s Test
1. Transfer 2.0 mL of your test solutions into a clean test tube.
2. Add 2-3 drops of α- naphthol solution into each tubes.
3. Very gently, pipette 1.0 mL of concentrated H2SO4 along the side of the test tube so that
the 2 distinct layers are formed.
4. Carefully observe any colour change at the junction of 2 layers.
5. An appearance of purple colour indicates the presence of carbohydrates in the sample
preparation or the test solution.
6. Note the colour of the rings obtained after some time. The first tube serves as control. In
some cases, especially polysaccharides, it may require a longer time for the colour to
appear.
7. Take a photo of your results and attach the images on your Lab Sheet.

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BC 102: Biochemistry Activity 02: Qualitative Analysis of Carbohydrates

B. Iodine Test
1. Transfer 1.0 ml of your test solutions in a clean test tube.
2. Add a drop of iodine solution in each test tube. A blank is performed with water.
3. Shake the test tube and observe the colour formation.
4. Heat the test tube gently until a change is observed.
5. Cool the test tube and observe.
6. Take a photo of your results (for steps 3-5) and attach the images on your Lab Sheet.

C. Benedict’s Test
1. Transfer 1.0 mL of your test solutions into a clean test tube.
2. Add 2.0 mL (~10 drops) of Benedict’s reagent in your test tubes containing your test
solutions.
3. Heat your solutions using a hot water bath for 3-5 minutes and observe.
• TIP: You can put your test tubes inside a 250 mL beaker filled with water then place
them inside your water bath. Make sure the water inside your beaker will not leak
inside the test tubes when heating. Put your tubes in an upright position.
4. Cool your test tubes and observe.
5. Take a photo of your results (for steps 3 & 4) and attach the images on your Lab Sheet.

D. Fehling’s Test
1. Transfer 2.0 mL of given sample solution in a clean test tube.
2. Add 2.0 mL of Fehling’s solution A and 2.0 mL Fehling’s solution B to each tube. Take a
photo after addition of your Fehling’s reagents.
3. Keep the solution in a boiling water bath for about 10 minutes.
4. Check for the formation of a coloured precipitate.
6. Take a photo of your results and attach the images on your Lab Sheet.

LABORATORY STATION AFTERCARE

1. Clean your glassware with a diluted dish washing soap solution. Cleaning materials like
test-tube brush will be provided to you from the stockroom.
2. Sponge, soap, tissues, matches, and wiping cloth are items you should personally
bring/buy for lab use.

DISPOSAL
1. CONFIRM FIRST WITH THE LABORATORY IN CHARGE IF DIRECT FLUSHING IS ALLOWED.
2. Precipitate: Put your collected precipitates in a container with their label.
3. Filtrate: Pour your filtrates to their labeled collection container.
4. Broken Glassware: If your test tube broke while heating, put the broken pieces aside to
cool. After cooling, carefully collect with and wrap the broken pieces in a paper (about 2
to 3 sheets), label it as broken test tube, and hand it over to the SA together with the rest
of your borrowed materials. You will replace this broken item before the semester ends
as part of your clearance requirement.

REFERENCES:
1. Karki, G. Molisch's test: Objectives, principle, reagents, procedure and result.
https://www.onlinebiologynotes.com/molischs-test-objectives-principle-reagents-
procedure-and-result/ (accessed Jun 21, 2022).
2. Biochemistry Laboratory Manual - Isca. http://isca.co.in/BIO_SCI/lab_manual/IeP-BS-LM-
2013-003.pdf (accessed Jun 21, 2022).
3. Libretexts. (2022, July 4). Starch and iodine. Chemistry LibreTexts. Retrieved June 22, 2022,
from
https://chem.libretexts.org/Bookshelves/Biological_Chemistry/Supplemental_Modules_(Bi
ological_Chemistry)/Carbohydrates/Case_Studies/Starch_and_Iodine

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BC 102: Biochemistry Activity 02: Qualitative Analysis of Carbohydrates

Name: Score:
Section: Date Performed:
Group #: Date Submitted:

OBSERVATIONS

I. Physical Test
A. Solubility

Sample Water NaCl Ethanol Theoretical Explanation

Arabinose is soluble in water since it

Slightly was dissolved completely. Slightly
1. Arabinose Soluble Soluble Insoluble soluble in NaCl since some of the
particles are still present. Insoluble in
ethanol since the particles did not show
any chemical reaction.
Fructose is soluble in NaCl since the
Slightly particles dissolved. Insoluble in ethanol
2. Fructose Soluble Soluble Insoluble since the particles did not show any
chemical reaction. Slightly soluble in
water since some particles are still
present.
Glucose in water and NaCl is soluble
Slightly since it did not show any chemical
3. Glucose Soluble Soluble Soluble reaction. Slightly soluble in ethanol
since some of the particles are still
present.
Sucrose in water and ethanol is slightly
Slightly Slightly soluble since some of the particles are
4. Sucrose Soluble Soluble Soluble still present. Soluble in NaCl since it
was completely dissolved.
Starch in water, NaCl, and ethanol is
insoluble since it did not show any
5. Starch Insoluble Insoluble Insoluble chemical reaction.

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BC 102: Biochemistry Activity 02: Qualitative Analysis of Carbohydrates

II. Reactions of Carbohydrates

A. Molisch’s Test
Initial Final
Sample Theoretical Explanation
Observation Observation
After Adding After a min, the In our initial observation after
concentrated sulfur color started to adding concentrated sulfur it
1. 1% Glucose
acid it did not show appear. did not show any chemical
color reactions. After a while, color
purple started to appear.
The color is It showed a dark After adding the solution the
slightly visible shade of color. color is slightly visible. In our
2. 1% Fructose
final observation, it showed a
dark shade of color after a
while.
After adding the Same with the After adding the solution the
solution the color is initial observation color is noticeable. In the final
3. 1% Arabinose
noticeable observation, it did not show any
chemical reactions.
The purple color After a min the In the initial observation, after
was visible after purple color adding the solution the color is
4. 1% Sucrose
adding the solution darkens visible. After a while, the color
darkens.
After adding the After a min, the After adding the solution light
solution light green purple color started green color is observed. In the
5. 1% Starch
color appeared to appear. final observation, the color
purple started to appear after a
while.
The green color Same with the The green color is observed
was visible after initial observation after adding the solution. After
6. Water
adding the solution. a while, it showed no chemical
reaction and stays the same.

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BC 102: Biochemistry Activity 02: Qualitative Analysis of Carbohydrates

B. Iodine Test
Initial Final
Sample Theoretical Explanation
Observation Observation
Color yellow is After heating Since the color in the initial
visible the color vanish observation, which is yellow,
1. 1% Glucose
vanishes or becomes clear, this
indicates that glucose has no starch.
Color yellow is When it comes The color in the initial observation
visible to a boil it of fructose (yellow) vanishes or
2. 1% Fructose
slightly popped becomes clear. This indicates that
and the color fructose has no starch.
vanished
Color yellow is After heating A visible yellow color was initially
visible the color slightly observed in the arabinose solution.
3. 1% Arabinose
fades In our final observation, we have
concluded that the color vanished or
it became clear which means that
arabinose has no starch.
Color yellow is The color Our initial observation of the
visible vanished sucrose solution was that it displayed
4. 1% Sucrose
a yellow pigment. But they vanish or
become clear in our final
observation. Thus, concluding that
there is no starch in sucrose.
Dark yellow After heating The initial observation of the starch
color is visible the color had a yellow color visible, and after
5. 1% Starch
becomes clear adding the Iodine solution, our final
and after a observation resulted in blue color in
while, it turned the solution. Therefore, this indicates
to blue that only the starch has a reaction
among the solutions.
Color yellow is The color In water, our initial observation has
visible vanished a visible yellow color, but it vanishes
6. Water
or become clear in our final
observation, which means that water
has no starch.

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BC 102: Biochemistry Activity 02: Qualitative Analysis of Carbohydrates

C. Benedict’s Test
Initial Final
Sample Theoretical Explanation
Observation Observation
Color is light After heating, With the presence of heat and with
blue the color turned the 10 drops of Benedict’s reagent
1. 1% Glucose
brown.
the solution turned to the color
brown.
The color is The color The fructose turned color orange
light green turned dark after heating for 10 mins and when it
2. 1% Fructose
brown cooled down it changes to dark
brown.
Light blue The color With the presence of heat and
turned to light Benedict’s reagent from light blue, it
3. 1% Arabinose
green
developed to a slightly brown color.
Light blue The color With the presence of heat, light
turned to light brown in the solution is observed.
4. 1% Sucrose
brown

Light blue The color did Starch has no chemical reaction on

not change this test. No shifting of color.
5. 1% Starch

Dark blue The color With the presence of heat and

turned to light
6. Water Benedict’s reagent, the color lightens
blue
from dark to light blue.

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BC 102: Biochemistry Activity 02: Qualitative Analysis of Carbohydrates

D. Fehling’s Test
Initial Final
Sample Theoretical Explanation
Observation Observation
Has two layers Dark brown After adding solutions A and B it
color green at shows two layers. The color changes
1. 1% Glucose
the top and blue into a dark brown after boiling for
at the bottom 10 mins and it has solid particles
settled at the bottom.
The solution Brown After adding solutions A and B it
shown is color shows yellowish green color. It
2. 1% Fructose
yellowish green changes color into brown after
boiling for 10 mins and it has solid
particles settled at the bottom.
Color is blue Dark blue After adding solutions A and B color
blue is visible. The color changes into
3. 1% Arabinose
a dark blue after boiling for 10 mins
and it has solid particles settled at
the bottom.
Color is blue Dark blue After adding solutions A and B color
blue is visible. The color changes into
4. 1% Sucrose
a dark blue after boiling for 10 mins
and it has solid particles settled at
the bottom.
Color is blue Dark blue After adding solutions A and B color
blue is visible. The color changes into
5. 1% Starch
a dark blue after boiling for 10 mins
and it has solid particles settled at
the bottom.
Color is blue Dark blue After adding solutions A and B color
blue is visible. The color changes into
6. Water
a dark blue after boiling for 10 mins
and it has solid particles settled at
the bottom.

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 BC 102: Biochemistry Activity 02: Qualitative Analysis of Carbohydrates

RESULTS AND DISCUSSION

After doing all the procedures we’ve all notice changes on the physical and chemical
reactions to every test that we’ve made. From molisch’s, iodine, to benedict’s, and lastly fehling’s.
All of this test was tested with sample glucose, fructose, sucrose, arabinose, starch, and water. In
molisch’s test we added sulfuric acid to each sample after adding, we have notice some changes in
color and some sample takes time to see changes when exposed to moderate sunshine or kept at high
temperatures, it becomes yellow and brittle. The material's great flammability must also be
considered (Williams1994).
In iodine’s test it was a little different from molisch’s because in iodine after adding solutions
we can see sudden changes in colors the decreasing effect of the four provided carbs is examined.
According to predictions made using molecular structure, it will cause the iodine test solution to
decolorize relatively quickly and heated it to see the final changes. in benedict’s test we can see
different colors from blue to brown/dark brown after heating the concentration of reducing sugar in
the sample differs from the intensity and shade of the color of the reaction mixture. This shade of
color can be used to estimate the concentration of reducing sugar in the sample. Color may vary
from greenish to yellow to orange-red to brick-red. As the concentration of reducing sugar increases
color gradually changes from greenish to yellowish to orange to brick-red.
In fehling’s Fehling's A is a blue liquid containing copper(II) sulphate, while Fehling's B is a
clear liquid containing potassium sodium tartrate (Rochelle salt) and a strong alkali, generally
sodium hydroxide. During the test, these two solutions are created separately after adding these two
solutions. we can see the initial changes there were layering of colors after heating the layering of
colors disappeared.

All of these test have purpose and it is to determine if the sample have carbohydrates in
molisch’s, in iodine its to determine if the samples have starch, in benedict’s it’s to determine
whether there is a presence of carbohydrate in a unidentified analyte, lastly in fehling’s it is to
detection or estimate of reducing sugar and non-reducing sugars.

REFERENCES: Follow proper citation.

https://microbiologynote.com/fehlings-test/http://pubs.sciepub.com/wjce/7/2/3/index.html
https://www.researchgate.net/publication/340443893_Effectiveness_Evaluation_of_Molisch's_Test_for_th
e_Identification_of_Historical_Cellulose_Plastics
https://microbenotes.com/benedicts-test/