Expt 2: Estimation of glucose by Benedict’s quantitative reagent

AIM:
To estimate the amount of glucose present in the given unknown sample by Benedict’s
method.

PRINCIPLE:
Benedict’s quantitative reagent is the modified form of qualitative reagent. It consists of
cupric sulphate, sodium carbonate and sodium citrate, potassium thiocyanate, and potassium
ferrocyanide.
The alkali present in the Benedict’s reagent enolises the sugar, thereby causing them to
be a strong reducing agent. Ferrocyanide serves to dissolve the copper hydroxide while
thiocyanate helps to convert the red cuprous oxide to whit crystals of cuprous thiocyanate, which
gives the clear end point.

REAGENTS REQUIRED
(i)Benedict’s quantitative reagent (BQR)
(a)Cupric sulphate, (b)Sodium carbonate, (c)Sodium citrate, (d)Potassium
thiocyanate, (e)Potassium ferrocyanate.
(ii)Anhydrous sodium carbonate
(iii)Working standard glucose solution
(iv)Porcelain beads.

PROCEDURE:

Titration I: Standardisation of Benedict’s Qualitative Reagent

Accurately pipette out 5ml of Benedict’s quantitative reagent into a clean conical flask.
Two spatula full of 2g of sodium carbonate was added into the conical flask. Few pieces of
porcelain beads were also added in order to avoid bumping. The contents were brought to
temperature approximately 60-70 oC. The contents were titrated against the standard glucose
solution with regular shaking until the blue colour disappeared. The end point is the appearance
of chalky white precipitate. The titrations were repeated for concordant values.

Titration II: Estimation of Glucose

The given unknown sample solution was made up to 100ml with distilled water in a
standard flask. It was shaken well for uniform concentration. The burette was filled with this
unknown solution and the titrations were performed as mentioned above till the appearance of
chalky white precipitate. The titrations were repeated for concordant values.

Result:
The amount of glucose present in 100ml of the given solution =__________mg

EXPT 2
Titration I
Standardisation of Benedict’s Quantitative reagent

1
 Std Glucose Vs Benedict’s Quantitative reagent Indicator : Self
Contents in Burette reading Concordant
S.No. conical Value
flask Initial Final (ml)
(ml)

1.

2.

3.

Titration II
Estimation of Glucose
Standard Benedict’s Quantitative reagent Vs Unknown Glucose
Indicator : Self
Contents Burette Concordant
S.No. in conical reading Value
flask (ml)
(ml) Initial Final

1.

2.

3.

Calculation
100 of the unknown glucose solution contains = Standard value x 100
unknown value

2
QUALITATIVE ANALYSIS OF CARBOHYDRATES

PREPARATION OF REAGENTS

1. Molisch’s reagent
5% a naphthal in alcohol, i.e., 5g of anaphthal dissolved in 100ml of ethanol.
2. Iodine solution
0.005% in 3% KI, i.e., 3g of KI dissolved in 100ml water and then 5mg of iodine
is dissolved.
3. Benedict’s solution
17.3g of sodium citrate and 10g of sodium carbonate are dissolved in 75ml of
water. 1.73g of CuSO4.5H2O is dissolved in 20ml of water. Mix the CuSO4
solution with alkaline citrate with constant stirring, finally the whole volume is
made up to 100ml with water.
4. Barfoed’s reagent
13.3g of copper acetate in 200ml of water and add 2ml of glacial acetic acid.
5. Bial’s reagent
Dissolve 300mg of orcinol in 100ml of concentrated HCl.
6. Seliwanoff’s reagent
Dissolve 50g of resorcinol in 100ml of con.HCl in the ratio of 1:2.
7. Concentrated HCl
8. Concentrated H2SO4
9. Osazone Reagent
 Phenyl hydrazine hydrochloride
 Sodium acetate
 Acetic acid

PRINCIPLE OF REACTIONS

1. Molisch’s test
Con. H2SO4 dehydrates carbohydrates to form furfural and its derivatives. This
product combines with sulphonated a naphthal to give purple colour.
2. Iodine test
Iodine forms a coloured absorption complex with polysaccharides due to the
formation of micellae aggregate. Iodine will form a polysaccharide inclusion
complex.
3. Benedict’s test
Carbohydrates with a potential aldehyde or ketone group have reducing
property when placed in an alkaline solution. Cupric ions present in the
solution will be reduced to cuprous ion. This will give a red coloured
precipitate. Moreover, this test is more specific for reducing sugars.
4. Barfoed’ test

3
 Barfoed’s reagent is weakly acidic and it is only reduced by monosaccharides.
Prolonged boiling may hydrolyze the disaccharide
to give false positive test.
5. Bial’s test
When pentose is heated with con.HCl, furfural, which condenses with orcinol
in the prescence of ferric ion to give a blue green colour.
6. Seliwanoff’s test
Ketoses are dehydrated more rapidly than aldose to give a furfural derivatives,
which then condenses with resorcinol to form a red colour complex.
7. Mucic acid test
Monosaccharides are converted into respective dicarboxylic acid in the presence
of strong oxidizing agent con HNO3. Galactaric acid will form a colourless
broken glass piece shaped crystals.
8. Osazone test
Compounds containing aldehyde and keto groups form crystalline osazone with
phenyl hydrazine hydrochloride. Osazone crystals have characteristic shape and
melting point which helps in the identification of reducing sugar.

GENERAL PROCEDURE FOR QUALITATIVE ANALYSIS OF

CARBOHYDRATES

S.No. EXPERIMENT OBSERVATION INFERENCE

1. Molisch’s test
To 1ml of test solution, add 2 Violet coloured ring Prescence of carbohydrate.
drops of Molisch’s reagent. Then is formed at the
add con. H2SO4 carefully along the junction of the 2
sides of the test tube. layers.
2. Iodine test
To 1ml of the test solution, 2 (i) Deep blue colour Prescence of
drops of iodine is added and is obtained. polysaccharide.
observe the colour change. (ii)Dark brown Prescence of
colour polysaccharide.(Glycogen)
is obtained. Absence of
(ii)No characteristic polysaccharide.
colour change.
3. Benedict’s test
5ml of Benedict’s reagent is (i)Orange red Prescence of reducing
mixed with 1ml of test solution and precipitate is sugar.
the contents are boiled for a few obtained.
minutes. (ii)No characteristic Absence of reducing
colour change. sugar.
4. Barfoed’s test
To 2ml of test solution, 2ml of (i)Brick red Prescence of reducing
Barfoed’s reagent is added and precipitate is monosaccharide
boiled for 3 minutes and the colour obtained at the

4
 change is noted. bottom of test Absence of reducing
tube. monosaccharide.
(ii)No characteristic
colour change.

Bial’s test (i)Blue green colour Prescence of pentose

5. To 1ml of the test solution, 5ml is obtained. sugar.
of Bial’s reagent is added. The (ii)No characteristic Absence of pentose sugar.
contents are boiled and cooled. colour change.

6. Seliwanoff’s test
To 1ml of the test solution, 3ml (i)Cherry red colour Prescence of fructose.
of Seliwanoff’s reagent is added is obtained.
and the contents are boiled (ii)No characteristic Absence of fructose.
colour change.
7. Mucic acid test
1ml of test solution is mixed (i)Colourless Prescence of galactose or
with equal volume of con.HNO3, crystals formed. lactose.
then heated in boiling water bath The crystals are
for about an hour. observed through
a microscope.

Broken glass piece

shaped crystals are
observed
(ii)No characteristic Absence of galactose and
change. lactose.

8. Osazone test
To 1ml of the test solution, , (i)Yellow colour Prescence of fructose is
add 2-3 drops of glacial acetic acid, crystals are formed confirmed
followed by the addition of a pinch in 5 minutes, as
of phenyl hydrazine hydrochloride observed through a
and twice the amount of sodium microscope.
acetate and the contents are boiled

Haystack structure
form of fructosazone.
(ii) Yellow colour

5
crystals are formed Prescence of glucose is
in 5-10 minutes, as confirmed.
observed through a
microscope.

Haystack structure
form of glucosazone
(iii) Yellow colour
crystals are formed Prescence of galactose is
in 10 minutes. confirmed.

Broad fan structure

crystals of
galactosazone are
observed through
microscope.
(iv) Yellow colour
crystals are formed Prescence of Matose is
in 20-25 minutes, confirmed.

Sunflower shaped
crystals of
maltosazone are
observed through the
microscope.
(v)Yellow colour
crystals are formed Prescence of Xylose is
in 5-10 minutes, confirmed.

6
 scattered needles
shaped crystals of
Xylosazone are
observed through the
microscope.
(vi) Yellow colour
crystals are formed Prescence of Lactose is
in 20-30 minutes, confirmed.

Powderpuff shaped
crystals of
Lactosazone are
observed through the
microscope.

QUALITATIVE ANALYSIS OF GLUCOSE

S.No. EXPERIMENT OBSERVATION INFERENCE

1. Molisch’s test
To 1ml of test solution, add 2 Violet coloured ring Prescence of
drops of Molisch’s reagent. Then add is formed at the carbohydrate.
con.H2SO4 carefully along the sides of junction of the 2
the test tube. layers.
2. Iodine test
To 1ml of the test solution, 2 drops No characteristic Absence of
of iodine is added and observe the colour change. polysaccharide.
colour change.
3. Benedict’s test
5ml of Benedict’s reagent is mixed (i)Orange red Prescence of
with 1ml of test solution and the precipitate is reducing sugar.
contents are boiled for few minutes. obtained.
.
4. Barfoed’s test
To 2ml of test solution, 2ml of (i)Brick red Prescence of
Barfoed’s reagent is added and boiled precipitate is reducing

7
 for 3 minutes and the colour change is obtained at the monosaccharide
noted. bottom of test tube.

5. Bial’s test No characteristic Absence of

To 1ml of the test solution, 5ml of colour change. pentose sugar.
Bial’s reagent is added. The contents
are boiled and cooled.
6. Seliwanoff’s test
To 1ml of the test solution, 3ml of No characteristic Absence of
Seliwanoff’s reagent is added and the colour change. fructose.
contents are boiled.
7. Mucic acid test
1ml of test solution is mixed with No characteristic Absence of
equal volume of con.HNO3, then change. galactose and
heated in boiling water bath for about lactose.
an hour.
8. Osazone test
To 1ml of the test solution, , add Yellow colour Prescence of
2-3 drops of glacial acetic acid, crystals are formed in glucose is
followed by the addition of a pinch of 5-10 minutes, as confirmed.
phenyl hydrazine hydrochloride and observed through a
twice the amount of sodium acetate microscope.
and the contents are boiled

Haystack structure
form of glucosazone

QUALITATIVE ANALYSIS OF FRUCTOSE

S.No. EXPERIMENT OBSERVATION INFERENCE

1. Molisch’s test
To 1ml of test solution, add 2 Violet coloured ring Prescence of
drops of Molisch’s reagent. Then add is formed at the carbohydrate.
con.H2SO4 carefully along the sides of junction of the 2
the test tube. layers.

8
2. Iodine test No characteristic Absence of
To 1ml of the test solution, 2 drops colour change. polysaccharide.
of iodine is added and observe the
colour change.
3. Benedict’s test
5ml of Benedict’s reagent is mixed (i)Orange red Prescence of
with 1ml of test solution and the precipitate is reducing sugar.
contents are boiled for a few minutes. obtained.
.
4. Barfoed’s test
To 2ml of test solution, 2ml of (i)Brick red Prescence of
Barfoed’s reagent is added and boiled precipitate is reducing
for 3 minutes and the colour change is obtained at the monosaccharide
noted. bottom of test tube.
5. Bial’s test
To 1ml of the test solution, 5ml of No characteristic Absence of
Bial’s reagent is added. The contents colour change. pentose sugar.
are boiled and cooled.
6. Seliwanoff’s test
To 1ml of the test solution, 3ml of (i)Cherry red colour Prescence of
Seliwanoff’s reagent is added and the is obtained. fructose.
contents are boiled.
7. Mucic acid test
1ml of test solution is mixed with No characteristic Absence of
equal volume of con.HNO3, then change. galactose and
heated in boiling water bath for about lactose.
an hour.
8. Osazone test
To 1ml of the test solution, , add Yellow colour Prescence of
2-3 drops of glacial acetic acid, crystals formed in 5 fructose is
followed by the addition of a pinch of mins, as observed confirmed
phenyl hydrazine hydrochloride and through microscope.
twice the amount of sodium acetate
and the contents are boiled.

Haystack structure
form of fructosazone.

9
 QUALITATIVE ANALYSIS OF GALACTOSE

S.No. EXPERIMENT OBSERVATION INFERENCE

1. Molisch’s test
To 1ml of test solution, add 2 Violet coloured ring Prescence of
drops of Molisch’s reagent. Then add is formed at the carbohydrate.
con.H2SO4 carefully along the sides of junction of the 2
the test tube. layers.
2. Iodine test
To 1ml of the test solution, 2 drops No characteristic Absence of
of iodine is added and observe the colour change. polysaccharide.
colour change.
3. Benedict’s test
5ml of Benedict’s reagent is mixed (i)Orange red Prescence of
with 1ml of test solution and the precipitate is reducing sugar.
contents are boiled for few minutes. obtained.

4. Barfoed’s test
To 2ml of test solution, 2ml of (i)Brick red Prescence of
Barfoed’s reagent is added and boiled precipitate is reducing
for 3 minutes and the colour change is obtained at the monosaccharide
noted. bottom of test tube.

5. Bial’s test
To 1ml of the test solution, 5ml of No characteristic Abscence of
Bial’s reagent is added. The contents colour change. pentose sugar.
are boiled and cooled.
6. Seliwanoff’s test
To 1ml of the test solution, 3ml of No characteristic Absence of
Seliwanoff’s reagent is added and the colour change. fructose.
contents are boiled.
7. Mucic acid test
1ml of test solution is mixed with (i)Colourless crystals Prescence of
equal volume of con.HNO3, then formed. The galactose or
heated in boiling water bath for about crystals are lactose.
an hour. observed through a
microscope.

Broken glass piece

shaped crystals are
observed.

10
8. Osazone test
To 1ml of the test solution, , add Yellow colour Prescence of
2-3 drops of glacial acetic acid, crystals are formed in galactose is
followed by the addition of a pinch of 10 minutes, confirmed.
phenyl hydrazine hydrochloride and
twice the amount of sodium acetate
and the contents are boiled

Broad fan structure

crystals are observed
through microscope.

QUALITATIVE ANALYSIS OF XYLOSE

S.No. EXPERIMENT OBSERVATION INFERENCE

1. Molisch’s test
To 1ml of test solution, add 2 Violet coloured ring Prescence of
drops of Molisch’s reagent. Then add is formed at the carbohydrate.
con.H2SO4 carefully along the sides of junction of the 2
the test tube. layers.
2. Iodine test
To 1ml of the test solution, 2 drops No characteristic Absence of
of iodine is added and observe the colour change. polysaccharide.
colour change.
3. Benedict’s test
5ml of Benedict’s reagent is mixed (i)Orange red Prescence of
with 1ml of test solution and the precipitate is reducing sugar.
contents are boiled for few minutes. obtained.

4. Barfoed’s test
To 2ml of test solution, 2ml of Brick red Prescence of
Barfoed’s reagent is added and boiled precipitate is reducing
for 3 minutes and the colour change is obtained at the monosaccharide.
noted. bottom of test tube.
5. Bial’s test
To 1ml of the test solution, 5ml of Blue green colour Prescence of
Bial’s reagent is added. The contents is obtained. pentose sugar.
are boiled and cooled.
6. Seliwanoff’s test
To 1ml of the test solution, 3ml of No characteristic Absence of
Seliwanoff’s reagent is added and the colour change. fructose.
contents are boiled

11
7. Mucic acid test
1ml of test solution is mixed with No characteristic Absence of
equal volume of con.HNO3, then change. galactose and
heated in boiling water bath for about lactose.
an hour.
8. Osazone test
To 1ml of the test solution, , add (i)Yellow colour Prescence of
2-3 drops of glacial acetic acid, crystals are formed in Xylose is
followed by the addition of a pinch of 5-10 minutes, confirmed.
phenyl hydrazine hydrochloride and
twice the amount of sodium acetate
and the contents are boiled

Random needle
shaped crystals are
observed through a
microscope.

QUALITATIVE ANALYSIS OF MALTOSE

S.No. EXPERIMENT OBSERVATION INFERENCE

1. Molisch’s test
To 1ml of test solution, add 2 Violet coloured ring Prescence of
drops of Molisch’s reagent. Then add is formed at the carbohydrate.
con.H2SO4 carefully along the sides of junction of the 2
the test tube. layers.
2. Iodine test
To 1ml of the test solution, 2 drops No characteristic Absence of
of iodine is added and observe the colour change. polysaccharide.
colour change.
3. Benedict’s test
5ml of Benedict’s reagent is mixed (i)Orange red Prescence of
with 1ml of test solution and the precipitate is reducing sugar.
contents are boiled for few minutes. obtained.

4. Barfoed’s test
To 2ml of test solution, 2ml of No characteristic Absence of
Barfoed’s reagent is added and boiled colour change. reducing
for 3 minutes and the colour change is monosaccharide.
noted.
5. Bial’s test

12
 To 1ml of the test solution, 5ml of No characteristic Absence of
Bial’s reagent is added. The contents colour change. pentose sugar.
are boiled and cooled.

6. Seliwanoff’s test
To 1ml of the test solution, 3ml of No characteristic Absence of
Seliwanoff’s reagent is added and the colour change. fructose.
contents are boiled.
7. Mucic acid test
1ml of test solution is mixed with No characteristic Absence of
equal volume of con.HNO3, then change. galactose and
heated in boiling water bath for about lactose.
an hour.
8. Osazone test
To 1ml of the test solution, , add (i)Yellow colour Prescence of
2-3 drops of glacial acetic acid, crystals are formed in Maltose is
followed by the addition of a pinch of 20-25 minutes, confirmed.
phenyl hydrazine hydrochloride and
twice the amount of sodium acetate
and the contents are boiled

Sunflower shaped
crystals are viewed
through microscope.

QUALITATIVE ANALYSIS OF LACTOSE

S.No. EXPERIMENT OBSERVATION INFERENCE

1. Molisch’s test
To 1ml of test solution, add 2 Violet coloured ring Prescence of
drops of Molisch’s reagent. Then add is formed at the carbohydrate.
con.H2SO4 carefully along the sides of junction of the 2
the test tube. layers.
2. Iodine test
To 1ml of the test solution, 2 drops No characteristic Absence of
of iodine is added and observe the colour change. polysaccharide.
colour change.
3. Benedict’s test
5ml of Benedict’s reagent is mixed Orange red Prescence of
with 1ml of test solution and the precipitate is reducing sugar.
contents are boiled for a few minutes. obtained.

13
4. Barfoed’s test No characteristic Abscence of
To 2ml of test solution, 2ml of change. reducing
Barfoed’s reagent is added and boiled Monosaccharide.
for 3 minutes and the colour change is
noted.

5. Bial’s test
To 1ml of the test solution, 5ml of Blue green colour Prescence of
Bial’s reagent is added. The contents is obtained. pentose sugar.
are boiled and cooled.
6. Seliwanoff’s test
To 1ml of the test solution, 3ml of No characteristic Absence of
Seliwanoff’s reagent is added and the colour change. fructose.
contents are boiled.
7. Mucic acid test
1ml of test solution is mixed with (i)Colourless crystals Prescence of
equal volume of con.HNO3, then formed. The lactose.
heated in boiling water bath for about crystals are
an hour. observed
through a
microscope.

Broken glass piece

shaped crystals are
observed

8. Osazone test
To 1ml of the test solution, , add (i)Yellow colour Prescence of
2-3 drops of glacial acetic acid, crystals are formed in Lactose is
followed by the addition of a pinch of 20 –30 minutes confirmed.
phenyl hydrazine hydrochloride and
twice the amount of sodium acetate
and the contents are boiled

Powder puff or
hedgehog shaped as
observed through a
microscope.

14
 QUALITATIVE ANALYSIS OF SUCROSE

S.No. EXPERIMENT OBSERVATION INFERENCE

1. Molisch’s test
To 1ml of test solution, add 2 Violet coloured ring Prescence of
drops of Molisch’s reagent. Then is formed at the carbohydrate.
add con.H2SO4 carefully along the junction of the 2
sides of the test tube. layers.
2. Iodine test
To 1ml of the test solution, 2 No characteristic Absence of
drops of iodine is added and then colour change. polysaccharide.
observe the colour change.
3. Benedict’s test
5ml of Benedict’s reagent is No characteristic Absence of
mixed with 1ml of test solution and colour change. reducing sugar.
the contents are boiled for a few
minutes.

4. Hydrolysis
The non reducing sugar is converted into the reducing sugar by the
hydrolyzing the substance by adding few drops of con.HCl. Then it is heated for
10 minutes in boiling water bath. It is neutralized by the sodium carbonate
solution until the effervescence ceases. Then, reduction reactions are performed,
by using the hydrolyzed solution.
5. Benedict’s test
5ml of Benedict’s reagent is Orange red Prescence of
mixed with 1ml of test solution and precipitate is reducing sugar
the contents are boiled for few obtained. in the
minutes. hydrolysate.
6. Barfoed’s test
To 2ml of test solution, 2ml of Brick red Prescence of
Barfoed’s reagent is added and boiled precipitate is reducing
for 3 minutes and the colour change obtained. monosaccharide.
is noted.
7. Bial’s test
To 1ml of the test solution, 5ml of No characteristic Absence of
Bial’s reagent is added. The contents colour change. pentose sugar.
are boiled and cooled.

8. Seliwanoff’s test
To 1ml of the test solution, 3ml of (i)Cherry red colour Prescence of
Seliwanoff’s reagent is added and the is obtained. fructose.
contents are boiled.
9. Mucic acid test
1ml of test solution is mixed with No characteristic Absence of

15
 equal volume of con.HNO3, then change. galactose and
heated in boiling water bath for about lactose.
an hour.
10. Osazone test
To 1ml of the test solution, , add Yellow colour This confirms
2-3 drops of glacial acetic acid, crystals are formed in the prescence
followed by the addition of a pinch of 5-10 minutes, of glucose and
phenyl hydrazine hydrochloride and fructose in the
twice the amount of sodium acetate hydrolysate.
and the contents are boiled

Haystack crystals
observed through a
microscope.

QUALITATIVE ANALYSIS OF STARCH

S.No. EXPERIMENT OBSERVATION INFERENCE

1. Molisch’s test
To 1ml of test solution, add 2 Violet coloured ring Prescence of
drops of Molisch’s reagent. Then is formed at the carbohydrate.
add con.H2SO4 carefully along the junction of the 2
sides of the test tube. layers.
2. Iodine test
To 1ml of the test solution, 2 Deep blue colour Prescence of
drops of iodine is added and then is obtained polysaccharide.
observe the colour change.
3. Hydrolysis
The non reducing sugar is converted into the reducing sugar by the
hydrolyzing the substance by adding few drops of con.HCl. Then it is heated for
10 minutes in boiling water bath. It is neutralized by the sodium carbonate
solution until the effervescence ceases. Then, reduction reactions are performed,
by using the hydrolyzed solution.
4. Benedict’s test
5ml of Benedict’s reagent is Orange red Prescence of
mixed with 1ml of test solution and precipitate is reducing sugar.
the contents are boiled for few obtained.
minutes.
5. Barfoed’s test
To 2ml of test solution, 2ml of Brick red Prescence of
Barfoed’s reagent is added and boiled precipitate is reducing
for 3 minutes and the colour change obtained at the monosaccharide.
is noted. bottom of test tube.

16
6. Bial’s test No characteristic Absence of
To 1ml of the test solution, 5ml of colour change. pentose sugar.
Bial’s reagent is added. The contents
are boiled and cooled.

7. Seliwanoff’s test
To 1ml of the test solution, 3ml of No characteristic Absence of
Seliwanoff’s reagent is added and the colour change. fructose.
contents are boiled
8. Mucic acid test
1ml of test solution is mixed with No characteristic Absence of
equal volume of con.HNO3, then change. galactose and
heated in boiling water bath for about lactose.
an hour.
9. Osazone test Presence of
To 1ml of the test solution, , add Yellow colour glucose in the
2-3 drops of glacial acetic acid, crystals are formed in hydrolysate is
followed by the addition of a pinch of 5-10 minutes, as confirmed.
phenyl hydrazine hydrochloride and observed through a
twice the amount of sodium acetate microscope.
and the contents are boiled.

Haystack shaped
crystals.

QUALITATIVE ANALYSIS OF GLYCOGEN

S.No. EXPERIMENT OBSERVATION INFERENCE

1. Molisch’s test
To 1ml of test solution, add 2 Violet coloured ring Prescence of
drops of Molisch’s reagent. Then is formed at the carbohydrate.
add con.H2SO4 carefully along the junction of the 2
sides of the test tube. layers.
2. Iodine test
To 1ml of the test solution, 2 Dark brown colour is Prescence of
drops of iodine is added and then obtained polysaccharide.
observe the colour change. (GLYCOGEN)

17
3. Hydrolysis
The non reducing sugar is converted into the reducing sugar by the
hydrolyzing the substance by adding few drops of con.HCl. Then it is heated for
10 minutes in boiling water bath. It is neutralized by the sodium carbonate
solution until the effervescence ceases. Then, reduction reactions are performed,
by using the hydrolyzed solution.
4. Benedict’s test
5ml of Benedict’s reagent is Orange red Prescence of
mixed with 1ml of test solution and precipitate is reducing sugar.
the contents are boiled for few obtained.
minutes.
5. Barfoed’s test
To 2ml of test solution, 2ml of Brick red Prescence of
Barfoed’s reagent is added and boiled precipitate is reducing
for 3 minutes and the colour change obtained at the monosaccharide.
is noted. bottom of test tube.
6. Bial’s test
To 1ml of the test solution, 5ml of No characteristic Absence of
Bial’s reagent is added. The contents colour change. pentose sugar.
are boiled and cooled.

7. Seliwanoff’s test
To 1ml of the test solution, 3ml of No characteristic Absence of
Seliwanoff’s reagent is added and the colour change. fructose.
contents are boiled
8. Mucic acid test
1ml of test solution is mixed with No characteristic Absence of
equal volume of con.HNO3, then change. galactose and
heated in boiling water bath for about lactose.
an hour.

9. Osazone test Presence of

To 1ml of the test solution, , add Yellow colour glucose in the
2-3 drops of glacial acetic acid, crystals are formed in hydrolysate is
followed by the addition of a pinch of 5-10 minutes, as confirmed.
phenyl hydrazine hydrochloride and observed through a
twice the amount of sodium acetate microscope.
and the contents are boiled.

Haystack shaped
crystals.
.

18
 GENERAL REACTIONS OF PROTEIN (EGG PROTEIN)

S.N0 EXPERIMENT OBSERVATION INFERENCE

1) Solubility:
The solubility of
the given substance is
observed with the follow-
ing.

a) Water Colloidal solution is formed. Presence of protein.

b) Dilute NaOH solution Partially soluble. Presence of protein.

2) Precipitation by neutral
salt solution:
To 1ml of the
substance add equal volume White precipitate is formed. Presence of Globulin.
of a saturated solution of
Ammonium sulphate(half-
saturation).
The solution is saturated
with ammonium sulphate White precipitate is formed. Presence of Albumin.
(full saturation).
3) Precipitation by heavy
Metals:
To 1ml of the
add equal volume of 5% White precipitate is formed. Presence of protein.
Mercuric nitrate.
4) Precipitation by alcohol:
To 1ml of the
substance add equal volume White precipitate is formed. Presence of protein.
of alcohol.
5) Heat coagulation:
About 1ml of the Cloudy white precipitate is formed Presence of protein.
substance is taken in clean by coagulation.
test tube and heated.
6) Biuret test:
To 1ml of the
substance add few drops of
Biuret reagent. Purple colour is formed. Presence of protein.
7) Ninhydrin test:
To 1ml of the A purple colour is obtained. Presence of amino acid
test solution add few drops in the protein.
of Ninhydrin reagent and
heat for 2 minutes.

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8) Xanthoprotic test:
To 1ml of test Yellow colour is formed after the Presence of aromatic
solution add few drops of addition of conc. Nitric acid and Amino acid in the
conc. nitric acid and heat it. this turns red on the addition Protein.
Cool it. Then add few drops of NaOH.
of 40% NaOH.
9) Pauly’s test:
To 1ml of test Deep blue colour dye is obtained. Presence of Tyrosine
solution add few drops of and histidine units in
1% sulphanilic acid and protein.
Cool it in an ice bath. Then
add 1ml of 5% NaNO2 and
1ml of 1% Na2 CO3.
10) Millon’s test:
To 1ml of test Red colour is obtained. Presence of phenolic
solution add Millon’s group containing amino
reagent and heat it for few acid (tyrosine) in the
minutes. protein.
11) Morner’s test:
To 1ml of the Green colour is obtained.
test solution add Millons Presence of Tyrosine in
Reagent and heat it in a the protein.
boiling water bath.
12) Folin’s phenol test:
To 1ml of the Blue colour is obtained.
test solution add equal Presence of Tyrosine in
volume of Folin’s reagent the protein.
followed by the addition of
1% of Na2 CO3.
13) Aldehyde test:
To 1ml of the test A violet colour ring is formed at
solution add 2-3 drops of 1% the junction of the two layers. Presence of Tryptophan
HCHO and then care- in the protein.
fully add few drops of conc.
H2SO4 along the sides of the
test tube.
14) Ehrlisch’s test:
To 1ml of the test Pinkish red colour is obtained.
solution add few drops of Presence of Tryptophan
Ehrlisch’s reagent and heat it in the protein.
in a boiling water bath.
15) Sakaguchi’s test:
To 2ml of test solution Red colour is obtained.
add few drops of alpha-
naphthol in alcohol followed Presence of Arginine in
by the addition of 1ml of the protein.

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 20% NaOH and a few drops
of Bromine water.
16) Sulphur test:
To 1ml of test solution Dirty black precipitate is obtained. Presence of Cysteine in
add few drops of 45% NaOH the protein.
and boil it for 2 minutes cool
it, then add lead acetate.
17) Sodium nitroprusside test:
To 2ml of test solution Reddish purple colour is obtained. Presence of Methionine
add few drops of 20% NaOH in the protein.
.Then add 1ml of sodium –
nitroprusside followed by
the addition of 1.5ml of 1%
Glycine.Boil it for few
minutes. Then add 1ml of
6N HCl.
18) Molisch’s test:
To 1ml of the substance Violet colour ring is formed at the Presence of
add few drops of Molisch’s junction of two layers. Carbohydrate unit in the
reagent then add Conc.sul- protein.
phuric acid along the sides
of the test tubes.

Results:
The given protein (egg protein) contains the following,
Arginine, Tyrosine, Tryptophan, Cysteine, Methionine
and Carbohydrate.

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