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CORE PRACTICAL I
COMPILED BY
Dr. A.LIYAKATH ALI & Mr. V. MAGENDIRA MANI
DEPARTMENT OF BIOCHEMISTRY
ISLAMIAH COLLEGE (AUTONOMOUS)
VANIYAMBADI - 635751
Page 1
CALCULATION
Weight of oxalic acid = 1.576 g
Titration I
Standard oxalic acid Vs Sodium hydroxide
Indicator: phenolphthalein
S.NO
Burette readings
acid (ml)
Initial (ml)
20 ml
0 ml
20 ml
0 ml
Volume of Sodium
Final (ml)
(V 1)
20 ml
(N1 )
0.1 N
(V2)
-------- ml
(N2)
--------------------------- N
We know that,
hydroxide (ml)
V1N1 = V2N2
N2
= V1N1
V2
N2 =
20 x 0.1/---------
N2 =
-----------------
(N2)
Page 2
Ex. No :
Date
Aim
To estimate the amount of amino acid (Glycine) present in the whole of the given unknown
solution
Principle
Amino acid reacts with excess of formaldehyde to give free hydrogen ion and act as acidic
solution. This acidic solution is titrated against standard alkali (Sodium hydroxide) using phenolphthalein
as indicator.
Reagents required
i.
ii.
iii.
Phenolphthalein as indicator.
iv.
Formaldehyde
v.
Page 3
Burette readings
Initial (ml)
Volume of Sodium
hydroxide (ml)
Final (ml)
20 ml amino acid + 5 ml
0 ml
1
formaldehyde
20 ml amino acid + 5 ml
0 ml
2
formaldehyde
(Test value)
------------------ ml
Burette readings
Initial (ml)
Volume of Sodium
Final (ml)
hydroxide (ml)
20 ml distilled water + 5 ml
1
0 ml
formaldehyde
20 ml distilled water + 5 ml
0 ml
formaldehyde
------------------ ml
= ------------------------ ml
COMPILED BY Dr. A.LIYAKATH ALI & Mr. V. MAGENDIRA MANI
Page 4
Procedure
Titration I
Standard oxalic acid Vs Sodium hydroxide solution
Weighed accurately 1.576 g of crystalline oxalic acid and transfer into a 250 ml of standard flask
then the volume is made up to 250 ml using distilled water. Pipette out exactly 20 ml of this solution
into a clean conical flask and two drops of phenolphthalein as indicator is added. This is titrated against
the Sodium hydroxide solution taken in the burette. The end point is the appearance of pale permanent
pink colour. The titrations are repeated for concordant values. From the titre value the normality of
Sodium hydroxide solution is calculated.
formaldehyde and two drops of phenolphthalein as indicator is added and shaken well for 5 minutes.
This is titrated against the standardized Sodium hydroxide solution taken in the burette. The end point is
the appearance of pale permanent pink colour. The titrations are repeated for concordant values.
Titration III (Blank value)
Blank Vs Standardized Sodium hydroxide solution
Pipette out exactly 20 ml of distilled water into a clean conical flask to this 5 ml of formaldehyde
and followed by two drops of phenolphthalein as indicator is added. The contents are mixed well for 5
minutes. This is titrated against the standardized Sodium hydroxide solution taken in the burette. The
end point is the appearance of pale permanent pink colour. The titrations are repeated for concordant
values.
Page 5
Calculation
Volume of Sodium hydroxide solution
(V 1)
-------------- ml
(N1)
-------------- N
(V2)
20 ml
(N2)
We know that,
V1N1 = V2N2
N2
= V1N1
V2
N2 =
----------------
(N2)
= ------------------ N
75
The amount of amino acid (Glycine) present in the whole of the given unknown solution
----------------------------- grams
Page 6
Test value Blank value will give the actual amount of Sodium hydroxide consumed by the
amino acid solution. From this value the strength of amino acid is calculated, and from this strength the
amino acid present in the whole of the given unknown solution is calculated
75
Result
The amount amino acid (Glycine) present in the whole of the given unknown solution is
=
----------------------- grams
Page 7
Ex. No :
Date
:
CALCULATION
Standard Glucose
Titration I
Standard Glucose Vs Benedicts Reagent
S.NO
Volume of Benedicts
Burette readings
Reagent (ml)
Initial (ml)
5 ml
0 ml
5 ml
0 ml
Final (ml)
Volume of Standard
Glucose (ml)
Titration II
Unknown Glucose Vs Benedicts Reagent
S.NO
Volume of Benedicts
Burette readings
Volume of Unknown
Glucose (ml)
Reagent (ml)
Initial (ml)
5 ml
0 ml
5 ml
0 ml
Final (ml)
Page 8
Weighed accurately 250 mg of Glucose and transfer into a 250 ml of standard flask then the volume is
made up to 250 ml using distilled water. (concentration 1 mg/1 ml)
Procedure
Titration I
Standard Glucose solution Vs Benedicts quantitative Reagent
Pipette out exactly 5 ml of Benedicts quantitative reagent into a clean conical flask. To this add 1 gram
of sodium carbonate. The contents are mixed well and heated in boiling water bath till the first bubble
appearance. This is titrated against the Standard Glucose solution taken in the burette. The end point is
the disappearance of blue colour. The titrations are repeated for concordant values
Page 9
X
X 100
Y
= -------------------- mg
Page 10
Titration II
Unknown Glucose Vs Benedicts Reagent
The given unknown Glucose solution is made up to 100 ml standard flask using distilled water.
The burette is rinsed with unknown Glucose solution and filled with the same unknown Glucose
solution. Pipette out exactly 5 ml of Benedicts quantitative Reagent into a clean conical flask to this
1gram of sodium carbonate added. The contents are mixed well and heated till the first bubble
appearance. This is titrated against the unknown Glucose solution taken in the burette. The end point is
the disappearance of blue colour. The titrations are repeated for concordant values.
Precaution
i.
ii.
iii.
Result
The amount of Sugar (Glucose) present in the whole of the given unknown
Solution ------------------------ grams
Page 11
S.NO
Volume of Standard
Burette readings
Initial (ml)
10 ml
0 ml
10 ml
0 ml
Volume of Dye
(ml)
Final (ml)
--------------------------- ml
Titration II
Unknown Ascorbic acid Vs Dye
S.NO
Volume of Unknown
Burette readings
Initial (ml)
10 ml
0 ml
10 ml
0 ml
Volume of Dye
Final (ml)
--------------------------- ml
(ml)
Page 12
Ex. No :
Date
:
Aim
To estimate the amount of ascorbic acid present in the whole of the given unknown solution
Principle
Ascorbic acid is oxidized by the colourD dye 2, 6 Dichloro phenol indophenol to dehydro
ascorbic acid. At the same time the dye is reduced to colourless compound, so that, the end point of the
reaction can be easily determined.
Page 13
Calculation
Working standard ascorbic acid
-------------------- mg
The amount of ascorbic acid present in the whole of the given unknown solution
------------------ mg x 10
------------------- mg
Page 14
Reagent required
2, 6 Dichloro phenol indophenols
Dissolve 42 grams of Sodium bi carbonate and 52 grams of Dichloro phenol indophenols in 50 ml
of distilled water and finally dilute to 250 ml using distilled water.
Stock standard ascorbic acid solution
100 mg of ascorbic acid is weighed exactly and carefully transfer in to 100 ml standard flask and
made up to 100 ml using 0.6 % oxalic acid.
Working standard ascorbic acid solution
10 ml of Stock standard ascorbic acid solution is pipette out in to a 100 ml standard flask and
made up to 100 ml using 0.6 % oxalic acid.
Procedure
Titration I
Standard Ascorbic acid Vs Dye
Pipette out exactly 10 ml of working standard ascorbic acid solution into a clean conical flask
and it is titrated against the dye taken in the burette. The end point is the appearance of pale
permanent pink colour. The titrations are repeated for concordant values.
Titration II
Unknown Ascorbic acid Vs Dye
The given unknown ascorbic acid solution is made up to 100 ml standard flask using 0.6 % oxalic
acid. Pipette out exactly 10 ml of this unknown ascorbic acid solution into a clean conical flask and it is
titrated against the dye taken in the burette. The end point is the appearance of pale permanent pink
colour. The titrations are repeated for concordant values.
Page 15
Result
The amount of ascorbic acid present in the whole of the given unknown
Solution ------------------------ mg
Page 16
Burette readings
acid (ml)
Initial (ml)
20 ml
0 ml
20 ml
0 ml
Final (ml)
(V 1)
20 ml
(N1 )
0.1 N
(V2)
-------- ml
(N2)
--------------------------- N
Volume of Potassium
hydroxide (ml)
We know that,
V1N1 = V2N2
N2 = V1N1
V2
N2 =
20 x 0.1/---------
N2 =
-----------------
(N2)
Page 17
Ex. No :
Date
Aim
To estimate the amount of Acid no of the given Fat
Principle
During storage of fat become rancid. As a result the peroxide formation of the double bond by
atmospheric oxygen and or hydrolyzed by micro organism with liberation of free fatty acids. The amount
of acid present gives the indication of age and quality of the fat.
Acid value is the number of milligrams of KOH required to neutralize the free fatty acids in one gram of a
fat or oil. It is a measure of free fatty acid contents in a fat or oil.
Reagents required
i.
Fat
ii.
Fat solvent
iii.
iv.
v.
Phenolphthalein as indicator.
vi.
Methanol / Ethanol
Procedure
Titration I
Standard oxalic acid Vs Potassium hydroxide solution
Weigh accurately 1.575 g of oxalic acid and transfer into a 250 ml of standard flask then the
volume is made up to 250 ml using distilled water. Pipette out exactly 20 ml of this solution into a clean
conical flask and two drops of phenolphthalein as indicator is added. This is titrated against the
Potassium hydroxide solution taken in the burette. The end point is the appearance of pale permanent
pink colour. The titrations are repeated for concordant values. From the titre value the normality of
Potassium hydroxide solution is calculated.
Page 18
(-)
Titration II
Test value
S.NO
Burette readings
Initial (ml)
Volume of Potassium
hydroxide (ml)
Final (ml)
of alcohol + 3 drops of
0 ml
Phenolphthalein
Titration III
Blank Value
Contents taken in the conical
Burette readings
Volume of Potassium
S.NO
flask
Initial (ml)
50 ml of alcohol + 3 drops of
0 ml
Final (ml)
hydroxide (ml)
Phenolphthalein
By Oil alone
------------------ ml
Page 19
Titration II
Test Value
Weigh about one gram of edible oil and carefully transfer in to a clean dry conical flask. Then 50
ml of alcohol is added followed by two drops of phenolphthalein as indicator is added. The contents are
mixed well for 20 minutes. This is titrated against the standardized Potassium hydroxide solution taken
in the burette. The end point is the appearance of pale permanent pink colour and persisting up to
20 30 seconds.
Titration III
Blank Value
50 ml of alcohol is taken in a conical flask and three drops of phenolphthalein as indicator is
added. The contents are mixed well. This is titrated against the standardized Potassium hydroxide
solution taken in the burette. The end point is the appearance of pale permanent pink colour and
persisting up to 20 30 seconds.
Page 20
Acid no of Fat =
--------------------- grams
Result
------------------------------
Page 21
Volume of potassium
Burette readings
Initial (ml)
20 ml
0 ml
20 ml
0 ml
Final (ml)
(V 1)
20 ml
(N1)
0.1 N
(V2)
-------- ml
(N2)
--------------------------- N
We know that,
V1N1 = V2N2
N2 = V1N1
V2
N2 =
20 x 0.1/---------
N2 =
-----------------
(N2)
Page 22
Ex. No :
DETERMINATION OF IODINE NO OF FAT
Date
Aim
To estimate the amount of Iodine no of the given Fat
Principle
Iodine no of fat is defined as the no of grams of iodine absorbed by 100 gram of fat or oil. It is
a measure of degree unsaturation of the fatty acids in a fat or oil. Unsaturated fatty acids, either free or
combined in lipids react with halogens like bromine and iodine which get decolorized. These halogens
add at the carbon carbon double bond.
Hanes method is used for the determination of Iodine number. About 1 gram of the fat is taken in a well
cleaned dry iodine flask. To this 20 ml of chloroform is added to dissolve the fat. The contents are
shaken well and kept for 30 minutes. Then 20 ml of potassium iodide is added to liberate the iodine and
it is titrated against standard sodium thio cyanate solution. From this titration iodine number of fat is
calculated.
Reagents required
i.
Hanes solution
ii.
Fat
iii.
iv.
v.
10 % potassium iodide
vi.
1 % Starch
vii.
Chloroform
Page 23
Titration I
Standard potassium dichromate solution Vs Sodium thio cyanate solution
Weighed accurately 1.225 g of potassium dichromate solution and transfer into a 250 ml of
standard flask then the volume is made up to 250 ml using distilled water. Pipette out exactly 20 ml of
this solution into a clean conical flask to this 5 ml of Conc. Hydrochloric acid is added, followed by 10 ml
of 10 % potassium iodide is added. This contents are mixed well and titrated against the Sodium thio
cyanate solution taken in the burette, the titration is continued until a pale brown colour is appears. At
the time 1 ml of 1 % Starch solution is added. And the titration is continued till to get the end point
appearance of emerald green colour, it is the end point. The titrations are repeated for concordant
values. From the titre value the normality of Sodium thio cyanate solution is calculated.
Titration II
Determination of iodine no of fat (Test value)
Weigh about one gram of edible oil and carefully transfer in to a clean dry iodine flask. Then
20 ml of Chloroform is added, the contents are mixed well to dissolve the oil. To this 20 ml of Hanes
solution is added, shaken well and kept in dark for 30 minutes with occasional shaking. Then the flask is
taken out to this 20 ml of 10 % potassium iodide is added to liberate iodine. Except the iodine that is
absorbed by the oil. To this mixture 100 ml of distilled water is added, so the liberated iodine is nicely
disturbed in the solvent then it is titrated against the Sodium thio cyanate solution taken in the burette,
the titration is continued until a pale brown colour is appears. At the time 1 ml of 1 % Starch solution is
added. And the titration is continued till to get the end point disappearance of blue colour it is the end
point.
Blank value
Blank value is also done without oil
Equivalent weight of iodine
127
Page 24
Titration II
Determination of iodine no of fat (Test value)
Burette readings
Titration
Volume of Sodium
Final (ml)
0 ml
+ 100 ml of distilled water + 20 ml of
10 % Potassium iodide + 1 ml of 1 %
Starch
Blank
20 ml of chloroform + 20 ml of Hanes
Solution + 100 ml of distilled water +
0 ml
20 ml of 10 % Potassium iodide + 1 ml
of 1 % Starch
(-)
-------------------- ml
Page 25
Equivalent weight of iodine x Blank value - Test value x Normality of Sodium thio cyanate solution x 100
1000 x Weight of the Oil
Result
------------------------------
Page 26
OILS
ACID VALUE
SAPONIFICATION VALUE
IODINE VALUE
Coconut oil
5 13
250 264
8.0 9.5
Sesame oil
1 10
188 193
103 117
Castor oil
0.3 4
178 188
80 88
Linseed oil
18
190 196
170 203
26
186 196
83 105
0.4 2
220 241
26 38
Ghee (cow)
225.5 236
31.5 45
Ghee (Buffalo)
228.5 236
26.5 - 44
Butter
Page 27