Method

The reaction between alpha-amino acid and ninhydrin involved in the development of color are described by the following five mechanistic steps:

  alpha-amino acid + ninhydrin ---> reduced ninhydrin + alpha-amino acid + H2O

  alpha-amino acid + H2O ---> alpha-keto acid +NH3

  alpha-keto acid + NH3 ---> aldehyde + CO2

Step (1) is an oxidative deamination reaction that removes two hydrogen from the alpha-amino acid to yield an alpha-imino acid. Simultaneously, the original ninhydrin is reduced and loses an oxygen atom with the formation of a water molecule. In Step (2), the NH group in the alpha-imino acid is rapidly hydrolyzed to form an alpha-keto acid with the production of an ammonia molecule. This alpha-keto acid further undergoes decarboxylation reaction of Step (3) under a heated condition to form an aldehyde that has one less carbon atom than the original amino acid. A carbon dioxide molecule is produced here. These first three steps produce the reduced ninhydrin and ammonia that are required for the production of color in the last two Steps (4) and (5). The overall reaction for the above reactions is simply (slightly inaccurately) expressed in Reaction (6) as follows:

alpha-amino acid + 2 ninhydrin ---> CO2 + aldehyde + final complex(BlUE) + 3H2O

In summary, ninhydrin, which is originally yellow, reacts with amino acid and turns deep purple. It is this purple color that is detected in this method.Ninhydrin will react with a free alpha-amino group, NH₂-C-COOH. This group is contained in all amino acids, peptides, or proteins. Whereas, the decarboxylation reaction will proceed for a free amino acid, it will not happen for peptides and proteins. Thus, theoretically only amino acids will lead to the color development. However, one should always check out the possible interference from peptides and proteins by performing blank tests especially when such solutions are readily available. For example, one can simply add the ninhydrin reagent to a solution of only proteins and see if there is any color development. There is no excuse for failing to perform such a vital test when the sample mixture contains both proteins and amino acids. There are also reports that chemical compounds other than amino acids also yield positive results.

This test can be used routinely for the detection of glycine in the absence of other interfering species. Although this is a fast and sensitive test for the presence of alpha-amino acids, because of the nonselectivity, it cannot be used to analyze the relative individual contents of a mixture of different amino acids. Furthermore, the color intensity developed is dependent on the type of amino acid. Finally, it does not react with tertiary or aromatic amines.

Note that since ninhydrin is a strong oxidizing agent, proper caution should be exercised in handling this compound. It is especially potent at the elevated temperature under which the reaction is carried out. The ninhydrin reagent will stain the skin blue and cannot be immediately washed off completely if it comes in contact with the skin. However, as in any other stain on the skin, the color will gradually rub off after about a day.

List of Reagents and Instruments

A. Equipment

Test tubes
Pipets
Spectrophotometer

B. Reagents

Ninhydrin Reagent Solution
Ninhydrin: 0.35 g
Add ethanol to: 100 ml (See Note 1.)

Procedures