Experiment #11: Analysis of Non-Protein Nitrogen

I. Objectives:

At the end of the experiments, students should be able to:

1. Know the mechanism of identifying non-protein nitrogenous substances.

2. Gain an understanding of the biological importance of these substances in certain metabolic
processes.

II. Materials:
Beaker, heating set-up, filter paper, litmus paper, test tubes and water bath.

III. Reagents
Solid urea, 40% NaOH, 1% Urea, Sodium hypobromite reagent, phenolphthalein indicator,
Urease suspension, 10% NaOH, 1% CuSO4, Uric acid solution, 20% Phosphotungstic acid, Ammonium
Silver nitrate solution, 0.1% Creatinine and saturated picric acid.

IV. Procedure:

A. Urea: Generation of Ammonia

1. To a pinch of Urea in a test tube, add 1mL of 40% NaOH and heat. Note the smell of
Ammonia.
2. Test the vapor by holding a piece of moistened red litmus paper at the mouth of the test
tube. Observe the reaction.

B. Hypobromite test
To 2mL of 1% Urea solution, add 4-5 drops of Sodium hypobromite reagent. Note the brisk
effervescence.

C. Urease test
1. Label 2 test tubes as test and control. To both, add 5mL of Urea solution.
2. To the test, add 2mL of Urease suspension.
3. To the control, add the enzyme preparation (Urease suspension is heated to boiling.)
4. Incubate the tubes for 15 minutes at room temperature.
5. Add 2 drops of Phenolphthalein indicator to each tube.
6. Observe the color and reaction to phenolphthalein.

D. Biuret formation
1. Heat a pinch of Urea in a dry test tube until it melts and solidifies. Note the evolution of
Ammonia.
2. Cool and dissolve the residue in 1mL of 10% NaOH.
 3. Add 1 or 2 drops of 1% Copper Sulfate solution. Note the color change.

E. Uric acid Phosphotungstic acid Reduction test

To 2mL of uric acid solution (100mg in 100mL with 0.5g of Sodium carbonate), add few drops of
20% Phosphotungstic acid. Note the color change.

F. Schiff’s test
1. Wet a piece of filter paper with a few drops of Ammonium Silver nitrate solution.
2. Add 2 drops of Uric acid solution on the same paper. Note the color reaction.

G. Jaffe’s test
1. To 2mL of 0.1% Creatinine solution, add 2mL of saturated picric acid solution and a few drops
of 10% NaOH.
2. Perform a control test.
3. Observe the change in color.
 Experiment 11
Non-Protein Nitrogen
Name: Date:
Group/Section: Instructor:

I. Data and Results

A. Generation of Ammonia
1. Observation:

2. Equation:

B. Hypobromite test
1. Observation:

2. Equation:

C. Urease test
1. Observation:

2. Appearance of colored solution indicates:

3. Equation:

D. Biuret reaction
1. Observation:
 2. Equation:

E. Phosphotungstic acid Reduction test

1. Observation:

2. Major products:

F. Schiff’s test
1. Observation:

2. Is it applicable to any reducing compound?

G. Jaffe’s test
1. Observation:

2. What is the reason why alkaline picrate solution should be freshly prepared?

II. Questions

A. state the implications of a high value of non-protein nitrogen (NPN) in blood and urine.
 B. What does the amount of Urea, Ammonia and Creatinine in the urine tell about renal
function.

C. What food substances can lead to an increased value of NPN in the blood.