Ninhydrin detection

The polyamines putrescine, cadaverine, spermidine, and spermine, which are seen at elevated levels in some victims of cancer, were separated on a Technicon (The Technicon Company Chauncey, NY) TSM Amino Acid Analyzer packed with an 8% divinylbenzene-co-polystyrene sulfonated resin with post-column ninhydrin detection.111 Amines such as ethanolamine, noradrenaline, hexamethylene diamine, methoxytryptamine, spermine, and spermidine were separated from amino acids on a DC-4A cation exchange resin.112 A similar approach, using a Beckman Model 121M amino acid analyzer equipped with an AA-20 column, was also successful.113 A Polyamin-pak strong cation exchange column (JASCO) was eluted with a citrate buffer for the detection of putrescene, spermine, cadaverine, and 1,5-diaminohex-ane from rat thymus.114 A post-column o-phthaldehyde detection system was used. 

Figure 10.19 Amino acid analyser trace. Separation of a complex physiological standard mixture of amino acids in 3.5 hours using lithium citrate buffers and ninhydrin detection 10 nmol of each amino acid, including the internal standard, nor-leucine, were applied to the column (0.3 X 35 cm) in a total volume of 50 ml. 

For analyses of synthetic peptides that are generally available in larger amounts than isolated natural peptides and proteins, the ninhydrin detection method is fully sufficient. The alternative detection method with orthophthaldialdehyde, which improves the sensitivity by a factor of five, was originally proposed for post-column derivatization, 34 but is presently used mainly in pre-column derivatization techniques. 35,36 ... 

The use of chemiluminescence reactions for the detection of metal ions by liquid chromatography was recently reported [59,60]. The detectors made use of the chemiluminescence produced in the reaction between luminol and hydrogen peroxide which is catalyzed by transition metals. The column effluent was mixed with the reagents in order to yield the chemiluminescence. The reaction was fast and was carried out at room temperature. By varying the pH of the buffer, selectivity towards certain metals was also achieved. For example, at pH 10-11 nickel could be analyzed but lead and aluminium were inactive at pH 13-14, the converse was true [59]. Aminco-Bowman has marketed a liquid chromatographic system in which amino acids and amines are analyzed by means of the fluorescence produced on reaction with the reagent fluorescamine. Fluorescamine does not fluoresce, but it does react with primary amino groups to produce fluorescent derivatives. The reaction is instantaneous and may be carried out at room temperature, usually at pH 9. This detection system promises to be far more sensitive than the ninhydrin detection system and is much more easily adapted to HPLC. 

Side Note 9.3. The Ninhydrin Detection of a-Amino Acids... 

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. 

Preparative HPLC strongly acidic cation exchange resin volatile buffers ninhydrin detection... 

Analytical procedures involving reduction and determination of mercaptan are not accurate determinations of cystine in permanent-waved hair or in hair treated with mercaptan because mixed disulfide is reduced to mercaptan during analysis adsorbed mercaptan can also interfere in the determination. Procedures that do not involve reduction of hair such as ninhydrin detection (alpha-amino group) or dinitrofluorobenzene (DNFB) reaction followed by chromatographic separation [1, 58] discriminate between mercaptans and, therefore, should be better analytical procedures for detecting the different types of mercaptans and disulfides actually present in permanent-waved hair. 

Pratically quantitative yields of couplings were obtained within 24 hours as indicated by ninhydrin detection (12). A more critical test was used to prove that the coupling was quantitative indeed After each coupling step some of the polymer carrying growing peptide was cleaved and subject to TLC test to observe contamination that could have arisen from previous incomplete couplings. No such contaminations were observed. However, by increasing the concentration of the "shadchan" the reaction time could be shortened. 

An amino acid analyser with post-column ninhydrin detection can give specific and accurate measurement of sulfur-containing amino acids, such as cystine in plasma and urine. However, analysis times are long (hours) and thiol-containing amino acids, such as cysteine, cannot be measured since the necessary elevated column temperatures lead to their rapid oxidation. In an attempt to circumvent this problem, Brigham et al. derivatised cysteine with iodoacetate to give the stable analyte S-carboxymethylcysteine. Subsequently, A-ethylmaleimide and iodoacet-amide have been used to derivatise amino-thiols prior to amino acid analysis. 

Proprietary dual wavelength detection for ninhydrin-detectable amino acids and 2-pyridyl resorcinol-detectable transition metals. 

Ion exchange chromatography with ninhydrin detection. This time-consuming method requires only sample preparation, has high specificity but low sensitivity. An internal control of the sample is provided by other amino acids with the same analysis. 

Macchi, F. D., Shen, F. J., Keck, R. G. and Harris, R. J. Amino acid analysis, using postcolumn ninhydrin detection, in a biotechnology laboratory. Methods Mol. Biol. 159 9-30, 2001. (Totowa, N. J). 

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