Method

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

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

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

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

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

1. Add 1 ml of the ninhydrin solution to 5 ml of sample. Cover the test tube with a piece of paraffin film to avoid the loss of solvent due to evaporation. A capped test tube can also be used instead.
2. With gentle stirring, react at 80-100ºC for 4-7 minutes. (How would one find out the amount of time needed to ensure a complete reaction?) If a large heated water bath is used for the entire class and if there is no good provision for holding the test tube in the hot water bath, the test tube may be held with a piece of wire and hang on the side of the water container. A clamp usually does not work too well.
3. After cooling to room temperature in a cold water bath, record the absorbance with a spectrophotometer. (How would you find out the best wavelength to use for this purpose? Hint: the final purple colored complex of ninhydrin absorbs the most amount of light at the wavelength of 570 nm.)

            1ml                                              O.D.
   
        starch soln -->----+                       +-------> at 570nm
                         | | |                   | | |
                         |-+-|                   |-+-|
                         |   |        heat       |   |
                         |   |      -------->    |   |
                         |   |                   |   |
                         |   |                   |   |
                         +---+                   +---+
   
                    5ml sample soln
   

Notes

1. Iso-propanol or a 1:1 mixture of acetone/butanol may be used in lieu of ethanol in preparing the ninhydrin reagent.

Questions

1. Why was the sample cooled to room temperature before measuring the absorbance?
2. What properties of the ninhydrin-amino acid reaction other than color can be used to detect the presence of amino acids?
3. List and describe briefly a representative method used to measure a specific amino acid in a mixture. Choose three amino acids and find a selective analytical method for each of the three.

References

1. Hwang, M. and Ederer, G. M., Rapid hippurate hydrolysis method for presumptive identification of group B streptococci, J. Clin. Microbiol., 1, 114, 1975.