Ammonium sulphate saturation test:

Significance:

Ammonium sulphate saturation test is a confirmatory test. Half saturation (50% saturation) with ammonium sulphate will result in precipitation of globulins, whereas albumins are precipitated after full saturation (100% saturation) of the test sample with ammonium.

Rragents:

Ammonium sulphate crystals.

Principle:

Protein is precipitated out by ammonium sulphate crystals due to dehydration and neutraliazation reactions and the process is known as salting out.

Albumin due to large surface area holds more water than gelatin and globulins. Therefore the albumin requires much higher concentration of salt to be precipitated out.

Procedure:

A. 50% Ammonium Sulphate saturation test:

• Take 2ml of original solution (Protein solution) in the test tube.
• Add 2ml of saturated ammonium sulphate solution in the test tube.
• Mix thoroughly.
• The solution is now half saturated.
• A bulky precipitate of globulin is formed.
• Allow the precipitate to settle down and then filter through a filter paper into clean and dry beaker,
• Filtrate if cloudy, should be re-filtered through the same filter paper, to get a clear filtrate.
• No precipitation will indicate the presence of peptones in the test tube.

B. 100% Ammonium Sulphate saturation test:

• Take 2ml filtrate from 50% ammonium sulphate saturation test in a test tube.
• Add ammonium sulphate crystals in the test tube.
• Shake vigorously to dissolve crystals,
• Continue adding ammonium sulphate crystals to the filtrate until some crystals remain undissolved at the base of the test tube.
• The solution is now fully saturated, and a white precipitate of albumin is formed.
• Filter out the precipitated albumin.
• The absence of precipitation indicates the presence of peptones.

Observation:

• When white precipitate is formsed in 50% ammonium saturated solution, then Gelatin and Globulin is present in the solution.
• If a precipitate is not formed with 50% saturated solution, then add more ammonium sulphate crystals to make it 100% saturated solution. The White precipitate is formed in this 100% saturated solution, “Albumin” is present.
• An absence of precipitation indicates the presence of peptones.

Precautions:

• Wash the apparatus before and after the experiment.
• Carefully handle the chemicals while performing the experiment.