Ruhemann’s purple

FIGURE 4.10 The pathway of the ninhy-drin reaction, which produces a colored product called Ruhemann s Purple that absorbs light at 570 nm. Note that the reaction Involves and consumes two molecules of nlnhydrln. 

Ninhydrin Amino acid Ruhemann s purple Simplified reaction between ninhydrin and primary amino acids... 

Rubredoxin(s) 858[s ], 859 Fe-S cluster 857s Ruhemann s purple 121s Rusticyanin 883 Rusts 20... 

Detection of amino acids is typically by UV absorption after postcolumn reaction with nin-hydrin. Precolumn derivatization with ninhydrin is not possible, because the amino acids do not actually form an adduct with the ninhydrin. Rather, the reaction of all primary amino acids results in the formation of a chromophoric compound named Ruhemann s purple. This chro-mophore has an absorption maximum at 570 nm. The secondary amino acid, proline, is not able to react in the same fashion and results in an intermediate reaction product with an absorption maximum at 440 nm. See Fig. 5. Detection limits afforded by postcolumn reaction with ninhydrin are typically in the range of over 100 picomoles injected. Lower detection limits can be realized with postcolumn reaction with fluorescamine (115) or o-phthalaldehyde (OPA) (116). Detection limits down to 5 picomoles are possible. However, the detection limits afforded by ninhydrin are sufficient for the overwhelming majority of applications in food analysis. 

Ninhydrin (1,2,3-triketohydrindene monohydrate). Widely available chemical, light-sensitive. Ninhydrin reacts with free amines (2 1 molar ratio), giving a purple product (Ruhemann s purple resonance structure). Used for a qualitative test to determine the presence of aliphatic amines on the agarose beads as a 0.2% w/v solution in ethanol (see Note 4). Hazards Harmful if swallowed skin, eye, and respiratory irritant. Toxicity data LD50 78 mg/kg intraperitoneal, mouse. Should be handled in a fume hood with safety glasses and gloves. 

Ninhydrin is a common reagent for visualizing spots or bands of amino acids that have been separated by chromatography or electrophoresis. When ninhydrin reacts with an amino acid, one of the products is a deep violet, resonance-stabilized anion called Ruhemann s purple. Ninhydrin produces this same purple dye regardless of the structure of the original amino acid. The side chain of the amino acid is lost as an aldehyde. 

Use resonance forms to show delocalization of the negative charge in the Ruhemann s purple anion. 

Amino groups can be quantified based on their reaction with ninhydrin (13, see Table 3) to produce Ruhemann s purple.F -" Testing for Pro and Cys using this procedure should be avoided because they give a different response with ninhydrin. 

Ninhydrin is allowed to react with a-amino acids in methanol at room temperature in the presence of N-phenylmaleimide to give stereoselective cycloadducts 104 in good yields, confirming the ylide intermediacy (84CC180). The N-unsubstituted azomethine ylide intermediates 103, when no dipolar-ophile is present, are transformed into a purple dye 106, called Ruhemann s purple, through a transamination into imine tautomers 105. This ninhydrin... 

Reaction with ninhydrin Ninhydrin is a strong oxidizing agent. When a solution of amino acid is boiled with ninhydrin, the amino acid is oxidatively deaminated to produce ammonia and a ketoacid. The keto acid is decarboxylated to produce an aldehyde with one carbon atom less than the parent amino acid. The net reaction is that ninhydrin oxidatively deaminates and decarboxylates a-amino acids to C02, NH3 and an aldehyde. The reduced ninhydrin then reacts with the liberated ammonia and another molecule of intact ninhydrin to produce a purple coloured compound known as Ruhemann s purple. 

Amino acid analysis. There are some 20 amino acids found in proteins and these are released by overnight hydrolysis in 6M HCl. Plasma and urine contain an even larger number of amino acids or related compounds. At low pH, amino acids are cations and for 40 years have been separated by cation exchange column, chromatography. The problem with amino adds is that in general they possess no chromophores by which they can all be detected. In the traditional amino add analyser, their detection was accomplished by a post-column reaction with nin-hydrin which forms a purple colour on heating with an amino acid at pH 5.5. This colour, Ruhemann s purple, is formed with all primary amino acids and can be detected at 570 nm. Secondary amino acids such as proline form a yellow chromophore measurable at 440 nm. 

An aldehyde shorter by one C-atom and hydrindantin, the reduced form of ninhydrin, are formed in this reaction. Hydrindantin reacts with ammonia and a second molecule of ninhydrin to form a red dye called Ruhemann s purple. This dye has an absorption maximum at 570 run (see Fig. 4-25). 

Procedure for the Ninhydrin Test [235,236]. The most convenient method for rapid monitoring of the coupling process is the ninhydrin test [235]. Ninhydrin reacts with primary amines to give the dye known as Ruhemann s purple. 

Fig. 4. Formulae of ninhydrine (I), Ruhemann s purple (II), fluorescamine (III) and fluorescent reaction product (IV)...

A principal question, the monitoring of the effluent from the column remains to be discussed here. Small amounts of the individual amino acids emerging in the sequence of their elution have to be revealed and quantitated. In earlier procedures these amounts were in the range of 10 to 100 nanomoles but with improvements in the methodology much smaller samples can now be applied. The classical color reagent for the detection of nanomole quantities is ninhydrin, which reacts with amino acids in a transamination-decarboxylation reaction to yield Ruhemann s purple ... 

---