Amino acids reaction with ninhydrin

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. 

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

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. 

The reaction of amino acids with ninhydrin can detect amino acids on a wide variety of substrates. For example, if a kidnapper touches a ransom note with his fingers, the dermal ridges on his fingers leave traces of amino acids from skin secretions. Treatment of the paper with ninhydrin and pyridine causes these secretions to turn purple, forming a visible fingerprint. 

Spray the paper chromatogram lightly with ninhydrin reagent and heat for 5 min in a 100°C oven. The amino acids will appear as blue or purple spots (except proline, which will appear yellow). Figure 6-9 shows the reaction that amino acids undergo with ninhydrin. 

Figure 6-9 Reaction of amino acids with ninhydrin.

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. 

Ninhydrin Assay for Adsorbed Proteins. Measurements were made by a colorimetric procedure based on the reaction of ninhydrin with amino acids (25). The films were hydrolyzed in 5 ml of 2.5N NaOH for 2 hrs in capped plastic tubes in a boiling water bath. Then 1.5 ml of glacial acetic acid was added and mixed next I ml of ninhydrin reagent was added and mixed. [The reagent was three times more concentrated in ninhydrin, SnCb, and citrate than prescribed by Moore and Stein (25)]. The tubes were capped and boiled 20 mins more. The solution was clarified by centrifugation, and the absorbance read immediately at 570 nm on a Beckman DB spectrophotometer. If necessary, the sample was diluted with 50-50 2-propanol-water. Calibration curves (absorbance vs. fig of protein) were constructed in the 0-30 and 0-100 fig range with known amounts of each type of protein subjected to this same analysis procedure. 

In a typical experiment, 35 ml of aqueous solution into which both CO2 and NH3 or ammonium carbonate had been dissolved (pH = 8.9) was sealed in a quartz ampule and irradiated at 90 °C for 100 hr. The resulting solution was heated at 90 °C to dryness under the reduced pressure in order to remove the remaining NH3 or ammonium carbonate and the residue (white) was hydrolyzed at 105 °C for 48 hr in 6 M HCl. After evaporating the HCl solution at 90 °C to dryness, the residue was dissolved with 10 ml of water. Amino acids in the solution were separated by HPLC (SCL-6A, product of Shimadzu Co.) equipped with a column of cation exchange resin (Shim-pack 1SC-07/S1504L1, a product of Shimadzu Co.) and detected based on the ninhydrin reaction [13]. Nucleic acid bases and organic acids such as oxalic and oxamic acids were separated by HPLC with an CDS reversed phase column (Shim-pack CLC-ODS) and detected spectrophotometrically. 

The reaction itself is analogous to the reaction of ninhydrin with amino acids ... 

Saifer, A., and Oreskes, I., Color reaction of amino acids with alloxan, isatin, and ninhydrin in circular paper chromatography. Anal. Chem. 28, 501-504 (1956). 

The reactions of amino acids with ninhydrin, carbon dioxide, metal ions, and glucose are described below. The last three are of physiological importance. 

Aminoacylation A portion of the resin (0.9 g, 0.44 mmol) was transferred into a Wheaton glass vial. To a solution of a protected Fmoc-amino acid (1.5 mmol) and HOBt (203 mg, 1.5 mmol, 3.41 equiv.) in DMF (5 mL) was added DIG (237 pL, 1.5 mmol, 3.41 equiv.) and the reaction mixture was shaken for 20 min at rt. The activated amino acid solution was then added to the resin, and the mixture was shaken at rt for 3 h, or until a ninhydrin test was negative. The resin was then washed with DMF (twice), CH2GI2 (once), and DMF (once). The Fmoc group was removed by treatment with 50% piperidine in DMF (twice, 1 -r 15 min), and the resin was washed with DMF (four times). The substitution with amino acid, as determined by UV measurement of the dibenzofulvene-piperidine adduct (Xmax 302 nm) formed during the deprotection, was approximately 0.41 mmol g f... 

Very few ions are directly detectable by uv/vis absorption spectroscopy. However, if a postcolumn reaction is performed to chelate a cation with a chromophore (color producing group), to add a fluorescing agent, or to simply react the compound with another compound, such as using ninhydrin with amino acids, then uv/vis spectroscopy can be used. An example of such a system is the separation of lanthanides shown in the cation section. 

The reaction of amino acids (and peptides) with ninhydrin has been known for nearly a century. Ninhydrin reacts rapidly with all primary amines to produce characteristic blue-colored spots. 

A number of other reagents have been used to give colored derivatives of amino acids, among these being Folin s reagent, 1,2-naphthoquinon-4-sulfonic acid and o-phthalaldehyde. However, the ninhydrin reaction is so easy to perform and sensitive that other reactions are of very limited importance. Derivatization of amino acids to give colored and/or fluorescent products subsequently subjected to chromatographic procedures can be mentioned in connection with the discussion of color reactions for amino acids. 

---