1. Salt formation: Amino acids form salts (-COONa) with bases and esters (-COOR') with alcohols.
2. Decarboxylation: Amino acids undergo decarboxylation to produce corresponding amines.
R-CH(NH3+ )-COO ® R-CH2(NH3+) + CO2
This reaction assumes significance in the living cells due to the formation of many biologically important amines. These include histamine, tyramine and g-amino butyric acid (GABA) from the amino acids histidine, tyrosine and glutamate, respectively.
3. Reaction with ammonia: The carboxyl group of dicarboxylic amino acids reacts with NH3 to form amide
Aspartic acid + NH3 ® Asparagine
Glutamic acid + NH3 ® Glutamine
Reactions due to -NH2 group:
4. Acts as bases: The amino groups behave as bases and combine with acids (e.g. HCI) to form salts (-NH3+Cl-).
5. Reaction with ninhydrin: In the pH range of 4-8, all α- amino acids react with ninhydrin (triketohydrindene hydrate), a powerful oxidizing agent to give a purple colored product (diketohydrin) termed Rhuemann’s purple. All primary amines and ammonia react similarly but without the liberation of carbon dioxide. The imino acids proline and hydroxyproline also react with ninhydrin, but they give a yellow colored complex instead of a purple one. Besides amino acids, other complex structures such as peptides, peptones and proteins also react positively when subjected to the ninhydrin reaction (Note:Proline and hydroxyproline give yellow color with ninhydrin).
Reaction with Proline:
Ninhydrin partially reacts with proline to give yellow product as given below.
A reference set of amino acids solutions subjected to ninhydrin and heat is created. Unique and identifiable colors spectrums are created for each amino acid. Spectral analysis can now be used to create a set of reference spectrums for the amino acids. Such reference spectrums were created for 1. Arginine, 2. Cysteine, 3. Glutamine, 4. Glycine, 5. Histidine, 6. Lysine. A reference spectrum was also created for 7. Aspartame; a particular dipeptide, and 8. Ninhydrin solution
6. Color reactions of amino acids: Amino acids can be identified by specific color reactions:
a. Xanthoproteic acid test
Aromatic amino acids, such as Phenyl alanine, tyrosine and tryptophan, respond to this test. In the presence of concentrated nitric acid, the aromatic phenyl ring is nitrated to give yellow colored nitro-derivatives. At alkaline pH, the color changes to orange due to the ionization of the phenolic group.
b. Pauly's diazo Test
This test is specific for the detection of Tryptophan or Histidine. The reagent used for this test contains sulphanilic acid dissolved in hydrochloric acid. Sulphanilic acid upon diazotization in the presence of sodium nitrite and hydrochloric acid results in the formation a diazonium salt. The diazonium salt formed couples with either tyrosine or histidine in alkaline medium to give a red coloured chromogen (azo dye).
c. Millon's test
It is a test specific for tyrosine, the only amino acid containing a phenol group, a hydroxyl group attached to benzene ring. In Millon’s test, the phenol group of tyrosine is first nitrated by nitric acid in the test solution and Then the nitrated tyrosine complexes mercury (I) and mercury (II) ions in the solution to form either a red precipitate or a red solution, both positive results (Note that any compound with a phenol group will yield a positive test, so you should be certain that the sample that you are testing doesnot contain any phenols other than those present in tyrosine).
| Red Precipitate or Red solution |
| Tyrosine |
| Nitrated tyrosine |
d. Nitroprusside test:
The nitroprusside test is specific for cysteine, the onle amino acid containing a sulfhydryl group (-SH). The group reacts with nitroprusside in alkaline solution to yield a red complex.
e. Histidine test
This test was discovered by Knoop. This reaction involves bromination of histidine in acid solution, followed by neutralization of the acid with excess of ammonia. Heating of alkaline solution develops a blue or violet coloration.
f. Hopkins cole test
This test is specific test for detecting tryptophan. The indole moiety of tryptophan reacts with glyoxilic acid in the presence of concentrated sulphuric acid to give a purple colored product. Glyoxilic acid is prepared from glacial acetic acid by being exposed to sunlight.
g. Sakaguchi test
Under alkaline condition, α- naphthol (1-hydroxy naphthalene) reacts with a mono-substituted guanidine compound like arginine which upon treatment with hypobromite or hypochlorite produces a characteristic red color.
h. Lead sulphide test
Sulphur containing amino acids, such as cysteine and cystine upon boiling with sodium hydroxide (hot alkali), yield sodium sulphide. This reaction is due to partial conversion of the organic sulphur to inorganic sulphide, which can be detected by precipitating it to lead sulphide, using lead acetate solution.
i. Folin's McCarthy Sullivan Test
Imino acids such as Proline and hydroxyproline condense with isatin reagent under alkaline condition to yield blue colored adduct. Addition to sodium nitroprusside [Na2Fe(CN)5NO] to an alkaline solution of methionine followed by the acidification of the reaction yields a red color. This reaction also forms the basis for the quantitative determination of methionine.
j. Isatin test
Imino acids such as Proline and hydroxyproline condense with isatin reagent under alkaline condition to yield blue colored adduct.
7. Transamination: Transfer of an amino group from an amino acid to a keto acid to form a new amino acid is a very important reaction in amino acid metabolism.
8. Oxidative deamination: The amino acids undergo oxidative deamination to liberate free ammonia.
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