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Tests to be done:
Heavy metals
Silver nitrates
 Lead acetate
Organic acids
 Picric acid
Trichloro Acetic Acid (TCA)
Neutral salts (e.g. Ammonium sulphate)
Precipitation of Proteins
I. By
At
Heavy metals:
pH 7 and above, proteins are
usually
negatively
positively
neutralize
charged,
the
metal
ions
charged
the charges
of protein
causing precipitation of the protein.
Precipitation
by
heavy
React with Alkali (OH-)
metals
Dipolar ion
is
therefore most effective at neutral to
slightly alkaline pH value.
NOTE: The solution must not be too
alkaline; otherwise there is a risk of
precipitation of metal hydroxides.
Negatively Charged
Precipitation of Proteins
I. By Heavy metals:
Heavy metals such as Ag+, Pb2+, Hg2+, etc form a complex with the alkaline
proteins and precipitate.
Precipitation of Proteins
Procedure for precipitation by Heavy metals:
To 2 ml of protein (e.g., albumin or solution of egg white, gelatin, casein, add a
few drops of :
1. Silver nitrate (Ag+)
2. Lead acetate (Pb2+)
Observe the extent of precipitate in each experiment.
Proteins precipitation
by Heavy metals
Few drops of B
Mix
2 mL of A
C
Result
Unknown
A
Reagent
B
Proteins (e.g. Albumin)
1.
Silver nitrate (Ag+)
or
1.
Observation
C
Lead acetate (Pb2+)
White Precipitation
Proteins
Precipitation of Proteins
II. Precipitation by organic acids: e.g. Picric acid & TCA
Organic acids carry a large negative charges which neutralize positively charged
protein to form an insoluble salt. The acidic reagents are most effective at acid pH
value where proteins are positively charged. The precipitation of proteins by this
method is irreversible.
Procedure:
a. To 2ml of albumin solution or egg white solution, add from 5-8 drops of picric acid or
Trichloro Acetic Acid (TCA solution).
b. Precipitate is formed in each tube.
c. Slowly add dilute NaOH and observe the results as the pH increases.
Proteins precipitation by
Picric Acid or TCA
Few drops of B
Mix
2 mL of A
C
Result
Unknown
A
Proteins (e.g. Albumin)
Reagent
B
1- Picric Acid
or
2. TCA
Observation
C
1.
2.
Yellow Precipitation
White precipitation
Proteins
Precipitation of Proteins
III. Precipitation by neutral salts: e.g. Ammonium sulphate
The charges on a protein solution can also be neutralized by the addition of neutral
salt and this also has been used for purification of proteins. Theoretical, any salt
can be used but generally ammonium sulphate (NH4)2SO4 is preferred because it
has high solubility and its dissolution in water is endothermic.
Procedure:
a. To 2 ml of albumin solution (egg white + NaCl solution).
b. Add equivalent volume of saturated ammonium sulphate solution or salt.
c. The solution becomes milky or turbid due to the precipitation of globulin, ovamucin
and lysozomes.
Proteins precipitation
by Neutral salts
2 mL of B
Mix
2 mL of A
C
Result
Unknown
A
Proteins (e.g. Albumin)
Reagent
B
Saturated ammonium sulphate or salt
Observation
C
White Precipitation or white turbidity
Proteins
Precipitation of proteins
Proteins can be precipitated by:
Heavy metals : Silver nitrate or Lead acetate
Organic acids: Picric acid or Trichloracetic acid
Neutral salts : Ammonium sulfate
Test
Observation
Result
Heavy metals
Sample solution (2 ml) + Silver nitrate (drops)
White ppt
Protein precipitates
Heavy metals
Sample solution (2 ml) + Lead acetate (drops)
White ppt
Protein precipitates
Organic acids
Sample solution (2 ml) + Picric acid (drops)
Yellow ppt
Protein precipitates
White ppt
Protein precipitates
White ppt
Protein precipitates
Organic acids
Sample solution (2 ml) + Trichloro acetic acid (drops)
Neutral salts
Sample solution (2 ml) + Fully saturated ammonium
sulfate solution (2 ml) or Ammonium sulfate salt (2-3
pinches)
Lipids
Lipids:
– Fats
– Oils
– Cholesterols
– Vitamins
Lipids
I. Simple lipids:
They are esters of fatty acids with various alcohols. They include oils & fats which are
esters of fatty acids with glycerol (i.e. triglycerides).
• As chains become longer and/or more saturated, the triglyceride is more likely to be
a fat (solid at room temperature ,RT)
• As chains become shorter and/or more unsaturated, the triglyceride is more
likely to be an oil (liquid at RT)
Unsaturated oil
Saturated fat
Lipids
II. Complex lipids:
They contain (in addition to fatty acids and alcohols) additional groups as
phosphate (e.g. phospholipids), carbohydrates (e.g. glycolipids) and
proteins (e.g. lipoproteins).
Lipids
III. Precursor and derived lipids:
They include fatty acids, cholesterol, vitamins (vitamin A, D, E & K), etc.
I. Solubility of
Lipids (oil & fat)
1. Test for solubility of lipids in polar solvents (e.g. Water):
− Add about 2 ml of the provided oil sample to 2 ml of water in a test tube and try to
mix oil with water. By shaking, oil and water mix initially; however, they gradually
separate out to form 2 layers. Oils & water DO NOT mix.
Solubility of Oil in Polar
Solvents (e.g. Water)
2 mL of B
Leave it
Mix
for 2 min
2 mL of A
Result
Unknown
A
Any Fat or Oil
Reagent
B
Polar solvent (e.g. Water)
Observation
C
Phase separation
any Fat or Oil (due to fat & oil do not mix
with polar solvents)
Solubility of Lipids (oil & fat)
in Non Polar solvent
2. Test for solubility of lipids in non polar solvents (e.g. chloroform):
- Add about 2 ml of the provided oil sample to 5 ml of chloroform in a test tube and
try to mix oil with chloroform. By shaking, oil and chloroform mix well and they form
one layer.
Solubility of Oils in Non Polar
Solvents (e.g. Chloroform)
5 mL of B
Mix
Leave it for 2 min
2 mL of A
Result
Unknown
A
Any Fat or Oil
Reagent
B
Non Polar solvent (e.g. chloroform)
Observation
C
One phase
any Fat or Oil (due to fat & oil mix well with
non polar solvents)
II. Un saturation test
Test for unsaturation of lipids
This test is specific for the double bonds in the lipid’s chain.
- Prepare about 2 ml of the provided oil/fat sample
- Add few drops of bromine (Orange color) in chloroform or iodine in chloroform.
- Mix them well and observe the color:
if no change in color
it is saturated lipid
If it becomes colorless
it is unsaturated lipid
II. Unsaturation test
Drops of B
Mix
2 mL of A
C
Result
Unknown
A
Any Fat or Oil
Reagent
B
Bromine or Iodine in chloroform
Observation
C
Colorless solution
No change in color
Unsaturated fat or oil due to double bonds
Saturated fat or oil due to no double bonds
III. Cholesterol
II. Precursor and derived lipids:
Cholesterol is a member of the group of
steroid known as sterols which have in common
a hydroxyl group at carbon number 3 and a long
saturated eight carbon at carbon number 17. It is
the source of steroid hormones.
Imbalance in lipid metabolism can lead
to major clinical problems such as
obesity and atherosclerosis.
III. Detection of Cholesterol
1. Salkowski test
-
2 ml of the provided chloroformic extract of canned food,
-
Add an equal volume of concentrated sulfuric acid;
-
A yellow to brick-red color is formed indicating the presence of cholesterol
1. Salkowski’s test
2 mL of B
2 mL of A
C
Result
Unknown
A
Choloroformic extract of Cholesterol
Reagent
B
Concentrated H2SO4
Observation
C
Yellow to brick red color
Cholesterol
II. Detection of Cholesterol
2. Libermann-Burchard’s test
-
2 ml of the provided chloroformic extract of canned food,
-
Add 2 mL of acetic anhydride + 5 drops of concentrated sulfuric acid;
-
A bluish-green color is formed indicating the presence of cholesterol
2. Libermann-Burchard’s
test
B
2 mL of A
C
Result
Unknown
A
Choloroformic extract of Cholesterol
Reagent
B
2 mL of acetic anhydride + 5 drops of
concentrated H2SO4
Observation
C
Bluish-green color
Cholesterol
IV. Saponification of
oil/fat
Saponification of oils:
Saponification of oils means the hydrolysis of esters (triglycerides) in the oil in presence
of an alkaline medium (e.g. NaOH) into glycerol and fatty acids’s sodium salt “soap”.
Triglycerides
Glycerol
Soap
IV. Saponification of
oil/fat
Saponification of oils:
- Warm 1 ml of the provided oil sample in a test tube + 3 ml of alcoholic NaOH
solution.
-Mix them together and stir while warming in a water bath from time to time using a
glass rod until a semi-solid mass is formed.
- Put a small amount of this mass in a test tube containing about 5 ml of warm soft
water and shake well; a froth is obtained indicating the presence of soap.
-Put a small amount of this mass in a test tube containing about 5 ml of warm hard
water (containing heavy metals : e.g. Mg, Ca, Cu, etc) and shake well;
- OR add few drops of CaCl2 or MgSO4 to this solution and shake well; note the
formation of insoluble Ca or Mg soaps without any froth. This explains why
soap is not effective in hard water which contains calcium and magnesium;
for soap to be effective, it must be soluble in water.
IV. Saponification of oil/fat
Why does soap clean grease, oils & fats?
When we agitate or rub Soap with Grease, Oils & Fats, soap cleans by bringing in
grease, oils & fats inside its non polar (hydrophobic) part.
IV. Saponification of oil/fat
Why mineral oils do not saponify?
Saponification is the reaction between triesters and
alcohol. Mineral oil is composed of paraffins and
alkanes , it does'nt contain ester group hence it does
not undergo saponification.
IV. Saponification of oil/fat
C
3 mL of B
Glass rod
Stir till
semi-solid
Stir with
glass rod
D
is formed
1 mL of A
Result
Unknown
A
Warm fat or Oil
Reagent
B
Warm NaOH in alcohol
Observation
C
froth is obtained
Soap in soft water
Observation
D
No froth is obtained
Soap in hard water
I. Solubility of lipids (fat/oil)
 Lipids are soluble in non polar solvents (e.g. chloroform) and they form one phase.
 Lipids are insoluble in polar solvents (e.g. water) and they form two phase.
II. Unsaturation test
III. Detection of Cholesterol: * Salkowski’s test
IV. Saponification of fat/oil
Test
Lipids (fat/oil)
Sample solution (2 ml) + Chloroform (5 ml) then mix well and
leave it for 2 min
Lipids (fat/oil)
Sample solution (2 ml) + Water (5 ml) then mix well and leave
it for 2 min
Unsaturation of fat/oil
Sample solution (2 ml) + Iodine in chloroform (2 ml) then mix
well
Salkowski
Sample solution (2 ml) + Concentrated H2SO4 (2 ml)
*Libermann-Burchard’s test
Observation
Result
One phase
Lipids (fat/oil)
Two phases
Lipids (fat/oil)
Colorless
Unsaturated fat/oil
Yellow to brickred color
Cholesterol
Bluish-green color
Cholesterol
froth
Lipids (oil/fat)
Libermann-Burchard
Sample solution (2 ml) + [Concentrated H2SO4 (2 ml) + acetic
anhydride (5 drops)]
Saponification
Sample solution (1 ml) + Alcoholic NaOH (3 ml) then warm
well and stir.
Take a piece of the product in H2O (2 ml) and mix well