Qualitative Analysis of Amino

Acids, Proteins, Carbohydrates,

Lipids

Lipids
LIPIDS are biomolecules consist mostly of
nonpolar group
SIMPLE: True fats
EXAMPLE: ESTERS OF FATTY ACID
COMPOUND: contains parts of other
biochemical
compounds in
addition to the glycerol and
fatty acid
components
EXAMPLE: PHOSPHOLIPIDS
MISCELLANEOUS: fat-like substances
EXAMPLE: FAT-SOLUBLE VITAMINS

 Esters of saturated fatty acids are solids FATS

Esters of unsaturated fatty acids are liquids
OILS
RANCIDITY: (UNSATURATED FATTY ACIDS DEVELOP
RANCID ODOR)
HYDROLYSIS OF FATTY ACID BY ACID yield:
Glycerol and Fatty acid
HYDROLYSIS OF FATTY ACID BY ALKALINE yield:
Glycerol and Metallic salts of fatty

Qualitative tests for lipids

Solubility test
Principle: Fats and fatty acids (long chain)
are insoluble in water as they are
hydrophobic in nature. However, they are
soluble in organic solvents.

Observation: (solids) do not dissolve in water,

while (liquid) separates into two phases. All the
test samples dissolve clearly in organic
solvents.

Qualitative tests for

lipids
Acrolein test
Principle: Glycerol or glycerol
containing compounds on heating with
potassium bisulfate undergo dehydration
to yield acrolein or propenal, which
has a characteristic pungent odor.
Observation: Detection of a pungent
smell confirms the presence of glycerol.

Qualitative tests for

lipids
Halogenation Reactions
Reaction with bromine
Principle:
The double bonds present in the
unsaturated fats and fatty acids
undergo halogenation (bromination)
to yield di-halo adducts. The extent of
decolorization is a measure of the
degree of unsaturation.

 Observation: Decolorization of
bromine water is observed for
unsaturated fats or fatty acids, while
no decolorization occurs in saturated
fats and fatty acids

Qualitative tests for

lipids
Hubls (hanus) iodine test
Observation: Decolorization of iodine is
observed, confirming the presence of
unsaturated fats or fatty acids.

However, further addition of the reagent

results in retention of the color, indicating
the completion of iodination reaction.

Qualitative tests for

lipids
Salkowski test : cholesterol
Observation: The acid layer shows a
yellow color with green fluorescence,
while the chloroform layer is cherry
red.

 TEST FOR UNSATURATION FATTY

ACIDS
pp.35-38

AMINO ACIDS
PROTEINS : the building block of proteins
are amino acids
Polymers of amino acids held by
covalent linkages
the peptide bonds
AMINO ACID:
20 common naturally occurring amino
acids
In nature it exist as a L-amino acids

 AMINO ACID:
amphoteric - contain both acidic and
basic groups
zwitterion- neutral molecule with a
positive and a negative electrical charge
(dipolar ion)
Isoelectric point - is the pH at which a
particular molecule or surface carries no
net electrical charge (neutral zwitterion)

Nonpolar side chain

The side chains of
alanine, valine,
leucine, and
isoleucine
tend to cluster
together within
proteins, stabilizing
protein structure by
means of
hydrophobic
interactions

Nonpolar side chain

Methionine,
one of the two sulfur-containing amino acids,
Has a nonpolar thioether group in its side chain.

Proline
has an aliphatic side chain with a distinctive
cyclic structure.
The secondary amino (imino) group of proline is
held in a rigid conformation that reduces the
structural flexibility of polypeptide regions
containing proline.

Aromatic R groups chain

Aromatic R Groups
Phenylalanine,
tyrosine, and
tryptophan, with
their aromatic side
chains,
are relatively nonpolar
(hydrophobic). All can
participate in
hydrophobic
interaction

Polar, Uncharged R Groups

Polar, Uncharged R
Groups
The R groups of
these amino acids
are soluble in water,
or hydrophilic,
because they contain
functional groups
that form hydrogen
bonds with water

Polar, Uncharged R Groups

Cysteine
is readily oxidized to form a covalently
linked dimeric amino acid called cystine,
in which two cysteine molecules are
joined by a disulfide bond.
The disulfide-linked residues are
strongly hydrophobic (nonpolar)

Positive charged R Groups

Positively Charged (Basic) R Groups
The most hydrophilic R groups are those
that are either positively or negatively
charged.
The amino acids in which the R groups
have significant positive charge at pH
7.0 are
lysine
which has a second primary amino
group at the a position on its
aliphatic chain;

Positive charged R Groups

arginine
which has a
positively
charged
guanidinium
group;
histidine
which has an
aromatic
imidazole
group.

Negatively Charged (Acidic) R

Groups
Negatively Charged
(Acidic) R Groups
The two amino acids
having R groups with a
net negative charge
at pH 7.0 are aspartate
and glutamate, each of
which has a second
carboxyl group.

Qualitative Tests for Amino Acid

Ninhydrin test
Principle: Amino acids react with
ninhydrin to yield a purple colored
complex (Ruhemanns purple),
while, imino acids such proline give
yellow color.

 Observation: Appearance of purple to

blue color indicates the presence if amino acids. In case of imino acids, a
yellow color is observed.

Qualitative Tests for Amino Acid

Xanthoproteic test
Principle: The aromatic groups of
either the free amino acids or proteins
Observation: In acid medium the
solution is yellow and upon treatment
with alkali, the color changes to
orange.

Qualitative Tests for Amino Acid

Hopkins-Cole test
Principle: The indole moiety
of tryptophan condenses
with aldehydes in acidic
condition to yield purple or
violet colored compounds.

 Observation: A purple-violet
ring appears at the junction
of the amino acid solution
and the conc. sulfuric acid.
The formation of this colored
ring confirms the presence of
tryptophan.

Qualitative Tests for Amino Acid

Test for SH group
Principle: Sulfur containing amino
acids, such as cysteine and cystine
upon boiling with NaOH yield lead
acetate hydroxide (hot alkali) yield
sodium sulfide, which can be
detected by precipitating it to lead
sulfide, using lead acetate solution

 Observation: The
appearance of a black
precipitate of lead sulfide
confirms the presence of
cysteine or cystine.

Qualitative Tests for Amino Acid

Millons test
Principle: The phenolic group of
tyrosine reacts with mercuric ions in
acidic condition in the presence of
sodium nitrite, yielding a red colour
complex.
Observation: Formation of a red
colour confirms the presence of
tyrosine.

Tests for Proteins

Biuret test
Principle: The peptide bonds of the
protein react with cupric copper,
under alkaline condition to yield a
purple color.
Observation: Formation of a violetpurple colour indicates the presence
of protein.

Tests for Proteins

Heat coagulation test
Principle: Proteins in acidic medium,
upon heating undergo thermal
denaturation, leading to their
aggregation and precipitation.
Observation: The appearance of a
precipitation (coagulum) indicates
presence of protein(s).