Precipitation by acids

Principle:
When HNO3 is added to the protein solution it gets denatured. At the
junction of the two layers white ring is formed after shaking total
solution changes to precipitate. It indicates the presence of protein.
Procedure:
To 2ml of Conc. HNO3 solution, add protein solution slowly and

shake the contents. The experiment is repeated by using Conc.H 2SO4

Precipitation by alkali
Principle:
When NaOH is added to the protein solution it gets denatured. At the
junction of the two layers white ring is formed after shaking and total
solution changes to precipitate. It indicates the presence of protein.
Procedure:
To 2ml of NaOH solution add protein solution slowly and shake the
contents.
Precipitation by metals
Principle:
Metals having positive charge combine with negative charge of the protein and
form precipitate. It is white in colour.
Procedure:
To 3ml of protein solution, add lead acetate solution and observe the color change.
Precipitation by acidic reagents
Principle
Negatively charged ions like picric acid, TCA and sulphosalicilate are protein
precipitants. They neutralize the positive charges on the proteins and cause
denaturation, resulting in the precipitation of complexes like protein picrate.
Procedure:
To 2ml of protein solution add acidic reagents drop by drop until precipitate is
formed.
Precipitation by alcohols
Principle:
Organic solvents like acetone or ethanol when added to a protein solution
in water, reduce the concentration of water available for the proteins in the
solution and precipitate is formed.
Procedure:
To 3ml of protein solution add absolute alcohol until precipitate is formed
Precipitation by salts
Principle:
When ammonium sulphate is added to a solution
of protein, the effective concentration of water
available for protein decreases and the protein gets
precipitated. This phenomenon is called “Salting out”
Procedure:
Take 5ml of protein solution and add weighed
quantity of ammonium sulphate in minute quantities
until precipitate is formed. Again, weigh the
remaining amount of salt and calculate the amount of
salt required to precipitate of 1 gm protein.
Determination of Acid number in oil
 Determination of Acid number
Aim:
To determine the acid number of an edible oil.
Principle:
The acid number of an oil is defined as the number of milligram
of KOH to neutralize free fatty acids present in 1 gram of Fat or
oil. During Storage fat or oil may be hydrolyzed by
microorganisms to free fatty acids. Free fatty acids gives an
indication of the age and quality of the oil. Thus the high acid
Number will indicate that the oil is old or rancid.
Reagents Required:
i)Oxalic acid(0.1N) :
It is prepared by dissolving 630mg of oxalic acid in 100 ml of
distilled water in a standard flask.
ii) KOH (potassium hydroxide0.1 N): It is prepared by
dissolving
5.6 gms in one litre of distilled water.
iii) Phenolphthalein indicator
iv) Ethyl alcohol
Determination of Acid number

Titration I:
Standardization of potassium
hydroxide
The burette is filled with potassium
hydroxide (KOH) solution. Pipette out
10 ml of oxalic acid into a clean conical
flask and titrate it against potassium
Hydroxide using phenolphthalein
indicator. The endpoint is
appearance of pale permanent the
colour. Titrations are repeated pink
concordant
for values the titrated
value
from the normalityof KOH is
determined.
Pale Permanent Pink colour

Excess solution may

develop dark pink
colour

Pale
permanent
pink
colour
 Determination of Acid number

Titration II:
Determination of acid number
Take 50 ml of ethyl alcohol (equal amounts) and add one
drop of phenolphthalein indicator (1% solution in
alcohol). If any pink colour develops, add very little
amount of diluted hydrochloric acid (HCl) till the colour
just disappear. Weight out 10 gram of oil and suspend in
the above mixture. Add 1 ml of phenolphthalein. Boil
the contents Titrate against 0.01 KOH solutions till a pink
colour forms stays for 30 minutes. The same procedure is
followed for blank, without adding the oil.
From the amount of alkali added, calculate the amount
of free fatty acid present in the oil. It is calculated in
milligram of KOH required neutralizing the free fatty acid
present in 1 gram of oil. This is called acid number.

Result:
The acid number of oil is-------------
Pale Permanent Pink colour

Excess solution may

develop dark pink
colour

Pale
permanent
pink
colour
Determination of Acid number
Calculation
Titration I standardization of K O H
Standard Oxalic acid Vs KO H

S.No Volume of Burette reading volume concordant Indicator

Oxalic acid of K O H value
(ml) (ml)
Initial Final
(ml) (ml)

1 phenolphthalein
2

Calculation :

Volume of oxalic acid V1=

Normality of oxalic acid N1=

Volume of KOH V2=

Normality of KOH N2= ?

N2=V1N1
V2
Normality of N2= -----------N
KOH
Determination of Acid number
Titration II

Determination of iodine n um be r

S t a n d a r d K O H V s Te s t o i l

Te s t Va l u e :

S.No Burette reading volume of concordan t Indicator

Volume of KOH(ml) value
P i p e t t e solution (ml)
Initial Final (ml)
in ( m l )
(ml)

1 1 0 g r a m of oil phenolphthalein
+50 ml
of ethyl alcohol
.
Determination of Acid number

Calculation:
Acid value = 56.1 V X N/W
Where,
V = Volume in ml of standard KOH or
NaOH used
N = Normality of the KOH or NaOH and
W = Weight in gm of the sample
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