MOLISCH TEST
for?
Molisch’s test is a general test for all carbohydrates.
In this test, carbohydrates when reacted with conc.
H2SO4 (Sulfuric acid) get dehydrated to form
furfural and its derivatives.
When monosaccharide are treated with conc H2SO4
or conc HCl, -OH group of sugar are removed in the
form of water and furfural is formed from pentose
sugar and hydroxymethyl furfural is formed from
hexose sugar. These products reacts with
sulphonated α- naphthol to give a purple (violet red)
colored complex.
PROCEDURE OF MOLISCH’S TEST:
1. Take 2ml of sample in dry test tube.
2. Take 2ml of distilled water in another tube
as control.
3. Add 2-3 drops of Molisch’s reagent to the
solution.
4. Gently pipette 1ml conc. H2SO4 along the
side of the tube so that two distinct layers
are formed.
5. Observe color change at the junction of two
layers.
6. Appearance of purple color indicates the
presence of carbohydrates.
RESULT INTERPRETATION OF MOLISCH’S
TEST:
 Positive Molisch’s test: purple color
complex
 Negative Molisch’s test: no purple color
MOLISCH TEST RESULT
Water –
Glucose –
Sucrose –
Starch –
What does Molisch’s test is used
Molisch’s test is a chemical test that detects the presence of
carbohydrates in an analyte. This test is named after Czech-
Austrian botanist Hans Molisch, who discovered it.
What is the principle of Molisch’s
Test?
Molisch’s test is based on the dehydration of sulphuric acid
into furfural. One hydroxyl group is removed from a sugar
molecule when a sample containing carbohydrate
molecules is treated with sulphuric acid and concentrated
hydrochloric acid. Water is used to eliminate the hydroxyl
group. After the hydroxyl group is removed from the sugar
molecule, furfural is formed. The resulting furfural reacts
with Molisch’s reagent (sulphonated – naphthol) to produce
a purplish red product.
Which carbohydrate gave a positive
result with Molisch’s test?
All carbohydrates (monosaccharides, disaccharides, and
polysaccharides) pass the Molisch test. It is based on
Sulphuric acid dehydrating the carbohydrate to produce an
aldehyde, which condenses with two molecules of α-
naphthol, resulting in the appearance of a purple ring at the
interface.
What is the negative result of
Molisch test?
The purple-coloured ring forms at the interface between the
sulphuric acid and the test solution. Because the sulphuric
acid is denser than the test solution, it remains above it. The
lack of colour indicates a negative outcome.
What is the composition of Molisch’s
reagent?
3.75 gm of α-naphthol in 25 ml of Ethanol 99% is the
composition of Molisch’s reagent.
To learn more about Molisch’s test and other important
chemical tests, such as the biuret test, register with BYJU’S
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SELIWANOFFS TEST
Seliwanoff’s test is used to differentiate between TOLLEN’S PHLOROGLUCIN
sugars that have a ketone group (ketose) and sugars REACTION
that have an aldehyde group (aldoses). This test is a
timed color reaction specific to ketohexoses.
 To distinguish reducing sugars from non-
Objectives of Seliwanoff’s test reducing sugars.
• To detect the presence of ketohexoses in a given  To detect the presence of aldehyde containing
sample. carbohydrates and differentiate them from
ketone containing carbohydrates.
• To distinguish ketoses from aldoses.
Water –
Shows positive test for:
Glucose – POSITIVE
Ketoses
Arabinose –
Reactions:
The test reagent dehydrates
ketohexoses to form 5-
hydroxymethylfurfural. 5-
hydroxymethylfurfural further reacts
with resorcinol present in the test
reagent to produce a red product within
two minutes (reaction not shown).
Aldohexoses react to form the same
product, but do so more slowly.

Seliwanoff’s test with aldose and ketose

 Ketose sugars react with the Seliwanoff's
reagent to give immediately a deep cherry red
color.
 Aldose sugars react with the Seliwanoff's
reagent to give faint pink color which intensifies
with time.

Reactions with;
Water – Urine Color
Glucose – Pinkish - aldose
Fructose – Red - ketoses
Arabinose – Urine color
  The intensity of the color decreases with the
increase in temperature and the presence
of water-miscible organic compounds like
ethanol.
REACTION WITH IODINE  On heating, the blue color amylase-iodine
complex dissociates but is formed again
Iodine Test Definition on cooling because the helical structure is
The iodine test is a chemical test used to distinguish disrupted; thereby amylose loses its iodine
mono- or disaccharides from certain polysaccharides like binding capacity and the blue color.
amylase, dextrin, and glycogen. This test has a variation  The blue color reappears on cooling due to
termed starch-iodine test that is performed to indicate the
the recovery of iodine binding capacity
presence of glucose made by plants in the leaves.
due to regaining of the helical structure.
Objectives of Iodine Test
 To detect the presence of polysaccharides, RESULT AND INTERPRETATION
primarily starch.
Principle of Iodine Test The appearance of blue-black or purple color
 The iodine test is based on the fact that represents a positive test, indicating the presence of
polyiodide ions form colored adsorption starch.
complex with helical chains of glucose
residue of amylase (blue-black), dextrin
(black), or glycogen (reddish-brown).
 Monosaccharides, disaccharides,
branched polysaccharides like cellulose
and FEIHLING’S TEST
remain colorless. Amylopectin produces Fehling’s solution is prepared by mixing two
an orange-yellow hue. solutions Fehling’s A and Fehling’s B. Fehling’s A
 The reagent used in the iodine test is Lugol’s contains copper sulfate solution whereas Fehling’s
iodine, which is an aqueous solution of B contains potassium hydroxide and potassium
elemental iodine and potassium iodide. sodium tartrate.
 Iodine on its own is insoluble in water.
Addition of potassium iodine results in a 1mL starch solution + 1mL Fehling’s A + 1mL
reversible reaction of the iodine ion with Fehling’s B = Blue Color
iodine to form a triiodide ion, which
further reacts with an iodine molecule to With starch solution, a blue color was seen
form a pentaiodide ion. indicating that it is a non-reducing sugar.
 Bench iodine solution appears brown, While in the case of starch, it does not possess any free
whereas, the iodide, triiodide, and aldehyde group or ketone group which can open up the
pentaiodide ion are colorless. starch structure. As it lacks a free ketone or aldehyde
 It is observed that the helix (coil or spring) group, it cannot give out a free electron and thus it
structure of the glucose chain is the key to cannot work as a reducing agent.
this test.
 Further, the resulting color depends on the
length of the glucose chains.
 The triiodide and pentaiodide ions formed
are linear and slip inside the helix
structure.
 It is believed that the transfer of charge
between the helix and the polyiodide ions
results in changes in the spacing of the
energy levels, which can absorb visible
light, giving the complex its color.
 a. it does not possess any free aldehyde
group or ketone group which can open
up the starch structure. As it lacks a free
ketone or aldehyde group, it cannot give
out a free electron and thus it cannot
HYDROLYSIS OF work as a reducing agent.

STARCH
FEHLING’S B – Benedict’s Reaction
Starch is a condensation polymer made from
glucose monomer units. When these large starch NOTE THAT:
molecules react with water they break down into
The primary application of Benedict's test is to
smaller sugar molecules. The starch is said to be
detect the presence of simple carbohydrates in an
hydrolysed.
unidentified analyte. This test can be used to check
1. 1 mL starch solution + 5 mL for reducing sugars that hold free aldehyde or
concentrated hydrochloric acid + ketone functional groups. The reducing sugar can be
800C temperature + a drop of Iodine either a monosaccharide or a disaccharide.
= Brown Color
1. Glucose – Positive
2. 1 mL starch solution + 5 mL
2. Galactose – Positive
concentrated hydrochloric acid + 5
3. Fructose – Positive
drops of Iodine + 3 mL Fehling’s
4. Lactose – Positive
solution + boil + 10% Sodium
5. Maltose – Positive
hydroxide = Non-reducing sugar
6. Sucrose – Negative
7. Starch - Negativ

FEHLING’S TEST
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
What is Fehling's test? One of the most popular solution. Negative Benedict's Test: No color change
tests used for the estimation or detection of reducing (Remains Blue). Reducing sugars absent.
sugars and non-reducing sugars is the Fehling's test.
FEHLING’S A TROMMERS TEST
1. Glucose – Reducing Sugar
Trommer's test involves treating a sample with a
2. Galactose – Reducing Sugar
strong acid (sulfuric acid) which results in the acid
3. Fructose – Reducing Sugar
hydrolysis of disaccharides into mono-saccharides.
4. Lactose – Reducing Sugar
The solution is then neturalized (usally with potash
5. Maltose – Reducing Sugar
or soda, but hydroxide could be used, as in the
6. Sucrose – Non - reducing Sugar
benedict's test) and a solution of copper sulfate is
a. Sucrose is a non-reducing sugar because
added.
the two monosaccharide units are held
together by a glycosidic linkage between 1. Glucose – Positive
C1 of α-glucose and C2 of β-fructose. 2. Galactose – Positive
Since the reducing groups of glucose 3. Fructose – Positive
and fructose are involved in glycosidic
4. Lactose – Positive
bond formation, sucrose is a non-
reducing sugar.
5. Maltose – Positive
7. Starch – Non - reducing Sugar 6. Sucrose – Negative
7. Starch - Negative
Trommer's Test for reducing sugars in 1. Glucose – Positive
urine, blue indicates no sugars present. While, blue 2. Galactose – Positive
turning to red/orange indicates high amount of 3. Fructose – Positive
sugars present. 4. Lactose – Positive
5. Maltose – Positive
6. Sucrose – Negative
7. Starch – Negative

BARFOED’S TEST NYLANDER’S TEST

Shows positive test for: The black precipitates form within a short time (bismuth
Reducing monosaccharides subnitrate is reduced to bismuth black), indicates the
positive test.
Reactions: Nylander’s Test is a type of chemical test used to
Reducing monosaccharides are oxidized by determine the presence of reducing sugars.
the copper ion in solution to form a
carboxylic acid and a reddish precipitate of
copper (I) oxide within three minutes. Principle of Nylander’s Test 
Reducing disaccharides undergo the same
reaction, but do so at a slower rate. Aldehyde group or free ketone of carbohydrate
reduces and forms black precipitates bismuth
1. Glucose – Positive subnitrate reduced to black bismuth. A very tiny
2. Galactose – Positive amounts of glucose (0.08 percent) can be identified
3. Fructose – Positive through this test.
4. Lactose – Negative
5. Maltose – Negative 1. Glucose – Positive
6. Sucrose – Negative 2. Galactose – Positive
7. Starch - Negative 3. Fructose – Positive
4. Lactose – Positive
5. Maltose – Positive
 Positive Barfoed’s test: development of brick
red color ppt within 3-5 minutes 6. Sucrose – Negative
 Negative Barfoed’s test: absence of red color 7. Starch – Negative
This gives us the idea that Sucrose an starch are
non-reducing sugar.
PICRIC ACID TEST
Positive result in the picric acid reaction is the
appearance of a mahogany red colour in the solution.
This is a sign of the presence of reducing sugar within
the solution.
Procedure:
 Add 1mL of the above reagent to 1mL of
the test solution followed by 0.5 mL of 10%
Sod. Carbonate solution.
 Heat the test tube in a boiling water bath.
 Observe the color of the solution.
SELIWANOFF’S TEST ANTHRONE’S TEST
1. Galactose – Positive Objectives of Anthrone Test
2. Glucose – Positive
 To detect the presence of carbohydrates in a
3. Maltose – Positive
given solution.
4. Lactose – Positive  To quantify the concentration of free and bound
5. Sucrose – Positive carbohydrates in a solution.
6. Fructose – Positive
Principle of Anthrone Test
The red precipitate seen in each solution indicates
that they have a positive reaction and are If carbohydrate is present in the form of free
carbohydrate as poly- or monosaccharide or bound as in
saccharides or a sugar solution.
a glycoprotein or a glycolipid, the concentrated acid in
the Anthrone reagent first hydrolyses it into component
monosaccharide. Similarly, the concentrated acid then
PHOROGLUCINOL TEST catalyzes the dehydration of the monosaccharides to
form furfural (from pentoses) or hydroxyl furfural (from
This test is specifically to detect galactose and lactose in hexoses).
a solution.
1. Galactose – Positive
Principle 2. Glucose – Positive
3. Maltose – Positive
This test also involves the formation of furfural derivates
in the presence of concentrated HCL. The furfural 4. Lactose – Positive
derivatives formed by galactose then condense with the 5. Sucrose – Positive
phloroglucinol to form a red-colored compound.  6. Fructose – Positive

1. Galactose – Negative Since all of the solution gives a color change signifying
2. Glucose – Negative the presence of carbohydrates and the concentration of
free and bound carbohydrates in the solution.
3. Maltose – Negative
4. Lactose – Positive
5. Sucrose – Negative
6. Fructose – Negative
Lactose is basically a reducing sugar, it is known as milk
sugar, because it is found to occur in the milk of cows
and also in other mammals. Lactose is found to be
lowest in sweetness among all others found in nature.
When lactose undergoes acid hydrolysis then two
monosaccharides are formed, one molecule of D-glucose
and one molecule of D-galactose.
 The bond from the anomeric carbon of the first
monosaccharide unit is in upward direction.
 This process also involves the catalytic action of
enzyme lactase.
 Hence, we can conclude that the option (a)is
correct that lactose is made up of galactose and
glucose units.
MUCIC TEST
Proteins bind copper at higher pH, generating the biuret
complex in particular. Proteins mobilise copper ions in a
pH-dependent manner.
Why is biuret purple in colour?
Mucic acid test definition
Mucic acid test is a test that is highly specific and is used Biuret reagent is normally blue in colour. When peptide
for the detection of the presence of galactose and lactose. bonds — the chemical bonds that hold amino acids
It is also termed galactaric acid that is named after the together — are present, the reagent turns violet.
product of the reaction.
What is the role of albumin in the Biuret test?
Objectives of Mucic acid test
 To detect the presence of galactose and lactose Positive control for the protein test is egg albumin +
in a given sample. Biuret’s reagent. It demonstrates that the test result was
 To distinguish between the galactose containing positive (protein is present).
saccharides and other sugars.
Uses What is the substance that reacts with biuret?

The most important use of the mucic acid test is to The biuret method works on the principle that proteins
identify the presence of galactose or its derivatives in the (and, by extension, all molecules containing two or more
food sample and in synthetics manufacture. peptide linkages) react with copper to generate a
colourful complex whose absorption (λmax= 454 nm) is
This test can also be used to detect the presence of proportional to the amount of protein present in the
lactose or agar-agar. presence of excess copper.
1. Glucose – No crystals
2. Galactose – Has crystals Principle of Biuret Test
3. Fructose – No crystals This test’s technique is based on a number of ideas. The
4. Lactose – Has crystals following are some of them:
5. Maltose – No crystals
6. Sucrose – No crystals  When Biuret is treated with dilute copper sulphate
in the presence of alkaline, a purple-coloured
material is generated. The creation of a chelate
 The formation of crystal at the bottom of the complex, or copper coordination complex, is
tube indicates a positive result which means that responsible for its colour.
the sample solution has galactose or its
 Using water’s oxygen and the unshared electron
derivatives.
pairs of peptide nitrogen, Cu(II) or cupric ions
 The absence of such crystals indicates a negative form a violet-colored chelate complex.
result and represents that the sample doesn’t
have galactose or its derivative. The solution  This compound appears violet since it absorbs light
might still have other carbohydrates. at 540 nm. Its colour shifts from blue to violet
when protein is present.

BIURET TEST 1. Albumin – Pink Lavender

2. Peptone – Light Purple

Why do we need the Biuret test? XANTHOPROTEIX REACTION

The biuret test is a chemical procedure for determining
Objective:
whether or not a substance has peptide bonds. As a
result, the biuret test can be used to determine the  this test is used to differentiate aromatic
amount of protein in analyte. amino acids which give positive result
What is the purpose of the biuret reagent’s high pH? from other amino acids

Principle:
  Xanthoproteic test is used to detect amino
acids containing an  aromatic nucleus
(tyrosine, tryptophan and
phenylalanine) in a protein solution
which gives yellow color nitro derivatives
on heating with conc. HNO3. The
aromatic benzene ring undergoes nitration
to give yellow colored product.
Phenylalanine gives negative or weakly
positive reaction though this amino acid
contains aromatic nucleus because it is
difficult to nitrate under normal
condition. On adding alkali to these  nitro
derivative salts, the color change fro
yellow to orange.
negative Moranella morganii and Providencia
rettgeri.
 The production of hydrogen sulphide is a useful
diagnostic test in the identification of enteric
bacteria.
ADAMKLEWKZ REACTION
The Adamkiewicz reaction is part of a biochemical test
used to detect the presence of the amino acid tryptophan
in proteins. When concentrated sulfuric acid is combined
with a solution of protein and glyoxylic acid, a
red/purple colour is produced.
Principle:

1. 1 ml concentrated casein + 1 mL
sulfuric acid + boil for 5-10 mins = Red
wine
2. 1 ml concentrated casein + 1 mL
sulfuric acid + boil for 5-10 mins + 4
mL sodium = Murky white at top, with
vinegar-like color at the bottom
INFERENCE:
The compounds that have indole ring can condense with
aldehydes (more readily with formic aldehyde) to form
colorful condensation products. Among protein amino
acids, only tryptophan undergoes this reaction. The
indole group of tryptophan reacts with glyoxylic acid in
the presence of conc.  H2SO4 to give a purple colored
complex.
Interpretation:

1. Vinegar-like color indicates the presence of  Purple color at the interface in Positive Hopkin’s
aromatic amino acid (Tyrosine and tryptophan) cole test.
2. The heating of it resulted to murky-white ring
formation of yellow derivative.

REDUCED-SULFUR TEST
Objective
 To test for the ability of an organism to reduce
sulphur
 To differentiate gram-negative enteric bacilli on
the basis of sulfide production
Expected Results
 Positive: Darkening of the medium (a black
precipitate) or blackening of the line of
inoculation indicates the presence of bacteria
producing hydrogen sulfide.
 Negative: A negative H2S test is denoted by the
absence of blackening.
Uses
 It is used to differentiate sulfur reducing
members of the genera Salmonella,
Shigella and Proteus from the