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... 

The mechanism of the fluorescence reaction has not been elucidated. See Volume 1 a Ninhydrin — Collidine Reagent for the ninhydrin reaction. 

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. 

VK Sarin, SBH Kent, JP Tam, RB Merrifield. Quantitative monitoring of solid-phase peptide synthesis by the ninhydrin reaction. Anal Biochem 117, 147, 1981. 

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 ninhydrin reaction requires heat and therefore the stream of column eluate plus ninhydrin reagent must pass through a coil of narrow-bore tubing (approximate diameter 1 mm) held in a 100°C heating bath. It is important to ensure that the flow is not restricted because excessive heating may cause the ninhydrin to precipitate in these micro-bore tubes, resulting in complete stoppage of the analyser. 

The c-phthalaldehyde reaction compares favourably with the ninhydrin reaction in several respects. The reagent is stable and is in an aqueous form, which eliminates the use of potentially toxic chemicals and storage under nitrogen. Because the reaction proceeds quickly at room temperature there is no need for the 100°C heating bath with all its inherent problems, and the increased sensitivity permits detection at the picomole level. 

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. 

Whitaker (1970) reported that E. parasitica protease has its maximum stability between pH 3.8 and 4.8. Below pH 2.5, activity losses were associated with increase in ninhydrin reaction groups, which suggests autolysis of the molecule. Above pH 6.5 activity was rapidly lost, accompanied by decreased solubility and no increase in ninhydrin reaction groups. Thunell et al. (1979) found that E. parasitica protease is easily destroyed in whey at 68°C at all pH values from 5.2 to 7.0. 

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. 

Basic Protocol 1 Ninhydrin Reaction to Determine Degree of... 

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. 

NINHYDRIN REACTION TO DETERMINE DEGREE OF PROTEIN HYDROLYSIS... 

Ninhydrin reaction, protein hydrolysis measurement, 142-144, 153 N-lodoacetyl- N -(5-sulfo-1-... 

Protein hydrolysis, evaluating degree of fluorescamine reaction, 146-147 formol titration, 147-148 ninhydrin reaction, 142-144, 153 pH-stat, 149-151, 153 TCA solubility index, 152 TNBS reaction, 144-146... 

Sucrose, ninhydrin reaction, effect on, 153 Sugars, see also Disaccharides Monosaccharides ... 

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. 

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