Undergraduate First Year Practicals Manual

RAMAKRISHNA MISSION VIVEKANANDA COLLEGE
EVENING COLLEGE, CHENNAI-4

DEPARTMENT OF BIOCHEMISTRY
Quantitative Analysis
Expt 1:Estimation of Amino Acid by Sorenson’s Formol Titration
AIM:
To estimate the amount of amino acid present in the whole of the given solution.
PRINCIPLE:
The acid group present in the glycine can be titrated with NaOH. It is not easy
in this case because the amino group present will interfere at the end point. To prevent
it excess of formaldehyde is used by which the amino group is blocked by the
formation of methylene glycine. Then it is titrated with NaOH using phenolphthalein
indicator.
REAGENTS REQUIRED:
(i) Standard oxalic acid 0.1 N
(ii) Link solution NaOH
(iii) Formaldehyde
(iv) Phenolphthalein indicator
(v) Amino acid solution
PROCEDURE:
Standardisation of NaOH
Rinse a clean burette first with distilled water and then with the link solution of
NaOH. Rinse a clean 20ml pipette first with distilled water and then with the given
standard oxalic acid. Pipette out 20ml of oxalic acid into a clean conical flask. Add a
few drops of phenolphthalein indicator and titrate against NaOH taken in the burette. The
end point is the appearance of permanent pale pink colour. Repeat the titration for
concordant values.
Estimation of Amino acid

Make up the given amino acid solution with distilled water to 100ml in a
volumetric flask, observing the usual precautions. Shake the solution well for uniform
concentration. Pipette out 20ml of amino acid solution into a clean conical flask. Add
5 ml of HCHO and keep it for 2 minutes. Then titrate it against the NaOH solution taken
in the burette. Phenolphthalein is used as the indicator. The end point is the appearance
of permanent pale pink colour. Repeat the titration for concordant values.
Blank titration
Pipette out 20ml of distilled water in a clean conical flask. Add 5ml of HCHO
and keep it aside for a few minutes. Add 1-2 drops of phenolphthalein indicator. Titrate
it against the NaOH solution taken in the burette. The end point is the appearance of
permanent pale pink colour. Repeat the titration for concordant values.
Result:
The amount of amino acid present in the whole of the given solution = _______ g
EXPT 1
Titration I
Standardisation of NaOH
Concentration of Oxalic acid = 0.1 N
Standard oxalic acid Vs NaOH

Volume of Burette reading Concordant
S.No. Oxalic acid Value
(ml) Initial Final (ml)
1.
2.
3.
Calculation
Volume of oxalic acid, V1 = ml
Normality of oxalic acid, N1 = N
Volume of NaOH, V2 = ml
Normality of NaOH, N2 = N
Titration II
Estimation of amino acid
Amino acid + HCHO Vs NaOH

Contents in Burette reading Concordant
S.No. conical Value
flask Initial Final (ml)
1.
2.
3.
Calculation
Volume of NaOH, (Test value) = ml
Volume of NaOH, (Blank value) = ml
Volume of NaOH used to titrate amino acid = Test value – Blank value
= ml
Titration III
Blank titration
Standard NaOH Vs HCHO + H2O

S.No. conical Value
1.
2.
3.
Calculation
Volume of amino acid, V1 = ml
Normality of amino acid, N1 = N
Volume of NaOH, V2 = ml
Normality of NaOH, N2 = N
Strength of amino acid = V(NaOH) x N(NaOH) = V(amino acid) x N(amino acid)
Amount of amino acid present in 100ml of the given solution
= Equivalent weight x Normality x 100
1000
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
Std Glucose Vs Benedict’s Quantitative reagent Indicator : Self

S.No. conical Value
(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
Expt: 3 Estimation of Ascorbic acid by Titrimetric Method
AIM:
To estimate the amount of ascorbic acid present in the given solution.
PRINCIPLE:
Ascorbic acid is a strong reducing agent. It gets oxidized to dehydro ascorbic acid
by 2, 6 dichlorophenol indophenol dye. At the same time the dye gets reduced to a
colourless compound. So the reaction with end point can easily be determined.
REAGENTS REQUIRED
(i)Std Indophenol solution : 42mg of sodium bicarbonate and 52mg of
2, 6 dichlorophenol indophenol were dissolved in 50ml of water and diluted to
200ml. The solution was filtered and stored.
(ii)Std Ascorbic acid : 50mg ascorbic acid was dissolved and made up to 250ml
using 0.6% oxalic acid.
(iii)Preparation of test solution : Given solution was made up to 100ml with 0.6%
oxalic acid.
PROCEDURE:
Titration I: standardisation of dye

The burette was rinsed and filled up to the convenient mark with dye solution.
10ml of std ascorbic acid solution was pipetted out into a clean conical flask. It was
titrated against the dye taken in the burette. The end point was the appearance of
permanent pale pink colour which remains for 10 minutes. The titrations were repeated
for concordant value. The dye equivalent of std ascorbic acid was calculated.
Titration II: Estimation of Ascorbic acid

10ml of made up solution was pipetted out into a clean conical flask. It was
titrated against the dye taken in the burette, till the permanent pale pink colour was
obtained. The titrations were repeated for concordant value.
Using the dye equivalent of ascorbic acid, the amount of ascorbic acid in the
given solution was calculated.
RESULT:
The amount of ascorbic acid present in 100ml of given solution=_______mg
EXPT 3
Titration I
Standardisation of dye
Standard Ascorbic acid Vs Dye Indicator : Self

Volume of Burette Concordant
S.No. Ascorbic reading Value
acid (ml)
(ml) Initial Final
(ml) (ml)
1.
2.
3.
Concentration of standard
= 100µg/ml
Titration II
Estimation of Ascorbic acid
Standardised dye Vs Ascorbic acid Indicator : Self
S.No. Unknown reading Value
ascorbic (ml)
acid Initial Final
(ml) (ml) (ml)
1.
2.
3.
Expt 4 : Determination of acid number of an edible oil
AIM:
To determine the acid number of the given edible oil.
PRINCIPLE:
The Acid number is defined as the number of milligram of KOH required to
neutralize the free fatty acid present in one gram of oil or fat.
Fat may become rancid during the storage for a long time. Fat or oil are
hydrolysed by micro organisms with the formation of free fatty acids. The amount of
free fatty acid present in the oil in the indicator of the age and quality of that oil. Thus
the high acid number will indicate that the oil is old and rancid.
REAGENTS REQUIRED:
(i)Oxalic acid (0.1N)
(ii)KOH
(iii)Fat solvent (Ethanol and ether in 1:1 ratio)
(iv)Phenolphthalein
PROCEDURE:
Titration I: Standardisation of KOH

10ml of the standard oxalic acid (0.1N) is pipetted out into a clean conical flask.
Add 2 drops of phenolphthalein indicator and titrate against KOH taken in the burette
till the appearance of permanent pale pink colour. Repeat the titration for concordant
values.
Titration II: Determination of Acid number

Weigh around 1g of the oil and transfer into a clean conical flask. Add 20ml of
lipid solvent to dissolve the oil and shake well. Add a few drops of phenolphthalein
indicator and titrate against standardized KOH taken in the burette. End point is the
appearance of permanent pale pink colour.
Titration III: Blank titration

Pipette out 20ml of lipid solvent into a clean conical flask, add 2 drops of
phenolphthalein indicator and titrate it against KOH taken in the burette. The end point
is the appearance of permanent pale pink colour. Repeat the titration for concordant
values.
RESULT:
Acid number of given edible oil is __________mg
EXPT 4
Titration I
Standardisation of KOH
Standard oxalic acid Vs KOH Indicator : Phenolphthalein
S.No. unknown reading Value
oxalic (ml)
acid Initial Final
(ml) (ml) (ml)
1.
2.
3.
Calculation:
Volume of oxalic acid, V1 = ml
Normality of oxalic acid, N1 = N
Volume of KOH, V2 = ml
Normality of KOH, N2 = N
V1x N1 = V2x N2
Titration II
Determination of acid number
Standard KOH Vs Oil Indicator : Phenolphthalein
Contents of Burette Concordant
S.No. the conical reading Value
flask (ml)
(ml) Initial Final
(ml) (ml)
1.
Volume of KOH consumed of

oil = Test value – Blank value = ml
Titration III
Blank titration
Determination of acid number
Standard KOH Vs lipid solvent Indicator : Phenolphthalein
S.No. lipid Reading Value
solvent (ml)
(ml) Initial Final
(ml) (ml)
1.
2.
3.
Acid number = Strength of
KOH x Volume of KOH consumed x Equivalent weight of KOH x 1000
Weight of oil x 1000
Expt 5 :Determination of Saponification number of an edible oil
AIM:
To determine the saponification number of the given edible oil.
PRINCIPLE:
Saponification value is defined as the number of milligram of KOH required to
neutralise the fatty acid resulting in the complete hydrolysis of 1g of oil or fat. It gives
an indication of the nature of the fatty acid present in the fat.
On refluxing the fat or oil with alkali, the ester of glycerol is hydrolysed into
glycerol and potassium salt of free fatty acid.
If the length of the free fatty acid chain is longer, the acid is lesser in the utilization of
KOH is less and vice-versa.
A weighed amount of fat is saponified with known amount of alcoholic KOH,
excess alkali is back titrated with standardized acid and the saponification value of the
oil is determined.
REAGENTS REQUIRED:
(i)Alcoholic KOH 0.5N
(ii)Hydrochloric acid 0.5N
(iii)Sodium carbonate 0.5N
(iv)Phenolphthalein
(v)Methyl orange
PROCEDURE
Titration I: Standardisation of Hydrochloric acid
10ml of standard sodium carbonate is pipetted out into a clean conical flask. Add
2 drops of methyl orange indicator and titrate it against HCl taken in the burette. End
point is the change of yellow to red colour. Repeat the titration for concordant values.
Titration II: Determination of saponification value

Weigh around 1g of oil and transfer into a clean conical flask. Add 5ml of lipid
solvent and 25ml of alcoholic KOH and heat in the water bath for about 30 minutes.
Add two drops of phenolphthalein indicator and titrate it against standardised HCl taken
in the burette. End point is the disappearance of pink colour.
Blank titration:
Pipette out 20 ml of alcoholic KOH and add 5ml of lipid solvent, in a clean
conical flask. Add 2 drops of phenolphthalein indicator, and titrate against HCl taken in
the burette. The end point is the disappearance of permanent pale pink colour. Repeat
the titration for concordant values.
Result:
The saponification number of the given edible oil is ___________mg
EXPT 5
Titration I
Standardisation of hydrochloric acid
Standard Sodium carbonate Vs HCl Indicator : Methyl orange
S.No. sodium reading Value
carbonate (ml)
(ml) Initial Final
1.
2.
3.
Calculation
Volume of sodium carbonate, V1 = ml
Normality of sodium carbonate, N1 = N
Volume of HCl, V2 = ml
Normality of HCl, N2 = N
V1x N1 = V2x N2
Titration II
Determination of saponification value
Oil+Lipid solvent+KOH Vs HCl Indicator : Phenolphthalein

Contents of Burette Concordant Volume of HCl
S.No. the conical Reading Value (ml)
flask (ml)
(ml) Initial Final
1.
2.
Saponification = Amount of KOH x Equivalent weight x Normality

2.542 x 1000
Titration III
Blank titration
Lipid solvent+KOH Vs HCl Indicator : Phenolphthalein

Contents of Burette Concordant
S.No. the conical Reading Value
flask (ml)
(ml) Initial Final
1.
2.
3.
Expt 6: Estimation of chloride by Mohr’s method

AIM:
To estimate the amount of chloride present in the given solution.
PRINCIPLE:
When a solution of silver nitrate is added to a solution of sodium chloride, white
precipitate of silver chloride is formed. Potassium chromate is used as an indicator and
produces the silver chromate. The end point is the change from yellow to reddish brown
due to the formation of silver chromate.
REAGENTS USED:
(i)NaCl (0.29g of sodium chloride dissolved in 100ml of water in a 100ml

standard flask.
(ii)Silver nitrate (0.85g of silver nitrate dissolved in 100ml of water)
(iii)Indicator : Potassium chromate solution (2%)
PROCEDURE:
Titration I: Standardisation of silver nitrate

Pipette out 10ml of standard sodium chloride into a clean conical flask. Add a
drop of potassium chromate indicator and titrate against the silver nitrate solution taken
in the burette. The end point is the appearance of permanent reddish brown precipitate.
Note the reading. Repeat the titration for concordant values.
Titration II: Estimation of chloride

Make up the given solution upto 100ml in a standard flask. Pipette out 10ml of
the unknown solution into a clean conical flask. Add 1 drop of potassium chromate
indicator and titrate against silver nitrate solution taken in the burette. The end point is
the appearance of reddish brown precipitate. Repeat the titration for concordant values.
RESULT:
Amount of chloride present in 100ml of the given solution ________g
Expt : 6
Titration I
Standardisation of Silver nitrate
Standard AgNO3Vs unknown Chloride Indicator : Potassium chromate
S.No. NaCl reading Value
(ml) (ml)
Initial Final
1.
2.
3.
Calculation
Volume of NaCl, V1 = ml
Normality of NaCl, N1 = N
Volume of HCl, V2 = ml
Normality of HCl, N2 = N
V1x N1 = V2x N2
Titration II
Estimation of Chloride
Standard AgNO3Vs unknown Chloride Indicator : Potassium chromate
S.No. chloride reading Value
(ml) (ml)
Initial Final
1.
2.
3.
Calculation
Volume of chloride, V1 = ml
Normality of chloride, N1 = N
Volume of silver nitrate, V2 = ml
Normality of silver nitrate, N2 = N
V1x N1 = V2x N2
Amount of chloride present in 100ml of given solution=Equivalent weight x Normality x
Weight/litre
Expt 7 : Estimation of Chloride by Volhard’s method
AIM:
To estimate the amount of chloride present in the given solution.
PRINCIPLE:
Silver nitrate reacts with potassium thiocyanate to form a precipitate of silver
thiocyanate.
When all silver nitrate has been converted to silver thiocyanate, this results in
dark brown colour due to the formation of ferric thiocyanate in the prescence of ferric
alum. From this the chloride equivalent to silver nitrate in terms of potassium
thiocynate, can be calculated.
REAGENTS USED
(i)Silver nitrate 0.05N
(ii)Potassium thiocyanate 0.05N
(iii)Ferric alum
(iv)6N nitric acid
PROCEDURE:
Titration I: Standardisation of potassium thiocyanate

The link solution potassium thiocyanate is taken in the burette. 10ml of standard
0.05N silver nitrate solution is pipetted out into a clean conical flask and 10ml of 6N
nitric acid is added and titrated against the thiocyanate solution taken in the burette. The
indicator is 1ml of ferric alum. The end point is the appearance of permanent reddish
tinge. Repeat the titration for concordant values.
Titration II: Estimation of chloride

10ml of unknown is taken in the conical flask and to this, 10ml of 6N nitric acid,
10ml of silver nitrate and 1ml of ferric alum are added. Then it is titrated against
potassium thiocyanate taken in the burette. The end point is the appearance of
permanent reddish tinge. Repeat the titration for concordant values.
RESULT:
Amount of chloride present in the given solution is ___________g
Expt 7
Titration I
Standardisation of Potassium thiocyanate
Indicator : Ferric alum
Burette Concordant
S.No. Contents in reading Value
the conical (ml)
flask Initial Final
(ml)
1.
2.
3.
Calculation
Volume of sodium chloride, V1 = ml
Normality of silver nitrate in terms of potassium thiocyanate, N1 = N
Volume of unreacted silver nitrate, V2 = ml
-
Normality of unreacted silver nitrate taken by Cl , N2 = N
V1x N1 = V2x N2
Titration II
Estimation of chloride
Indicator : Ferric alum
Contents in Burette Concordant
flask (ml)
(ml) Initial Final
1.
2.
3.
Amount of chloride present in
100ml of given solution=Equivalent weight x Weight/litre
Expt 8 : Determination of iodine number of an edible oil by Hanu’s method
AIM:
To determine the iodine number of the given oil by Hanu’s method.
PRINCIPLE:
Iodine number is defined as the number of grams of iodine taken up by 100g of
oil or fat. In this case, addition reaction takes place across the double bonds of
unsaturated fatty acids present in the fat by the addition of a halogen, such as iodine.
Thus, the iodine number gives the indication of the degree of unsaturation of fats.
Iodine value is directly proportional to the degree of unsaturation. Determination
of iodine number is used for the assessment of its purity.
In Hanu’s method, the oil is treated with Hanu’s reagent. (Iodine bromide in
chloroform) for a period of time. The unreacted iodine is titrated against standardized
thiosulphate solution.
REAGENTS REQUIRED:
(i)Sodium thiosulphate 0.1N
(ii)Potassium dichromate 0.1N
(iii)Potassium iodide 10%
(iv)Hanu’s solution(With the aid of heat 13.2g of iodine was dissolved in 1litre of glacial
acetic acid and to this, 3ml of bromine water was added and mixed thoroughly.)
(v)Hydrochloric acid – 20%
(vi)Starch : Indicator
PROCEDURE:
TitrationI: Standardisation of sodium thiosulphate
Pipette out 10ml of standard (0.1N) potassium dichromate in a clean conical
flask, add 10ml of 10% potassium iodide and 5 ml of 20% hydrochloric acid followed
by 75ml of water. This solution was titrated against thiosulphate solutiontaken in the
burette till the pale yellow colour is formed. Then add a few drops of starch indicator
and titrated with thio solution till the blue colour disappears. Repeat the titrations for
concordant values.
Titration II: Determination of iodine number
1g of the oil is weighed and transferred into a clean conical flask and incubate for
about 30 minutes, remove the stopper and add 10ml of 10% potassium iodide follwed by
80ml of water. The liberated iodine is titrated with standardized thiosulphate till the
pale yellow colour is formed. Then add a few drops of starch indicator, and the titration
is continued till the disappearance of the blue colour. The contents present in the flask
is mixed up thoroughly while the titration is carried. A duplicate is also conducted by
the same method.
The blank titration is also performed without the oil.
RESULT: Iodine Number of the given edible oil is ___________g.
Expt 8
Titration I
Standardisation of sodium thiosulphate
Indicator : Starch
flask (ml)
(ml) Initial Final
1.
2.
3.
Calculation
Volume of potassium dichromate, V1 = ml
Normality of potassium dichromate, N1 = N
Volume of thiocyanate, V2 = ml
Normality of thiocyanate, N2 = N
V1x N1 = V2x N2
Titration II
Determination of iodine number
Indicator : Starch
flask (ml)
(ml) Initial Final
(ml) (ml)
1.
Volume of iodine not

consumed by oil = Volume of Hanu’s solution that reacted with sodium
thiosulphate .
QUALITATIVE ANALYSIS OF CARBOHYDRATES
PREPARATION OF REAGENTS
1. Molisch’s reagent
5% α naphthal in alcohol, i.e., 5g of αnaphthal 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 α
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
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 Violet coloured ring Prescence of carbohydrate.
2 drops of Molisch’s reagent. is formed at the
Then add con. H2SO4 carefully junction of the 2
along the sides of the test tube. layers.
2. Iodine test
To 1ml of the test solution, (i) Deep blue colour Prescence of
2 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 precipitate is sugar.
and the contents are boiled for obtained.
a few minutes. (ii)No characteristic Absence of reducing
colour change. sugar.
4. Barfoed’s test
To 2ml of test solution, 2ml (i)Brick red Prescence of reducing
of Barfoed’s reagent is added precipitate is monosaccharide
and boiled for 3 minutes and obtained at the
the colour 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, is obtained. sugar.
5ml of Bial’s reagent is added. (ii)No characteristic Absence of pentose sugar.
The contents are boiled and colour change.
cooled.
6. Seliwanoff’s test
To 1ml of the test solution, (i)Cherry red colour Prescence of fructose.
3ml of Seliwanoff’s reagent is is obtained.
added and the contents are (ii)No characteristic Absence of fructose.
boiled colour change.
7. Mucic acid test
1ml of test solution is (i)Colourless Prescence of galactose or
mixed with equal volume of crystals formed. lactose.
con.HNO3, then heated in The crystals are
boiling water bath for about an observed through
hour. a microscope.
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.
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.
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.
1) Millon’s Reagent:
Dissolve 15g of Mercuric Sulphate in 100ml of 15% of Sulphuric
acid.
2) Sulphanilic Acid:
1g of Sulphanilic Acid in 100ml of 10% HCl (1% solution).
3) Sodium Nitrate(5%):
5g of NaNO2 is dissolved in 100ml of water.
4) Sodium Carbonate: (1%)

Dissolve 1g of Na2CO3 in 100ml of water (1% solution)
5) Ehrlisch’s Reagent:
Dissolve 10g of p-dimethyl amino benzaldehyde in 100ml of 10%
HCl (10%solution).
6) α-Naphthol:
1g of α-Naphthol is dissolved in 100ml of alcohol.
7) Sodium hydroxide (40%)

40g of NaOH is dissolved in 100ml of water (40% solution)
8) Bromine Water:
Few drops of Bromine in 100ml of water.
9) Lead Acetate(1%):
1g of lead dissolved in 100ml of water.
10) Sodium hydroxide (1%)

1g of NaOH dissolved in 100 ml of water. (1% solution).
11) Con.Sulphuric acid
12) Glacial acetic acid
1) Ninhydrin Test:
Ninhydrin is a powerful oxidising agent reacts with α-amino-
acids,between pH 4-8 to give a purple color complex.Ninhydrin reagent is reduced
to hydrindantin during reaction with α-amino-acids.The amino acid in turn is
converted into an aldehyde. Ammonia&Carbon dioxide are evolved.Hydrindantin
and ammonia interact with another molecule of ninhydrin to form Ruhemann’s
purple colored complex.
2) Xanthoproteic Test:
Amino acid containing aromatic chains will form Xanthoproteic acid when it is
treated with Con.HNO3 salts of these derivatives are orange in color when treated with
alkali.
3) Pauly’s Test:
Diazotised sulphanilic acid couples with amino phenol and immidazole to form a
coloured azo compound in cold condition.
4) Millon’s Test:
Phenolic amino acid on treatment with Millon’s reagent gives red color.
Mercuric sulphate forms a colored compound with hydroxyl group of amino acid
(Tyrosine).
5) Morner’s Test:
Amino acid containing aromatic hydroxyl group reacts with this reagent to give
green color. This test is to specify for amino acid containing aromatic hydroxyl
group.(tyrosine)
6) Folin’s Test:
Amino acid containing aromatic ring reacts with this reagent to give blue
colour.
7) Hopkin’s Cole Test:
This reaction is answered by tryptophan.This reaction is due to the prsence of
indole group in tryptophan.
8) Ehrlisch Test:
Indole group containing amino acid reacts with this reagent to give purple
color or pinkish red colored complex.
9) Sakaguchi Test:
This reaction is specific for guanidino group of Arginine or protein containing
Arginine.
10) Sulphur Test:
Amino acids containing the thiol or sulphydryl group reacts with sodium
plumbate to form a dark grey or black precipitate which is insoluble in dil.HCl.
11) Sodium Nitroprusside (Bollin’s) Test:
Amino acids containing the free thiol group (S—H) (due to cysteine) yields a
red color, with sodium nitroprusside in an ammoniacal environment. Cystine which
contains disulphide linkage (S—S) may be reduced to cysteine using reducing agent
such as sodium cyanide, sodium brohydride or sodium bisulphate which then yields a
positive result.
S.No Experiment Observation Inference

1 Ninhydrin Test: To 1ml of A purple color is observed Presence of α amino
this test solution add a few acid
drops of Ninhydrin and heat
for 2 min.
2 Xanthoproteic Test: To a few Yellow Color is formed Presence of
drops of the test solution add after the addition of Aromatic amino
1ml of con.HNO3 and heat it con.HNO3 and this turns to acid.
in a boiling water bath.Cool red color while NaOH is
it.Then add few drops of added
40% NaOH
No characteristic color Absence of aromatic
change amino acid.
3 Pauly’s Test: To 2ml of the Deep red color dye is Presence of
test solution add 1ml of 5% obtained Histidine and
sulphanilic acid and Tyrosine.
cool.Then add 1ml of 5%
sodium nitrite and after 5 Absence of
min add 2ml of 1% sodium No characteristic colored Histidine,Tyrosine
cabonate dye and
Tryptophan.
4 Millon’s Test: To few drops Red color is obtained Presence of phenolic
of the test solution add a few group containing
drops Mercuric Sulphate amino acid.
Sodium nitrite. Then add a No characteristic red color Absence of phenolic
few drops of 5% NaNO2. group containing
amino acid.
5 Morner’s Test: To few drops Green color is formed. Presence of
of the test solution add a few Tyrosine.
drops of Morner’s reagent
and heat the solution in a No characteristic color
boiling water bath. change. Absence of
Tyrosine.
6 Folin’s Phenol Test: To 2ml Blue color is obtained. Presence of
of the test solution add equal Tyrosine.
1ml of Folin’s Phenol
reagent and then add 1% No characteristic color
Na2CO3 change Absence of
Tyrosine.
7 Aldehyde Test: To few drops A violet color ring is Presence of
of the test solution add 1ml formed at the junction of Tryptophan.
of 1% HCHO and then add two layers.
1ml of con.H2SO4
No characteristic color Absence of
change. Tryptophan.
8 Hopkin’s Cole Test: To few A violet color ring is Presence of
drops of the test solution add formed at the junction of Tryptophan.
1ml of glyoxallic acid and two layers.
then add con.H2SO4 carefully
along the sides of the test No characteristic color Absence of
tube. change. Tryptophan
9 Ehrisch’s Test: To 2ml of the Pinkish red color is Presence of
test solution add equal obtained. Tryptophan.
volumes of ehrlish’s reagent
and heat the solution in a
boiling water bath for afew Absence of
minutes. No characteristic color Tryptophan
change
10 Sakaguchi’s Test: To 8 drops Red color is obtained Presence of
of the test solution add 1ml Arginine.
of 20%NaOH followed by
the addition of a few drops of No characteristic color
Molisch reagent in change Absence of
alcohol.Then add 2ml of Arginine.
20% Bromine water and
observe the color change.
11 Sodium Nitroprusside Test: Presence of

To 2ml of test solution and Redddish purple color is Methionine.
1ml of 20% NaOH followed obtained.
by the addition of sodium
nitroprusside and 1ml of
glycine.Now heat the
mixture.Cool it.Then add 6N
HCl slowly in drops through
the sides of test tube. No characteristic color Absence of
change. Methionine.
12 Sulphur Test: To few drops Dirty colored black Presence of
of test solution add equal precipitate is obtained. Cysteine.
volumes of 45%NaOH and
then heat for 2 min in boiling
water bath.Cool it.Then add No characteristic change Absence of
5ml of lead acetate solution. Cysteine.
_______________________________________________________________________
_

1 Ninhydrin Test: To 1ml of the A purple color is obtained. Presence of α-
test solution add a few drops amino acid.
of ninhydrin and heat it for 2
min.
2 Xanthoproteic Test: To a few No characteristic color Absence of aromatic
drops of the test solution add change. amino acids.
1ml of con.HNO3 and heat it
in a boiling water bath.Cool
it.Then add few drops of
40% NaOH
3 Pauly’s Test: To the No characteristic color Absence of
substance add 1ml of 1% change. Histidine,Tyrosine
sulphanilic acid and cool. and Tryptophan.
Then add 1ml of 5% sodium
nitrite and after 5 min add
2ml of 1% sodium carbonate
4 Millon’s Test: To few drops No characteristic color Absence of phenolic
of the test solution add a few change. group containing
drops Mercuric Sulphate amino acids.
5% Sodium nitrite.
5 Morner’s Test: To few drops No characteristic color Absence of
of the test solution add a few change. Tyrosine.
drops of Morner’s reagent
boiling water bath for a few
minutes.
6 Folin’s Phenol Test: To few No characteristic color Absence of
drops of the test solution add change. Tyrosine.
few drops of Folin’s Phenol
reagent and then add 1%
Na2CO3.
7 Aldehyde Test: To 1ml of the No characteristic color Absence of
test solution add 2-3 drops of change. Tryptophan.
1%HCHO and then carefully
add few drops of con.H2SO4
along the inner sides of the
test tube.
8 Hopkin’s Cole Test: To few No characteristic color Absence of
drops of the test solution add change. Tryptophan.
few drops of glyoxallic acid
and then add con.H2SO4
carefully along the sides of
the test tube.
9 Ehrisch’s Test: To 2ml of the No characteristic color Absence of
test solution add equal change. Tryptophan.
volumes of ehrlish’s reagent
boiling water bath for a few
minutes.
10 Sakaguchi’s Test: To 2 ml of A red color is observed Presence of
the test solution add few Arginine.
drops of α-naphthol in
alcohol followed by the
addition of 1ml of
20%NaOH and then add few
drops of 10% Bromine
Water.
11 Sulphur Test: To 1ml of test No characteristic color Absence of
solution add few drops of change. Cysteine.
45%NaOH and boil it for 2
min. Cool it.
12 Sodium Nitroprusside Test: No characteristic color Absence of
To 2ml of test solution and change. Methionine.
add few drops of of 20%
NaOH.Then add 1ml sodium
nitroprusside followed by the
addition of 1.5ml of 1%
glycine.Boil it for few
minutes. Then add 1ml of 6N
HCl.
_______________________________________________________________________
_

min.
2 Xanthoproteic Test: To a few No characteristic color Absence of aromatic
drops of the test solution add change. amino acid.
1ml of con.HNO3 and heat it
40% NaOH
3 Pauly’s Test: To the No characteristic color Absence of
substance add 1ml of 1% change. Histidine.
sulphanilic acid and cool.
Then add 1ml of 5% sodium
nitrite and after 5 min add
2ml of 1% sodium carbonate.
4 Millon’s Test: To few drops No characteristic color Absence of
of the test solution add a few change. Tyrosine.
drops Millon’s reagent and
heat it for few minutes.
5 Folin’s Phenol Test: To 1ml No characteristic color Absence of
of the test solution add few change. Tyrosine.
drops of Folin’s Phenol
reagent and then add few
drops of 5% NaNO2.
test tube.
7 Ehrlisch’s Test: To 1ml of the No characteristic color Absence of
test solution add few drops change. Tryptophan.
of Ehrlish’s reagent and heat
it in a boiling water bath for
a few minutes.
8 Sakaguchi’s Test: To 2 ml of No characteristic color Absence of Arginine
the test solution add few change.
addition of 1ml of
Water.
9 Sulphur Test: To 1ml of test Dirty black precipitate is Presence of
solution add few drops of obtained. Cysteine.
min. Cool it.
HCl.

min.
2 Xanthoproteic Test: To a few No characteristic color Absence of
drops of the test solution add change. Aromatic amino
1ml of con.HNO3 and heat it acid.
40% NaOH
3 Pauly’s Test: To few drops of A red color is obtained. Presence of
the the test solution add few Histidine
drops of 1% sulphanilic acid
and cool it in an ice
bath..Then add 1ml of 5%
sodium nitrite and after 5
min add few drops of 1%
sodium cabonate
of the test solution add change. Tyrosine.
Millon’s reagent and heat it
for a few minutes.
drops of 5% NaNO2.
test tube.
a few minutes.
8 Sakaguchi’s Test: To 2 ml of No characteristic color Absence of
the test solution add few change. Arginine.
addition of 1ml of
Water.
min. Cool it.
HCl.

min.
2 Xanthoproteic Test: To a few No characteristic color Absence of
drops of the test solution add change. Aromatic amino
40% NaOH.
3 Pauly’s Test: To few drops of No characteristic color Absence of
the the test solution add few change. Histidine.
min add 1ml of 1% sodium
cabonate.
for a few minutes.
drops of 5% NaNO2.
test tube.
a few minutes.
addition of 1ml of
Water.
9 Sulphur Test: To 1ml of test No characteristic colored Absence of
solution add few drops of precipitate. Cysteine.
min. Cool it.
10 Sodium Nitroprusside Test: Reddish Purple color is Presence of
To 2ml of test solution and obtained. Methionine.
HCl.

mins.
2 Xanthoproteic Test: To a few Yellow color formed after Presence of
drops of the test solution add addition of Con.HNO3. aromatic amino
40% NaOH.
3 Pauly’s Test: To few drops of No characteristic color Absence of
the test solution add few change. Histidine and
drops of 1% sulphanilic acid Tyrosine
cabonate.
for a few minutes.
reagent and add few drops of
5% NaNO2.
6 Aldehyde Test: To 1ml of the Violet ring is formed at the Presence of
test solution add 2-3 drops of junction of two layers. Tryptophan.
1% HCHO and then
carefully add few drops of
con.H2SO4 along the inner
sides of the test tube.
7 Ehrlisch’s Test: To 1ml of the Pinkish red color is Tryptophan is
test solution add few drops obtained. confirmed.
a few minutes.
alcohol followed by the 1ml
of 20% NaOH and add a few
drops of 10% bromine Water.
min. Cool it.
HCl.

min.
2 Xanthoproteic Test: To a few Yellow color formed after Presence of
drops of the test solution add addition of Con.HNO3. aromatic amino
40% NaOH.
3 Pauly’s Test: To few drops of No characteristic color Prescence of
the the test solution add few change. Tyrosine
cabonate.
4 Millon’s Test: To few drops Red color is obtained. Tyrosine is
of the test solution add confirmed.
for a few minutes.
5 Folin’s Phenol Test: To 1ml Blue color is obtained. Presence of
of the test solution add few Tyrosine.
drops of 5% NaNO2.
test tube.
a few minutes.
addition of 1ml of
drops of 10% Bromine water.
min. Cool it.
NaOH. Then add 1ml
sodium nitroprusside
1.5ml of 1% glycine.Boil it
for few minutes. Then add
1ml of 6N HCl.

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RAMAKRISHNA MISSION VIVEKANANDA COLLEGE

EVENING COLLEGE, CHENNAI-4

Estimation of Amino acid

Standard oxalic acid Vs NaOH

Amino acid + HCHO Vs NaOH

Standard NaOH Vs HCHO + H2O

Titration I: Standardisation of Benedict’s Qualitative Reagent

Titration II: Estimation of Glucose

Std Glucose Vs Benedict’s Quantitative reagent Indicator : Self

Titration I: standardisation of dye

Titration II: Estimation of Ascorbic acid

Standard Ascorbic acid Vs Dye Indicator : Self

Titration I: Standardisation of KOH

Titration II: Determination of Acid number

Titration III: Blank titration

Volume of KOH consumed of

Titration II: Determination of saponification value

Oil+Lipid solvent+KOH Vs HCl Indicator : Phenolphthalein

Saponification = Amount of KOH x Equivalent weight x Normality

Lipid solvent+KOH Vs HCl Indicator : Phenolphthalein

Expt 6: Estimation of chloride by Mohr’s method

(i)NaCl (0.29g of sodium chloride dissolved in 100ml of water in a 100ml

Titration I: Standardisation of silver nitrate

Titration II: Estimation of chloride

Expt 7 : Estimation of Chloride by Volhard’s method

Titration I: Standardisation of potassium thiocyanate

Titration II: Estimation of chloride

Expt 8 : Determination of iodine number of an edible oil by Hanu’s method

Volume of iodine not

QUALITATIVE ANALYSIS OF CARBOHYDRATES

GENERAL PROCEDURE FOR QUALITATIVE ANALYSIS OF

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

S.N0 EXPERIMENT OBSERVATION INFERENCE

4) Sodium Carbonate: (1%)

7) Sodium hydroxide (40%)

10) Sodium hydroxide (1%)

11) Con.Sulphuric acid

12) Glacial acetic acid

S.No Experiment Observation Inference

11 Sodium Nitroprusside Test: Presence of

S.No Experiment Observation Inference

S.No Experiment Observation Inference

S.No Experiment Observation Inference

S.No Experiment Observation Inference

S.No Experiment Observation Inference

S.No Experiment Observation Inference

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