EXPERIMENT 10

Amines, Amino Acids and Proteins

INTRODUCTION

Amines are organic derivatives of ammonia where one, two, or three hydrogens are
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replaced by alkyl or aryl groups. Alkylamines have their nitrogens attached to an sp -
2
hybridized carbon atom while arylamines have their nitrogens attached to the sp -hybridized
carbon of a benzene ring. An important property of amines is their basicity which can be
attributed to the presence of a pair of electrons on nitrogen. The more available the electron
pair, the more basic the amine.

In the diazotization reaction, diazonium salts are formed from the reaction of primary
aromatic amines with nitrous acid. The salts obtained are among the most versatile
intermediates in organic synthesis. Products obtained are used as acid-base indicators, food
coloring, etc.

Proteins are made up of units of amino acids linked together by peptide bonds. Most
qualitative tests for proteins depend on the presence of certain characteristic chemical groups
or specific linkages in the side chains which differentiate amino acids from each other. These
groups react with a variety of reagents forming colored products. Different color reactions will
yield products varying in color intensity in accordance with the nature and amount of the
groups contained in a particular protein. However, other tests should be done together with
color reaction tests because substances other than proteins respond to some of the color
tests.

The ninhydrin test is a general test for protein and protein hydrolysates. The Biuret test is
positive for all compounds containing two or more peptide bonds.

OBJECTIVES

1. To observe the general properties of amines, amino acids and proteins.

2. To synthesize diazonium salts.
3. To perform qualitative tests to differentiate proteins and amino acids.

PROCEDURE

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.

QUESTIONS:

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.