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Tests for Amino Groups


An amine functional group contains a lone pair on a central nitrogen atom with one or more alkyl or aryl groups attached to this nitrogen atom. Amines are structurally similar to ammonia in which nitrogen can link up to three hydrogen atoms.

Tests for Amino Groups

Amines can be distinguished by a number of properties depending on connections made by carbon with other groups. A variety of tests are performed to identify amine compounds among a given set of organic compounds. Following is the list of qualitative tests that are most commonly performed to determine the presence of an amino group in an organic molecule:

  • Solubility test
  • Litmus test
  • Carbyl-amine test
  • Nitrous acid test
  • Azo-dye test
  • Hinsberg test

Tests for Amino Groups:

Let us look into each one of them thoroughly:

1. Solubility Test:

Aim: To determine the presence of amino group in an organic compound.

Theory: Compounds containing amino groups like Amino acids are soluble in water and insoluble in non-polar organic solvents like hydrocarbons. Both the Amino group and Carboxyl group, are present in amino acids, this allows the amino acid to accept and donate the protons to the aqueous solution. Thus, we can say that the amine group is one that acts as a base and due to this, the amino acids can dissolve in polar solvents like hydrochloric acids.

Note: A solubility test can show the possibility of the presence of an amine group in a compound but it is not a confirmatory test.


  • The given organic compound to be tested is taken in a clean test tube.
  • 2 to 3 drops of concentrated hydrochloric acid are added carefully to this compound.
  • The solution is shaken well and finally, the solubility of the compound is observed.

Chemical Reaction:

C6H5NH2 + HCl → C6H5NH+3 Cl-

Observations: If the given compound is soluble in Hydrochloric acid (a mineral acid), then it may be an amine.

2. Litmus Test:

Aim: To determine the presence of an amine group in an organic compound.

Theory: Since the amine group is basic in nature, it can turn the red litmus paper to blue. This test can prove the basic nature of amines.


  • A moist red litmus paper is used for this test.
  • One to two drops of the given organic compound are allowed to be placed on the red litmus paper.
  • Change in the color of red litmus paper is later observed.

Chemical Reaction:

CH3CH2NH2 + H2O → CH-3CH-2 NH+3 + OH-

Observations: The possibility of the presence of the Amine group is confirmed by a change in the color of litmus paper from red to blue.

Note: This test is not a confirmatory test for the presence of an amine group in an organic compound. Hence, this test indicates the possibility of the presence of an amine group in a compound but it does not confirm its presence. To confirm the presence following tests are performed.

3. Carbylamine Test:

Aim: To determine the presence of the primary amine group.

Theory: Particularly carbylamine test is used to identify a primary amine group from a given set of compounds. Both the aliphatic and aromatic amines are positive for carbylamine test. Thus, we can briefly tell that the carbylamine test is a test used to determine whether an organic compound contains an aliphatic or aromatic primary amine group. In this test, the given organic compound containing the amine group is treated with chloroform in the presence of alkali (Potassium Hydroxide) and heated, as a result, isocyanide is formed. Hence, this test is also known as the Isocyanide test.

Note: Secondary and tertiary amines are negative for this test.

Chemical Reaction:

R-NH2 + 3KOH + CHCl3 → RNC (isocyanide) + 3KCl + 3H2O


  • 2-3 drops of the compound are placed in a test tube.
  • Now 2-3 drops of chloroform are added to the test tube followed by the addition of an equal volume of 0.5 M alcoholic potassium hydroxide solution (alkali).
  • Contents in the test tube are then heated gently.
  • An obnoxious smell of carbylamine confirms the presence of the primary amino group in the given compound.
  • Never inhale the vapors, instead, always destroy the product in the test tube immediately by carefully adding concentrated hydrochloric acid and simply flush it into the sink.

Observations: The presence of Primary amine is determined by a pungently bad smell, vapors of carbylamines are mostly toxic so do not purposefully smell the vapors.

4. Nitrous Acid Test:

AIM: To determine the presence of different forms of amine group in an organic compound and to be able to differentiate between the aliphatic and aromatic forms of primary, secondary and tertiary amines.

THEORY: The reaction of amines with nitrous acid (HNO2) is another test that classifies the amine not only as primary, secondary, or tertiary but also as aliphatic or aromatic.

Chemical Reaction:

C6H5NH2 + HNO2 (in presence of HCl) → C6H5-N≡N+Cl-

(Di-azonium compound)

C6H5-N≡N+Cl- + H2O (at 5oC) → C6H5-OH + N2(g)


  • A5 Molar amine solution is prepared by adding 0.5 g of the amine in a mixture of 5 ml of concentrated hydrochloric acid and 2.5 ml of water in a test tube.
  • It is then allowed to cool to 0°C in a beaker of ice.
  • In another test tube, 0.5 g of sodium nitrite is dissolved in 2.5 ml of water.
  • This solution is added dropwise with constant shaking to the cold solution of the amine hydrochloride.
  • From this mix 2 ml of the final solution is moved to another test tube, gently warmed, and ready to be examined for evolution of gas.


  • Primary Amines: Rapid bubbling or foaming is observed as the aqueous sodium nitrite solution is added at 0°C to the cold amine solution. This bubbling indicates the presence of a primary aliphatic amine in the compound under observation. Here, nitrogen gas is seen as bubbles.

R-NH2 + HONO → R-OH + H2O + N2

If no such thing is observed then it indicates the presence of a primary aromatic amine. This is confirmed by the evolution of gas (which appears like bubbling) upon warming the mixture to room temperature.

  • Secondary Amine: If a pale yellow oil or low-melting solid, is formed with no evolution of gas, the original amine is a secondary amine.

R2NH + HONO → R2N-NO + H2O

  • Tertiary Amine: If a dark-orange solution or an orange crystalline solid (nitrite salt) is formed, which is the hydrochloride salt of the C-nitrosamine, the amine is tertiary aromatic. Treating 2 ml of this orange-colored solution with a few drops of 10 % sodium hydroxide or sodium carbonate solution produces the bright green or blue nitrosamine base.

R3N + HONO → R3NH+NO-2

5. Azo-dye Test:

Aim: To distinguish between the aliphatic and aromatic forms of amine from the given set of organic compounds containing amine groups.

Theory: The Azo compounds generally show dark shades of color, thus they are capable of being used as a dye in manufacturing industries like textiles etc. Hence we call this test as Azo Dye Test. The azo dye test is used to distinguish the aromatic and aliphatic amines. In this test, a given compound is reacted with nitrous acid and a di-azonium salt is formed. The N2 of di-azonium salt of an aromatic amine act as an electrophile so, another aromatic amine attacks this electrophile and N2N2get bridged between two aromatic amines.

Chemical Reaction:

C6H5-N≡N+Cl- + C6H5-OH → C6H5-N≡NC6H5-OH + Cl- + H2O

p-Hydroxyazobenzene (orange dye)


  • Three test tubes marked as A, B and C are taken simultaneously.
  • The given organic compound is dissolved in 2ml of dilute hydrochloric acid in the first test tube labeled as A.
  • An aqueous solution of sodium nitrite solution is prepared in the second test tube labeled as B.
  • Some amount of Naphthol is dissolved in dilute sodium hydroxide in the third test tube labeled as C.
  • All three test tubes are placed in an ice bath.
  • The contents in test tube B are now added to the mixture in test tube A.
  • The resulting mixture is added to the contents in test tube C.
  • This final mixture is stirred with a glass rod and the changes in the test tube are observed.

Note: Temperature for the Azo-dye test should be maintained between 0-5o C.

Observations: The appearance of a red-orange dye confirms the presence of aromatic amines. If no such observation is seen then the given amine is supposed to be an aliphatic amine.

6. Hinsberg Test:

Aim: To determine the presence of different forms of amine.

Theory: The Hinsberg test is a qualitative method in organic chemistry that is used to distinguish between primary, secondary, and tertiary amines. In the Hinsberg test, amines act as nucleophiles and attack electrophiles. This leads to chloride substitution and sulfonamide formation. This sulfonamide product formed from a primary and secondary amine is insoluble in the solution and precipitates out as a solid from the solution. This property of secondary and primary amines to form sulfonamide is the basic principle behind this test.

Hinsberg reagent is used in this test, it can be described as an alternate name for benzene sulfonyl chloride. This reagent is an organosulfur compound, the chemical formula of Hinsberg reagent is C6H5SO2Cl. The Hinsberg reagents is a colorless oil, which is viscous in nature, and it is generally soluble in organic solvents. This reagent undergoes a reaction with compounds containing N-H, and O-H bonds, which are reactive in nature. We can also use it in the preparation of sulfonamides (via reaction with amines).

Chemical Reaction:

  • The sulfonamides resulting from primary and secondary amines are poorly soluble and precipitate as solids from solution.

PhSO2Cl + 2 RR'NH → PhSO2NRR' + [RR'NH2+]Cl-

  • For primary amines (R' = H), the initially formed sulfonamide is deprotonated by the base to give a water-soluble sulfonamide salt (Na[PhSO2NR]).

PhSO2N(H)R + NaOH → Na+[PhSO2NR-] + H2O

  • Tertiary amines promote hydrolysis of the sulfonyl chloride functional group, which affords water-soluble sulfonate salts.

PhSO2Cl + R3N + H2O → R3NH+[PhSO-3] + HCl


  • The given organic compound is taken to be tested in a clean test tube.
  • 2ml of sodium hydroxide solution and 1ml of benzene sulfonyl chloride is added to the test tube containing the given compound.
  • The mixture is shaken properly and water is added to dilute the prepared solution.
  • The mixture is allowed to cool under tap water and the formation of precipitate is observed.
  • If a precipitate is formed then it is treated with concentrated hydrochloric acid. This step should be performed very carefully.


  • The primary amines contain 2 active hydrogen ions, so they react with benzene sulphonyl chloride and can produce salt, which can be dissolved in NaOH because of the remaining active hydrogen. Hence if the precipitate formed dissolves in NaOH then the given compound is a primary amine.
  • The secondary amines contain only 1 hydrogen ion, so it reacts with benzene sulphonyl chloride but does not dissolve in NaOH. Hence if the precipitate formed does not dissolve in NaOH then the given compound is a secondary amine.
  • Tertiary amines do not hold active hydrogen, and thus they don't react with benzene sulphonyl chloride. Thus there is no formation of precipitate in cases of tertiary amines.

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