Ninhydrin and

In the ion-exchange technique, separated amino acids exiting (eluting) from the end of the chromatography column mix with a solution of ninhydrin and undergo a rapid reaction that produces an intense purple color. The color is detected by a spectrometer, and a plot of elution time versus spectrometer absorbance is obtained. 

Modras (51) reported spot test reactions to differentiate hydralazine from closely related drugs. Reagents used were aqueous copper (I) chloride, aqueous ammonium molybdate, iodine in potassium iodide solution, aqueous cobalt (II) nitrate, alcoholic ninhydrin, and alcoholic bromophenol blue. The tests were performed on paper or on Silica Gel G. 

High-voltage electrophoresis and subsequent paper chromatography of the fractions obtained made possible the isolation from the analyzed mixture of twenty-two components giving colored spots with ninhydrin and isatin. Among these, fourteen were identified as peptides and their amino acid composition established (Table 5). In the case of eight peptides, also N- and C-terminal amino acids were determined (Table 6). 

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

Ninhydrin Proline Yellow chromophore Simplified reaction between ninhydrin and secondary amino adds... 

Reversed Pre- o-phthal- OPA, unlike ninhydrin (and in common with other... 

Separation and quantitation of the toxic components of A. muse aria and related mushrooms can be achieved by HPLC, or by TLC using ninhydrine and heating for detection. With ninhydrine, muscimol develops a yellow spot and has a limit of detection of 0.1 pg. 

Three gradients of 0.0-0.5 M sodium chloride were run consecutively at 4°C in 0.05 M sodium acetate-acetic acid, 1 mM sodium azide, pH 5.25, followed by 0.05 M sodium acetate-acetic acid, 1 mM sodium azide, pH 3.5, and finally by 0.05 M sodium dihydrogen phosphate-disodium hydrogen phosphate (approx. 1 3), 1 mM sodium azide, pH 7.0. After sample application, the column was washed with the starting buffer to remove any non-bound compounds. Elution was continued with the high salt buffer. Fractions of 4 ml were collected and assayed for reactivity towards ninhydrin and for electric conductivity (salt concentration) after 75-fold dilution of a 100-pl aliquot. Ninhydrin-positive fractions were pooled for each peak, concentrated, and desalted by size exclusion chromatography (see above). 

Cyclobutanecarboxylic acid, 98%, is from Aldrich Chemical Company, Inc., and is vacuum distilled before use. Tetrahydrofuran is freshly distilled from sodium-benzophenone under nitrogen. N-Methyiraorpholine, 99%, is from Aldrich Chemical Company, Inc., and is pre-dried over barium oxide, distilled from ninhydrin, and stored over sodium hydroxide pellets. Ethyl chloroformate, 97%, is from Aldrich Chemical Company, Inc., and is freshly distilled prior to use under a nitrogen atmosphere. Anhydrous ammonia (99.99% min) is from a Matheson lecture bottle. The silica gel used for flash chromatography is... 

The ninhydrin test will not interfere with the Ehrlich reagent. Therefore, a plate can be examined under UV light, immersed in iodine vapors, treated with ninhydrin, and the Pauly or Ehrlich reagents successively. 

The classical post-column derivatization, as developed by Moore and Stein, 3 uses ninhydrin and detection is achieved at 570 nm for primary amine derivatives and at 440 nm for secondary amine derivatives with detection limits that strongly depend upon the instrumentation used. 

Ninhydrin to Detect Lipids on TLC Plates Ninhy-drin reacts specifically with primary amines to form a purplish-blue product. A thin-layer chromatogram of rat liver phospholipids is sprayed with ninhydrin, and the color is allowed to develop. Which phospholipids can be detected in this way ... 

The Chemistry of Ninhydrins and Other Cyclic 1,2,3-Tricarbonyl Compounds A. Schoenberg and E. Singer, Tetrahedron, 1978, 34, 1285-1300. 

The synthesis of the Saa-peptide conjugates in solution follows standard solution-phase synthesis using, for example, IIDQt117 or EDC as the coupling agent. The reaction is checked by TLC (ninhydrin and/or 10% H2S04 in MeOH dip). The product is usually purified by flash chromatography. 

Exercise 25-8 The reactions that lead to the blue color produced between ninhydrin and a-amino acids are examples of reactions discussed previously in the context of carbonyl chemistry (see, for instance, Section 16-4C). Write mechanisms, based insofar as possible on analogy, for each of the steps involved in the ninhydrin test, using glycine as an example. Would you expect ammonia or methanamine to give the blue color Explain. 

A typical graph of the progress of protein hydrolysis by the pH-stat technique is shown in Figure B2.2.5. The pH-stat technique offers reliability, reproducibility, and simplicity. In addition, the technique has the advantage that no secondary reaction is needed. However, if the equipment needed to perform a pH-stat experiment is not available, the ninhydrin and TNBS techniques are good alternatives. 

RL Cunico, T Schlabach. Comparison of ninhydrin and o-phthalaldehyde post-column detection techniques for high-performance liquid chromatography of free amino acids. J Chromatogr 266 461-470, 1983. 

A stoichiometric mixture of ninhydrin and L-proline 2 (200 g) was milled in a 2-L horizontal ball-mill (Simoloyer ) with steel balls (100Cr6, 2 kg, diameter 5 mm) at 1100 min 1 for 40 min until the liberation of C02 was complete. The temperature varied from 15 °C at the water cooled walls to 21 °C in the center. The power was 800 W. Quantitative reaction to give 3 was secured by weight (146 g, 100%) and by spectroscopic techniques. The product was not separated in a cyclone but the milling-out towards the end was completed with 4 times 250 mL of water, each. This part of the highly disperse (< 1 pm) pure azo-methine ylide 3 was obtained after centrifugation and drying in a vacuum. The combined water phase contained 0.2 g of 3. 

Amino acids have been detected fluorimetrically after ion-exchange chromatography by monitoring the fluorescent derivatives produced on treatment with o-phthalaldehyde and 2-mercaptoethanol [57]. An Aminco-Bowman fluoromicrophotometer was used for the detection. An advantage of this technique is that only 2 min were required at room temperature for formation of the products, thus avoiding the lengthy reaction coils of the ninhydrin and cerium(IV) systems. 

The reduction of 2, either catalytically or with zinc in acetic acid, leads to 3-amino-4-hydroxy-2-quinolone 20 [72TH000], These amino compounds are rather unstable they dimerize with loss of ammonia to "bis-amines", which in turn are readily oxidized to dyes similar to those obtained from ninhydrin and primary amines [68M1205] [68M1543], The amino derivatives 20 are therefore conveniently converted into 0,N-diacetyl derivatives, the N-acetyl derivative 21, or its dehydrated form, the oxazolo derivative 22 [95MI000], The variety of biological activity of oxazolo-quinolines of type 22 has been detected only in recent years [94JHC1647],... 

Cyclohexylamine Extraction from water on C18 microcolumn then TLC Ninhydrin and exposure to UV [46]... 

M. M. Joullie, T. R. Thompson, Ninhydrin and Ninhydrin Analogs. Syntheses and Applications, Tetrahedron 1991, 47, 8791-8830. 

---