By ninhydrin

Thin-Layer Chromatography (tic). Tic (126) is used widely for quahtative analysis and micro-quantity separation of amino acid mixtures. The amino acids detected are developed by ninhydrin coloring, except for proline and hydroxyproline. Isatia has been recommended for specific coloring of pToline (127). 

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. 

Singh utilized anionic dyes to detect gramicidin on paper chromatograms184. Paris and Theallet described three paper chromatographic systems for gramicidin185. Ritschel and Lercher described two solvent systems for antibioticsiOD. The solvent systems were butanol-pyridine-acetic acid-water (15 10 3 12) and water saturated butanol-water saturated ethyl ether-acetic acid (5 1 1) on Schleicher and Schull 2043b paper. The antibiotics were visualized by ninhydrin. 

The reaction was monitored by TLC (eluent petroleum ether-ethyl acetate, 1 1). Visualized by ninhydrin dip, the product stained brown-orange, R 0.43. The 4-methoxystyrene (visualized by UV) has R 0.72. 

Problems such as diffusional limitations and the analysis of catalyst composition occur with solid-phase catalysts. Much work has been done on diffusion in bound enzymes (for reviews, see 24 and 88). In our work we used ninhydrin, which is a reagent ideal for surface analysis amino acid analysis is used wherever possible. Amine depletion as followed by ninhydrin is not exact, but some quantitative guides are obtained. Certainly synthetic catalysts must be made with bonds other than amide bonds and components other than those compounds that are detectable on the amino acid analyzer. 

Separation of a purified hydrolyzate of demineralized bovine dentin by cation exchange chromatography at pH 5.25. Gradient 0.0 - 0.5 M NaCl in 0.05 M HAc/NaAc, 1 mM NaNs, pH 5.25. Column SP Sephadex C25 (34 x2.6cm). Flow rate approx. 30 ml/h. The 4-ml fractions were assayed for amino acids by ninhydrin reaction (—) and for NaCl by electric conductivity measurement after 75-fold dilution (—). Fractions collected for further characterization are denoted by bars and Roman numerals. 

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. 

Amino acid analyzers have improved the analysis of free amino acids to a great extent. They offer superior sensitivity, speed, and accuracy to conventional methods. Many such systems are based on IEC. Postcolumn detection is done by ninhydrin derivatization followed by photometric measurement at 570 and 440 nm for primary and secondary amino acids, respectively. Amino acid analyzers are now common and are being manufactured by many companies (e.g., Hitachi, Beckman, PerkinElmer, HP, Pharmacia, etc.). Numerous authors have used amino acid analyzers to monitor proteolysis in several kinds of cheeses (Ardo and Gripon, 1995 Edwards and Kosikowski, 1983 Fenelon et al., 2000 Gardiner et al., 1998 Kaiser et al., 1992 Yvon et al., 1997). A comparison of amino acid analyzers and several other methods for amino acid analysis is available from Biitikofer and Ardo (1999) and Lemieux et al. (1990). 

Control by Ninhydrin test if (+), return to 5 if (-) follow step 1 forward following amino acid. 

Figure 1. Calibration curve for amino group (dl-leucine) and ammonium ion (ammonium chloride) analysis by ninhydrin method.

Monitor the coupling steps by ninhydrin assay (26) or Bromophenol blue assay (27) in the case of proline to assess the presence of free amines. 

More recent studies by Schmir and Cohen (1961) have led to a better understanding of the details and to improved yields in the tyrosine cleavage Phloretylglycine was oxidized with three equivalents of NBS in various buffer mixtures and the extent of cleavage determined both by ninhydrin assay for glycine and by the intensity of the dienone peak at 260 m/u in the ultraviolet. From the results (Table VIII) it is evident that cleavage is favored by increasing the acidity of the reaction mixture. 

After removal of excess of alkylating agent by ether extraction the reaction mixture was heated for 1 hr at 100°C. The liberated amino acid was determined by ninhydrin assay (Moore and Stein, 1948). 

Reaction mixture was analyzed directly by ninhydrin method (Moore and Stein,... 

Determined by ninhydrin assay for the cleaved amino acid. 

Fmoc-cleavage were monitored by Ninhydrin reaction [4] and TLC. The integrity of the protected peptides was proven by ID and 2D iH-NMR spectroscopy [19], The correct masses were established by FAB-MS. 

Thin layer chromatography is also used for direct enantiomeric resolution of D,L-arginine, D,L-histidine, d,l-lysine, D,L-valine, and D,L-leucine on silica gel plates impregnated with optically pure (IR, 3R, 5R)-2-azabicy-clo[3,3,0]octan-3-carboxylic acid, which serves as a chiral selector in the pharmaceutical industry. To successfully resolve D,L-amino acids, various combinations of aceto-nitrile-methanol-water were proposed. The spot was detected by ninhydrin (0.2% in acetone). 

Post-column derivatization—IEC is used to separate free amino acids followed by ninhydrin or o-phthaldehyde (OPA) post-column derivatiza-... 

Acetyl DL-methionine which is used as a substrate for amino acylase activity determination was prepared by acetylation of DL-methionine with acetic anhydride in acetic acid [5]. The rate of enzymatic hydrolysis was determined by measuring the liberated amino acid by ninhydrin method [6] where ascorbic acid was used as oxidizing agent instead of sodium cyanide. The activity curve of pure amino acylase enzyme is shown in Fig. 1 as a continuous line. For determining the effect of metal ions on the activity of amino acylase the following procedure was adopted. 

The freeze-dried peptide is hydrolyzed in 6 N HCl at 110°C for 1 h, HCl is removed by evaporation, and the residue is dissolved in H2O, mixed with unlabeled phosphoamino acid markers, and electrophoresed on the Kodak cellulose sheets (Erdodi et al., 1987). The phosphoamino acids are identified by ninhydrin staining and autoradiography (Rg. 2). The procedure is only qualitative, because of the incomplete hydrolysis of the phosphopeptide and the partial destruction of the liberated phosphoserine and phosphothreonine. 

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