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Amines, Amino Acids and Proteins

A. Solubility in Water
1. Place a few crystal of solid samples or 10 drops of liquid samples in a test tube.
2. Add 2 mL of distilled water.
3. Shake to mix. Observe.
4. Test the mixture with red and blue litmus paper.
5. Save the aqueous mixtures of amines for the next solubility test.

B. Solubility in Aqueous Acid

1. To the aqueous mixtures of amines, add 5 mL of concentrated HCl.
2. Shake to mix. Observe.

C. Preparation and Reactions of Diazonium Salts

1. Preparation of diazonium salt

a. Add 1 g sulfanilic acid to the mixture of 8 ml 1% NaNO2 and 8 mL 2% HCl.
b. Shake well.
c. Allow to stand for 1 minute in an ice bath.
d. Keep the mixture in the ice bath and use them for parts 2 and 3.

2. Replacement Reaction of diazonium salt

a. Add 0.5 mL of 3 M H2SO4 to 5 mL diazonium salt solution prepared in part 1.
b. Heat for several minutes in a hot water bath.
c. Observe the contents closely.

3. Coupling reaction of diazonium salt

a. Place 4 drops of N,N-dimethylaniline in a test tube.
b. Add 2 mL water.
c. Add 6M HCl dropwise until the amine just dissolves.
d. Add 3 mL diazonium salt solution.
e. Add a slight excess of 50% sodium acetate solution.

D. Color Reaction of Amino Acids and Proteins

1. Ninhydrin test (Perform using aniline, alanine, and casein)

a. Spot a small amount of sample on a strip of filter paper.
b. Allow to dry. Dip the paper on 2% ninhydrin solution in acetone.
c. Evaporate the solvent. Allow color development.
d. Inspect the strip after 30 minutes.

2. Biuret test ( Perform using alanine, casein and albumin)

a. Place 2 mL of aqueous sample in a test tube.
b. Add 2 mL of 10% NaOH. Shake to mix.
c. Add 1 drop of 2% CuSO4.
d. Mix and examine the solution.
e. Run a water blank and compare.
3. Reaction with lead acetate
a. Place two strands of hair in a test tube.
b. Add 1 ml of 20% NaOH.
c. Add 2 drops of 10% lead acetate.
d. Boil the mixture for 5 minutes and examine.


1. Explain why milk and egg white are used as antidotes in heavy metal poisoning.
2. In diazotization, what is the purpose of cooling the system thoroughly?
3. Explain the difference in pH (if any) of the different amines.