EXPERIMENT 1: CARBOHYDRATES

Carbohydrate is an organic compound with a general formula 𝐶𝑚 (𝐻2 𝑂)n that consists only of carbon,
hydrogen and oxygen. It is an essential component of life, our main source of energy. Certain
carbohydrate derivatives are also used as drugs like cardiac glycoside/antibiotics.

Carbohydrates are classified into four major groups- monosaccharides, disaccharides, oligosaccharides
and polysaccharides. Monosaccharides also called “simple sugars” are those which cannot be
hydrolysed further into simpler forms and contain either an aldehyde group or a ketone group. When
two monosaccharides are joined by a glycosidic bond a disaccharide is formed. Examples of
disaccharides are maltose- which yields two molecules of glucose on hydrolysis, sucrose- table sugar,
fructose- fruit sugar and lactose- a sugar found in milk which yields two molecules of glucose and one
molecule of galactose on hydrolysis. Long chains of monosaccharides joined together are called
polysaccharides. The major storage polysaccharides are glycogen (found in animals), starch (in plants)
and dextran (in yeast and bacteria). Structural polysaccharide- cellulose, is found in plant cell walls.
Short chains of monosaccharides which yield to 3-10 units linked by glycosidic bonds are called
oligosaccharides.

Test I: Based on the Formation of Furfural and its Derivatives

Furfural is a colorless liquid but tends to turn brown upon prolonged exposure to air and moisture. It
consists of a formyl group attached to the 2-position of furan. It is a product of the dehydration of
sugars, as occur in a variety of agricultural byproducts including corncobs, oats, wheat bran and
sawdust. It is one of the oldest renewable chemicals and can be found in many processed foods and
beverages

Phenolic compounds will react with furfural or hydroxymethyl furfural in the presence of acid to form
colored condensation products. These colored compounds indicate a positive test for carbohydrates.

The following are the tests used to determine the presence of carbohydrates based on the formation of
furfural and its derivatives.

 Molisch’s Test
o It is the general test to detect the presence of carbohydrates, and some compounds
containing carbohydrates in a combined form. It is based on dehydration of the
carbohydrate by sulfuric acid allowing it to react with the acohol forming furfural or
hydroxymethyl-furfural. A positive result with the presence of a carbohydrate will show
the formation of a purple ring at the interface of the two liquids.

MATERIALS & PROCEDURE

o Five test tubes are prepared each containing glucose, xylose, lactose and starch. The
other one that serves as the control contains 4 mL of distilled water and 2 drops of
molisch’s reagent. Each solution is mixed with two drops of molisch’s reagent. After
gently shaking the test tubes, about 5 ml of concentrated sulfuric acid is slowly added in
an inclined position allowing the acid to run down the side of the tube. A positive
reaction is indicated by appearance of a purple ring formed at the junction of the two
liquids.
 RESULT

DESCRIPTION OF VISIBLE
SUBSTANCE TESTED RESULT
RESULTS
Control No formation -
Glucose Purple ring formation +
Xylose Purple ring formation +
Lactose Purple ring formation +
Starch Purple ring formation +

Glucose, lactose, xylose and starch are all carbohydrates which give positive result for
Molisch’s test. The control gives a negative result because it contains distilled water.

Bial’s Orcinol Test

o It is used to determine the presence of pentoses and nucleotides that contain pentose
sugars. It is also used to distinguish between pentoses and hexoses. The components
include orcinol, hydrochloric acid, and ferric chloride. Furfural from pentoses yields a
blue-green color when treated with orcinol in the presence of ferric ions. While on
hexoses, hydroxymethyl furfural is formed and gives yellow-brown condensation
products.

MATERIALS & PROCEDURE

o Three test tubes are prepared and each one contains 1 mL of 3% solution of xylose,
glucose and starch. 3 ml of bial’s reagent is added to each test tube. The test tubes are
then heated over a bunsen flame until the solution begins to boil and 2 drops of 10%
ferric chloride solution is added. A positive reaction for pentose indicates blue-green
color and yellow-brown to hexoses.

RESULT

DESCRIPTION OF VISIBLE
SUBSTANCE TESTED RESULT
RESULTS
Xylose Blue green +/hexose
Glucose Brown -/hexose
Starch Brown -/hexose

 Seliwanoff’s Test
o It is used to distinguish ketohexoses from aldohexoses. When treated with concentrated
hydrochloric acid, ketohexoses dehydrates rapidly than aldohexoses and forms a bright
cherry condensation product that yields a positive result, while the aldohexoses
produces a pale pink coloration, a negative result.

MATERIALS & PROCEDURE

o 1 ml of each 3% xylose, glucose, fructose and sucrose are placed in separate test tubes.
4 ml of Seliwanoff’s reagent are added to each test tube. The test tubes are placed in a
 boiling water bath for exactly 1 minute. A change in colour will identify each solution
whether it is a ketohexose or an aldohexose. Continued heating and observe the color
changes at 1 minute intervals.

RESULT

DESCRIPTION OF VISIBLE
SUBSTANCE TESTED RESULT
RESULTS
Xylose Pale-pink -
Glucose Pale-pink -
Fructose Bright cherry +
Sucrose Light cherry +

Fructose and sucrose yields positive result. The fructose gives a bright cherry color
which identifies it as a ketohexose and the sucrose has a property of aldohexose but
further hydolyzation gives a ketohexose property and yields a positive result.

Test II: Based on the Reducing Property of Sugars

Reduction of sugars plays an important role in biochemistry. Some sugars are called reducing sugars
because they are capable of transferring hydrogen to other compounds. Reducing sugars have a free
carbonyl group, one that can react with an oxidizing agent. Several oxidation reactions of sugars are of
some importance in laboratory practice because they can be used to identify sugars. Aldehyde groups
can be oxidized to give the carboxyl group, and this reaction is the basis of a test for the presence of
aldoses. When the aldehyde is oxidized, some oxidizing agent must be reduced. Aldoses are called
reducing sugars because of this type of reaction; ketoses can also be reducing sugars because they
isomerize to aldoses.

 Benedict’s Test
o It is a deep blue alkaline solution used to test for the presence of simple carbohydrates
and used to identify monosaccharide and some disaccharide reducing properties which
have free ketone or aldehyde functional groups. The reagent contains cupric sulphate,
sodium carbonate and sodium citrate. When the reagent is added to the solution, a
formation of a brick red precipitate of cupric sulphate will yield a positive result. As the
concentration of reducing sugar increases, the nearer the colour is to brick-red and
greater precipitate is formed. The sodium carbonate provides the alkaline conditions
which are required for the oxidation-reduction reaction. Sodium citrate complexes with
the copper (II) ions so that they do not deteriorate to copper (I) ions during storage.

MATERIALS & PROCEDURE

o 1 ml of each 3% solutions of glucose, xylose, fructose, lactose, and sucrose are placed
separately in labelled test tubes and 5 ml of Benedict’s reagent is added to each
solution. The test tubes are heated in a boiling water bath for approximately 2-3
minutes. Colour change will determine the percentage of sugar present in the solution.
 RESULT

SUBSTANCE TESTED RESULT EXPLANATION

Glucose + Reducing sugar is present
Xylose + Reducing sugar is present
Fructose - Reducing sugar is present
Lactose + Reducing sugar is present
Sucrose - Nonreducing disaccharide

Note:
Positive Result: Formation of reddish precipitate within 3 minutes
Negative Result: No color change

Benedict’s test is used to detect the presence of reducing sugars. The test shows that
glucose, xylose, and lactose, are all reducing sugars. They all have a free reactive
carbonyl group. Lactose which is a disaccharide has an exposed carbonyl group thus
making it a reducing sugar. Sucrose is a disaccharide and yields negative result.

 Barfoed’s Test
o It is a test used to determine between monosaccharide, disaccharides, and
oligosaccharides. It is based on the reduction of cupric acetate which produces cuprous
oxide and gives back a brick red precipitate. Monosaccharides react in about 1-5
minutes and produce a red precipitate. Disaccharides are less easily oxidized and take 7-
10 minutes to produce a red precipitate. This is due to prolonged heating which causes
hydrolysis of disaccharides and then react with the reagent cupric acetate to produce
cuprous oxide and make it into a monosaccharide.

MATERIALS & PROCEDURE

o Six test tubes are prepared- the first one serves as control which contains distilled
water, and the rest contains 1 ml of each 3% solutions of glucose, xylose, fructose,
lactose, and starch in separately labelled test tubes. 3 ml of Barfoed’s reagent are added
on each solution. All the tubes are then placed in a boiling water bath for 10 minutes.

RESULT

SUBSTANCE TESTED RESULT EXPLANATION

Control - No presence of carbohydrate
Glucose + Presence of reducing sugar
Xylose + Presence of reducing sugar
Fructose + Presence of reducing sugar
+ Disaccharide with an exposed
Lactose
carbonyl group
Starch - Polysaccharide
 Positive result for glucose, xylose, and fructose yields an appearance of red precipitate
which indicates the presence of monosaccharides. Lactose is a disaccharide component
that has an exposed carbonyl group making it a reducing sugar.

 Tollen’s Test
o Also known as the silver-mirror test, is used to identify between an aldehyde and a
ketone. Tollen’s test reagent oxidizes an aldehyde into the corresponding carboxylic
acid. The reaction is accompanied by the reduction of silver ions in tollen’s reagent into
metallic silver which forms a silver mirror on the test tube. On the other hand, ketones
are not oxidized by tollen’s reagent. Tollen’s test uses a reagent which is a colorless,
basic, aqueous solution containing silver ions coordinated to ammonia.

MATERIALS & PROCEDURE

o Place 5 drops of 3% solutions of glucose, xylose, and sucrose in separate test tubes and
add 2 ml of tollen’s reagent into each tube. Boil for about 5 minutes. A silver mirror test
yields a positive result.

RESULT

SUBSTANCE TESTED RESULT EXPLANATION

Glucose + With presence of aldehyde
Xylose + With presence of aldehyde
Sucrose - With presence of ketone
References:

M.N. Chatterjea, Rana Shinde. (2012) Textbook of Medical Biochemisty, (8th edition). Jaypee Brothers
Medical Publishers. New Delhi Panama City, London. Available from: https://books.google.com.ph

M.K. Campbell, SO Farrell. Biochemistry, (8th Edition). Cengage Learning. United States. Available from:
https://books.google.com.ph

D. Hames, N. Hooper. (2005) Biochemisty, (3rd Edition). Taylor & Francis Group Publishing. 270 Madison
Avenue New York, NY 10016. Available from: https://books.google.com.ph

Zenaida T. Nucum, Ed. D. (2010) Laboratory Manual for Simplified Biochemistry. C&E Pubishing, Inc. 839
EDSA, South Triangle, Quezon City

Chegg Study Textbook Solutions. https://www.chegg.com/homework

Microbiology Info.com. https://microbiologyinfo.com/benedicts-test-principle-composition-preparation-

procedure-and-result-interpretation/

Bioscience Notes, One Biological Notes for Students. http://www.biosciencenotes.com/barfoeds-test/

Chemistry Libretexts. https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Supplemental_

Modules_(Organic_Chemistry)/Aldehydes_and_Ketones/