MODULE 3

LABORATORY EXERCISE 3:

CARBOHYDRATES

I. Introduction

Carbohydrates or also name as carbs, are sugar molecules. Along with proteins and fats, carbohydrates
are one of three main nutrients found in foods and drinks. Your body breaks down carbohydrates into glucose.
Glucose, or blood sugar, is the main source of energy for your body's cells, tissues, and organs.

There is no one-size-fits-all amount of carbohydrates that people should eat. This amount can vary, depending
on factors such as your age, sex, health, and whether or not you are trying to lose or gain weight. On average, people
should get 45 to 65% of their calories from carbohydrates every day. On the Nutrition Facts labels, the Daily Value for
total carbohydrates is 275 g per day. This is based on a 2,000-calorie daily diet. Your Daily Value may be higher or
lower depending on your calorie needs and health.

Benedict’s Test is used to test for simple carbohydrates. The Benedict’s test identifies reducing
sugars (monosaccharide’s and some disaccharides), which have free
ketone or aldehyde functional groups. Benedict’s solution can be used to
test for the presence of glucose in urine.

Some sugars such as glucose are called reducing sugars because

they are capable of transferring hydrogens (electrons) to other
compounds, a process called reduction. When reducing sugars are
Figure 1. Benedict Test
mixed with Benedicts reagent and heated, a reduction
reaction causes the Benedicts reagent to change color. The color varies from green to dark red (brick) or rusty-
brown, depending on the amount of and type of sugar.
 Figure 2. Structure Formula of the Benedict Test

Benedict’s quantitative reagent contains potassium thiocyanate and is used to determine how much
reducing sugar is present. This solution forms a copper thiocyanate precipitate which is white and can be used in a
titration. The titration should be repeated with 1% glucose solution instead of the sample for calibration.

Positive Benedict’s Test: Formation of a reddish precipitate within three minutes. Reducing sugars present.
Example: Glucose
Negative Benedict’s Test: No color change (Remains Blue). Reducing sugars absent. Example: Sucrose.

Barfoed’s test is a chemical test used to detect the presence of monosaccharides which detects reducing
monosaccharides in the presence of disaccharides. This reaction can be used for disaccharides, but the reaction
would be very slow.
The Barfoed reagent is made up of copper acetate in a dilute solution of acetic acid. Since acidic pH is
unfavorable for reduction, monosaccharides, which are strong reducing agents, react in about 1-2 min. However, the
reducing disaccharides take a longer time of about 7-8 minutes, having first to get hydrolyzed in the acidic solution and
then react with the reagent. Once the reaction takes place, thin red precipitate forms at the bottom of the sides of the
tube. The difference in the time of appearance of precipitate thus helps distinguish reducing monosaccharides from
reducing disaccharides.
Positive Barfoed’s Test: Formation of a red
precipitate within three minutes. Reducing sugars
present.
Negative Barfoed’s Test: No color change or there
are no red precipitates in the solution. Reducing sugars
absent.
Figure 3. Barfoed’s Test
 Seliwanoff’s test is used to differentiate between sugars that have a ketone group (ketose) and sugars
that have an aldehyde group (aldoses). This test is a timed color reaction specific to ketohexoses.

Figure 4. Positive and Negative result of Seliwanoff’s Test

Iodine test is a chemical test used to distinguish mono- or disaccharides from certain
polysaccharides like amylase, dextrin, and glycogen. This test has a variation termed starch-iodine test that is
performed to indicate the presence of glucose made by plants in the leaves.

Figure 4. Iodine Test

Disaccharides and polysaccharides can be hydrolysed under acidic conditions. Acid hydrolysis of disaccharides
and polysaccharides produces monosaccharides by breaking the glycosidic links (ether bonds) between monomer
units in the structure of the molecule.

Fermentation, chemical process by which molecules such as glucose are broken down anaerobically. More
broadly, fermentation is the foaming that occurs during the manufacture of wine and beer, a process at
least 10,000 years old. The frothing results from the evolution of carbon dioxide gas, though this was not recognized
until the 17th century. French chemist and microbiologist Louis Pasteur in the 19th century used the term
fermentation in a narrow sense to describe the changes brought
about by yeasts and other microorganisms growing in the absence of air (anaerobically); he also recognized that ethyl
alcohol and carbon dioxide are not the only products of fermentation.

II. Methodology
A. Procedure of Benedict’s Test

1. Approximately 1 ml of sample is placed into a clean test tube.

2. 2 ml (10 drops) of Benedict’s reagent (CuSO4) is placed in the test tube.
3. The solution is then heated in a boiling water bath for 3-5 minutes.
4. Observe for color change in the solution of test tubes or precipitate formation.

B. Procedure in Barfoed’s Test

1. Take 1 ml of a given sample in a clean, dry test tube. The concentration of disaccharides sample
(if used) should not exceed 1% (w/v).
2. Take control of 1 ml of distilled water in another tube.
3. Add about 2-3 drops of Barfoed’s reagent to both the tubes and mix them in a vortex.
4. Keep the test tubes in the water bath for 1-2 minutes. The boiling should not be done for more than
2 minutes as the disaccharides might hydrolyze into monosaccharides and give a positive result.
5. Observe the appearance of color in the test tubes.
6. Noted own the time taken for the appearance of color in the tubes.

C. Procedures in Seliwanoff’s Test

1. Take two clean, dry test tubes and add 1 ml of the test sample in one test tube and 1 ml of distilled
water in another as blank.
2. Add 2 ml of Seliwanoffs’ reagent to both the test tubes.
3. Keep both the test tubes in a water bath for 1 min.
4. Observe the formation of color and note it down.
D. Procedures in Iodine Test

1. Take 1 ml of a given sample in a clean, dry test tube.

2. Take control of 1 ml of distilled water in another tube.
3. Add about 2-3 drops of Lugol’s solution to both the tubes and mix them in a vortex.
4. Observe the appearance of color in the test tubes.
5. Heat the test tubes in the water bath until the color disappears.
 6. Take the test tubes out for cooling
7. Note down the appearance of color seen in the test tubes.

III. RESULTS AND OBSERVATIONS.

A.Structural Formula for Carbohydrates

Table 1.1. Structural Formulas of Monosaccharides

Structures
Monosaccharide
Fischer Haworth Chair

Glucose

Fructose

Table 1.2. Structural Formulas of Disaccharides (indicating the glycosidic bonds).

Structures
Disaccharide
Haworth Chair

Lactose
 Maltose

Sucrose

Table 1.3. Structural Formulas of Polysaccharides

Polysaccharides Representative Structures

Amylose

Amylopectin
B.Benedict’s Test for Reducing Sugars

Table 2. Results/Observations for Benedict’s Test

Compound Color Reducing Sugar (Yes/No)

Water blue No (Negative Control)

Glucose Orange/ red Yes

Fructose Orange / red Yes

Sucrose blue No

Maltose Orange/ red Yes

Lactose Orange / red Yes

Starch Green Yes (traces of reducing sugars)

C.Barfoed’s Test for Monosaccharides

Table 3. Results/Observations for Barfoed’s Test

Compound Color Monosaccharide (Yes/No)

Water blue Negative control

Glucose red Yes

Fructose red Yes

Sucrose blue No

Maltose blue No

Lactose blue No

Starch blue No

D.Seliwanoff’s Test for Ketohexoses

Table 4.1 Results/Observations for Seliwanoff’s Test

Compound Color Ketohexose/Aldohexose

Water No color Negative

 Glucose No color Aldohexose

Fructose Red Ketohexose

Sucrose (glucose + fructose) Red Ketohexose

Maltose No color Ketohexoses

Lactose No color Aldohexoses

Starch No color Aldohexoses

E.Iodine Test for Polysaccharides

Table 5.1 Results/Observations for Iodine Test

Compound Color Polysaccharide (Yes/No)

Water brown Negative control - No

Glucose brown No

Fructose brown No

Sucrose brown No

Maltose brown No

Lactose brown No

Starch Dark blue Yes

F.Hydrolysis of Di- and Polysaccharides

Direction: Write the observed color of the solution for each test. Table 6. Hydrolysis of
Sucrose and Starch
Carbohydrate Benedict’s Test Iodine Test

Sucrose + H20 blue brown

Sucrose + HCl
Red brown
(Hydrolysed)

Starch + H2O blue Dark-blue

 Starch + HCl (Hydrolysed)
blue brown

The difference occurs because hydrolysis frees up the reducing sites on the molecules, the carbons with 2
oxygens bound to them. Sucrose is not a reducing sugar because the reducing sites of both glucose and fructose are
bound to each other. In starch, there may be only one reducing site in a polymer of 1000 units, so hydrolysis opens up
1000-fold more of them. While when iodine is added to starch it converts to blue-black solution due to presence of
amylose of starch

G.Testing an Unknown Carbohydrate

Direction: If a positive result of the test is observed, write “+”. Otherwise, write “-”.

Test A B C D E

Benedict’s - + (traces) + + +

Barfoed’s - - + - +

Seliwanoff’s + - + - -

Iodine - + - - -

Identification
of Unknown

These positive results that are present in testing an unknown carbohydrate are:

A – Sucrose
B – Starch
C – Fructose
D – Maltose
E – Glucose

H.Fermentation Test
Table 7. Results/Observation of Fermentation Test.
Compound Observations Fermented (Yes/No)

Water No bubbles Negative control - No

Glucose Bubbles are present. Yes

 Sucrose Bubbles are present. Yes

Lactose No bubbles No

Fructose Bubbles are present. Yes

Starch No bubbles No

Guide Question: Why don’t lactose and starch undergo fermentation with yeast?
During the fermentation the enzymes in the yeast are unable to cause the lactose and starch to ferment.
References:
Britannica – Fermentation
https://www.britannica.com/science/fermentation

Medline Plus – Carbohydrates

https://medlineplus.gov/carbohydrates.html#:~:text=Carbohydrates%2C%20or%20carbs%2C%20are
%20sugar,cells%2C%20tissues%2C%20and%20organs.
Microbiology E-notes – Barfoed’s Test
https://microbenotes.com/barfoeds-test/

Microbiology Information – Benedict’s Test

https://microbiologyinfo.com/benedicts-test-principle-composition-preparation-procedure-and- result-
interpretation/

Microbiology E-notes – Seliwanoff’s Test

https://microbenotes.com/seliwanoffs-test/

Microbiology E-notes – Iodine Test

https://microbenotes.com/iodine- test/#:~:text=Iodine%20test%20is%20a%20chemical,by%20plants%20in%20the
%20leaves.