Release of nitrogen from amino acids with ninhydrin for 15N analysis

✍ Scribed by I.R. Kennedy

• Publisher: Elsevier Science
• Year: 1965
• Tongue: English
• Weight: 341 KB
• Volume: 11
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(65)90048-5

✦ Synopsis

The reaction with indane-1,2,3-trione-2-hydrate (ninhydrin) is firmly established for the calorimetric determination of a-amino acids (l-3). The intensity of the blue-violet color produced is measured at about 570 rnp. This color is most intense when the reaction with ninhydrin is carried out at about pH 5 (1). At this pH, the products of the reaction are usually carbon dioxide, an aldehyde containing one less carbon atom than the parent amino acid, and the blue pigment, diketohydrindylidinediketohydrindamine.

When the reaction is performed at pH less than 3, however, the pigment is not formed, and the a-amino group is released as ammonia. MacFadyen (4) describes a method for the quantitative assay of several amino acids with ninhydrin based on estimation of ammonia produced at pH 2.5. It is necessary to remove the ninhydrin completely while the solution is acid, since the complex is re-formed under neutral or alkaline conditions, and variable recoveries of ammonia result. MacFadyen achieved this by reduction of ninhydrin with hydrogen sulfide to insoluble hydrindantin, which was filtered. Adsorption on charcoal has also been employed ( 5), but the most convenient procedure is by oxidation with strong hydrogen peroxide (6).

In this paper a method for the release of amino groups as ammonia after formation of the colored complex at pH 5.4 is described. Performing the reaction at pH 5.4 extends the range of amino acids from which complete recovery of amino nitrogen as ammonia can be achieved. For most amino acids, the accuracy and precision is comparable to that achieved by Kjeldahl analysis; however the method is much less sensitive than the calorimetric procedures. It is easily adapted to the recovery of a-amino nitrogen as ammonia for mass spectrometry after calorimetric analysis or detection on paper chromatograms.