1. Faculty of Medicine Biochemistry Department Practical Biochemistry Precipitation of Proteins
A/Prof. Magdy Elnashar (Preparatory Year)

2. Precipitation of Proteins
Tests to be done:
Heavy metals
Silver nitrates
Lead acetate
Organic acids
Picric acid
Trichloro Acetic Acid (TCA)
Neutral salts (e.g. Ammonium sulphate)
A/Prof. Magdy Elnashar (Preparatory Year) 2

3. Precipitation of Proteins
I. By Heavy metals:
At pH 7 and above, proteins are usually negatively charged, the positively charged metal ions neutralize the charges of protein causing precipitation of the protein. Precipitation by heavy metals 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.
React with Alkali (OH-)
Dipolar ion
Negatively Charged
A/Prof. Magdy Elnashar (Preparatory Year) 3

4. Precipitation of Proteins
By Heavy metals:
Heavy metals such as Ag+, Pb2+, Hg2+, etc form a complex with the alkaline proteins and precipitate.
A/Prof. Magdy Elnashar (Preparatory Year) 4

5. 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 :
Silver nitrate (Ag+)
Lead acetate (Pb2+)
Observe the extent of precipitate in each experiment.
A/Prof. Magdy Elnashar (Preparatory Year) 5

6. Proteins precipitation by Heavy metals
Few drops of B
Mix
2 mL of A C
Result
Unknown A
Proteins (e.g. Albumin)
Reagent B
Silver nitrate (Ag+) or
Lead acetate (Pb2+)
Observation C
White Precipitation
Proteins
A/Prof. Magdy Elnashar (Preparatory Year)

7. Precipitation of Proteins
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.
A/Prof. Magdy Elnashar (Preparatory Year) 7

8. 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
Yellow Precipitation
White precipitation
Proteins
A/Prof. Magdy Elnashar (Preparatory Year)

9. 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.
A/Prof. Magdy Elnashar (Preparatory Year) 9

10. 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
A/Prof. Magdy Elnashar (Preparatory Year)

11. 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
Sample solution (2 ml) + Lead acetate (drops)
Organic acids
Sample solution (2 ml) + Picric acid (drops)
Yellow ppt
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)
A/Prof. Magdy Elnashar (Preparatory Year)

12. Faculty of Medicine Biochemistry Department Practical Biochemistry Lipids (Fats, Oils, Cholestroles & Vitamins) & Enzymes
A/Prof. Magdy Elnashar (Preparatory Year)

13. Lipids Lipids: Fats Oils Cholesterols Vitamins
A/Prof. Magdy Elnashar (Preparatory Year) 13

14. 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
A/Prof. Magdy Elnashar (Preparatory Year) 14

15. 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).
A/Prof. Magdy Elnashar (Preparatory Year) 15

16. Lipids III. Precursor and derived lipids:
They include fatty acids, cholesterol, vitamins (vitamin A, D, E & K), etc.
A/Prof. Magdy Elnashar (Preparatory Year) 16

17. I. Solubility of Lipids (oil & fat)
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.
A/Prof. Magdy Elnashar (Preparatory Year) 17

18. Solubility of Oil in Polar Solvents (e.g. Water)
2 mL of B
Leave it
for 2 min
Mix
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)
A/Prof. Magdy Elnashar (Preparatory Year)

19. 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.
A/Prof. Magdy Elnashar (Preparatory Year) 19

20. 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)
A/Prof. Magdy Elnashar (Preparatory Year)

21. 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
A/Prof. Magdy Elnashar (Preparatory Year) 21

22. 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
A/Prof. Magdy Elnashar (Preparatory Year)

23. 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.
A/Prof. Magdy Elnashar (Preparatory Year) 23

24. III. Detection of Cholesterol
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
A/Prof. Magdy Elnashar (Preparatory Year) 24

25. 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
A/Prof. Magdy Elnashar (Preparatory Year)

26. 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
A/Prof. Magdy Elnashar (Preparatory Year) 26

27. 2. Libermann-Burchard’s test
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
A/Prof. Magdy Elnashar (Preparatory Year)

28. 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
A/Prof. Magdy Elnashar (Preparatory Year) 28

29. 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.
A/Prof. Magdy Elnashar (Preparatory Year) 29

30. 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.
A/Prof. Magdy Elnashar (Preparatory Year) 30

31. 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.
A/Prof. Magdy Elnashar (Preparatory Year) 31

32. IV. Saponification of oil/fat
3 mL of B
Glass rod
Transfer little semi-solid to hot
soft water & MIX
Stir till semi-solid
is formed
Stir with
glass rod D
Transfer little semi-solid
to hot hard water & MIX
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 D
No froth is obtained
Soap in hard water
A/Prof. Magdy Elnashar (Preparatory Year)

33. Yellow to brick-red color
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 *Libermann-Burchard’s test
IV. Saponification of fat/oil
Test
Observation
Result
Lipids (fat/oil)
Sample solution (2 ml) + Chloroform (5 ml) then mix well and leave it for 2 min
One phase
Sample solution (2 ml) + Water (5 ml) then mix well and leave it for 2 min
Two phases
Unsaturation of fat/oil
Sample solution (2 ml) + Iodine in chloroform (2 ml) then mix well
Colorless
Unsaturated fat/oil
Salkowski
Sample solution (2 ml) + Concentrated H2SO4 (2 ml)
Yellow to brick-red color
Cholesterol
Libermann-Burchard
Sample solution (2 ml) + [Concentrated H2SO4 (2 ml) + acetic anhydride (5 drops)]
Bluish-green color
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
froth
Lipids (oil/fat)
A/Prof. Magdy Elnashar (Preparatory Year)