AMINO ACID ASSAY BY NINHYDRIN COLORIMETRIC 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
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
References: Hwang, M. and Ederer, G. M., Rapid hippurate hydrolysis method for presumptive identification of group B streptococci, J. Clin. Microbiol., 1, 114, 1975.