Amino ninhydrin reaction

The automated amino acid analy2er depends on ion-exchange chromatography (117) and is now a routine tool for the analysis of amino acid mixtures (118). This most advanced machine can detect as Htde as 10 pmol in ninhydrin reaction analysis. One-half to two hours are required for each analysis. An analysis chart is shown in Figure 2. 

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 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. 

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... 

From 1951, Moore and Stein at the Rockefeller Institute refined the quantitative separation of amino acids on Dowex-50 which led to fully automated amino acid analyses. In early models two columns were needed one of 100 cm to separate most of the acidic and monobasic monocarboxylic acids between pH 3-11 and a short, 15 cm column for the basic amino acids which were eluted at pH <7. The columns operated above room temperature to give more rapid results, and the elution was monitored automatically by quantitative ninhydrin reactions. By the late 1950s a protein hydrolysate could be analyzed overnight. 

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. 

Moore S (1968) Amino acid analysis aqueous dimethyl sulfoxide as solvent for the ninhydrin reaction. J Biol Chem 243, 6281-6283. 

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. 

Both the ninhydrin reaction and pyridoxal phosphate-catalyzed decarboxylation of amino acids (Chapter 14) are examples of the Strecker degradation. Strecker reported in 1862 that alloxan causes the decarboxylation of alanine to acetaldehyde, C02, and ammonia.c... 

Ninhydrin Reaction. A neutral solution of an amino acid will react with ninhydrin (triketohydrindene hydrate) by heating to cause oxidative decarboxylation. The central carbonyl of the tnketone is reduced to an alcohol. This alcohol further reacts with ammonia formed from the amino add and causes a red-purplish color. Since the reaction is quantitative, measurement of the optical density of the color produced is an indication of amino add concentration. Imino acids, such as hydroxyproline and proline, develop a yellow color in the same type of reaction. 

The ninhydrin reaction (see Basic Protocol 1), the TNBS reaction (see Alternate Protocol 1), the fluorescamine reaction (see Alternate Protocol 2), and formol titration (see Alternate Protocol 3) all evaluate released amino groups by comparing the amounts of free amino groups before and after hydrolysis. The first three methods are spectro-photometric techniques, whereas the fourth is a potentiometric technique. The first and second are chromogenic techniques, whereas the third is fluorometric. These techniques are usually performed as time-course experiments. As the hydrolysis reaction proceeds, aliquots (samples) of the reaction are taken periodically and treated with a test reagent. Products of this reaction are proportional to the amount of free amino groups at each time point. 

Reaction of ninhydrin with an amino acid yields a colored complex. The ninhydrin reaction permits qualitative location of amino acids in chromatography and quantitative assay of separated amino acids. 

Usually the amino acid analyzer is first standardized by running through it a sample containing known quantities of amino acids to account for any differences in their ninhydrin reaction properties. In this way it is possible to relate directly the amount of amino acid present to the amount of colored product formed, as measured by the area under the peak produced on the strip-chart recorder (see fig. 3.15). Similarly, the amino acid hydrolysate of a protein of unknown composition can be run through the analyzer, and the relative peak areas can be used to estimate the ratios of the different amino acids present. 

Enzymic hydrolysis of PGA by crude extracts of papain and pancreatin was carried out as described by Miller 27). Samples withdrawn at intervals were analyzed for free amino groups using a ninhydrin reaction 41). Values were corrected for the ninhydrin reaction of an enzymic digest which contained no PGA. 

This approach has been used extensively for amino acid analysis using low-pressure ion-exchange chromatography and post-column ninhydrin reaction. Spraying, dipping and vapour-treatment techniques are well known as post-separation reactions in TLC, but these are considered only briefly since the majority of them are not quantitative. While the problems of pre-separation techniques are quite similar for TLC and HPLC, they differ considerably for post-separation reactions. 

The post-column derivatization of amino acids by the ninhydrin technique is a well known method for routine analysis of amino acids [7-9]. The amino acids are usually separated by ion-exchange chromatography and then converted into UV-absorbing derivatives for quantitation. The ninhydrin reaction is often used for TLC detection of amino acids and proteins. 

Deprotection. The BOC group is easily removed under quite mildly acidic conditions, a feature which underlines its value in selective deprotection in peptide synthesis. Typically, treatment at room temperature for 30-60 minutes with a 1 m solution of hydrogen chloride in acetic acid, or with neat trifluoroacetic acid, is used. It is of interest that after t.l.c. of BOC-amino acids, brief exposure of the plates to hydrogen chloride fumes enables the ninhydrin reaction to be used to detect the presence of the liberated free amino acids. 

Friedman, M. (2004). Applications of the ninhydrin reaction for analysis of amino acids, peptides, and proteins to agricultural and biomedical sciences. ]. Agric. Food Chem. 52, 385-406. 

In our studies the ninhydrin-reaction- revealed a number of new fractions, one of which is fluorescent. There are two fractions with a mobility greater than albumin, and two with a mobility lower than y-globulin (Fig. 62b). Some of these are present in many sera, some are rare. They are present in the ultrafiltrate of serum where also a central fraction can be seen, which has the mobility of the y-globulin. They are also present in normal cerebrospinal fluid and were found in hemoglobin preparations. It is not clear if only amino acids are involved, because larger molecules also react with ninhydrin. 

You have probably been introduced to a process known as the ninhydrin reaction for the detection of a-amino acids. This test reacts to any primary amines. Due to their biological relevance ... 

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. 

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. 

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