UNIT-6

TITRIMETRIC ANALYSIS
(REDOX REACTIONS)

T
HE oxidation and reduction reactions in aqueous solutions involve the
transfer of electrons from one species to another. In the oxidation of a
substance electron(s) is (are) transfered from the species and in reduction,
electron(s) is (are) gained by the species. Oxidation and reduction reactions occur
simultaneously. A reaction, which involves simultaneous oxidation and
reduction, is called a redox reaction. The titrations involving redox reaction are
called redox titrations. You know that in acid-base titrations, indicators which
are sensitive to pH change are employed to note the end point. Similarly, in
redox titrations there is a change in oxidation potential of the system.
The indicators used in redox reactions are sensitive to change in oxidation
potential. The ideal oxidation-reduction indicators have an oxidation potential
intermediate between the values for the solution being titrated and the titrant
and these show sharp readily detectable colour change.

EXPERIMENT 6.1

Aim
To determine the concentration/molarity of KMnO4 solution by titrating it against
a 0.1 M standard solution of oxalic acid.

Theory
In the present experiment, potassium permanganate acts as a powerful
oxidising agent. Although KMnO4 acts as an oxidising agent in alkaline medium
also, for quantitative analysis mostly acidic medium is used. The oxidising
action of KMnO4 in the acidic medium can be represented by the following
equation:
– –
MnO4 + 8H+ +5e → Mn2+ + 4H2O
The acid used in this titration is dilute sulphuric acid. Nitric acid is not used
as it is itself an oxidising agent and hydrochloric acid is usually avoided because
it reacts with KMnO4 according to the equation given below to produce chlorine
and chlorine which is also an oxidising agent in the aqueous solution.
2KMnO4 + 16 HCl → 2KCl + 2 MnCl2 + 5Cl2 + 8 H2O
 TITRIMETRIC ANALYSIS (REDOX REACTIONS)

Since, oxalic acid acts as a reducing agent, it can be titrated

against potassium permanganate in the acidic medium according
to the following equation:

Reactions of oxalic acid

A. Chemical equations

Reduction half reaction : 2KMnO4 + 3H2SO4 → K2SO4 + 2MnSO4 + 3H2O + 5 [O]

60°C
Oxidation half reaction : H2 C2 O4 + [O]  → 2CO2 + H2 O] × 5
________________________________________________________________________________
2KMnO4 + 3H2SO4 + 5H2C2O4 → K 2SO 4 + 2MnSO4 + 8H2O + 10 CO2
________________________________________________________________________________

B. Ionic equation

– – + 2+
Reduction half reaction : MnO4 + 5e + 8H → Mn + 4H2O] × 2

– –
Oxidation half reaction : C2O4 → 2CO2 + 2e ] × 5
_____________________________________________________________________
– 2– + 2+
2 MnO4 + 5C2O4 + 16H → 2Mn + 10CO2 + 8H2O
_____________________________________________________________________

– 2+ 2–
In these equations, MnO4 is reduced to Mn and C2O4 is
2–
oxidised to CO2. The oxidation number of carbon in C2O4 changes
from +3 to +4.
In these titrations, potassium permanganate acts as a self-
indicator. Initially colour of potassium permanganate is discharged
due to its reduction by oxalic acid. After complete consumption of
oxalate ions, the end point is indicated by the appearance of a
light pink colour produced by the addition of a little excess of
unreacted potassium permanganate. Further, during the titration
of oxalic acid against potassium permanganate, warming of oxalic
acid solution (50°–60°C) along with dilute H2SO4 is required.
This is essential because the reaction takes place at higher
temperature. During the titration, first manganous sulphate is
formed which acts as a catalyst for the reduction of KMnO4 by
oxalic acid. Therefore, in the beginning the reaction rate is slow
and as the reaction proceeds, the rate of the reaction increases.

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 LABORATORY MANUAL CHEMISTRY

Material Required

• Measuring flask (250 mL): One

• Burette (50 mL) : One
• Burette stand : One
• Pipette : One • Oxalic acid : As per need
• Conical flask : One • Potassium permanganate
• Funnel : One solution : As per need
• Weighing bottle : One • 1.0 M Sulphuric acid : As per need
• Glazed tile(white) : One
• Burner : One
• Wire gauze : One
• Chemical balance : One

Procedure
A. Preparation of 0.1 M standard solution of oxalic acid
Prepare 0.1M oxalic acid solution as mentioned in experiment
Oxalic acid 2.1(Unit 2, Class XI, Laboratory Manual)
B. Titration of oxalic acid solution against potassium
Potassium
permanganate permanganate solution
(i) Rinse and fill a clean burette with potassium permanganate
Sulphuric acid solution. Remove the air bubble, if any, from the nozzle of the
burette by releasing some solution through it. The burette
used in the permanganate titration must have a glass stop
cock as rubber is attacked by permanganate ions.
(ii) Take 10 mL of 0.1 M oxalic acid solution in a conical flask
and add half of the test tube full ( ≈ 5 mL) of 1.0 M H2SO4
to it to prevent the formation of any precipitate of
manganese dioxide during the course of the titration.
(iii) Heat the oxalic acid solution upto 50°– 60°C before titrating
it with potassium permanganate solution taken in the
burette. To increase the visibility of the colour change, place
the conical flask containing the solution to be titrated over
a white glazed tile kept below the nozzle of the vertically
fitted burette.
(iv) Note the initial reading of the volume of permanganate
solution in the burette and add it in small volumes to the
hot oxalic acid solution while swirling the contents of the
flask gently. The violet colour of permanganate solution is

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 TITRIMETRIC ANALYSIS (REDOX REACTIONS)

discharged on reaction with oxalic acid. The end point is

indicated by the appearance of permanent light pink colour
due to a slight excess of permanganate solution.
(v) Repeat the titration till three concordant readings are
obtained. Since the solution of KMnO4 is of dark colour,
the upper meniscus should be considered for noting the
burette readings.
(vi) Record the readings as shown in observation Table 6.1 and
calculate the strength of potassium permanganate solution
in mols/litre.

Table 6.1 : Titration of potassium permanganate solution

against standard oxalic acid solution

Burette readings Volume (V) of

Sl. No. Volume of Oxalic
acid in mL KMnO4 used
Initial (x) Final (y) V = (y–x) mL

Calculations
(i) The strength of the unknown solution in terms of molarity
may be determined by the following equation.
a1M1V1 = a 2 M2 V2 (6.1)
For oxalic acid vs potassium permanganate titration:
a1 = 2, (the number of electrons lost per formula unit of
oxalic acid in a balanced equation of half cell reaction)
a2 = 5, (the number of electrons gained per formula unit
of potassium permanganate in the balanced equation
of half cell reaction)
M1 and M2 are the molarities of oxalic acid and potassium
permanganate solutions used in the titration.
V1 and V2 are the volumes of oxalic acid and potassium
permanganate solutions.

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On putting the value of a1 and a2 in equation 6.1 we get

Oxalic acid KMnO4
2M1V1 = 5M2V2
2 M1V1
M2 = (6.2)
5 V2
We can calculate the molarity of potassium permanganate
solution by using equation 6.2. Strength of the solution is given by
the following equation:

Strength = Molarity × Molar mass

Result
(i) Molarity of KMnO4 solution is _______.
(ii) Strength of KMnO4 solution is _______.

Precautions
(a) Always rinse the burette and the pipette with the solutions to be taken in them.
(b) Never rinse the conical flask with the experimental solutions.
(c) Remove the air gaps if any, from the burette.
(d) Never forget to remove the funnel from the burette before noting the initial reading
of the burette.
(e) No drop of the liquid should hang at the tip of the burette at the end point and
while noting reading.
(f) Always read the upper meniscus for recording the burette reading in the case of
all coloured solutions.
(g) Never use pipette and burette with a broken nozzle.
(h) Lower end of the pipette should always remain dipped in the liquid while sucking
the liquid.
(i) Do not blow out the last drop of the solution from the jet end of the pipette.
(j) The strength of the solution must be calculated up to the fourth decimal place.
(k) Do not forget to heat the mixture of oxalic acid and H2SO4 solutions between 50°–60° C
while titrating it against potassium permanganate.

Discussion Questions

(i) What specific name is given to the permanganate titrations?

(ii) Which indicator is used in the permanganate titration?

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 TITRIMETRIC ANALYSIS (REDOX REACTIONS)

(iii) Why is a burette with pinch-cock regulator not used for the permanganate titration ?
(iv) Why do we heat oxalic acid solution containing sulphuric acid up to 50–60°C in the
permanganate titration?

EXPERIMENT 6.2

Aim
To determine the concentration/molarity of KMnO4 solution by
titrating it against standard solution of ferrous ammonium
sulphate.

Theory
Like oxalic acid, ferrous ammonium sulphate also acts as a
reducing agent in the titration against potassium permanganate.
The reaction which takes place is given below :

(a) Chemical equation

Reduction half reaction : 2 KMnO4 + 3H2SO4 → K2SO4 + 2 MnSO4 + 3H2O + 5 [O]
Oxidation half reaction : 2 FeSO4 (NH4)2 SO4 . 6H2O + H2SO4 + [O] → Fe2 (SO4) 3 + 2 (NH4)2 SO4
+ 13 H2O] x 5
_______________________________________________________________________________________
2KMnO4 + 8H2SO4 + 10FeSO4 (NH4)2 SO4 . 6H2O → K2SO4 + 2MnSO4 + 5Fe2(SO4)3
+ 10(NH4)2SO4 + 68H2O
_______________________________________________________________________________________

(b) Ionic equation

– – + 2+
Reduction half reaction : MnO4 + 5e + 8H → Mn + 4H2O
2+ 3+ –
Oxidation half reaction : Fe → Fe + e ] × 5
_______________________________________________________________________________________
MnO4– + 5Fe2+ + 8H+ → Mn2+ + 5Fe3+ + 4H2O
_______________________________________________________________________________________

The oxidation number of iron in Mohr’s salt is +2. Iron is

oxidised during the reaction and its oxidation number changes
from +2 to +3. In this titration heating of ferrous ammonium
sulphate solution is not required because reaction rate is very
high even at room temperature. Also, at high temperatures, ferrous
ions may be oxidised to ferric ions by oxygen of air and error may
be introduced in the experiment.

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Material Required

• Measuring flask (250 mL) : One

• Burette (50 mL) : One • Potassium
• Burette stand : One permanganate solution : As per need
• Pipette : One • Dilute sulphuric acid : As per need
• Conical flask : One • Ferrous ammonium
• Glazed tile (white) : One sulphate : As per need
• Funnel : One
• Weighing bottle : One

Procedure
A. Preparation of 0.05 M, standard solution of ferrous
Potassium
permanganate
ammonium sulphate
–1
(Molar mass of FeSO4(NH4)2SO4 . 6H2O = 392 g mol ).
(i) Weigh 4.9000 g of ferrous ammonium sulphate and
Sulphuric acid
transfer it into a 250 mL measuring flask through a funnel.
(ii) Transfer the solid sticking to the funnel with the help of
distilled water into the flask and add dilute H 2 SO4 into the
flask drop wise to get the clear solution.
(iii) Shake the flask till the substance dissolves and make the
solution upto the mark.

B. Titration of ferrous ammonium sulphate against

potassium permanganate solution
(i) Rinse and fill the clean burette with potassium
permanganate solution. Remove air bubbles if any, from
the burette tip by releasing some solution through it.
(ii) Take 10 mL of 0.05 M ferrous ammonium sulphate solution
in a conical flask and add half test tube (≈ 5 mL) full of
(1.0 M) H2SO4 to it.
(iii) Titrate the above solution with potassium permanganate
solution till the colour of the solution changes to permanent
pink. Swirl the content of the flask during the titration.
(iv) Repeat the titration, until three concordant readings are
obtained.
(v) Record the readings as shown in observation Table 6.2 and
calculate the strength of potassium permanganate solution
in mols/litre.

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 TITRIMETRIC ANALYSIS (REDOX REACTIONS)

Table 6.2 : Titration of potassium permanganate solution against

standard ferrous ammonium sulphate solution

Volume of ferrous Burette Readings Volume (V) of

ammonium sulphate
Sl. No. KMnO4 used
solution used for
Initial (x) Final (y) V = (y–x) mL
each titration in mL

Calculations
The strength of unknown solution in terms of molarity may be
determined by the following equation :
a1M1V1 = a2M2V2
M1 and M2 are the molarities of ferrous ammonium sulphate
and potassium permanganate solutions and V1 and V2 are volumes
of ferrous ammonium sulphate and potassium permanganate
solutions, respectively.
a1 = 1, (the number of electrons lost per formula unit of ferrous
ammonium sulphate in the half cell reaction)
a2 = 5, (the number of electrons gained per formula unit of
potassium permanganate in a half cell reaction)
Strength can be calculated by the formula given below :
Strength = Molarity × Molar mass

Result
The strength of the given potassium permanganate solution is _____ g/L.

Precautions

(a) Always use a fresh sample of ferrous ammonium sulphate to prepare its standard
solution.
(b) Other precautions are same as that in Experiment 6.1.

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Discussion Questions

(i) Why is ferrous ammonium sulphate solution not heated before titration?
(ii) Why is nitric acid or hydrochloric acid not used in permanganate titration? Explain.
(iii) Why is dilute sulphuric acid added while preparing a standard solution of ferrous
ammonium sulphate ?
(iv) How will you prepare 100 mL of 0.1 M standard solution of ferrous ammonium sulphate?
(v) Why is KMnO4 not regarded as a primary standard?
(vi) What type of titrations are given the name redox titrations? Name some other redox
titrations?

48
UNIT-8
TESTS FOR FUNCTIONAL GROUPS
IN ORGANIC COMPOUNDS

EXPERIMENT 8.1

Aim
To identify the functional groups present in an organic compound.

I. TESTS FOR UNSATURATION

Theory
Organic compounds containing > C = C < and / or — C ≡ C – bonds are called
unsaturated compounds. These compounds undergo addition reaction with
bromine water or the solution of bromine in carbon tetrachloride, chloroform or
glacial acetic acid. Addition of bromine to an alkene results in the formation of
vicinal dibromide. The reddish orange colour of the solution of bromine in carbon
tetrachloride disappears on reaction with an alkene. The reaction is as follows :

Alkenes decolourise the neutral/alkaline KMnO4 solution and vicinal glycols

are formed (Bayer’s test). Reaction takes place as follows :

Both the above reactions are used as tests for unsaturation.

 TESTS FOR FUNCTIONAL GROUPS IN ORGANIC COMPOUNDS

Material Required

• Potassium hydroxide
solution : 1–2 mL
• Carbon tetrachloride/
chloroform : 2 mL
• Test tubes : Two
• Bromine water/solution
• Test tube holder : One
of bromine in CCl4 or
chloroform : 2 mL
• Potassium permanganate
solution : As per need
• Compound to be tested : As per need

Procedure
A. Bromine water test
Dissolve 0.1 g or 5 drops of organic compound in 2 mL of carbon Potassium
tetrachloride in a test tube and add 2% solution of bromine in hydroxide
carbon tetrachloride or bromine water drop by drop with
continuous shaking. Decolourization of bromine solution indicates
the presence of unsaturation in organic compound. Carbon
tetrachloride

B. Bayer’s test
Chloroform
Dissolve 25-30 mg of organic compound in 2 mL of water or
acetone (free of alcohol) and add 1% potassium permanganate
solution containing equal volume of 1% sodium carbonate Bromine
solution. The discharge of the colour of more than one drop of
potassium permanganate indicates the presence of unsaturation Potassium
in the organic compound. Carrying out the reaction under alkaline permanganate

conditions removes the possibility of confusion due to substitution

in aromatic compounds.

Note: (i) Unsaturation in an organic compound is confirmed only when

both of the above tests are positive.
(ii) In place of CCl4 any other solvent such as CHCl3/dioxan and
even water can be used to dissolve the organic compound for
carrying out the reaction.

Precautions

(a) The tests should be performed at room temperature.

(b) Handle bromine solution carefully. Do not inhale the vapours and also avoid its
contact with the skin.

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 LABORATORY MANUAL CHEMISTRY

II. TEST FOR ALCOHOLIC (R–OH) GROUP

Theory
Alcoholic compounds on reaction with ceric ammonium nitrate
give a red colouration due to the formation of a complex.
(NH4)2 [Ce(NO3)6] + 3ROH → [Ce(NO3)4(ROH)3] + 2NH4NO3
Ceric ammonium Red complex
nitrate
Distinction between primary, secondary and tertiary alcohols
can be done on the basis of iodoform test and Lucas test.
Ethanol and secondary alcohols which contain CH3—CH(OH)R
group (iodoform reaction) give positive iodoform test. To carry out
reaction, potassium iodide and sodium hypochlorite solution are
added to the compound in the presence of sodium hydroxide
solution. Probably sodium hypochlorite first oxidses potassium
iodide into potassium hypoiodite, which oxidises CH3—CH(OH)R
group to CH3COR group and then iodinates it in the alkaline
medium of the reaction mixture by replacing the α-hydrogen
attached to the carbon atom adjacent to carbonyl group by iodine.
Iodoform is formed after cleavage of C—C bond.

Potassium Potassium
hypoiodite hypoiodite
NaOH
CH3CH2OH → CH3CHO → CI3CHO  → CHI3 + HCOONa

Lucas Test
Lucas reagent contains zinc chloride and concentrated
hydrochloric acid. This reagent reacts with primary, secondary
and tertiary alcohols at different rates. Tertiary alcohols react
almost instantaneously, secondary alcohols react in about 1-5
minutes and primary alcohols react very slowly. The reaction may
take 10 minutes to several days.
ZnCl
2
RCH2OH+HCl → No reaction/Slow reaction

ZnCl
2
R 2CHOH+HCl → R 2CHCl+H2O

ZnCl
2
R 3COH+HCl → R 3CCl+H2O
Alcohols are soluble in Lucas reagent but the formed alkyl
halides are not soluble. Therefore, formation of two layers in the
reaction medium indicate the occurrence of the reaction.
Primary alcohols – Layers do not separate
Secondary alcohols – Layers separate within 1-5 minutes
Tertiary alcohols – Layers separate immediately

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 TESTS FOR FUNCTIONAL GROUPS IN ORGANIC COMPOUNDS

Material Required

• Ceric ammonium
nitrate solution : As per need
• Test tube holder : One • Sodium hydroxide : As per need
• Test tubes : As per need • Iodine solution : As per need
• Lucas reagent : As per need
• Dioxan : As per need

Procedure
A. Ceric ammonium nitrate test
Take 1 mL solution of organic compound dissolved in a suitable Sodium
solvent. Add a few drops of ceric ammonium nitrate solution. hydroxide

Appearance of red colour shows the presence of alcoholic – OH group.

Iodine
Note : The red colour disappears after keeping the reaction mixture for
sometime. The colour also disappears if excess of ceric
ammonium nitrate solution is added. Therefore, use of excess
of ceric ammonium nitrate solution should be avoided.
B. Iodoform test

First method
Take 0.2 mL of the compound in a test tube, add 10 mL of 10%
aqueous KI solution and 10 mL of freshly prepared NaOCl solution.
Warm gently; yellow crystals of iodoform separate.

Second method
Dissolve 0.1 g or 4 to 5 drops of compound in 2 mL of water. If it
does not dissolve, add dioxane drop by drop to get a homogeneous
solution. Add 2 mL of 5% sodium hydroxide solution followed by
potassium iodide-iodine reagent* dropwise with continuous
shaking till a definite dark colour of iodine persists. Allow the
reactants to remain at room temperature for 2-3 minutes. If no
iodoform separates, warm the reaction mixture in a water bath at
60°C. Add more drops of potassium iodide–iodine reagent. If colour
of iodine disappears continue addition of reagent till the colour of
iodine persists even after two minutes of heating at 60°C. Remove
excess iodine by adding a few drops of sodium hydroxide solution
with shaking. Dilute the mixture with equal volume of water and
keep it at room temperature for 10-15 minutes. A yellow precipitate
of iodoform is obtained if test is positive.

* Potassium iodide-iodine reagent is prepared by dissolving 20 g of potassium

iodide and 10 g of iodine in 100 mL of water.

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 LABORATORY MANUAL CHEMISTRY

C. Lucas test
Take 1 mL of compound in a test tube. Add 10 mL of Lucas reagent.
Shake well and note the time for the separation of two distinct layers.

Note : Lucas test is applicable to only those alcohols which are soluble
in the reagent because the test is based on separation of alkyl
halides as separate layer.

III. PHENOLIC (AR-OH) GROUP

Theory
The –OH group attached directly to the ring carbon of an aromatic
ring is called phenolic –OH group. Phenols are weakly acidic,
therefore they are soluble in NaOH solution but at the same time
they are not sufficiently acidic to be soluble is sodium
hydrogencarbonate solution. Phenols give coloured complex with
neutral ferric chloride solution. For example, phenol gives a
complex of violet colour as follows :
3– +
6C6H5OH + FeCl3 → [Fe(C6H5O)6] + 3HCl + 3H
Violet complex
Resorcinol, o–, m– and p–cresol give violet or blue colouration,
catechol gives green colour which rapidly darkens. 1 and 2–Naphthol
do not give characteristics colours. Phenols condense with phthalic
anhydride in the presence of concentrated H2SO4, Phenol condeses to
give phenolphthalein which gives a dark pink colour with NaOH
solution. This is called phthalein dye test.

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 TESTS FOR FUNCTIONAL GROUPS IN ORGANIC COMPOUNDS

Table 8.1 : Colours produced by some other phenolic compounds in phthalein dye test

Compound Colour Compound Colour

o–Cresol red Catechol Usually blue takes

longer time to appear
m–Cresol bluish-purple
Resorcinol Green fluorescent
p–Cresol No colour
colour of fluorescein

Material Required

• Blue litmus paper

• Ferric chloride solution
• Conc. sulphuric acid
According
• Sodium hydroxide
• Test tube holder : One to
• Phthalic anhydride
• Test tubes : As per need requirement
• Organic compound
containing phenolic
–OH group

Procedure
A. Ferric chloride test
Sulphuric
Take 2 mL of aqueous or alcoholic solution of the organic acid
compound in a test tube, add neutral ferric chloride solution
dropwise and note the colour change. Appearance of a blue, green, Sodium
hydroxide
violet or red colour indicates the presence of phenolic –OH group.

B. Phthalein dye test Phthalic

anhydride
Take 0.1 g of organic compound and 0.1 g of phthalic anhydride
in a clean dry test tube and add 1-2 drops of conc. H2SO4. Heat
the test tube for about 1 minute in an oil bath. Cool and pour the
reaction mixture carefully into a beaker containing 15 mL of dilute
sodium hydroxide solution. Appearance of pink, blue, green, red
etc. colours indicates the presence of phenolic –OH group in the
compound. However, the colour disappears on addition of large
excess of sodium hydroxide solution.

Note : (i) Neutral ferric chloride solution is prepared by adding dilute sodium hydroxide solution to
ferric chloride solution drop by drop till a small but permanent brown precipitate appears.
Solution is filtered and the clear filtrate is used for the test.
(ii) Some phenols like 2,4,6 – trinitrophenol and 2,4 – dinitrophenol, which contain electron
withdrawing groups are strong acids and dissolve even in sodium hydrogencarbonate solution.

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 LABORATORY MANUAL CHEMISTRY

Precautions
(a) Always use freshly prepared, neutral and very dilute solution of ferric chloride.
(b) Phenol is toxic and corrosive in nature and should be handled with care.

O
IV. ALDEHYDIC AND KETONIC GROUPS (–CHO and –C–)

Theory
Both aldehydes and ketones contain carbonyl group (>C = O) and
are commonly known as carbonyl compounds. Identification of
aldehydes and ketones is done by two important reactions of
carbonyl group i.e.
(i) addition reaction on double bond of >C = O group and
(ii) oxidation of carbonyl group.
Addition reactions of derivatives of ammonia are important from
the point of view of identification of carbonyl compounds. Addition
is generally followed by elimination resulting in the formation of
unsaturated compound.

(R = alkyl, aryl or C6H5NH etc.)

These reactions are catalysed by an acid or a base and do not
occur under strongly acidic or basic conditions. Each reaction
requires an optimum pH for its occurrence. Therefore, maintenance
of pH is very important while carrying out these reactions.
As far as oxidation is concerned, aldehydes are easily oxidised
to carboxylic acids while ketones require relatively stronger
oxidising agents. Distinction can be made between these two types
of carbonyl compounds on the basis of difference in their reactivity.
Following tests are performed for the identification of aldehydic
and ketonic groups:
(i) On reaction with 2,4-dinitrophenylhydrazine (2,4-DNP), they
form the respective 2,4–dinitrophenyl hydrazones.

2, 4-Dinitrophenylhydrazine 2, 4 – Dinitrophenyl hydrazone of

carbonyl compound

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 TESTS FOR FUNCTIONAL GROUPS IN ORGANIC COMPOUNDS

These two carbonyl compounds (aldehydes and ketones) are

distinguished on the basis of tests using mild oxidising reagents,
like Tollen’s reagent and Fehling’s reagent or Benedict’s reagent.
Tollen’s reagent is an alkaline solution of silver cation complexed
with ammonia, and Fehling’s and Benedict’s reagents are alkaline
solutions containing cupric ions complexed with tartarate and
citrate ions respectively. Fehling’s reagent is freshly prepared by
mixing equal amounts of Fehling’s solution A and Fehling’s
solution B. Fehlings reagent deteriorates on keeping while Fehling’s
solutions A and B are quite stable. Fehling’s solution A is an
aqueous copper sulphate solution while Fehling’s solution B is
an alkaline solution of sodium potassium tartarate (Rochelle’s salt).
The reagent contains Cu2+ ion complexed with tartarate ions. The
structure of the complex is given below :

Copper tartarate complex

Benedict modified the original Fehling’s test by using a single
solution which is more convenient for the test. Benedict’s solution
is more stable than Fehling’s reagent and can be stored for a long
time. It is an alkaline solution containing a mixture of copper
sulphate and sodium citrate (2Na3C6H5O7.11H2O).
Complex formation decreases the cupric ion concentration
below that necessary for precipitation of cupric hydroxide. These
two reagents oxidize aldehydes while ketones remain unaffected.
The chemistry of these tests is as follows:
+ –
RCHO + 2[Ag (NH3)2] + 2OH → 2Ag + 3NH3 + H2O + RCOONH4
From Tollen’s reagent
2+ – –
RCHO + 2Cu (complexed) + 5OH → RCOO + Cu2O + 3H2O
Fehling’s solution
However, aromatic aldehydes do not give positive Fehling’s test.
2+ +
In Benedict test also, Cu ions are reduced to Cu ions in the
same manner as in the case of Fehling’s reagent.
Aldehydes also give pink colour with Schiff’s reagent (the
reagent is prepared by decolourising aqueous solution of
p–rosaniline hydrochloride dye by adding sodium sulphite or by
passing SO2 gas). Ketones do not respond to this test.

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 LABORATORY MANUAL CHEMISTRY

Material Required
• Schiff’s reagent : As per need
• Fehling’s solutions A and B: As per need
• Beaker (250 mL) : One • Silver nitrate : As per need
• Test tube holder : One • Dilute ammonium
• Test tubes : As per need hydroxide solution : As per need
• 2,4-Dinitrophenyl-
hydrazine reagent : As per need

Procedure
Silver nitrate
A. Test given by both aldehydes and ketones
Ammonia
solution 2,4-Dinitrophenylhydrazine test (2,4-DNP test)

Take 2-3 drops of the liquid compound in a test tube or in case of

Ammonia gas solid compound, dissolve a few crystals of it in 2-3 mL
alcohol. Add a few drops of an alcoholic solution of
2,4-dinitrophenylhydrazine. Appearance of yellow, orange or
orange-red precipitate confirms the presence of carbonyl group.
If precipitate does not appear at room temperature, warm the
mixture in a water bath for a few minutes and cool.

B. Tests given by aldehydes only

Following tests namely Schiff’s test, Fehling’s test and Tollen’s test
are given by aldehydes only.

Schiff’s test

Take 3-4 drops of the liquid compound or dissolve a few crystals

of organic compound in alcohol and add 2-3 drops of the Schiff’s
reagent. Appearance of pink colour indicates the presence of an
aldehyde.

Fehling’s test

Take nearly 1 mL of Fehling’s solution A and 1 mL of Fehling’s

solution B in a clean dry test tube. To this add 2-3 drops of the
liquid compound or about 2 mL of the solution of the solid
compound in water or alcohol. Heat the content of the test tube
for about 2 minutes in a water bath. Formation of brick red
precipitate of copper (I) oxide indicates the presence of an aldehyde.
This test is not given by aromatic aldehydes.

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 TESTS FOR FUNCTIONAL GROUPS IN ORGANIC COMPOUNDS

Benedicts test

Add 5 drops of the liquid compound or the solution of the solid

organic compound in water or alcohol to 2 mL Benedict’s reagent.
Place the test tube in boiling water bath for 5 minutes. An orange-
red precipitate indicates the presence of an aldehyde.

Tollen’s test

(i) Take 1 mL of freshly prepared (~ 2 %) silver nitrate solution

in a test tube. Add 1-2 drops of sodium hydroxide solution
to it and shake, a dark brown precipitate of silver oxide
appears. Dissolve the precipitate by adding ammonium
hydroxide solution drop-wise.
(ii) To the above solution, add an aqueous or an alcoholic
solution of the organic compound.
(iii) Heat the reaction mixture of step (ii) in a water bath for about
5 minutes. Formation of a layer of silver metal on the inner
surface of the test tube which shines like a mirror, indicates
the presence of an aldehyde.

Precautions

(a) Always use freshly prepared reagents to perform the tests.

(b) Do not heat the reaction mixture directly on a flame.
(c) After performing the test, destroy the silver mirror by adding dilute nitric acid
and drain off the solution with excess of water.

V. CARBOXYL GROUP (—COOH)

Theory
Organic compounds containing carboxyl functional groups are
called carboxylic acids.
The term carboxyl, derives its name from the combination of
words carbonyl and hydroxyl because carboxylic functional group
O
contains both of these groups ( —C—OH ). These acids turn blue
litmus red and react with sodium hydrogencarbonate solution to
produce effervescence due to the formation of carbon dioxide.
This is a test that distinguishes carboxylic acids from phenols.

RCOOH + NaHCO3 → RCOONa + H2O + CO2

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 LABORATORY MANUAL CHEMISTRY

These react with alcohols in the acidic medium to produce esters.

Conc.H SO
RCOOH 2 4 → RCOOR' + H O
+ R'OH 
∆ 2

Carboxylic Alcohol Ester

acid

Material Required

• Blue litmus paper

• Test tube holder : One /solution : As per need
• Glass rod : One • Ethyl alcohol : As per need
• Test tubes : As per need • Sodium hydrogencarbonate
solution : As per need

Procedure

A. Litmus test

Ethyl alcohol Put a drop of the liquid compound or a drop of the solution of the
compound with the help of a glass rod on a moist blue litmus paper.
If the blue colour of the litmus paper changes to red, the presence of
either a carboxylic group or a phenolic group is indicated.

B. Sodium hydrogencarbonate test

Take 2 mL of saturated aqueous solution of sodium
hydrogencarbonate in a clean test tube. Add a few drops of the
liquid compound or a few crystals of solid compound to it.
The evolution of brisk effervescence of CO2 indicates the presence
of carboxyl group.
C. Ester test
Take about 0.1 g compound in a test tube, add 1 mL ethanol or
methanol and 2-3 drops of concentrated sulphuric acid. Heat the
reaction mixture for 10-15 minutes in a hot water bath at about
50°C. Pour the reaction mixture in a beaker containing aqueous
sodium carbonate solution to neutralise excess sulphuric acid
and excess carboxylic acid. Sweet smell of the substance formed
indicates the presence of carboxyl function in the compound.

Precaution

Add the compound in sodium hydrogencarbonate solution slowly so that

effervescence is visible clearly.

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 TESTS FOR FUNCTIONAL GROUPS IN ORGANIC COMPOUNDS

VI. AMINO GROUP (—NH2)

Theory
Organic compounds containing amino group are basic in nature.
Thus they easily react with acids to form salts, which are soluble
in water.
Both, aliphatic and aromatic amines are classified into three
classes namely– primary(–NH2), secondary(-NH-) and tertiary (-N<),
depending upon the number of hydrogen atoms attached to the
nitrogen atom. Primary amine has two hydrogen atoms, secondary
has one while tertiary amine has no hydrogen atom attached to
nitrogen.
(i) Carbylamine test
Aliphatic as well as aromatic primary amines give carbylamine test
in which an amine is heated with chloroform.

∆→
R-NH2 + CHCl3 + 3KOH  RNC + 3KCl + 3H2O
(R=alkyl or aryl group) (Carbylamine)

Caution!
Carbylamine so formed is highly toxic and should be destroyed
immediately after the test. For this cool the test tube and add
carefully an excess of conc. HCl.

(ii) Azo dye test

Aromatic primary amines can be confirmed by azo dye test. Primary
amine e.g. aniline reacts with nitrous acid generated in situ by the
reaction of sodium nitrite with HCl at 0–5°C to produce diazonium
salt. This couples with β-naphthol to give a scarlet red dye, which
is sparingly soluble in water.

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 LABORATORY MANUAL CHEMISTRY

Material Required
• Chloroform
• Potassium hydroxide
• Sodium nitrite solution
• Test tubes : As per need
• Aniline
• Test tube holder : One As per need
• β-Naphthol
• Bunsen burner : One
• Dilute hydrochloric acid
• Sodium hydroxide solution
• Ice

Procedure
A. Solubility test
Chloroform
Take 1 mL of given organic compound in a test tube and add a few
Aniline
drops of dilute HCl to it. Shake the contents of the test tube well. If
the organic compound dissolves, it shows the presence of an amine.
+
–
Potassium C6H5NH2 + HCl → C6H5NH3 Cl
hydroxide (Anilinium chloride
soluble in water)
Sodium nitrite B. Carbylamine test
Take 2-3 drops of the compound in a test tube and add 2-3 drops of
β-naphthol chloroform followed by addition of an equal volume of 0.5 M alcoholic
potassium hydroxide solution. Heat the contents gently. An obnoxious
smell of carbylamine confirms the presence of primary amino group
in the compound.

Caution!
Do not inhale the vapours. Destroy the product immediately by
adding concentrated hydrochloric acid and flush it into the sink.
C. Azo dye test
(i) Dissolve nearly 0.2 g of the compound in 2 mL of dilute
hydrochloric acid in a test tube. Cool the content of the test
tube in ice.
(ii) To the ice cooled solution add 2 mL of 2.5% cold aqueous
sodium nitrite solution.
(iii) In another test tube, dissolve 0.2 g of β-naphthol in dilute
sodium hydroxide solution.
(iv) Add diazonium chloride solution prepared in step (ii) into
the cold β-naphthol solution slowly with shaking.
The formation of a scarlet red dye confirms the presence of
aromatic primary amine.

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 TESTS FOR FUNCTIONAL GROUPS IN ORGANIC COMPOUNDS

Precautions
(a) Do not expose yourself to the vapours while performing carbylamine test because
isocyanide is highly poisonous. Destroy it immediately as described above.
(b) Maintain the temperature of the reaction mixture below 5°C during diazotisation,
as diazonium chloride is unstable at higher temperatures.
(c) Always add diazonium chloride solution into the alkaline solution of β-naphthol
and not vice-versa.

Discussion Questions

(i) What is Bayer’s reagent?

(ii) Why do alkenes and alkynes decolourize bromine water and alkaline KMnO4?
(iii) Explain why for the confirmation of unsaturation in a compound both the tests namely
test with bromine water and test with Bayer’s reagent should be performed.
(iv) Why does phenol decolourize bromine water?
(v) How will you distinguish between phenol and benzoic acid?
(vi) Why does benzene not decolourise bromine water although it is highly unsaturated?
(vii) Why does formic acid give a positive test with Tollen’s reagent?
(viii) Outline the principle of testing glucose in a sample of urine in a pathological laboratory?
(ix) Why is Benedict’s reagent more stable than Fehling’s reagent?
(x) How would you distinguish an aldehyde from a ketone by chemical tests?
(xi) How would you separate a mixture of phenol and benzoic acid in the laboratory by using
chemical method of separation?
(xii) Write the chemistry of diazotisation and coupling reactions.
(xiii) How can you distinguish between hexylamine (C6H13NH2) and aniline (C6H5NH2)?
(xiv) How can you distinguish between ethylamine and diethylamine?
(xv) How can CH3OH and C2H5OH be distinguished chemically?
(xvi) Why is solution of iodine prepared in potassium iodide and not in water?
(xvii) What is haloform reaction? What type of compounds generally give this reaction?

(xviii) How can you distinguish the compounds and by simple

chemical test?

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UNIT-11
TESTS FOR CARBOHYDRATES
FATS AND PROTEINS
EXPERIMENT 11.1

Aim
To study the characteristics of carbohydrates, fats and proteins in pure form
and detection of their presence in the given foodstuffs.

I. TEST FOR CARBOHYDRATES, FATS AND PROTEINS IN PURE FORM

A. Tests for Carbohydrates

Theory
Carbohydrates are optically active polyhydroxy aldehydes, polyhydroxy ketones
or compounds, which give these units as hydrolysis product. Starch, cellulose
and sugars are the familiar examples of carbohydrates. Carbohydrates are
classified on the basis of number of polyhydroxy aldehyde or ketone units
obtained from them on hydrolysis. Three broad classes are as follows :
(i) Monosaccharides : These cannot be hydrolysed further to polyhydroxy
aldehydes or ketones.
(ii) Oligosaccharides : These yield 2-10 monosaccharide units on hydrolysis.
Common amongst these are disaccharides, which produce two
monosaccharide units.
(iii) Polysaccharides : These yield large number of monosaccharide units on
hydrolysis.
Monosaccharides are further classified on the basis of number of carbon atoms
and functional group present in them. If a monosaccharide contains aldehydic
group it is called aldose. If it contains keto group it is called ketose. Carbohydrates
of all classes give Molisch’s test. Carbohydrates, which are sweet in taste, are
called sugars. Glucose, fructose (fruit sugar) and sucrose (table sugar) are examples
of sugars. Sugars are classified into two major categories: reducing sugars and
non-reducing sugars. Reducing property of sugars is detected by the three tests
namely Fehling’s test, Benedict’s test and Tollen’s test.
Prepare 1% stock solution of glucose, fructose and sucrose in separate beakers
and divide each of the solutions into test tubes marked A, B, C and D etc. and
perform the following tests.
 TESTS FOR CARBOHYDRATES, FATS AND PROTEINS

I. Theory of Molisch’s test

On adding concentrated sulphuric acid to the aqueous solution
of carbohydrate containing alcoholic solution of 1-naphthol, a deep
violet colour appears at the junction of the two liquids.
Concentrated sulphuric acid hydrolyses glycosidic bonds of
carbohydrate to give monosaccharides which are dehydrated to
an aldehyde known as furfural which undergoes reaction with
1–naphthol to give a unstable condensation product of deep violet
colour. This test may be given by some other organic compounds
also. The following reaction takes place :

Material Required

• Glucose, fructose,
• Test tubes : As per need
sugar (sucrose) : As per need
• Test tube stand : One
• Test tube holder : One • Alcoholic solution of
1-Naphthol : As per need
• Beaker (100 mL) : One
• Concentrated H2SO4 : As per need

Procedure
Add 2-3 drops of alcoholic solution of 1% 1-naphthol in test tube
‘A’ and then pour 2 mL conc. H2SO4 down the sides of the test
tube so that it forms a separate layer at the bottom of the test
tube. The formation of a purple ring at the interface of the two
layers confirms the presence of carbohydrates.

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 LABORATORY MANUAL CHEMISTRY

II. Theory of test for reducing sugars

A. Fehling’s test and Benedict test
Black copper (II) oxide is formed on heating a suspension of copper
Alcohol hydroxide in alkaline solution.
Cu(OH)2 → CuO + H2O
Copper (II) oxide
H2SO4
(black)
If some reducing agent is present in the reaction medium, then
Hazard Warning
orange-red copper (I) oxide is precipated.
• In high concentration
1-Naphthol is extremely 2Cu(OH)2 → Cu2O + 2H2O + [O]
destructive to all body Copper (I) oxide
tissues. (Orange-red)
Reducing sugars contain aldehydic group or α-hydroxy ketonic
2+
group, therefore in alkaline medium reduce Cu ions. But if the
reaction is carried out directly in the presence of an alkali then,
copper (II) hydroxide gets precipitated. To overcome this problem,
copper (II) ions are complexed with tartarate ions (Fehling’s reagent) or
citrate ions (Benedict’s solution). Both the complex ions are soluble
2+
in alkaline medium and yield Cu ions in such a low concentration
that solubility product of cupric hydroxide is not reached.
Reducing sugars react with Fehling’s reagent according to the
following reaction:
– –
RCHO + 2 Cu2+ + 5OH → RCOO + Cu2O + 3H2O
(orange-red
precipitate)
The discharge of blue colour due to Cu2+ ions and appearance of
orange-red precipitate of Cu2O, indicates the reducing property of
sugars.

2+ Na2CO3 (aq )
D-Glucose + Cu (citrate)  → D-Gluconic acid + Cu2O
(orange-red precipitate)
2+ NaOH (aq )
D-Maltose + Cu (tartarate)  → D-Maltobionic acid + Cu2O
(orange-red precipitate)
Fehling's
Sucrose (disaccharide) 
solution→ No reaction

Sometimes, the cuprous precipitate comes down as yellow

cuprous hydroxide, but on warming this is converted to orange-
red copper (I) oxide.

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 TESTS FOR CARBOHYDRATES, FATS AND PROTEINS

In some cases, this reaction may be used as a quantitative

analytical process for the determination of reducing sugars in
blood and urine etc.
All monosaccharides are reducing sugars. Disaccharides which
contain a free hemi-acetal group ( ) are also mild reducing

sugars. Most naturally occurring disaccharides are reducing

sugars (sucrose is an exception).

B. Tollen’s test
Tollen’s reagent is ammoniacal solution of silver nitrate. A reducing
sugar, reduces silver ion to metallic silver which gets deposited on
the inner surface of the test tube in the form of silver mirror. The
reaction occurs as follows:
+ –
RCHO + 2 [Ag (NH3)2] + 2OH → 2Ag + RCOONH4 + H2O + 3NH3

Material Required

• Test tubes : As per need

• Test tube stand : One • Fehling’s solutions
• Test tube holder : One A and B : As per need
• Beaker (100 mL) : One • Benedict’s reagent : As per need
• Tollen’s reagent : As per need
• Water bath : One
• Bunsen burner : One

Procedure

A. Fehling’s test
Mix 1 mL each of Fehling’s solutions A and B in a test tube and
add the mixture to test tube B. Heat the content of the test tube on
a water bath. The formation of a orange-red precipitate indicates
the presence of reducing sugar.

B. Benedict’s test
Add 1 mL of Benedict’s reagent to test tube C and heat the mixture
to boiling in a water bath for 2 minutes. The formation of a orange-
red precipitate due to the formation of copper (I) oxide indicates
the presence of reducing sugar.

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 LABORATORY MANUAL CHEMISTRY

C. Tollen’s test
Prepare Tollen’s reagent by adding sodium hydroxide solution
dropwise to 1 mL aqueous silver nitrate solution to get the precipitate
of silver oxide. Now add ammonium hydroxide solution while shaking
Resorcinol the mixture so that initially formed silver oxide precipitate dissolves.
Add the reagent to the sugar solution contained in a test tube and
warm the reaction mixture on a water bath. Formation of silver mirror
Furfural
on the walls of the test tube shows the presence of reducing sugar.

Caution!
Never heat the test tube on direct flame as it may cause explosion.

III. Theory of test to distinguish Monosaccharide from

Disaccharide
Barfoed’s test
The reagent is cupric acetate in acetic acid solution. It is weakly
acidic and is reduced by only monosaccharides. Prolonged boiling
may hydrolyse disaccharides and false positive test may be
obtained. Monosaccharides react with this reagent within 5 minutes
to give a brick red precipitate of copper (I) oxide. Disaccharides
take a longer time to react because aldehyde function is masked in
the acetal linkage.
2+
RCHO + 2Cu → RCOOH + Cu2O
Monosaccharide Brick-red

The precipitate of cuprous oxide obtained is less dense and its

colour is brick-red instead of orange-red.

Material Required

• Test tubes : As per need

• Test tube stand : One
• Test tube holder : One • Glucose, fructose,
• Beaker (100 mL) : One sugar (sucrose) : As per need
• Water bath : One • Barfoed’s reagent : As per need
• Bunsen burner : One

Procedure
Take 10 drops of 1% sugar solution in a test tube and add
1 mL Barfoed’s reagent. Heat the the content of the test tube in a
water bath to boiling for five minutes. The formation of orange-

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 TESTS FOR CARBOHYDRATES, FATS AND PROTEINS

red precipitate indicates positive test for monosaccharides.

Disaccharides do not give this test.

Test for Sucrose

Hydrolyse the sucrose for performing the test by adding 5 drops
of concentrated HCl to 5 mL of 1% sucrose solution and heating
the mixture in a boiling water bath. Cool the mixture and add
NaOH solution to obtain neutral or slightly alkaline solution.
Perform the tests for reducing sugar and Seliwanoff’s test given
below with the hydrolysed product and record your results.

IV. Test to distinguish Ketose from Aldose

Seliwanoff’s test
Ketoses dehydrate very rapidly under acidic conditions to give
furfural, which reacts with resorcinol (1,3-dihydroxy benzene) to
give a coloured product.

Ketohexoses give red colour and ketopentoses give blue-green

colour. Aldoses take longer time to produce colour because under
the same conditions, aldoses form furfural slowly, probably
because β-elimination is required before dehydration to furfural.
Therefore prolonged heating should be avoided.

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 LABORATORY MANUAL CHEMISTRY

Material Required

• Test tubes : As per need

• Test tube stand : One
• Test tube holder : One • Glucose, fructose,
• Beaker (100 mL) : One sugar (sucrose) : As per need
• Water bath : One • Seliwanoff’s reagent : As per need
• Bunsen burner : One

Procedure
Add 2 mL of Seliwanoff’s reagent to 10 drops of 1% sugar solution
taken in a test tube. Heat the test tube in boiling water for two
minutes. Ketohexoses give red colour. Ketopentose gives blue-green
colour. Aldoses do not give colour within two minutes.

V. Theory of test for Polysaccharides (Starch)

Starch gives blue colour with iodine solution due to the formation
of a complex known as starch iodide complex. Starch is present in
wheat, rice, maize, potatoes etc.

Material Required

• Test tubes : As per need

• Test tube stand : One
• Test tube holder : One • Starch solution : As per need
• Beaker (100 mL) : One • Iodine solution : As per need
• Water bath : One
• Bunsen burner : One

Procedure

Iodine test
Make a suspension of (0.5 g) starch in 5 mL water and pour it in
Iodine
50 mL boiling water to get an aqueous colloidal solution. To this
add a few drops of aqueous iodine solution. The appearance of
blue colour indicates the presence of starch.

B. Test for Oils and Fats

Theory
These are the esters of glycerol and long chain fatty acids and are
known as triglycerides. Triglycerides which are liquids at room

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 TESTS FOR CARBOHYDRATES, FATS AND PROTEINS

temperature are oils and those that are solids are called fats. Oils
are of plant origin and fats are of animal origin. Triglycerides in
which three acyl groups are same are called simple triglycerides
and a triglyceride in which three acyl groups are different is called
mixed triglyceride. Many naturally occurring fatty acids contain
two or three double bonds. The fats from which these come are
called polyunsaturated fats or oils. while oils are glycerides of
unsaturated fatty acids. Fats and oils are insoluble in water.
On heating with potassium hydrogen sulphate, oils and fats
give characteristic odour of acrolein. This is the test for glycerol
present either free or combined as an ester. On heating with
potassium hydrogen sulphate, glycerol is dehydrated and acrolein
is formed which has a pungent odour. The reaction is as follows :

Material Required

• Test tubes : As per need

• Test tube stand : One
• Test tube holder : One • Mustard oil/ghee : As per need
• Potassium hydrogen-
• Beaker (100 mL) : One
sulphate : As per need
• Water bath : One
• Bunsen burner : One

Procedure
Add a few crystals (0.5g) of dry potassium hydrogen sulphate to 3
mL of mustard oil/ghee taken in a test tube and heat the content
of the test tube gently. A pungent smell confirms the presence of
an oil or a fat.

C. Tests for Proteins

Theory
Proteins are complex organic compounds containing nitrogen and
are made up of amino acids. Proteins are present in egg albumin,
soya beans, pulses, fish, milk etc. Their presence can be confirmed
by several tests. Due to the presence of characteristic side chains
in them, certain amino acids exhibit typical colour reactions that
form the basis for their identification. Proteins also respond to the
colour reactions of amino acids, but can be distinguished from
amino acids by biuret reaction, and coagulation reaction.

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 LABORATORY MANUAL CHEMISTRY

I. Biuret test for peptide bonds

Alkaline copper sulphate reacts with compounds containing two or
more peptide bonds to form complexes of violet colour.

2–
and [Cu (NHCONHCONH)2]

The name of the test comes from the name of the compound,
biuret, which gives this test. The reaction is not absolutely specific
for peptide bond because many compounds containing two carbonyl
groups linked through nitrogen or carbon atoms give a positive result.

II. Ninhydrin reaction

Ninhydrin is a powerful oxidizing agent and reacts with proteins to
give a blue-violet compound called Rhumann’s purple.

Note : Ammonia, primary amines, amoniacids and peptides also react

with ninhydrin.

III. Xanthoproteic reaction

Aromatic groups of either the free aminoacid or protein, undergo
nitration on heating with concentrated nitric acid. The salts of these
derivatives are orange in colour.

Material Required

• Test tubes : As per need

• Test tube stand : One • Egg albumin/casein
• Ninhydrin
• Test tube holder : One
• Copper reagent : As per need
• Beaker (100 mL) : One • Concentrated HNO3
• Water bath : One • Alcohol
• Bunsen burner : One

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 TESTS FOR CARBOHYDRATES, FATS AND PROTEINS

Procedure
A. Biuret test
Prepare 0.5% (w/V) solution of casein or egg albumin in
0.1 M NaOH solution. Take 2-3 mL of the solution and add about HNO 3
2 mL of 10% sodium hydroxide solution to it. Add a few drops of
copper reagent and warm the mixture for about 5 minutes.
Appearance of violet colour due to the formation of a complex Alcohol
species of Cu2+ ions with - CONH – group confirms the presence
of protein in the sample.
B. Ninhydrin reaction
Take 2-3 mL of an aqueous solution of egg albumin in a test tube.
Add 3-4 drops of ninhydrin solution to it and heat. Appearance of
blue colour indicates the presence of protein.
C. Xanthoproteic test
Take 1 mL of an aqueous solution of egg albumin in a test tube and
add a few drops of concentrated nitric acid. Heat the reaction mixture
for a few minutes on a Bunsen flame. A yellow colour appears. Cool
the test tube under running tap and add a few drops of 10M sodium
hydroxide solution, an orange colour appears.

II. TEST FOR CARBOHYDRATES, FATS AND PROTEINS IN FOODSTUFFS

(i) Take the samples of milk, wheat flour, rice and gram flour
powder of legumes to test for the presence of carbohydrates,
fats and proteins.
(ii) Take 0.5 mL sample of milk to carry out each of the tests.
(iii) For wheat flour, rice flour, gram flour and legume powder,
add 100 mg of the sample in 10 mL of distilled water and boil
the suspensions, to get a colloidal solution. Perform the tests
with this collidal solution and record the results in Table 11.1.
Table 11.1 : Test for carbohydrates, fats and proteins in the
different samples of food materials

Sample Carbohydrates Fats Protein

Present/Absent Present/Absent Present/Absent

Milk

Wheat flour

Rice flour

Gram flour

Legumes

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 LABORATORY MANUAL CHEMISTRY

From the experiments, it will be observed that food materials

like wheat flour, gram flour and legumes contain carbohydrates and
proteins. The rice flour contains carbohydrates, while milk contains
fats and proteins. Similarly, other food materials may be tested for
the presence of carbohydrates, fats and proteins.

Precautions

(a) Shake the mixture thoroughly while preparing the extract of gram, wheat and
rice flour.
(b) Always use fresh reagents to carry out the tests.
(c) Use only required quantities of reagents.

Discussion Questions

(i) How will you distinguish between sucrose and glucose?

(ii) Explain why does fructose reduce Fehling’s solution and Tollen’s reagent inspite of the
presence of ketonic group?
(iii) What is the role of tartarate and citrate ions in Fehling’s reagent and Benedict’s reagent
respectively?

124