Introduction:-

Benedict Test:-
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 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.

Benedict’s quantitative reagent:-

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.
 CARBOHYDRATES:-

A carbohydrate is a biomolecule consisting of carbon (C),

hydrogen (H) and oxygen (O) atoms, usually with a hydrogen–
oxygen atom ratio of 2:1 (as in water) and thus with the
empirical formula Cm(H2O)n (where m may be different from
n). This formula holds true for monosaccharides. Some
exceptions exist; for example, deoxyribose, a sugar component
of DNA,[1] has the empirical formula C5H10O4.[2] The
carbohydrates are technically hydrates of carbon; structurally it
is more accurate to view them as aldoses and ketoses.
The term is most common in biochemistry, where it is a
synonym of saccharide, a group that includes sugars, starch,
and cellulose. The saccharides are divided into four chemical
groups: monosaccharides, disaccharides, oligosaccharides, and
polysaccharides. Monosaccharides and disaccharides, the
smallest (lower molecular weight) carbohydrates, are
commonly referred to as sugars.[3] While the scientific
nomenclature of carbohydrates is complex, the names of the
monosaccharides and disaccharides very often end in the suffix
-ose, as in the monosaccharides fructose (fruit sugar) and
glucose (starch sugar) and the disaccharides sucrose (cane or
beet sugar) and lactose (milk sugar).
Carbohydrates perform numerous roles in living organisms.
Polysaccharides serve for the storage of energy (e.g. starch and
glycogen) and as structural components (e.g. cellulose in plants
and chitin in arthropods). The 5-carbon monosaccharide ribose
is an important component of coenzymes (e.g. ATP, FAD and
NAD) and the backbone of the genetic molecule known as
RNA. The related deoxyribose is a component of DNA.
Saccharides and their derivatives include many other important
biomolecules that play key roles in the immune system,
fertilization, preventing pathogenesis, blood clotting, and
development.[5]
They are found in a wide variety of natural and processed
foods. Starch is a polysaccharide. It is abundant in cereals
(wheat, maize, rice), potatoes, and processed food based on
cereal flour, such as bread, pizza or pasta. Sugars appear in
human diet mainly as table sugar (sucrose, extracted from
sugarcane or sugar beets), lactose (abundant in milk), glucose
and fructose, both of which occur naturally in honey, many
fruits, and some vegetables. Table sugar, milk, or honey are
often added to drinks and many prepared foods such as jam,
biscuits and cakes.
Cellulose, a polysaccharide found in the cell walls of all plants,
is one of the main components of insoluble dietary fiber.
Although it is not digestible, insoluble dietary fiber helps to
maintain a healthy digestive system[6] by easing defecation.
Other polysaccharides contained in dietary fiber include
resistant starch and inulin, which feed some bacteria in the
microbiota of the large intestine, and are metabolized by these
bacteria to yield short-chain fatty acids.
 Carbohydrates are the most abundant class of organic
compounds found in living organisms.
 They originate as products of photosynthesis, an
endothermic reductive condensation of carbon dioxide
requiring light energy

General Information:

n H20 + Energy→CnH2nOn + n 02
 The formulas of many carbohydrates can be written as
carbon
hydrates, Cn(H20)n, hence their name.
 The carbohydrates are a major source of metabolic energy,
both for plants and for animals that depend on plants for
food.
 Aside from the sugars and starches that meet this vital
nutritional role, carbohydrates also serve as a structural
material (cellulose), a component of the energy transport
compound ATP, recognition sites on cell surfaces, and one
of three essential components of DNA and RNA.
Carbohydrates are called saccharides or, if they are
relatively small, sugars.
 OBJECTIVE:-

Benedict's reagent is used to test for the presence of glucose in

urine. Once a reducing sugar is detected in urine, further tests
have to be undergone in order to ascertain which sugar is
present. The copper sulphate in Benedict's solution reacts with
reducing sugars and the cupric ions to cuprous ions, these are
precipitated as red copper oxide, which is insoluble in water.
Alkaline medium is provided to the reaction by sodium
carbonate present in the reagent. The original colour of
Benedict's reagent is blue. It changes to green, yellow, orange
or red, according to the concentration of glucose present in
urine.
When Benedict’s solution and simple carbohydrates are
heated, the solution changes to orange red/ brick red. This
reaction is caused by the reducing property of simple
carbohydrates. The copper (II) ions in the Benedict’s solution
are reduced to Copper (I) ions, which causes the colour
change.
The red copper(I) oxide formed is insoluble in water and is
precipitated out of solution. This accounts for the precipitate
formed. As the concentration of reducing sugar increases, the
nearer the final colour is to brick-red and the greater the
precipitate formed. Sometimes a brick red solid, copper oxide,
precipitates out of the solution and collects at the bottom of
the test tube.
Sodium carbonate provides the alkaline conditions which are
required for the redox reaction. Sodium citrate complexes
with the copper (II) ions so that they do not deteriorate to
copper(I) ions during storage.
Complex carbohydrates such as starches DO NOT react
positive with the Benedict’s test unless they are broken down
through heating or digestion (try chewing crackers and then
doing the test). Table sugar (disaccharide) is a non-reducing
sugar and does also not react with the iodine or with the
Benedict Reagent. Sugar needs to be decomposed into its
components glucose and fructose then the glucose test would
be positive but the starch test would still be negative.

Composition and Preparation of Benedict’s

Solution :-

Benedict’s solution is a deep-blue alkaline solution used to

test for the presence of the aldehyde functional group, – CHO.
Anhydrous sodium carbonate = 100 gm
Sodium citrate – 173 gm
Copper(II) sulfate pentahydrate = 17.3 gm
One litre of Benedict’s solution can be prepared from 100 g of
anhydrous sodium carbonate, 173 g of sodium citrate and 17.3
g of copper(II) sulfate pentahydrate.
 SCOPE AND LIMITATION:-
Scope:-
Benedict's reagent can be used to test for the presence of
glucose in urine. Glucose in urine is called glucosuria and can
be indicative of diabetes mellitus, but the test is not
recommended or used for the diagnosis of diabetes mellitus. A
false positive reaction can be due to the presence of other
reducing substances in urine such as ascorbic acid, drugs and
homogentisic acid.

Limitation:-
If blood glucose level cross renal threshold, than it excreted
in urine. E.g. in diabetes Mellitus. If Renal threshold for
glucose decrease in renal failure, so in this case also benedict
test come positive. Each reducing substance gives positive
test, So Following substance can gives false positive test E.g.
Vitamin – C, B-Complex vitamin, Salicylic acid.
 PRINCIPLE AND THEORY
Principle of Benedict’s Test:-
When Benedict’s solution and simple carbohydrates are
heated, the solution changes to orange red/ brick red. This
reaction is caused by the reducing property of simple
carbohydrates. The copper (II) ions in the Benedict’s solution
are reduced to Copper (I) ions, which causes the color change.
The red copper(I) oxide formed is insoluble in water and is
precipitated out of solution. This accounts for the precipitate
formed. As the concentration of reducing sugar increases, the
nearer the final colour is to brick-red and the greater the
precipitate formed. Sometimes a brick red solid, copper oxide,
precipitates out of the solution and collects at the bottom of
the test tube.
Sodium carbonate provides the alkaline conditions which are
required for the redox reaction. Sodium citrate complexes
with the copper (II) ions so that they do not deteriorate to
copper(I) ions during storage.
Complex carbohydrates such as starches DO NOT react
positive with the Benedict’s test unless they are broken down
through heating or digestion (try chewing crackers and then
doing the test). Table sugar (disaccharide) is a non-reducing
sugar and does also not react with the iodine or with the
Benedict Reagent. Sugar needs to be decomposed into its
components glucose and fructose then the glucose test would
be positive but the starch test would still be negative.
Theory of benedict test:-
Carbohydrates:-
General Information:
n H20 + Energy→CnH2nOn + n 02
 The formulas of many carbohydrates can be written as
carbon
hydrates, Cn(H20)n, hence their name.
 The carbohydrates are a major source of metabolic energy,
both for plants and for animals that depend on plants for
food.
 Aside from the sugars and starches that meet this vital
nutritional role, carbohydrates also serve as a structural
material (cellulose), a component of the energy transport
compound ATP, recognition sites on cell surfaces, and one
of three essential components of DNA and RNA.
Carbohydrates are called saccharides or, if they are
relatively small, sugars.
Simple Sugars :
 Contain the elements carbon, hydrogen, and oxygen.
 The name carbohydrate literally means water compounds
of carbon. The general formula for simple sugars is
Cn(H20)n.
 This class of compounds is better described as
Polyhydroxy aldehydes and ketones.
 The simplest carbohydrates are glyceraldehyde and
dihydroxyacetone.
Classification of Carbohydrates:-

Methods of Classification:-

Several methods are used to classify carbohydrates.

Method of classification is based on whether the

carbohydrate can be broken down into smaller units:-
• Monosaccharides cannot be broken down into smaller units
by hydrolysis.Sometimes called simple sugars.
• Disaccharides can be broken down (hydrolyzed) into two
monosaccharide units.
• Oligosaccharides can be broken into three to six
monosaccharide units.
• Polysaccharides composed of 7 or more mono-saccharide
units.

Method is based on the number of carbons

found in a simple sugar.
• If it has 3 carbons it is called a triose.
• If it has 4 carbons it is called a tetrose.
• If it has 5 carbons it is called a pentose.
• If it has 6 carbons it is called a hexose.
Method uses the kind of carbonyl group:

Aldose: A monosaccharide with an aldehyde group.

Ketose: A monosaccharide with a ketone group.

Stereoconfigurations Of Simple Sugar:

Carbohydrates contain many stereocenters.
1. If the OH group is found on the right side of the carbon
chain, the sugar is designated as a D sugar.
2. If the OH group is found on the left side of the chain of
carbons, the sugar is designated as an L sugar.

Cyclic Structures:-
 Five membered sugar rings are known as furanose rings.
 Six membered sugar rings are known as pyranose rings.
Carbohydrate Anomers:-

 Formation of either of the cyclic form has created a new

stereocenter.
 These stereoisomeric ring forms of carbohydrates are
called Anomers.
 Anomers are carbohydrates that differ by the stereo-
configuration of the carbon involved in ring formation.
  The greek letters α and ß are used to describe the
configuration about the ring forming carbon.
 The α anomer always has the OH group oriented in a
downward fashion on the anomeric carbon of a D-sugar.
 The ß anomer always has the OH group oriented in an
upward fashion on the anomeric carbon of a D-sugar.

Important Carbohydrates:-

A. Monosaccharides composed of three to seven carbon

atoms:--
1. Glucose:-
• The most abundant hexose in our diet.
• The building block of complex carbohydrates.
• Component of the disaccharides: sucrose, maltose and lactose.
• Found in the polysaccharides: starch, cellulose and glycogen.

2. Galactose:-
 Found in the disaccharide, lactose.
 Found in the cellular membranes of the brain and
nervous system.
 Galactose is the C-4 epimer of glucose.
3. Fructose:-
• Sweetest of the carbohydrates.
• Component of the disaccharide sucrose.
• Fructose is a keto sugar.
 B. Disaccharides composed of 2 monosaccharide units:-

1. Maltose (malt sugar):-

• Used in cereals, candies and the brewing of beverages.
 Composed of two D-glucose sugars joined by an α-1,4
linkage.

2. Lactose (milk sugar):-

• Found in milk and milk products.
• Composed of one galactose and one glucose unit joined
by a β-1,4 linkage.

3. Sucrose (table sugar):-

• Product of sugar cane and sugar beets.
• Composed of one glucose and one fructose unit.
• Linkage is at both anomeric carbons.

C. Polysaccharides composed of many (more than 10)

monosaccharide units:-
1- Cellulose:-
• Major structural material of plant cells.
• Consists of many glucose units joined by β-1,4 linkages.

2. Starch:-
• Storage form of glucose found in rice wheat, potatoes,
grains and cereals.
 • Consists of many glucose units joined by α-1,4 linkages.
• Maltose is the disaccharide starting material.

3. Glycogen:-
• Animal starch. Storage form of glucose found in the liver
• and muscle of animals.
• Contains many highly branched glucose units.
• Joined by α-1,4 linkages and branched by α-1,6 linkages
4. Dextrin:-
• Mixture of branched and un-branched soluble
polysaccharides produced by partial hydrolysis of starch
by acids or amylases.

Importance of Carbohydrates:-
Now as we previously discussed, carbohydrates are
absolutely essential for life on the planet. Let us take a
more detailed look at the importance of carbohydrates.
Carbohydrates are responsible for storing chemical energy
in living organisms. You must hear all the time when
athletes carbo-load before a game. This is so they can
provide themselves with extra energy. They are also an
important constituent for supporting tissues in plants and
even in some animals.
As I am sure you are already aware of photosynthesis. It is
the process by which plants utilize solar energy to generate
energy for themselves and food for us. Through this
process, plants fix CO2 and synthesize carbohydrate. Let us
 take a look at the chemical reaction occurring during
photosynthesis.

x(CO2) + y(H2O) + Solar energy ⇒ Cx (H2O)y + O2

So carbohydrates due to photosynthesis are the repository
of solar energy in plants, Then when plants or animals
metabolize the said carbohydrate this energy releases. The
metabolizing equation is just the reverse of the
photosynthesis equation
Cx (H2O)y + O2 ⇒ x(CO2) + y(H2O) + Energy

Reducing sugars :-

• Any sugar that contains either:

1- A free aldehyde group.

2- An α-hydroxy ketone group.
3- A hemiacetal linkage

• The presence of any of these groups allows the

carbohydrate to undergo easy oxidation.
• If the sugar gets oxidized it causes reduction.
• Thus the name "reducing sugar”.
 QUALITATIVE TESTS FOR
CARBOHYDRATES

Benedict’s Test:-

Benedict 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 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.

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.
Experiment:-
Aim:- To test the presence of sugar in the given sample of
urine.

Principle:- In normal urine, practically there is no glucose.

Presence of glucose in urine is called glucosuria. To detect
reducing sugars, such as glucose, fructose etc. in urine
Benedict's is done. reducing CuSO4 substances present in
Benedict's solution or Fehling's solution is reduced on boiling
by the (glucose, fructose etc.) to form the coloured precipitate
of cuprous oxide. The light green, green, yellow and brick red
precipitates of cuprous oxides depend on the concentration of
reducing substances present in urine. Glucose reduces the blue
cupric sulphate of Benedict's reagent or Fehling's reagent to a
coloured insoluble precipitate.
CH2OH(CHOH)4 CHO + 2Cu++ SO4—+2H2O heating
(reducing sugar) (blue solution)
CHGluconic 2OH(CHOH)acid 4 COOH cuprous + Cu2O oxide
+ 4H+ + SO4--
(oxidised sugar) (red precipitate)
Requirement: Glasswares: Test tubes, beakers, spirit lamp,
pipette; Chemicals: Benedict's solution, Fehling's solution A
and B, Seliwanoff's reagent, Miscellaneous: Test tube holder,
test tube stand, urine sample.
Preparation of Reagents
Benedict's reagent Mix: 173 g of sodium citrate and 100 g of
anhydrous sodium carbonate in 600 mL of water in a beaker
and warm gently (solution water (solution A). Dissolve B).
Add 17.3 solution g of hydrated B to solution CuSOA 4 with
in 100 constant mL of distilled stirring. Cool and transfer to a
one litre flask and make upto the mark with water.

Proce
dure
Benedict's
test
• Take 5 mL of Benedict's reagent in a test
tube. Add 0.5 mL (8 drops) of freshly
passed urine to it.
• Boil for 2 minutes holding the test tube
firmly with a test tube holder (during
boiling, the contents of the test tube get a
tendency to spurt out. Hence, it is wise to
keep shaking the test tube after holding it in
the inclined position near the flame to avoid
overboiling).
• A light green, green, yellow and brick red
precipitate indicates the presence of
reducing substances in urine.
• The various coloured precipitates depend
on the concentration of reducing sugars in
urine which gives a rough estimate of the
concentration given below:

Colour of precipitate % of reducing suger

present
 Light green ----- 0.1 to 0.5 Green ----
-- 0.5 to 1.0 Yellow ------ 1.0 to 2.0
Brick red ------ above 2

Note: Benedict's test are not necessarily

indicative of only glucose in urine but it may
also indicates the presence of other reducing
sugars, such as lactose (in case of pregnant
woman and lactating mothers), fructose (in
fructosuria), galactose (in galactosuria),
homogentisic acid (in alkaptonuria),
glucuronates and mucin.

Principle of Benedict Test:-

When Benedict’s solution and simple carbohydrates are
heated, the solution changes to orange red/ brick red. This
reaction is caused by the reducing property of simple
carbohydrates. The copper (II) ions in the Benedict’s solution
are reduced to Copper (I) ions, which causes the color change.

The red copper(I) oxide formed is insoluble in water and is

precipitated out of solution. This accounts for the precipitate
formed. As the concentration of reducing sugar increases, the
nearer the final colour is to brick-red and the greater the
precipitate formed. Sometimes a brick red solid, copper oxide,
precipitates out of the solution and collects at the bottom of
the test tube.
Sodium carbonate provides the alkaline conditions which are
required for the redox reaction. Sodium citrate complexes
with the copper (II) ions so that they do not deteriorate to
copper(I) ions during storage.

Complex carbohydrates such as starches DO NOT react

positive with the Benedict’s test unless they are broken down
through heating or digestion (try chewing crackers and then
doing the test). Table sugar (disaccharide) is a non-reducing
sugar and does also not react with the iodine or with the
Benedict Reagent. Sugar needs to be decomposed into its
components glucose and fructose then the glucose test would
be positive but the starch test would still be negative.

Composition and Preparation of Benedict’s

Solution:-
Benedict’s solution is a deep-blue alkaline solution used to
test for the presence of the aldehyde functional group, – CHO.

Anhydrous sodium carbonate = 100 gm

Sodium citrate – 173 gm
Copper(II) sulfate pentahydrate = 17.3 gm
One litre of Benedict’s solution can be prepared from 100 g of
anhydrous sodium carbonate, 173 g of sodium citrate and 17.3
g of copper(II) sulfate pentahydrate.

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.

Result Interpretation of Benedict’s Test:-

If the color upon boiling is changed into green, then there
would be 0.1 to 0.5 percent sugar in solution.
If it changes color to yellow, then 0.5 to 1 percent sugar is
present.
If it changes to orange, then it means that 1 to 1.5 percent
sugar is present.
If color changes to red,then 1.5 to 2.0 percent sugar is present.
And if color changes to brick red,it means that more than 2
percent sugar is present in solution.