CARBOHYDRATES

• Carbohydrates are the main energy source for the human body. Chemically, carbohydrates are
organic molecules in which carbon, hydrogen, and oxygen bond together in the ratio: Cx(H2O)y,
where x and y are whole numbers that differ depending on the specific carbohydrate to which
we are referring.
• Animals (including humans) break down carbohydrates during the process of metabolism to
release energy. For example, the chemical metabolism of the sugar glucose is shown below:

• Animals obtain carbohydrates by eating foods that contain them, for example potatoes, rice,
breads, and so on. These carbohydrates are manufactured by plants during the process of
photosynthesis. Plants harvest energy from sunlight to run the reaction just described in
reverse:

CHEMICAL PROPERTIES OF CARBOHYDRATES

1. Dehydration
- Carbohydrates when mixed with non-oxidizing acids undergo dehydration reaction.
Seliwanoff’s and Molisch reagents are the two common dehydrating agents in the
laboratory. Seliwanoff’s reagent is employed to differentiate reactions between aldoses
and ketoses.
2. Hydrolysis
- The process by which polysaccharides or disaccharides are broken down into separate
sugars either by inorganic acids or enzymes. Disaccharides which have one glycosidic
bond are fully hydrolysed in an acid solution. On the other hand, polysaccharides
undergo only partial hydrolysis.

COLOR REACTION OF CARBOHYRDATES

Test Description Result
Molisch’s Used to check for the presence of carbohydrates in a given Purple or a purplish-red
Test analyte/sample. ring at the point of contact
(General test)
Involves the addition of Molisch’s reagent (a solution of α-
naphthol in ethanol) and the subsequent addition of a few
drops of concentrated H2SO4 to the mixture
Benedict’s Used to check for the presence of reducing sugars in a Formation of a brick-red
Test given analyte. precipitate
(Qualitative
test) Based on Benedict’s reagent (also known as Benedict’s
solution), which is a complex mixture of sodium citrate,
sodium carbonate, and the pentahydrate of copper(II)
sulfate.
Barfoed’s Used to detect the presence of monosaccharides which Red precipitate
Test detects reducing monosaccharides in the presence of
(Qualitative disaccharides
test)
Barfoed’s reagent is the reagent used in this test. It is
prepared by adding a 0.33 molar solution of neutral cupric
(II) acetate to a 1% acetic acid solution.
Bial’s Test Chemical test to detect the presence of pentoses. Blue-green precipitate or
(Qualitative bluish precipitate
test) The components for this test are orcinol, hydrochloric
acid, and ferric chloride.

This test is based on the principle in which pentoses are

dehydrated into furfural which in turn condense with
orcinol
Seliwanoff’s Used to differentiate between sugars that have a ketone Cherry red-colored
Test group (ketose) and sugars that have an aldehyde group complex
(Qualitative (aldoses)
test)
This test is a timed color reaction specific to ketohexoses

The reagent of this test consists of resorcinol and

concentrated HCl.
Fehling’s Used for the estimation or detection of reducing sugars Reddish-brown precipitate
Test and non-reducing sugars
Fehling’s test consists of Fehling’s A and Fehling’s B.

Fehling’s A is a solution containing copper(II) sulphate,

which is blue.

Fehling’s B is a clear liquid consisting of potassium

sodium tartrate (Rochelle salt) and a strong alkali, usually
sodium hydroxide.

Iodine Test Used to distinguish mono- or disaccharides from certain Blue-black or purple color
polysaccharides like amylase, dextrin, and glycogen.

The reagent used in the iodine test is Lugol’s iodine, which

is an aqueous solution of elemental iodine and potassium
iodide.
Tollen’s Test Used to differentiate reducing sugars from non-reducing Dark grey precipitate or
sugars. silver mirror on the
bottom and sides of the
The Tollen’s reagent is the alkaline solution of silver test
nitrate (AgNO3) mixed with liquid ammonia (NH3), which
results in the formation of a complex
Mucic Acid Highly specific and is used for the detection of the Formation of crystal at the
Test presence of galactose and lactose. bottom of the tube

Monosaccharides upon treating with potent oxidizing

agents like nitric acid yield saccharic acids (dicarboxylic
acids)
Osazone Test Used to detect reducing sugars. Osazone appears as
yellow-colored crystals
This test can distinguish between different types of
reducing sugars by the appearance time of the complex.

The reagent for this test consists of phenylhydrazine in

acetate buffer

PROTEINS
• Proteins are made up of amino acid residues joined by peptide bonds
• Due to their polypeptide structures and different amino acid residues, protein reacts with a
variety of reagents to form colored products
• Proteins are large, complex molecules that play many critical roles in the body. They do most of
the work in cells and are required for the structure, function, and regulation of the body’s tissues
and organs.
• Proteins are made up of hundreds or thousands of smaller units called amino acids, which are
attached to one another in long chains. There are 20 different types of amino acids that can be
combined to make a protein. The sequence of amino acids determines each protein’s unique 3-
dimensional structure and its specific function.
• Milk is a mixture of many types of proteins, most of them present in very small amounts. Milk
proteins are classified into three main groups of proteins on the basis of their widely different
behaviors and forms of existence. They are caseins (80%), whey proteins and minor proteins
• Casein is a heterogeneous mixture of phosphorous containing proteins in milk. Casein is
present in milk as calcium salt and calcium caseinate. It is a mixture of alpha, beta and kappa
 caseins to form a cluster called micelle. These micelles are responsible for the white opaque
appearance of milk.
• Principle: Most proteins show a minimum solubility at their isoelectric pH and this principle is
used to isolate casein by adjusting the pH of the milk to 4.6, its isoelectric point. The main bulk
of the precipitate is the casein. The entrapped residual fat can be removed by repeated washing
with the solvents such as ethanol and ether. Casein is insoluble in these solvents and this
property can be advantageously used to remove the unwanted fat from the preparation.

CHEMICAL PROPERTIES OF PROTEINS

1. Hydrolysis
- This refers to the breaking of the peptide bonds that connect amino acids to compose
protein. Strong acid solution is used to hydrolyze proteins. Inside our body, hydrolysis
takes a faster rate because of the help of hydrolysing enzymes.
2. Denaturation
- Denaturation of proteins involves the disruption and possible destruction of both the
secondary and tertiary structures. Since denaturation reactions are not strong enough to
break the peptide bonds, the primary structure (sequence of amino acids) remains the
same after a denaturation process. Denaturation disrupts the normal alpha-helix and
beta sheets in a protein and uncoils it into a random shape.
- Denaturation occurs because the bonding interactions responsible for the secondary
structure (hydrogen bonds to amides) and tertiary structure are disrupted. In tertiary
structure there are four types of bonding interactions between "side chains" including:
hydrogen bonding, salt bridges, disulfide bonds, and non-polar hydrophobic
interactions which may be disrupted. Therefore, a variety of reagents and conditions
can cause denaturation. The most common observation in the denaturation process is
the precipitation or coagulation of the protein.

COLOR REACTION OF PROTEINS

Test Description Target group/amino acid Result
Ninhydrin Test Ninhydrin is a powerful Alpha amino group A blue-purple
oxidizing agent and causes coloured compound
oxidative decarboxylation of
α-amino acids producing an
aldehyde with one carbon
less than the parent amino
acid

The reduced Ninhydrin

(Hydrin-dantin) then reacts
with ammonia which has
been liberated and one
molecule of ninhydrin
Biuret Reaction When urea is heated it forms Peptide bonds Purple-violet
biuret

If a strongly alkaline solution

of biuret is heated with very
dilute copper sulphate a
colour is obtained.

The colour depends upon the

presence of 2 or more
peptide linkages.
Xanthoproteic The amino acids such as Phenylalanine, Tyrosine, Yellow precipitate
Test phenylalanine, tyrosine and Tryptophan
tryptophan present in the
protein give color when
heated with conc. HNO3.

On addition of alkali, the

precipitate turns orange due
 to nitration of the aromatic
ring.

Collagen and gelatin do not

give a positive reaction
Millon’s Test The phenol group of Phenylalanine & Tyrosine Red-pink colored
phenylalanine and tyrosine mercury phenolate
containing proteins, when
heated with mercuric sulfate
in sulfuric acid and sodium
nitrite (or, mercurous and
mercuric nitrates in nitric
acid) form color
Hopkin’s-Cole Protein containing Tryptophan Purple ring
Test tryptophan gives this test
positive

Gelatin, collagen do not

contain tryptophan and
hence do not give this test
positive.
Sakaguchi’s Test Sakaguchi reagent consists of Arginine Red color
alcoholic α-naphthol and a
drop of sodium hypobromite.
Guanidine group of arginine
Sullivan & Color is produced when Cysteine and cystine Red color
McCarthy’s cysteine containing protein is
Reaction heated with sodium 1, 2
naphthoquinone -4-sulfonate
in the alkaline medium in
presence of Na2S2O4.
Sulfur Test Few amino acids containing Cysteine Brown or black color
sulphur such as methionine, on the lead acetate
cysteine and cysteine can be paper
tested using lead acetate
paper

The reaction between H2S

gas and lead acetate paper
yield to a lead (II) sulphide
Nitroprusside Proteins with free -SH group Cysteine Reddish color
Test of cysteine give color with
sodium nitroprusside in
ammoniacal solution.

Many proteins give this test

positive after heat
coagulation or denaturation
indicating the liberation of
free -SH groups