General and Specific Test for Carbohydrates

Experiment No. 5

Group No. 6

College of Science, University of Santo Tomas, Manila, Philippines

Abstract

The aim of the experiment was to characterize each carbohydrate and sugar. This was done by
conducting general and specific tests. The general tests conducted were molisch test, anthrone test,
and iodine test using the carbohydrates glycogen, amylose, and cellulose. Here, the characteristics of
the carbohydrates were distinct and these proved how similar or different these polymers are. For the
specific test, five different tests were conducted, mucic acid test, benedict’s test, barfoed’s test, bial’s-
orcinol test, and seliwanoff’s test. Through these tests and inferences, it was concluded that unknown
A was xylose and unknown B was fructose.

Introduction

Carbohydrates, also known as saccharides, are organic compounds which are

made of carbon, hydrogen, and oxygen. Its basic formula is (CH2O)n, with hydrogen

and oxygen present at a ratio of 2:1. Carbohydrates are the largest source of energy

used by our bodies. Without it, the body cannot function properly; it is a macronutrient

therefore it is needed by organisms in large quantities in order to grow and develop

properly. It is used in human bodies mainly by the central nervous system, especially

the brain, and by the muscles. Aside from being a source of energy, carbohydrates

can also be used for structure and as molecular tags. Carbohydrates consists of a

great amount of hydroxyl groups, aldehydes or ketones. Hydroxyl groups allows

carbohydrates to interact with the aqueous environment and to participate in hydrogen

bonding, both within and between chains (The Medical Biochemistry Page, 2017).

Carbohydrates that consist of aldehydes are referred to as aldoses and those of the

ketone group are referred to as ketoses. Having at least one chiral carbon,

carbohydrates are optically active and can form one of the two conformations, D-
conformation, which is the conformation of physiologically significant carbohydrates,

and L-Conformation, confirmations are mirror to each other (enantiomers).

Carbohydrates are classified into four groups: monosaccharides, disaccharides,

oligosaccharides, and polysaccharides.

Monosaccharides are the simplest forms of carbohydrates as it only consists of a

single sugar, thus “mono”. The n in the formula for monosaccharides can be either 3,

5 or 6. Monosaccharides are used by cells since it can go through the cell easily due

to its small size. Aldoses in the form of monosaccharides are reducing sugars, sugars

that can not be hydrolyzed but can be oxidized and can reduce another substance,

and ketoses in the form of monosaccharides are non reducing, not capable of being

oxidized and reducing another substance. The two forms that can be developed by

pentoses (five carbons) and hexoses (six carbons) through reacting with alcohol

groups spontaneously are cyclic and noncyclic, which may contain either a ketone or

an aldehyde forming intramolecular hemiketals or hemiacetals. In turn, pentoses,

resembling furan (an organic molecule), would be called furanoses and hexoses,

resembling another organic molecule called pyran, would be termed pyranoses. Some

examples of monosaccharides are glucose, galactose, fructose, xylose, ribose, and

deoxyribose. Glucose is the main source of energy of most living things because when

metabolized, it releases energy in a process called respiration (C6H12O6 + 6O2

6CO2 + 6H2O). Galactose, on the other hand, has a similar structure to glucose and

can be α or β. Fructose, another hexose like glucose and galactose, is a non-reducing

sugar, meaning it contains a ketose group, unlike glucose which is a reducing sugar,

meaning it contains an aldose group. Xylose is a pentose which contains an aldehyde

group and is usually found in plants. The reduced product of xylose is D-xylitol, which
is an artificial sweetener. Lastly, ribose and deoxyribose are also pentoses and they

make up RNA and DNA.

Disaccharides are not commonly found in nature. These are formed when two

monosaccharides, or when a monosaccharide and an alcohol, form covalent bonds

between their hydroxyl groups through condensation reaction, which uses energy and

releases water. These bonds are called glycosidic bonds and the product of forming

glycosidic bonds are called glycosides. Though soluble in water, disaccharides are not

used directly by cells; it is large in size thus it needs to be broken down before it can

pass through a cell. Disaccharides are broken down and releases energy through

hydrolysis reaction (C12H22O11 + H2O C6H12O6 + C6H12O6), the opposite of

condensation reaction, which happens during digestion, specifically when it passes

through the small intestine. Sucrose, lactose, and maltose the the three important

disaccharides needed for physiological function. Sucrose is a non-reducing sugar and

is made through combining α-glucose and α-fructose with an α–(1,2)–β-glycosidic

bond. These are usually found in sugar canes and sugar beets. Lactose are reducing

sugars found in mammals’ milk. Its alpha (α) form is a combination of α-glucose and

β-galactose and its beta (β) form is composed of β-galactose and β-glucose and is

bonded by α–(1,2)–β-glycosidic bond. Lastly, maltose, which is also a reducing sugar,

is made of 2 α-glucose bonded by an α–(1,4) glycosidic bond.

Oligosaccharides are made out of two to ten monosaccharides. These are formed

through the condensation process, which forms the glycosidic bonds. These are not

digested by the human body. Some forms of oligosaccharides are raffinose,

stachyose, and ajugose.

 Polysaccharides are much larger and heavier than oligosaccharides as it contains

hundreds of monosaccharides which are bonded through condensation

polymerisation. Each unit of the polymer is called a monomer which is usually the

monosaccharide D-glucose. Polysaccharides made up of only one kind of

monosaccharides are called homopolysaccharides and those that are composed of

more than one kind of polysaccharide are called heteropolysaccharides.

Polysaccharides function as energy storage and structural units. Polysaccharides that

function for structure usually link with other macromolecules like proteins, forming

glycoproteins or mucoproteins, and lipids, forming lipopolysaccharides. The function

of a polysaccharide relies on its length, branching, folding, and whether it is straight or

coiled. The three polymers with glucose polymers are starch, glycogen, and cellulose.

Starch is the main energy store of plants and its structure is similar to the structure of

glycogen with the exception of the degree of branching at every 20-30 residue and

being low. Starch has two forms, amylose and amylopectin. Amylose is unbranched

and coiled in a helical structure while amylopectin is branched and, when placed in

water, is insoluble. Glycogen is the main carbohydrate store of animals. This crucial

molecule is a homopolymer of glucose in α–(1,4) linkage; it is also highly branched,

with α–(1,6) branch linkages occurring every 8-10 residues (The Medical Biochemistry

Page, 2017). Since its structure is compact, it can store huge amounts of energy in a

small volume of space that barely affects cellular osmolarity. Glycogen produces

glucose through hydrolyzation. Lastly, cellulose is made out of β-glucose and straight

polymer molecules. It is a structural component for cell walls of plants and its durability

is due to hydrogen-bonding of the chains of polymers. Cellulose can only be digested

through an enzyme called cellulase, but otherwise, it cannot be hydrolyzed, therefore

it can not be digested.

General tests for carbohydrates are used to detect starch (amylose), glycogen,

and cellulose. Molisch test is a test for all carbohydrates. The reagents used for this

test are molisch reagent (5% solution of α-naphthol in alcohol), which acts as a

condensation reagent, and conc. sulphuric acid (H2SO4), which dehydrates pentoses

and hexoses to create furfural and its derivatives, hydroxymethylfurfural. When sulfuric

acid is added to the carbohydrate, it dehydrates the carbohydrate thus producing an

aldehyde (furfural or hydroxymethylfurfural),which condenses with the α-naphthol of

the molisch reagent. This forms the purple ring in the middle of a clear solution, which

is the positive characteristic of this test. Anthrone test is another test for all

carbohydrates and it determines how many carbohydrates are there in a solution. Its

only reagent is the anthrone reagent (0.2 % anthrone in conc. Sulfuric acid). The

sulfuric acid present in the reagent dehydrates the carbohydrate and forms furfural.

This furfural, when it reacts with the anthrone which is also present in the reagent,

gives out the green hue of the positive result of this test. The iodine test determines if

starch is present in a solution. Its main reagent is the iodine reagent which is

composed of iodine solution in an aqueous potassium iodine solution, which makes it

more soluble in water. A starch-iodine complex is made through the adsorption

complex of starch which gives out the blue-black hue of the positive result of this test.

Specific tests for carbohydrates determine which monosaccharides or

disaccharides are present in a solution. Mucic acid test is used mainly to determine if

there is a presence of galactose in a solution. Therefore, lactose, which is a

combination of glucose and galactose, would also test positive for this test. The

reagent for this conc. nitric acid (HNO3), which when heated with galactose forms white

crystals or a white precipitate. This white precipitate is insoluble dicarboxylic acid when

the substance tests for positive but when the sugar does not contain galactose, it is

soluble, thus no precipitate will form. Benedict’s test is used to determine if the sugar

is reducing or non-reducing. Its reagents are sodium carbonate (Na2CO3), which

encourages redox reaction by making the conditions of alkaline; sodium citrate

(Na3C6H5O7) which creates a complex with copper(II) sulfate pentahydrate

(CuSO4.5H2O), another reagent, and reduces this into copper (I) ion, giving the positive

result the red precipitate as copper is no longer soluble. Some examples of reducing

sugars are glucose, fructose and galactose. When the sample sugar is non-reducing,

the solution will not have a red precipitate. Barfoed test is similar to benedict’s test,

being a copper reduction test, except it is carried out in an acidic condition. It is a test

that determines if the substance is a reducing monosaccharide or a disaccharide. Its

only reagent is the barfoed’s reagent, which is composed of copper(II) acetate

(Cu(CH3COO)2) in acetic acid (CH3COOH). Similar to the benedict’s test, when the

sugar containing the reducing monosaccharide is mixed with the reagent and heated,

red precipitate will also form because the copper is reduced and has become insoluble.

Disaccharides test negative on this test because it takes more time for disaccharides

to respond to the reagent, thus not having the red precipitate. Bial’s-orcinol test if the

sugar is a pentose, containing 5 carbons. Its reagent is the bial’s orcinol reagent that

contains resorcinol (C6H6O2), hydrogen chloride (HCl), and ferric chloride (FeCl3).

Heating with strong acid converts the pentose to furfural which then reacts with the

coloured compound produced when orcinol and ferric chloride react with each other

(Kumar, n.d.). The blue-green hue is the qualitative sign that the sugar is positive or a
pentose and when the solution turns brown, this shows that the sugar is a hexane,

negative. Seliwanoff’s test is a test for fructose and sucrose or a test to differentiate

ketoses from aldoses. Its reagent is the seliwanoff’s reagent which contains 0.5%

resorcinol in hydrochloric acid (3N HCl). On treatment with conc. Acid, ketoses are

dehydrated more rapidly to give furfural derivatives and on condensation with

resorcinol give cherry red complex (Karki, 2018). Sugars hat test negative for this test

are clear solutions.

Results

General Test

Amylose Glycogen Cellulose

Molisch Test 3 layers: Pale pink solution Dark Pink solution

● Top- Clear with black precipitate with dark red
● Middle- Pink precipitate
● Bottom-
Clear

Anthrone Test Lime green solution Lime green solution Lime green solution

Iodine Test Dark Blue Solution Peach/orange Yellow cloudy

Before, During, solution solution with white
After precipitate

Clear solution Clear solution Cloudy pale yellow

 solution with
precipitate

Clear solution Pale yellow solution Cloudy pale yellow

solution with
precipitate

In the molisch test, though different in appearance, all the carbohydrates tested

are positive. Amylose is the perfect example of a positive result as it formed a pink-

purple ring in the middle of clear solutions. For glycogen and cellulose, though it

appeared as pink solutions, these are also positive because the pink hue signals that

the aldehyde has condensed with the α-naphthol of the molisch reagent. The

precipitate of glycogen and cellulose are the byproducts of the reaction.

For the anthrone test, the results of the three carbohydrates are uniformly lime

green. This green hue is a sign that the furfural, made from the dehydration of the

carbohydrate when it interacted with the sulfuric acid, has interacted with the anthrone

found in the reagent. The green hue is also a sign that the samples are definitely

carbohydrates.

The results of the iodine is where the three carbohydrates varied in terms of the

result. Amylose, which is known to be the branched starch, tested positive. Initially,

when the reagent was placed on the sugar, the solution has already turned blue, which

is already a sign that it is positive for the test. When heated, it turned clear because

the starch-iodine compound is known to be unstable so heating the solution broke

amylose chains. The result of glycogen and cellulose tested negative as the solutions

formed did not turn blue nor clear at the end of the test. The yellow solution is a sign
that the carbohydrates are negative and the precipitate formed in the cellulose batch

are the byproducts of the reaction from the reagent.

Specific Test

Test Positive Negative Unknown A Unknown B

Control Control

Mucic Acid Galactose Glucose ✗ ✗

Test Cloudy Solution Clear Solution Clear Solution Clear solution
with crystals

Benedict’s Glucose Sucrose ✓ ✓

Test Bright red Bright blue Rust colored Rust colored
solution with red solution solution with solution with
precipitate muddy green muddy green
suspension and suspension and
red precipitate red precipitate
(Time: 2 (Time: 2
minutes) minutes)

Barfoed’s Test Glucose Maltose ✓ ✓

Blue Solution Bright red Blue solution Blue Solution
with red solution with red with red
 precipitate precipitate precipitate
(Time: 2 (Time: 2
minutes and 30 minutes and 30
seconds) seconds)

Bial’s-Orcinol Xylose Glucose ✓ ✗

Test Blue-green Bright yellow Blue-green Brown Solution
solution solution solution

Seliwanoff’s Fructose Glucose ✗ ✓

Test Cherry colored Clear solution Clear Solution Cherry colored
solution solution

The unknowns were inferred using the following tests. Unknown A and B in the

mucic acid test, which identifies galactose, both tested negative. Both turned out clear,

unlike the positive control, which formed crystals. For the benedict’s test, which

determines if the sugars are reducing or non reducing, both unknowns tested positive,

deeming the two as reducing sugars; both formed red precipitate meaning it had

reduced the copper from +2 to +1, making it precipitate. Barfoed’s test confirmed that

the two unknowns as monosaccharides because both also formed a red precipitate,

which similarly with the benedict’s test, reduced the copper from +2 to +1. In the bial’s-

orcinol test, the two unknowns started to differentiate. Bial’s-orcinol tests if the sugars

tested are pentoses and here, unknown A tested positive by turning blue green and

unknown B tested negative by turning into a brown solution, making unknown A a

pentose and unknown B a hexose. Lastly, the seliwanoff’s test confirmed that unknown

B was fructose because it tested positive for this test. Other factors that led to this

conclusion are that unknown B is reducing (benedict’s test), a monosaccharide

(barfoed’s test), and a hexose (bial’s-orcinol test). Unknown A is xylose because it was

confirmed that unknown A is reducing (benedict’s test), a monosaccharide (barfoed’s

test), and a pentose (bial’s-orcinol test) and is not galactose (mucic acid test) nor

fructose (seliwanoff's test), which fits the description of a xylose.

Experimental

The general test for carbohydrates consisted of 3 separate tests. Molisch test was

conducted by first, placing 10 drops of each carbohydrate, amylose, glycogen, and

cellulose, in small test tubes. 2 drops of molisch reagent was then added to each test

tube and these solutions were mixed thoroughly. 10 drops of conc. sulfuric acid were

then dropped in the slightly inclined test tubes containing the mixed solutions. The

results were then taken note of. For the anthrone test, 10 drops of the anthrone

solutions were placed on 3 different spots on a spot plate. 1-2 drops of each

carbohydrate were then placed on the spots where the anthrone solution was and this

solution was mixed thoroughly. The results were then taken note of. Lastly, the iodine

test was conducted starting with placing 10 drops of each carbohydrate were placed

in small test tubes and one drop of the iodine solution was placed on each of the test

tubes. These were heated in a boiling water bath for two minutes and the appearance

before, during, and after heating were recorded.

The specific test for carbohydrates consisted of 5 different tests. Before

proceeding, half of the unknowns were placed in separate test tubes and were

dissolved in 5 mL of distilled water. Four medium sized test tubes were prepared for

each test. First, the mucic acid test was conducted.. 10 drops of the sugars, galactose,

glucose, and the two unknowns, were placed on the test tubes and were plugged with

cotton. These were subjected to a water bath for an hour. After heating, the surface

where the substance touched the glass was scratched using a stirrer. The results were

recorded 2 days after. Second, the benedict’s test was conducted starting by placing
10 drops of benedict's reagent in each test tube. Then, 5 drops of fructose, sucrose,

and the two unknowns. These were then heated until a muddy green suspension and

a red precipitate appeared. The solutions were then cooled and the time was taken

note of. Third, the barfoed’s test was conducted by placing 10 drops of the barfoed’s

reagent on each test tube then 5 drops of glucose, maltose, and the unknowns were

placed on the test tube following this. These were then subjected into a water bath

until a red precipitate formed. The time was noted and the characteristic of solution as

well. Fourth, the bial’s-orcinol test was conducted. 5 drops of xylose, glucose and the

unknowns were placed in the test tube and was followed by placing 10 drops of the

bial’s-orcinol reagent. These were subjected to a water bath until blue-green. The time

and characteristic of the substances was taken down. Lastly, the seliwanoff’s test was

conducted. 10 drops of the seliwanoff’s reagent were placed in the test tubes. Then 5

drops of fructose, glucose, and the unknowns were placed with the reagent. These

were heated in a water bath until the color changed to cherry red. The results were

noted down,

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