Name: Date Performed:

Course and Year: Group #: 1 Date Submitted:

Exercise No. 3

Carbohydrates

OBJECTIVES:

At the end of the experiment, student should be able to:

1. Use of chemical tests to identify some physical and chemical

characteristics of typical carbohydrates

2. Differentiate between monosaccharide, disaccharide and

polysaccharide using different tests

3. Identify an unknown carbohydrate

RESULTS:

A. Structural formulas for Carbohydrates

STRUCTURES

MONOSACCHARIDE FISCHER HAWORTH CYCLIC CHAIR

GLUCOSE

FRUCTOSE
 STRUCTURES

DISACCHARIDE HAWORTH CYCLIC CHAIR

LACTOSE

MALTOSE

SUCROSE

POLYSACCHARIDE STRUCTURE

HAWORTH CYCLIC CHAIR

AMYLOSE
 GLYCOGEN

EXPERIMENTAL DATA

I. Benedict’s Test, Barfoed’s Test, Seliwanoff’s Test, Iodine Test

Samples Benedict’s Test Barfoed’s Test Seliwanoff’s Iodine Test

Test
+/- Description +/- Description +/- Description +/- Description
Glucose + Red + Red - Colorless - Light
yellow
Fructose + Brick red + Red + Red;fast - Light
rxn yellow
Sucrose - Remains - Remains blue - Colorless - Light
blue yellow
Galactose + Maroon + Red - Colorless - Light
yellow
Starch - Remains - Remains blue + Light + Black
blue yellow
Water - Remains - Remains blue - colorless - Light
blue yellow
Cereal - Brownish - Remains blue + Brown red + Black
blue
Honey + Red orange + Initial + Brick red No
effervescence; changes
red
Sugar + Tomato red + Red + Yellow + Dark
orange brown

II. Hydrolysis

Benedict’s Test Iodine Test

Sample +/- description +/- description
Sucrose + Brick red - Wine red color
Starch - Light brown - Light yellow-
brown
Unknown + Brick red - Wine red color
 III. Fermentation

Sample Description
Glucose Moderate formation of bubbles
Fructose Moderate formation of bubbles
Sucrose Fast CO2 bubble formation; CO2 bubble
overflowed in tube
Starch No bubbles
Unknown Fast CO2 bubble formation; CO2 bubble
overflowed in tube

IV. Unknown Sample

Test +/- Description

Benedict - blue

Barfoed - blue

Seliwanoff + Brick red

Iodine - Bright yellow

DISCUSSION:

I. Benedict’s Test, Barfoed’s Test, Seliwanoff’s Test, Iodine Test

A. Benedict’s Test

All reducing sugars possess an aldehyde functional group. When these sugars are

combined with Benedict's solution and subjected to heat, they exhibit the ability to reduce

copper (II) ions, resulting in the formation of copper (I) ions. The resultant outcome is the

formation of Cu2O, which is responsible for the alterations in color observed in the solutions.

The quantity of precipitate generated is directly proportional to the quantity of aldehyde

groups in the solution. The alteration in color serves as a reliable measure of the quantity of

reducing sugar available. A red hue signifies the maximum concentration of reducing sugars,
while a green hue suggests a low sugar concentration. The solution first exhibits a blue tint.

In the absence of any reaction, there is no observable alteration in color, hence confirming

the absence of reducing sugars in the solution.

Since glucose and galactose were aldehydes possessing carbohydrates, a reducing

sugar, they react with Benedict’s test with red precipitate. Fructose, on the other hand, is not

an aldehyde but rather a ketone but because benedict’s reagent is an essential solution; it

gives a positive result due to an equilibrium that exists between ketoses and aldoses in a

reaction known as tautomerism (Chem Libretexts, n.d.). Therefore, fructose is a reducing

sugar. For the reaction of honey in Benedict’s test, honey contains fructose, a reducing sugar,

which reacts with Benedict’s test. In the sugar reaction in Benedict’s test, although most sugar

does not react with Benedict’s reagent because it is a disaccharide and not a reducing sugar,

some sugar possesses a free group, which gives a positive result in Benedict’s test. The

sucrose, starch, cereal, and water do not react because these are not reducing sugar. For

water, it does not react because there is no sugar present in the solution.

B. Barfoed’s Test

The reaction of Barfoed's test is predicated upon the reduction of cupric acetate by

monosaccharides and disaccharides possessing reducing properties. The cupric acetate

undergoes reduction, forming cuprous oxide precipitating as a brick-red solid.

Monosaccharides often exhibit a reaction time ranging from 1 to 5 minutes, forming a red

precipitate. The disaccharides undergoing reduction have a significantly prolonged duration,

ranging from 7 to 12 minutes before forming a red precipitate occurs. The reaction rate

involving disaccharides is comparatively lower due to the prerequisite hydrolysis of

disaccharides, followed by their subsequent reaction with the reagent cupric acetate,

resulting in the formation of cuprous oxide.

Since glucose, fructose, and galactose were monosaccharides and reducing sugar, it

gives a positive result in the barfoed’s test. It also reacts because these were reducing

monosaccharides. For honey, it was positive in barfoed’s test because of the presence of

fructose in honey. Although most sugars do not react or give a negative result for barfoed’s

because it is a disaccharide, in the presence of reducing sugar, it gives a red precipitate in the

test. The disaccharides remain blue because they slowly react with the reagent without being

hydrolyzed to react faster.

C. Seliwanoff’s Test

The Seliwanoff test distinguishes between ketose sugars, which possess a ketone

group, and aldose sugars, which possess an aldehyde group. The present examination is a

time-constrained color reaction specifically applicable to ketohexoses.

Since ketohexoses were present in fructose and honey (fructose-containing), it gives

a fast reaction and a positive result in Seliwanoff’s test. Colorless contains negative results

like glucose and galactose because it contains aldose. However, sucrose should react with

Seliwanoff’s test as it contains ketohexoses containing monosaccharides (fructose). Personal

error during the experiment was the cause of its incorrect result for sucrose. The observed

positive outcome, characterized by a yellow-orange color, potentially resulted from the

interaction between table sugar (sucrose) and other chemicals present in the sample, which

reacted with the reagent.

D. Iodine Test
 The 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 (Sapkota, 2022). Iodine (iodine-potassium iodide, I2KI) staining distinguishes starch

from monosaccharides, disaccharides, and other polysaccharides. The basis for this test is that

starch is a coiled polymer of glucose — iodine interacts with these coiled molecules and

becomes bluish-black (Biology Libretexts, n.d.)

In the experimental data, only the starch and cereal reacted in the iodine test because

these were the only polysaccharide samples. Since monosaccharides were not reactive in the

iodine test, they gave a negative result and a light-yellow precipitate.

II. Hydrolysis

The process of hydrolyzing di- and polysaccharides involves the introduction of water

molecules to the glycosidic link, resulting in its cleavage. When a di- or polysaccharide

undergoes hydrolysis, it will revert back to its constituent monosaccharide units. The

hydrolysis reaction of sucrose yielded a positive outcome in Benedict’s test as the

disaccharides broke down into two monosaccharides (glucose and fructose), which were a

reducing sugar causing the sucrose when hydrolyzed. On the other hand, starch gave a

negative result in benedict’s test; however, the presence of sugar molecules in the starch was

the reason for its light brown precipitate. Sucrose and starch had no observable alteration in

color during the iodine test, suggesting a negative outcome. Without a hydrolysis reaction,

the starch would exhibit a color change to black when subjected to an iodine test, indicating

a favorable outcome.
 III. Fermentation

The fermentation reactions involving glucose, fructose, and glucose were

observed to have an effervescence effect. The observed phenomenon might be

attributed to the metabolic breakdown of sugar by yeasts facilitated by the enzyme

zymase. This enzymatic activity is crucial in fermentation, wherein alcohol and carbon

dioxide are generated as end products. The effervescence observed in the reaction can

be attributed to the release of carbon dioxide. On the other hand, starch has no

effervescence because it comprises many glucose units joined together, but yeast cannot

digest starch unless it is broken down into glucose units.

IV. Unknown Samples

The unknown sample yielded a negative outcome when subjected to both Benedict's and

Barfoed's tests, indicating its non-reducing sugar and monosaccharide status. The outcome

of Seliwanoff's test was positive, indicating that the substance in question is classified as a

ketose. Furthermore, the iodine test had a negative outcome, indicating the absence of

starch.

The unknown sample showed a favorable outcome in Benedict's test after undergoing

hydrolysis, suggesting the presence of a minimum of one intricate carbohydrate that can be

broken down into reducing sugars. Additionally, the fermentation test yielded a good

outcome, suggesting the presence of at least one sugar that can undergo fermentation. The

test outcomes provide compelling evidence indicating that the unidentified sample is a

complex carbohydrate containing ketose. Sucrose can be considered as a potential example

of such a chemical. Sucrose is a disaccharide comprising a glucose molecule and a fructose

molecule. Glucose is classified as an aldohexose, whereas fructose is categorized as a

ketohexose. Hence, sucrose may yield a subtle positive outcome in the Seliwanoff test,

particularly when the specimen is diluted. The remaining test outcomes exhibit conformity

with sucrose. Sucrose is classified as a non-reducing sugar due to its inability to yield a positive

outcome when subjected to Benedict's or Barfoed's tests. Furthermore, this substance does

not include starch, yielding a negative outcome when subjected to the iodine test.

Nevertheless, the hydrolysis of sucrose can result in the formation of glucose and fructose,

which have lower properties. Consequently, after hydrolysis, the unidentified sample

exhibited a good outcome in the context of Benedict's test. The unknown substance exhibited

a favorable outcome in the fermentation test, aligning with the characteristics of sucrose.

Yeast can ferment sucrose, resulting in the production of ethanol and carbon dioxide.

In summary, the test findings indicate a significant likelihood that the unknown sample is

composed of sucrose.

CONCLUSION:

Chemical tests can ascertain several physical and chemical attributes of a common

carbohydrate. The Benedict's test was used to identify reducing sugars. The Barfoed test was

used to identify monosaccharides. Seliwanoff's test was used to distinguish between

ketohexoses and aldohexoses. The iodine test was employed to detect the presence of

polysaccharides. The hydrolysis reaction of di- and polysaccharides involves the cleavage of

glycosidic bonds connecting monosaccharide units, converting these complex carbohydrates

into their constituent simpler components. The fermentation process involves using yeast,

which possesses the enzyme zymase, to facilitate the breakdown of carbohydrates, producing

carbon dioxide and alcohol as secondary products. By performing all the tests in the

experiment, it was observed that the unknown sample was sucrose.

REFERENCES:

(n.d.). Retrieved from Chem Libretexts:

https://chem.libretexts.org/Courses/Sacramento_City_College/SCC%3A_Chem_309_-
_General_Organic_and_Biochemistry_(Bennett)/Text/14%3A_Carbohydrates/14.5%3A_Prop
erties_of_Monosaccharides

Biology Libretexts. (n.d.). Retrieved from Carbohydrates:

https://bio.libretexts.org/Bookshelves/Biotechnology/Bio-
OER_(CUNY)/02%3A_Chemistry/2.07%3A_Carbohydrates

Sapkota, A. (2022, March 18). Retrieved from Microbe Botes: https://microbenotes.com/iodine-test/