CHAPTER ONE.

INTRODUCTION TO FOOD CHEMISTRY.

 Food chemistry is the study of chemical processes and interactions of all biological and
non-biological components of foods.

 It covers the basic composition, structure and properties of foods and the chemistry
changes occurring during processing and utilization.
It also covers the chemistry of water, carbohydrates, proteins, lipids, vitamins, minerals
and enzymes
 Food chemistry is the study of composition of foods and of the reactions which lead to
changes in their constitution and characteristics.

IMPORTANCE OF FOOD CHEMISTRY.

 Food chemistry allows for subjecting food materials to chemical scrutiny. It employs
chemistry tools to analyse food items so that they transform to nutritious, safe and
materials of commercial value. Instruments that are popular in the vicinity of chemistry
are employed in food chemistry.
 Flavors, preservatives, emulsifiers, thickeners, stabilizers, sweeteners, colors are some of
the materials that are produced from food chemistry
 Basic knowledge of the constituents of food.

 - Determination of appropriate processing and preservation method.

 - Aids understanding of microbiological reactions in food.

 - Information on chemical reactions involving food.

 - Useful information in New Food Product Development

 - Useful information to Engineers in design and fabrication of appropriate food

 Processing equipment.
  - Help in choice of packaging material, equipment and technique.

 - Useful in storage stability and shelf life studies of food and food products

Assignment.

 Discuss the roles of food chemistry in the society. (10marks).

CHAPTER TWO.

WATER IN FOOD

 Water as basic constituent of ALL foods.

FORMS OF WATER IN FOOD

 Most natural foods contain water up to 70% of their weight. \

 Water in foods is classified in to two types:

(a) Bound water

(b) Free water

Water that can be extracted easily from foods by squeezing or pressing or cutting or pressing is
called as free water

BOUND WATER

 Water that is held so tightly by another molecule (such as a protein)

 Not easily removed from the food is called bound water. •
 This water is not free to act as solvent for salts and sugars.
  It can be frozen only at very low temperatures. •
 Density is greater than water.
 The water molecules are bound to polar groups or ions on molecules such as starches,
pectin, and proteins.
 The bound water is of three type

i. Constitutional
ii. ii. Vicinal
iii. iii. Multilayer

i. Constitutional: They form an integral part of a non-aqueous constituent forming

<0.03%

-It is constituted by a monolayer of water molecules absorbed on the polar

absorption site of the molecule is almost immobilized and thus behaves, like part
of the solid or like water in ice.

ii. Vicinal: It is the bound water that strongly acts with specific hydrophilic sites of non-
aqueous constituents to form monolayer coverage; water-ion and water dipole bonds
forming 0.1 to 0.9%.

iii. Multilayer: Bound water that forms several additional layers around hydrophilic
groups, water-water and water-solute hydrogen bonds

Free or entrapped water •

Water that can be extracted easily from foods by squeezing or cutting or pressing is called as
free water

. • Free water is held within matrix or gel,

• Entrapped water is immobilized in capillaries or cells but if released during cutting or damage,

it flows freely.
ROLES OF WATER IN FOODS

 Serves as a solvent to disperse water soluble compounds present in foods and is used in
food Prep
 A good medium for heat transfer - cooking medium
 Hard to burn foods in water compared with frying, broiling, bbq etc
 Serves as a reaction medium for chemical reaction, hydrolysis and degradation of food
components (proteins, CHO, lipids etc)
 Influences and changes the texture and structure of foods
 Gels, emulsions, cream, ice etc
 Change the properties of food when dried or concentrated

MOISTURE CONTENT AND WATER ACTIVITY

 Water content— is a measurement of the total amount of water contained in a food,

usually expressed as a percentage of the total weight
 .It’s a useful measurement for determining the dry weight of your food and ingredients
and it helps calculate your total yield.
 It can also be used to confirm whether the drying process of your foods is finished.
  Food’s moisture content will have a direct effect on the way your food is processed,
mixed, and dried, as well as the mouthfeel, appearance, and texture of the final product.

 Water activity explains how the water in your food will react with microorganisms. The
higher the water activity, the faster microorganisms like bacteria, yeast, and mold will be
able to grow – resulting in higher standards of food storage.
 Water activity is calculated by finding the ratio of the vapor pressure in food to the vapor
pressure of pure water and is primarily used to determine the necessary food storage
requirements and shelf life of your products
EQUILIBRIUM RELATIVE HUMIDITY AND SORPTION ISOTHERMS

The equilibrium relative humidity (ERH) of a food product is defined as relative humidity of
the air surrounding the food that is in equilibrium with its environment. When the equilibrium is
obtained, the ERH (in percent) is equal to the water activity multiplied by 100, i.e. ERH (%) =
aw × 100.

Sorption isotherms

 The food sorption isotherm describes the thermodynamic relationship between water
activity and the equilibrium of the moisture content of a food product at constant
temperature and pressure.

 Water activity depends on the composition, temperature and physical state of the
compounds

 A plot of water content of a food (g water/g dry material) versus aw at constant

temperature is known as a moisture sorption isotherm (MSI).

 Information derived from MSIs are useful for concentration and dehydration processes,
formulation of food mixtures so as to avoid moisture transfer among the ingredients,
determination of moisture barrier properties needed in a packaging material,
determination of what moisture content will curtail growth of microorganisms of interest
and prediction of the chemical and physical stability of food as a function of water
content
 Resorption (or adsorption) isotherms are prepared by adding water to previously dried
samples.

 Desorption isotherms are isotherms prepared by removing water from samples

Isotherms with a sigmoidal shape are characteristic of most foods.

 Foods such as fruits, confections, and coffee extract that contain large amounts of sugar
and other small, soluble molecules and are not rich in polymeric materials exhibit a J-
type isotherm.

 An isotherm can be typically divided into three regions; the water in region A represents
strongly bound water, and the enthalpy of vaporization is considerably higher than the
one of pure water. The bound water includes structural water (H-bonded water) and
monolayer water, which is sorbed by the hydrophilic and polar groups of food
components (polysaccharides, proteins, etc.). Bound water is unfreezable and it is not
available for chemical reactions or as a plasticizer.

 In region B, water molecules bind less firmly than in the first zone, they usually present
in small capillaries. The vaporization enthalpy is slightly higher than the one of pure
water. This class of constituent water can be looked upon as the continuous transition
from bound to free water.

 The properties of water in region C are similar to those of the free water that is held in
voids, large capillaries, crevices; and the water in this region loosely binds to food
materials (12-15). Moreover, hysteresis is related to the nature and state of the
components of food, reflecting their potential for structural and conformational
rearrangements, which alters the accessibility of energetically favourable polar sites. The
presence of capillaries in food results in considerable decrease in water activity. The
explanation for the occurrence of moisture sorption hysteresis comprises the ink bottle
 theory, the molecular shrinkage theory, the capillary condensation, and the swelling

fatigue theory (16).

 Hysteresis in foods is the phenomenon by which at constant water activity (Aw) and
temperature, a food adsorbs a smaller amount of water during adsorption than during a
subsequent desorption process.

INTERACTION OF WATER MOLECULES WITH NON-AQUEOUS

COMPONENTS OF FOOD.

 Mixing of solutes and water alters properties of each other.

 Hydrophilic solutes cause changes in structure and mobility of water and water causes
changes in the reactivity, and structure, of hydrophilic solutes.
 Hydrophobic groups of solutes interact only weakly with wate
 . In interaction of solute with water, various bonding forces existing between water and
solutes.

Interaction between water and ions

 Ions and ionic groups of organic molecules hinder mobility of water molecules to a
greater extent than other types of solutes.
 The strength of water-ion interaction is greater than that of hydrogen bonds, between the
water molecules, however, it is much less than that of covalent bonds.
 Water and inorganic ions (e.g. NaCl) undergo dipole-ion interactions.
 The ions compete for water and alter water structure, influence the permittivity of the
aqueous medium and influence thickness of the electric layer around colloids particle
“degree of hospitality” provided to other no aqueous solutes and to substances suspended
in the medium.

Interaction between water and hydrophilic solutes forming hydrogen bond

 Interactions between water and nonionic, hydrophilic solutes are weaker than that of the
interactions between water and ions and of the almost same strength as that of the
hydrogen bonds between water molecules.
 Solutes capable of hydrogen bonding enhance or at least not disrupt the normal structure
of pure water. •
 However, in some instances solutes have a disruptive influence on the normal structure of
water.
 Urea is good example which markedly disrupts normal structure of water

Interaction between water and non-polar substances

 The mixing of water and hydrophobic substances (e.g. apolar groups of fatty acids, amino
acids, proteins, etc.) is thermodynamically unfavorable event (ΔG>0).
 Water forms a special structure in vicinity of the incompatible apolar entities.
 This process has been referred to as hydrophobic hydration.
 Since hydrophobic hydration is thermodynamically unfavorable, water tends to minimize
its association with the apolar entities.
 Therefore, the incompatible aqueous environment will encourage two separate apolar
groups to associate, to decrease water polar interfacial area. This process is termed as
“hydrophobic interaction”.

CHAPTER THREE.

CARBOHYDRATE.
Carbohydrates represent a broad group of substances which include the sugars, starches, gums
and celluloses. The common attributes of carbohydrates are that they contain only the elements
carbon, hydrogen and oxygen, and that their combustion will yield carbon dioxide plus one or
more molecules of Water.

The Nutrition Sources of Carbohydrates

 There are both healthy and unhealthy sources of carbohydrates.

 Healthy sources of carbohydrates include both food sources-animal and plant
products, such as fresh fruits, vegetables, corn, potatoes, milk and milk products
 . Unhealthy sources include soda, white bread, artificial sugar, pastries, and other highly
processed foods.
TYPES OF CARBOHYDRATE

Carbohydrates can be divided into two main types: simple and complex. Simple
carbohydrates are made up of just one or two sugar units, whereas complex carbohydrates are
made up of many sugar unit

1. Simple carbohydrates

Simple carbohydrates are sometimes called "sugars" or "simple sugars." There are 2 types of
simple carbohydrates: monosaccharides and disaccharides.

Monosaccharides
 Simplest group of carbohydrates and often called simple sugars since they cannot be
further hydrolyzed.
 Colorless, crystalline solid which are soluble in water and insoluble in a non-polar
solvent.
 These are compound which possesses a free aldehyde or ketone group.
 The general formula is Cn(H2O)nor CnH2nOn.
 They are classified according to the number of carbon atoms they contain and also on the
basis of the functional group present.
 The monosaccharides thus with 3,4,5,6,7… carbons are called trioses, tetroses, pentoses,
hexoses, heptoses, etc., and also as aldoses or ketoses depending upon whether they
contain aldehyde or ketone group

There are 3 monosaccharides:

1. Glucose
2. Fructose
3. Galactose

 Glucose (C6H12O6) is a common monosaccharide and an important source of

energy. During cellular respiration, energy is released from glucose and that
energy is used to help make adenosine triphosphate (ATP). Plants synthesize
glucose using carbon dioxide and water, and glucose, in turn, is used for energy
requirements for the plant.
 Galactose (a milk sugar) and fructose (found in fruit) are other common
monosaccharides. Although glucose, galactose, and fructose all have the same
chemical formula (C6H12O6), they differ structurally and stereochemically. This
makes them different molecules despite sharing the same atoms in the same
proportions, and they are all isomers of one another, or isomeric
monosaccharides. Glucose and galactose are aldoses, and fructose is a ketose

The second type of simple carbohydrates is disaccharides. They contain two sugar units
bonded together.

There are 3 disaccharides:

Maltose (glucose + glucose)

Sucrose (glucose + fructose)

Lactose (glucose + galactose).

Complex carbohydrates

Complex carbohydrates are also called polysaccharides, because they contain many sugars. (The
prefix "poly-" means "many.") There are 3 main polysaccharides:

1. Starch
2. Glycogen
3. Fiber

All three of these polysaccharides are made up of many glucose molecules bonded together, but
they differ in their structure and the type of bonds

Glycogen is the storage form of glucose in humans and other vertebrates. It is made up of
monomers of glucose. Glycogen is the animal equivalent of starch and is a highly branched
molecule usually stored in liver and muscle cells. Whenever blood glucose levels decrease,
glycogen is broken down to release glucose in a process known as glycogenolysis.

Cellulose is the most abundant natural biopolymer. The cell wall of plants is mostly made of
cellulose and provides structural support to the cell. Cellulose is made up of glucose monomers
that are linked by β 1-4 glycosidic bonds. Every other glucose monomer in cellulose is flipped
over, and the monomers are packed tightly as extended long chains. This gives cellulose its
rigidity and high tensile strength—which is so important to plant cells.
Starch is the stored form of sugars in plants and is made up of a mixture of amylose and
amylopectin (both polymers of glucose). Plants are able to synthesize glucose, and the excess
glucose, beyond the plant’s immediate energy needs, is stored as starch in different plant parts,
including roots and seeds. The starch in the seeds provides food for the embryo as it germinates
and can also act as a source of food for humans and animals. The starch that is consumed by
humans is broken down by enzymes, such as salivary amylases, into smaller molecules, such as
maltose and glucose. The cells can then absorb the glucose.

Starch is made up of glucose monomers that are joined by α 1-4 or α 1-6 glycosidic bonds. The
numbers 1-4 and 1-6 refer to the carbon number of the two residues that have joined to form the
bond. As illustrated in Figure 6, amylose is starch formed by unbranched chains of glucose
monomers (only α 1-4 linkages), whereas amylopectin is a branched polysaccharide (α 1-6
linkages at the branch points).

METABOLISMS OF CARBOHYDRATE.

Digestion and absorption of carbohydrate

From the Mouth to the Stomach

  The mechanical and chemical digestion of carbohydrates begins in the mouth. Chewing,
also known as mastication, crumbles the carbohydrate foods into smaller and smaller
pieces.
 The salivary glands in the mouth secrete saliva that coats the food particles.
 Saliva contains the enzyme, salivary amylase. This enzyme begins carbohydrate
digestion by breaking some of the bonds between individual units of disaccharides,
oligosaccharides, and starches.
 The salivary amylase breaks down amylose and amylopectin into smaller chains of
glucose, called dextrins and maltose. Only about five percent of starches are broken down
in the mouth.
 When carbohydrates reach the stomach, no further chemical breakdown occurs because
the amylase enzyme does not function in the acidic conditions of the stomach. But
mechanical breakdown is ongoing—the strong peristaltic contractions of the stomach mix
the carbohydrates into a semi-fluid mass of partly digested food known as chyme.

From the Stomach to the Small Intestine

 Most chemical digestion of carbohydrates occurs in the small intestine.

 Chyme from the stomach is gradually released into the upper part of the small intestine.
 Upon entry of the chyme into the small intestine, the pancreas releases pancreatic juice
through a duct into the small intestine.
 This pancreatic juice contains the enzyme, pancreatic amylase, which starts again the
breakdown of dextrins into shorter and shorter carbohydrate chains.
 Additionally, enzymes are secreted by the intestinal cells that line the villi.
 These enzymes, known collectively as disaccharidases, are sucrase, maltase, and lactase.
 Sucrase breaks sucrose into glucose and fructose molecules.
 Maltase breaks the bond between the two glucose units of maltose,
 Lactase breaks the bond between the galactose and glucose units of lactose.
 Once carbohydrates are chemically broken down into single sugar units they are then
transported into the inside of intestinal cells.
 When people do not have enough of the enzyme lactase, lactose is not sufficiently broken
down resulting in a condition called lactose intolerance. The undigested lactose moves to
the large intestine where bacteria are able to digest it. The bacterial digestion of lactose
produces gases leading to symptoms of diarrhea, bloating, and abdominal cramps.
Lactose intolerance usually occurs in adults and is associated with race. Some people
with lactose intolerance can tolerate a small amount of dairy products in their diet. The
severity of the symptoms depends on how much lactose is consumed and the degree of
lactase deficiency.

Absorption: Going to the Blood Stream

 The cells in the small intestine have membranes that contain many transport proteins in
order to get the monosaccharides and other nutrients into the blood where they can be
distributed to the rest of the body
 . The first organ to receive glucose, fructose, and galactose is the liver. The liver takes
them up and converts galactose to glucose, breaks fructose into even smaller carbon-
 containing units, and either stores glucose as glycogen or exports it back to the blood.
How much glucose the liver exports to the blood is under hormonal control and you will
soon discover that even the glucose itself regulates its concentrations in the blood.

If needed for energy, glucose is released from the liver to the bloodstream, and on to cells that
need it. Excess glucose is converted to glycogen in the liver and muscles and stored in those
organs. The glycogen stored in the liver maintains blood glucose between meals; muscle
glycogen provides immediate energy to the muscle during exercise. Enzymes in the liver and
muscles combine glucose molecules to form glycogen through a process known as glycogenesis.
Stored glycogen can be broken down into glucose when needed through glycogenolysis ("-lysis"
= break down). Once the storage capacity of the liver and muscles is reached, excess glucose is
stored as fat.

.Properties of Carbohydrates

Physical Properties of Carbohydrates

 Stereoisomerism – Compound having the same structural formula but they differ in
spatial configuration. Example: Glucose has two isomers with respect to the penultimate
carbon atom. They are D-glucose and L-glucose.
 Optical Activity – It is the rotation of plane-polarized light forming (+) glucose and (-)
glucose.
 Diastereo isomers – It the configurational changes with regard to C2, C3, or C4 in
glucose. Example: Mannose, galactose.
 Annomerism – It is the spatial configuration with respect to the first carbon atom in

aldoses and second carbon atom in ketoses.

Chemical Properties of Carbohydrates

 Osazone formation: Osazone are carbohydrate derivatives when sugars are reacted with
an excess of phenylhydrazine. eg. Glucosazone
  Benedict’s test: Reducing sugars when heated in the presence of an alkali gets converted
to powerful reducing species known as enediols. When Benedict’s reagent solution and
reducing sugars are heated together, the solution changes its color to orange-red/ brick
red.
 Oxidation: Monosaccharides are reducing sugars if their carbonyl groups oxidize to give
carboxylic acids. In Benedict’s test, D-glucose is oxidized to D-gluconic acid thus,
glucose is considered a reducing sugar.
 Reduction to alcohols: The C=O groups in open-chain forms of carbohydrates can be
reduced to alcohols by sodium borohydride, NaBH4, or catalytic hydrogenation (H2, Ni,
EtOH/H2O). The products are known as “alditols”.

Isomers in carbohydrate.

Types of Isomers:

 Constitutional Isomers,
 Stereoisomers
 Enantiomers
 Diastereomers

PRACTICAL PART

Tests for Carbohydrates

1. Molisch’s Test

It is a screening test for confirming the presence or absence of carbohydrates in a given solution.
It is a highly sensitive test for carbohydrates. Monosaccharides, oligosaccharides, and
polysaccharides all give positive Molisch’s test.

Principle

This test is based on the reaction of the alpha-naphthol with carbohydrate in the presence of
sulfuric acid. The sugars react with alpha-naphthol in an acidic environment to form purple-
colored furfural or hydroxymethylfurfural derivatives. The intensity of the color is directly
proportional to the amount of carbohydrate present in the solution.

Apparatus
  Test tube
 Dropper or pipette
 Solution to be tested
Reagent

 Molisch’s reagent: 5% alpha-naphthol solution in ethyl alcohol

Procedure

1. Take 2 ml of the given solution in a test tube

2. Add 1-2 drops of the Molisch’s reagent in the above solution
3. Mix the solutions
4. Incline the test tube
5. Add 2 ml sulfuric acid along the side of the test tube
Observations

A violet-colored ring is formed at the junction of the two liquids i.e. solution with Molisch’s
reagent and the sulfuric acid.

Result

The violet ring indicates the presence of carbohydrates in the solution.

Points to Remember

 To give a positive test, the carbohydrate must have at least five carbons. (It is so because
it involves the formation of furfural derivatives that contain five carbon atoms.)
 Impurities in the reagent give green color indicating a false-negative test.
 Oligosaccharides and polysaccharides are first broken down to monosaccharides by acid
which then give the Molisch’s test positive.
 Proteins and lipids having an attached carbohydrate can also give this test positive.
Precautions

 Don’t add too much Molisch’s reagent.

 Don’t pour sulfuric acid directly into the solution. Otherwise, charring of carbohydrates
will occur and a black ring will be formed, giving a false negative test.
2. Iodine Test

This test is specific for polysaccharides. This test is used to differentiate polysaccharides from
the rest of carbohydrates. It is given positive by starch and glycogen. It can also be used to
differentiate between glycogen, starch, and cellulose.

Principle

The iodine test is based on the absorptive properties possessed by large polysaccharide
molecules. The glucose chains in most of polysaccharides are organized to form helices. The
space between the turns of the helix can hold small iodine molecules. This is seen with amylase
chains found in starch. Glycogen and amylopectin can also absorb these iodine molecules on
their surface. The absorptive property of polysaccharides decreases upon heating.

Blue or red-colored iodine complexes are formed in this test having ill-defined chemical nature.

Apparatus

 Test tube
 Dropper or pipette
 Solution to be tested
Reagent

 Iodine Reagent: 0.5 ml iodine diluted in 5ml distilled water

Procedure

1. Take 2 ml of the given solution in a test tube

2. Add 2-3 drops of iodine reagent in the above test tube
3. Wait for some time
Observations

When the iodine is added to the solution, the color of the solution changes. It may give the
following colors;

 Blue
 Reddish-purple
 Reddish-brown
Result

If the color of the solution changes on adding iodine, it represents that polysaccharide is present
in the solution. The nature of polysaccharide is detected based on the color formed.

 If blue color appears, amylase or starch is present in the solution

 If reddish-purple color appears, dextrin is present
 If reddish-brown color appears, glycogen is present
Points to Remember

 The test is highly specific for polysaccharides in solution

 Cellulose does not give this test, but you will not found cellulose in solution
 The color disappears on heating and reappears when the solution is cooled
 If the color does not appear upon cooling, it indicates that iodine has vaporized during
heating
Precautions
  Don’t add too much iodine to prevent false results
3. Benedict’s test

It is a test for reducing sugars. Carbohydrates having free functional group that is not involved in
a glyosidic bond give this test positive. All monosaccharides and disaccharides except sucrose
give positive Benedict’s test. This test is negative for polysaccharides.

Principle

This test is based on the ability of reducing sugars to undergo oxidation in alkaline solutions. In
the presence of an alkali, reducing sugars undergo tautomerization to form enediols. These
enediols reduce the cupric ions (Cu+2) to form cuprous ions (Cu+). The cuprous ions from
cuprous hydroxide. Upon heating, it is converted to cuprous oxide that forms precipitates.
The citrate ions present in the reagent release the cuprous ions slowly for reduction and prevent
the formation of Cu(OH)2 until the oxidation-reduction process is completed.
Apparatus

 Test tube
 Test tube holder
 Dropper
 Pipette
 Stand
 Spirit or gas lamp
 Solution to be tested
Reagents

Benedict’s Qualitative Reagent is used that contains;

 Copper Sulfate (to provide cupric ions)

 Sodium Carbonate (to make solution alkaline)
 Sodium Citrate (to provide citrate ions)
Procedure

1. Take 5 ml of Benedict’s qualitative reagent in a test tube

2. Add 8 drops of given solution in the above test tube
3. Mix the solutions
4. Hold the test tube on flame and boil for 2 minutes
5. Allow the solution to cool
6. Look for the precipitates
Observations

Brick-red precipitate is formed at the bottom of the test tube.

Results
The precipitates of cuprous oxide indicate the presence of a reducing sugar in the test tube.

Points to Remember

 It is also a semi-quantitative test as the color of the precipitate is proportional to the

concentration of reducing sugar in the test tube. Maximum concentration that can be
tested in 2% at which brick-red precipitates are formed.
 This test is frequently used as a screening test for diabetes mellitus.
 The test is false positive for ascorbic acid, glutathione, uric acid, etc.
Precautions

 Adding too much Benedict’s reagent or test solution may give false results.
4. Bial’s Test

It is a general test for carbohydrates and is sensitive only for pentoses. Any compound that
contains a pentose sugar will give a positive Bial’s test.

Principle

Pentoses form furfural compounds in the presence of concentrated acid. The furfural compounds
formed by pentoses condense with orcinol to form blue colored compounds.

Apparatus

 Test tube
 Test tube holder
 Dropper
 Pipette
 Stand
 Spirit or gas lamp
 Water bath
 Solution to be tested
Reagents

Bial’s reagent is used that is made by dissolving 300 mg of orcinol in 100 ml of concentrated
HCL and 0.25 mL ferric chloride solution.

Procedure

1. Add 3 ml of Bial’s reagent in an empty test tube

2. Add 3 ml of test solution to the above test tube
3. Heat the test tube in boiling water bath
4. Allow the solution to cool at room temperature
Observations

A bluish color appears in the test tube upon heating.

Results

The blue color indicates the presence of pentose sugar in the test solution.

Points to Remember

 The test is positive for all the compounds that contain pentose sugar like DNA, RNA,
etc.
 Hexoses form green color with Bial’s reagent.
Precautions

 Don’t allow rapid cooling otherwise the results may be affected.

5. Barfoed’s Test

It is a differentiating test to distinguish between monosaccharides and disaccharides. Barfoed’s

test is also based on the reducing ability of sugar. However, sucrose also gives this test positive
as it undergoes hydrolysis in the presence of an acid. Monosaccharides give early positive test
while the disaccharides give late positive.

Principle

Reducing sugar undergo tautomerization in mildly acidic medium to form enediols. These
enediols reduce cupric ions to cuprous ions that form cuprous hydroxide. This cuprous hydroxide
us converted to cuprous oxide on heating and precipitates are formed.

Its principle is similar to Benedict’s test except the acidic environment. Monosaccharides being
strong reducing agents give this test much early.

Apparatus

 Test tube
 Test tube holder
 Dropper
 Pipette
 Stand
 Spirit or gas lamp
 Solution to be tested
Reagents

Barfoed’s reagent is used that contains:

  Copper Acetate in Glacial Acetic Acid
Procedure.

1. Take 2 ml of Barfoed’s reagent in a test tube

2. Add 2 ml of the test solution to the above test tube
3. Mix the solutions
4. Hold the test tube on flame and boil for minutes
5. Allow to cool at room temperature
6. Look for the precipitates
7. If no precipitates are formed, boil for an additional 10 minutes
8. Allow to cool and look for the precipitates
Observations

1. Red precipitates are formed after the first 5 minutes

2. Red precipitates are formed after additional boiling
Results

1. Formation of red precipitates after the initial first 5 minutes indicates the presence of a
monosaccharide
2. If precipitates are formed after 15 minutes, a disaccharide is present in the test solution
Points to Remember

 This test helps to differentiate among monosaccharides and disaccharides

 When the heating period is increased, the disaccharides are hydrolyzed to
monosaccharides that give the positive test
Precautions

 Keep proper track of the boiling time

 Allow gradual cooling at room temperature
 If reheating is necessary, do it after the solution has become cold
6. Seliwanoff’s Test

This test is used to detect monosaccharides with a ketonic functional group. It is widely used to
differentiate fructose, a keto sugar, from glucose and galactose

Principle

This test involves the formation of furfural derivatives by monosaccharides with hydrochloric
acid. The furfural derivatives formed by a sugar with ketonic functional group condense with
resorcinol to form a chromogen having cherry-red color.

Apparatus

 Test tube
 Test tube holder
  Dropper
 Pipette
 Stand
 Spirit or gas lamp
 Solution to be tested
Reagents

Seliwanoff’s reagent is used that contains in 100 ml of water;

 50 mg resorcinol
 33 ml of concentrated HCL
Procedure

1. Take 3 ml of Seliwanoff’s reagent in a test tube

2. Add 1 ml of test solution in the above test tube
3. Hold the test tube on flame and allow to boil for 30 seconds
4. Allow to cool at room temperature
Observations

A cherry red color forms in the test tube upon cooling.

Results

The given solution contains a keto-sugar.

Points to Remember

 Seliwanoff’s test is specific only for hexoses having a ketonic functional group
 Sucrose also gives a positive test because it is hydrolyzed to glucose and fructose
 It is highly sensitive for sucrose even at .1% concentration
Precautions

 Prolonged boiling will lead to the conversion of glucose to fructose resulting in a false
positive test
 Cooling must be gradual at room temperature
7. Phloroglucinol test

This test is specifically to detect galactose and lactose in a solution.

Principle

This test also involves the formation of furfural derivates in the presence of concentrated HCL.
The furfural derivatives formed by galactose then condense with the phloroglucinol to form a
red-colored compound.
Apparatus

 Test tube
 Test tube holder
 Dropper
 Pipette
 Stand
 Spirit or gas lamp
 Solution to be tested
Reagents

Tollen’s reagent is used that contains;

 10 ml concentrated HCl mixed with 8 ml of 0.5% phloroglucinol

Procedure

1. Take 2 ml of test solution in a test tube

2. Add 2 ml of Tollen’s reagent to the above test tube
3. Mix the two solutions thoroughly
4. Hold the test tube on flame and boil for some time
5. Allow to cool at room temperature
Observations

When the solution is boiled and allowed to cool, it turns yellow to red.

Results

The change of color to red indicates the presence of galactose in the solution.

Points to Remember

 Lactose also gives this test positive as it is hydrolyzed by acid to yield glucose and
galactose. To differentiate between the two, perform Barfoed’s test.
 The furfural compounds formed by pentoses having a keto group also form a similar red-
colored compound with phloroglucinol.
Precautions

 Boiling for a prolonged time will give a false-positive test due to the conversion of
glucose to galactose
8. Osazone Test

It is a confirmatory test for carbohydrates. It gives you the final inference about the type of
carbohydrate present in the solution. Osazone derivative of a carbohydrate form specific crystals
that are characteristic to it. The shape of the crystal tells us about the nature of carbohydrate
present.
Principle

Phenyl hydrazine reacts with reducing sugar in an acidic environment at high temperature to
form phenylhydrazone. Phenyl hydrazine further reacts with the sugar on heating and forms
crystals of osazone specific to that carbohydrate.

Apparatus

 Test tube
 Test tube holder
 Dropper
 Pipette
 Stand
 Spirit or gas lamp
 Light Microscope
 Slides
 Coverslip
 Solution to be tested
Reagents

Osazone mixture is used in this process. It is made by mixing;

 1 part Phenyl Hydrazine

 2 parts Sodium acetate
 Few drops of glacial Acetic acid
Procedure

1. Take 5 ml of the given solution in a test tube

2. Add 3 pinches of osazone mixture to the above test tube
3. Mix thoroughly
4. Hold the test on flame and boil for 5 minutes
5. Check for yellow crystals. If not formed boil further
6. Keep checking after every 5 minutes for yellow crystals
7. Once the crystals are formed, allow the solution to cool at room temperature
8. Take the crystals out and prepare slide
9. Observe the slide under the microscope
Observations

Osazone crystals formed when viewed under the microscope have different shapes depending on
the type of carbohydrate present.

Results

The following inferences are drawn from the shapes of osazone crystals.
  Needle shaped/ broom-stick crystals are formed in 5 minutes indicate glucose
 Needle shaped/ broom-stick crystals are formed in 2 minutes indicate fructose
 Cotton ball-shaped crystals are formed by lactose in 30 minutes
 Sunflower shaped crystals are formed by maltose in 30 to 40 minutes
Points to Remember

 Osazones are the crystalline substances that have a specific structure when viewed under
the microscope.
 They are formed by all reducing sugars.
 Non-reducing sugars do not form osazone crystals unless they are hydrolyzed.
 Boiling the nonreducing sugars for a longer time releases individual monosaccharides
that give the test positive

CHAPTER FOUR

PROTEINS.

 Proteins are complex, organic compounds composed of many amino acids linked
together through peptide bonds and cross-linked between chains by sulfhydryl bonds,
hydrogen bonds and van der Waals forces.
 There is a greater diversity of chemical composition in proteins than in any other group
of biologically active compounds.
 The proteins in the various animal and plant cells confer on these tissues their biological
specificity.

Sources of proteins:
- Animal proteins: milk proteins,
egg(white) proteins, animal by-
products(blood/gelatin)
- Plant proteins(legumins): soy
proteins, lupine proteins,
sunflower proteins
- Novel(plan) proteins: potato
proteins, algae proteins, ‘leafy’
proteins
 Function of proteins in nature:
- Nutrition(e.g. milk, soybeans,
amino acids)
 Source of nitrogen(N) and
essential amino acids
- Structure(e.g. collagengelatine)
- Metabolism(e.g. homeostasis,
enzymes, antibodies)
 Function of proteins in food:
- Nutrition(meat, cheese, nuts)
- Texture(gluten in bread)
- Taste(Maillard reaction)
 Proteins, peptides and amino
acids:
- All proteins are buildup of amino
acids
- React: l
Sources of proteins:

 Animal proteins: milk proteins, egg(white) proteins, animal by-products(blood/gelatin)

 Plant proteins(legumes): soy proteins, lupine proteins, sunflower proteins
 Novel(plan) proteins: potato proteins, algae proteins, ‘leafy’ proteins

Function of proteins in nature:

 Nutrition(e.g. milk, soybeans, amino acids) Source of nitrogen(N) and essential amino
acids
 Structure(e.g. collagengelatine)
 Metabolism(e.g. homeostasis, enzymes, antibodies)

Function of proteins in food:

 Nutrition(meat, cheese, nuts)

 Texture(gluten in bread)
 Taste(Maillard reaction)
Proteins can be classified as:

(a) Simple proteins. On hydrolysis they yield only the amino acids and occasional small
carbohydrate compounds. Examples are: albumins, globulins, glutelins, albuminoids, histones
and protamines.

(b) Conjugated proteins. These are simple proteins combined with some non-protein material in
the body. Examples are: nucleoproteins, glycoproteins, phosphoproteins, haemoglobins and
lecithoproteins.

(c) Derived proteins. These are proteins derived from simple or conjugated proteins by physical
or chemical means. Examples are: denatured proteins and peptides.