Transcript 19.-Salt-Saturation-Test

Background
 Proteins which are collide in nature are kept in
solution by two factors
1. Electric charge: A large no of electric charge are
present on the surface of protein molecules. The
similarly charged particles repel each other so this
prevents coalescence of particles
2. Shell of hydration: each molecule is surrounded by
film of water known as shell of hydration this also
prevents coalescence of particles
Principle
 When an inorganic salt like ammonium sulphate is
added to protein solution, the effective concentration
of water available for the protein is decreased and
protein is precipitated. The amount of ammonium
sulphate required to precipitate a collide depends
upon the surface area of the particles. Thus small
molecule like albumin having relatively large surface
area are precipitated only by full saturation with
ammonium sulphate. The large casein and gelatin
molecules have a smaller surface area, therefore both
are precipitated by half saturation with ammonium
sulphate.
Principle
 Albumin due to large surface area holds much more
water than globulin . Hence albumin requires much
higher concentration of the salt than globulin to be
precipitated. This property used for separation of
albumin from globulin. The general rule is higher the
molecular weight of protein lesser is the surface area
and lesser is hydration and lower is the concentration
of salt required for the precipitation.
Reagents
 Solid ammonium sulphate
 Saturated solution of ammonium sulphate
 40% NaOH
 1% CuSO4
Half Slat Saturation Test
Procedure:
 Take 5 ml of given solution and 5 ml of saturated solution
of ammonium sulphate in a test tube shake vigorously and
allow to stand for 5 minutes (because the volumes of given
solution and saturated ammonium sulphate solution are
equal so now the saturated ammonium sulphate solution
become half saturated)
 Filter and perform Biuret test with the filtrate using an
equal volume of 40% NaOH and 2-3 drops of CuSO4
Half Slat Saturation Test
Interpretation:
 Biuret test is positive i.e. violet color appears in case of
albumin and negative i.e. no change in case of
globulin.
Full Slat Saturation Test
Procedure:
 Take 5 ml of given solution and add solid ammonium
sulphate in a test tube while mixing until the solution
is saturated i.e. there should be some undissolved salt
at the bottom. shake vigorously and allow to stand for
5 minutes
 Filter and perform Biuret test with the filtrate using an
equal volume of 40% NaOH and 2-3 drops of CuSO4
Full Slat Saturation Test
Interpretation:
 Biuret test is negative i.e. no change
Precipitation of protein by
concentrated salt solution
Experiment
Observation
Inference
Half Slat Saturation Test:
5 ml of given solution + 5
ml of saturated solution
of ammonium sulphate
shake and stand for 5 min
Filter and perform Biuret
test
Violet color appears in
case of albumin and no
change in case of
globulin.
Protein contain Albumin
Full Slat Saturation Test: No change in case of
5 ml of given solution and albumin and globulin.
add solid ammonium
sulphate. shake and stand
for 5 minutes
Filter and perform Biuret
test
Albumin and Globulin
are not present.