Ninhydrin, reaction with amino

Nicotine, structure of, 30, 916 Ninhydrin, reaction with amino acids, 1030... 

Ninhydrin Reaction with Amino Acids, Biochemistry Survey Lecture. D. S. Moore, Department of Chemistry, Howard University,... 

The postcolumn methods usually provide detectivity and are run online. The classic example, which has been mentioned several times, is the ninhydrin reaction with amino acids. For best results, the reaction must be fast and the mixing chamber efficient without introducing excessive dead volume. Most of the examples are in LC. 

Harding, V.J., Warneford, F.H.S. (1916) The ninhydrin reaction with amino-acids and ammonium salts. Journal of Biological Chemistry, 25, 319-335. 

The easy access of a hydroxy group of ninhydrin (254) to its substitution and the presence of the carbonyl groups makes this highly reactive compound an interesting starting point for cascade reactions with amino compounds. These have been mechanistically investigated with AFM on six different faces of 254 and strictly relate to the crystal packing [94]. 

Two types of detection system are in common use with amino acid analysers, ninhydrin detection and fluorescence detection. The two systems differ in that fluorescence detection is more sensitive than ninhydrin detection, but it is more specific in that it does not detect amino acids such as proline. The detection reagent is mixed with the eluate from the column and the mixture passes into the fluorimeter or spectrophotometer. The system described here is based on the ninhydrin reaction with the separated amino acids. 

It is known that not all reactions proceed in the same manner on all adsorbent layers because the material in the layer may promote or retard the reaction. Thus, Ganshirt [209] was able to show that caffeine and codeine phosphate could be detected on aluminium oxide by chlorination and treatment with benzidine, but that there was no reaction with the same reagent on silica gel. Again the detection of amino acids and peptides by ninhydrin is more sensitive on pure cellulose than it is on layers containing fluorescence indicators [210]. The NBP reagent (. v.) cannot be employed on Nano-Sil-Ci8-100-UV2S4 plates because the whole of the plate background becomes colored. 

Differences in the materials employed for the layers can also become evident when chemical reactions are performed on them. Thus, Macherey-Nagel report that the detection of amino acids and peptides by reaction with ninhydrin is less sensitive on layers containing luminescent or phosphorescent indicators compared to adsorbents which do not contain any indicator [7]. 

Because the amount of time required for a given amino acid to elute from a standard column is reproducible, the identities of the amino acids in a peptide can be determined. The amount of each amino acid in the sample is determined by measuring the intensity of the purple color resulting from its reaction with ninhydrin. Figure 26.3 shows the results of amino acid analysis of a standard equimolar mixture of 17 a-amino acids. Typically, amino acid analysis requires about 100 picomoles (2-3 /xg) of sample for a protein containing about 200 residues. 

Tible 1 Color reactions of amino acids with fluorescein isothiocyanate — ninhydrin (extract from original table). 

The analysis of amino acids involves chromatographic issues similar to those encountered in analysis of simple amines. Underivatized amino acids have, with a few exceptions, weak UV absorbance and a strong tendency to interact with stationary phases in undesirable ways. Underivatized amino acids are normally separated with ion exchange chromatography, then visualized post-column by reaction with ninhydrin, o-phthaladehyde (OPA), or other agents. Underivatized tryptophan and the metabolites kynurenine, 3-hydroxykynurenine, kynurenic acid, and 3-hydroxyanthranilic acid, were separated on a Partisphere 5-p ODS column with fluorescent detection.121... 

The peptide/polypeptide product is usually hydrolysed by incubation with 6 mol l-1 HC1 at elevated temperatures (110 °C), under vacuum, for extended periods (12-24 h). The constituent amino acids are separated from each other by ion-exchange chromatography and identified by comparison with standard amino acid preparations. Reaction with ninhydrin allows subsequent quantification of each amino acid present. 

The post-column ninhydrin reaction proceeds in a reaction coil at elevated temperatures (ca. I30-I35°C). Ninhydrin reacts with primary amino acids to give a chromophore with a wavelength of maximum absorption of 570nm. 

Amines other than a-amino acids will also give a colour reaction with ninhydrin but without the production of carbon dioxide. Thus /3-, y-, 8- and e-amino acids and peptides react more slowly than a-amino acids, to give the blue complex, while imino acids result in the formation of a yellow-coloured product which can be measured at 440 nm. Removal of substances such as protein, ammonia and urea from biological samples may be necessary in quantitative work because they also react in a similar manner. 

Figure 10.11 The ninhydrin reaction. The overall reaction of amino acids with ninhydrin is ...

All primary amines react with fluorescamine under alkaline conditions (pH 9-11) to form a fluorescent product (Figure 10.12) (excitation maximum, 390 nm emission maximum, 475 nm). The fluorescence is unstable in aqueous solution and the reagent must be prepared in acetone. The secondary amines, proline and hydroxyproline, do not react unless they are first converted to primary amines, which can be done using A-chlorosuccinimide. Although the reagent is of interest because of its fast reaction rate with amino acids at room temperature, it does not offer any greater sensitivity than the ninhydrin reaction. 

Derivatization with phenyl isothiocyanate (97, R = Ph) followed by HPLC was compared with IEC followed by the ninhydrin reaction for over ninety compounds. The former method was favored for speed, sensitivity and equipment versatility257. Phenylth-iocarbamyl derivatives of amino sugars and amino sugar alcohols (reaction 10) were... 

Liquid/liquid partition chromatography was explored by Willstatter from 1913. The process was extensively developed by Martin and Synge (ca. 1941-1948) who partitioned amino acid derivatives between chloroform and water using precipitated silica as support for the aqueous phase. The preparations of silica were again very variable and it was difficult to prevent adsorption which interfered with the expected behavior of the aminoacids. At first methyl orange was added to the water phase to visualize the amino acids the separation of the acids then caused a red band to move down the columns. The quantitative reaction with ninhydrin was introduced by Moore and Stein in 1948 for both the detection and estimation of the amino acids. Consid-... 

Nickel that is absorbed is excreted primarily in the urine. In the urine, nickel is primarily associated with low molecular weight complexes that have free amino acids as indicated by the ninhydrin reaction (Sunderman and Oskarsson 1991). In humans nickel is also eliminated in hair, skin, milk, and sweat. 

There are no special requirements in the selection of an N-terminal amino acid residue in a segment with which the carboxy component of a segment is to be coupled, unless a highly hindered amino acid or a secondary amino acid is selected. If a Pro or Hyp residue is located at the N-terminus of the segment, monitoring of the coupling reaction with ninhydrin or fluorescamine is extremely difficult. 

Quantitation of the amino acids proceeds through the postcolumn reaction with ninhydrin at 120-135°C, yielding a purple complex for the primary amino acids (absorbance measured at 570 nm) and a yellow complex for the amino acids proline and hydroxyproline (absorbance at 440 nm). 

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