LABORATORY REPORT

FFT 1013 BIOCHEMISTRY

SEPTEMBER 2022/2023

EXPERIMENT 1

GROUP 7

Lecturer : Dr Suriyani Faliq Adeeba Programme: SEH Date: 10 November 2022

Student’s name matric number
1. Muhammad Haziq Fikri Bin Mohd Nazri J22A0134
2. Nasrul Syahiran Bin Asrolafizi J22A0192
3. Muhammad Aiman Hakimi Bin Rahimi J22A0052
1. OBJECTIVE

To introduce the students with the technique to categorize the carbohydrates sample into
their their properties through several different tests that can linked to their chemical
classifications.

2. INTRODUCTION

Carbohydrates are the most abundant bio molecules on Earth. Each year, photosynthesis
converts more than 100 billion metric tons of CO2 and H2O into cellulose and other plant
products. Certain carbohydrates (sugar and starch) are a dietary staple in most parts of the
world, and the oxidation of carbohydrates is the central energy-yielding pathway in most
non-photosynthetic cells. Insoluble carbohydrate polymers serve as structural and protective
elements in the cell walls of bacteria and plants and in the connective tissues of animals.

Carbohydrates are an essential component of our diet and an important source of energy
for us. Most of the things generally included in our diet have a large ratio of carbohydrates
present. Also the patients suffering from Diabetes excrete huge amounts of sugar in their
urine which needs to be estimated. The purity of carbohydrates can also be checked by
methods which can qualitatively estimate particular sugars. Hence the need for carbohydrate
estimation arose.

Six different tests had been carried out in the experiment molisch's test, lodine test,
barfoed's test, benedict's test, seliwanoff's test and bial's test. Molisch's test was used to
indicate presence of any kind of carbohydrates or compounds containing carbohydrates in
combined form. The test was based on the dehydration reaction of the carbohydrates and
the sulfuric acid to yield an aldehyde that would condenses with phenolic structure producing
a red or purple coloured compound. Iodine test was used to differentiate the presence of
starch and glycogen from other polysaccharides. Upon reaction with starch and glycogen the
iodine develops to blue violet color or brown wine color compounds, respectively. Barfoed's
test was used to differentiate between the simple monosaccharides and the disaccharides
based on the effective reduction reaction of the cupric acetate by the monosaccharides
which reduced the compound faster compared to the disaccharides. Benedict's test was
used to differentiate the reducing sugars and the non-reducing sugars. Upon reaction with
the reducing sugars, the benedict's reagents changes color from turquoise to orange. A
reducing sugar was sugars with ability to acted as reducing agents due to presence of
hemiacetal (free aldehyde or ketone group). Seliwanoff's test was used to distiguieshes
aldose and ketode based on their functionality. If the sugar contains a ketone group, it was a
ketose and if it contains an aldehyde group, it was an aldose. This test was based on the
fact that, when heated, ketoses were more rapidly dehydrated than aldoses. Bial's test was
used to checked on the presence of pentoses sugar (five- carbon sugar). In the presence of
pentose sugar the bial's reagent turns bluish in colour and may form precipitate.
3. MATERIAL

Molisch’s reagent, N/50 Iodine solution, Barfoed’s reagent, Seliwanoff’s reagent, Bial’s
reagent, Unknown solution of Carbohydrate, Sulfuric Acid (H2SO4), Boiling water, Starch
solution, 5% Sodium Hydroxide, Sugar sample.

4. METHODS

1) Molich’s Test

Placed 2ml of a carbohydrate sample in a test tube, and added 2-5 dropped of molisch's
reagent in a test tube and mixed thoroughly. Added gently through the side by tilting the
tube, about 2ml of concerated h2so4 so that it forms a layer at the bottom of the tube.
Observe the colour at the interface between the two layers

2) Iodine Test

Placed 2 ml of a carbohydrate sample in a test tube, and added one to two dropped of n/50
iodine solution. Observe the different colour changed. As a reference, you may repeated the
test with 2 ml of starch solution added with 1 ml 5% sodium hydroxide

3) Barfoed’s Test

Placed 2 ml of a carbohydrate sample in a test tube and added 2 ml of barfoed's reagent in a

test tube. Heat the sample in a boiling water bath for 3 minutes and then cool the sample.
Observe the sample for the formation of precipitation

4) Benedict’s Test

Placed 5 ml of the benedict's reagent in the test tube, and added dropped of sugar sample.
Heat the sample in a boiling water bath for 3 minutes and allowed to cool under running tap
water. Observe for information of precipitate

5) Seliwanoff’s Test

Placed 5 ml of seliwanoff's reagent in a test tube and added 1 ml of sugar sample solution.
Heat the mixture to boil for 2 minutes and let it cool. Observe for the information of
precipitate.

6) Bial’s Test

Placed 5 ml of bial's reagent in a test tube and added 0. 5 ml of carbohydrate solution. Heat
the sample to boil and let it cool. Observe the sample for changed of colour.
5. RESULTS

Test Sample A Sample B Sample C Sample D

Moslich A dark purple ring A purple ring A purple ring A purple ring
formed in formed in formed in between formed in
between H2SO4 between H2SO4 H2SO4 and between H2SO4
and Moslich’s and Moslich’s Moslich’s reagent and Moslich’s
reagent mixture reagent mixture mixture reagent mixture
Iodine A No changes. The No changes. The No changes. The Colour changes
colour of colour of colour of from yellow to
substanes substanes substanes black at the
remains yellow. remains yellow remains yellow bottom tube.
Iodine B No changes No changes No changes Colour changes
from yellow to
clear and smells
like chlorine
Barfoed Brick-red Brick-red No changes No changes
precipitation precipitation
formed formed
Benedict Brick-red Brick-red on the No changes No changes
precipitation top and bottom
formed surfaces
Seliwanoff Colour changes Colour changes Colour changes to Clear
to light yellow to deep yellow cheery red after
after heating after heating heating
Bial Changed to light Changed to Changed to Changed to
yellow muddy brown golden yellow
Result Glucose Fructose Sucrose Starch

6. DISCUSSION

A general test for carbohydrates called the Molisch' test looks for the presence of the
carbonyl group, which emits a purple-colored material. Sulfuric acid concentrate was added,
resulting in the production of a purple-colored material. When sulfuric acid is added, the
carbohydated goes through a process of dehydration in which water is liberated. Pentoses
and hexoses interact with sulfuric acid to affect the hue in a beneficial way. In our previous
experiment, the Molisch's test was effective in obtaining the colour change as tubes C and D
converted to purple product and there was no reaction.

To distinguish between the presence of starch and glycogen and other polysaccharides, the
Iodine test is employed. In the Iodine Test A, 2 ml of a carbohyrate is added, followed by one
to two drops of a sample of N/50 iodine solution. After observation, the sample's colour is
determined to be brown and yellow in Test Tube A, brown and yellow in Test Tube B, brown
and yellow in Test Tube C, and brown and grey in Test Tube D. In addition, the Iodine Test
B uses 2 ml of starch solution mixed with 1 ml of 5% Sodium Hydroxide to conduct the test.
After observing the change in colour, test tubes A, B, and C show no reaction, but test tube
D changes to a grey colour.

Additionally, the Barfoed test involves adding 2 ml of Barfoed's reagent to test tube A, B, C,
and D along with 2 ml of a sample of carbohydrates. The efficient reduction reaction of the
cupric acetate by the monosaccharides, which reduced the molecule more effectively than
the disaccharides, is then utilised to distinguish between the simple monosaccharides and
the disaccharides. As a result, tube A and tube B, which both had a blue tint, displayed the
test's positive results. Benedict's test is then employed to distinguish between reducing
sugars and non-reducing sugars. The Benedict's test reagents change colour into a red
product in tubes A and B when they react with the reducing sugars.

Additionally, the Seliwanoff test distinguishes aldose and ketone based on how they act. The
sugar is a ketose if it has a ketone group, and an aldose if it has an adddehyde group. This
experiment is based on the observation that when heated, ketoses dehydrate more quickly
than aldoses. As a result, the test's colour in tubes B and C changed to cherry. Lastly, the
existence of pentises sugars is checked using Bial's assay (five-carbon sugars). The Bial's
reagent changes colour from clear to murky brown in all four tubes when pentose sugar is
present.

7. CONCLUSION

Finally, the Benedict test was run on glucose, fructose, sucrose, and starch. Because the
colour changed from blue to brick-red, glucose and fructose show positive results. As a
result, the positive result indicates that reducing sugars were present. Sucrose and starch
produce negative results because the colour does not change. Starch was subjected to an
iodine test. When iodine is added to the starch solution, it should turn blue. When blue starch
is heated, it turns colourless. When cooled, it turns into a blue solution. When 1% NaOH was
added to the starch, it became colourless, and when acetic acid was added, the blue colour
reappeared. The changes occur because the bond has begun to break down, and the
amylose chains have broken. The colour of the starch solution changes from dark purple to
light yellow. From minute 13 to minute 40, the colour shifts from dark to light. The longer the
starch is boiled, the lighter the colour becomes when iodine solution is added. This is due to
the fact that it hydrolyzed the glycoside bond.

8. REFERENCE

Anupama Sapkota (2020,

November 18). Bial’s Test-
Definition, Principle,
Procedure, Result, Uses.
https://microbenotes.com/bials-
test/
Anupama Sapkota (2020,
November 18). Bial’s Test-
Definition, Principle,
Procedure, Result, Uses.
https://microbenotes.com/bials-
test/
1) Dreywood, R. (1946). Qualitative Test for Carbohydrate Material. Industrial &
Engineering Chemistry Analytical Edition, 18(8), 499-499.
http://dx.doi.org/10.1021/i560156a015

2) http://www.biologydiscussion.com/carbohydrates/test/qualitative-and-quantitative-
tests-for-carbohydrates/13042

3) https://fulltimes.wordpress.com/carbohydrates-test/