The Ninhydrin Reaction

These types of reactions are useful in temporarily modifying or protecting either of the two functional groups, especially during the controlled linldng of amino acids to form peptides or proteins. 

PROBLEM 17.8 Using eqs. 17.7 and 17.8 as models, write equations for the following reactions  

Ninhydrin is a useful reagent for detecting amino acids and determining the concentrations of their solutions. It is the hydrate of a cyclic triketone, and when it reacts with an amino acid, a violet dye is produced. The overall reaction, whose mechanism is complex and need not concern us in detail here, is as follows  

Only the nitrogen atom of the violet dye comes from the amino acid the rest of the amino acid is converted to an aldehyde and carbon dioxide. Therefore, the same violet dye is produced from all a-amino acids with a primary amino group, and the intensity of its color is directly proportional to the concentration of the amino acid present. Only proline, which has a secondary amino group, reacts differently to give a yellow dye, but this, too, can be used for analysis. 

PROBLEM 17.9 Write an equation for the reaction of alanine with ninhydrin. 

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

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... 

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... 

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. 

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. 

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. 

N-a-acety1-L-lysine (0.5 mmole) and glucose (1.0 mmole) are dissolved in 1 ml 0.1 M K2HP0 buffer solution. The solution is heated in a closed vessel for 5 h at 100°C. Then the solution is made 6 N with HC1 and heated at 105° for 24 h. The hydrolysate is evaporated in vacuo. A solution of the residue in 20 ml water can be used for the TLC production of furosine spots. For quantitative evaluation the same absorbance was assumed for the ninhydrin reaction product both arginine and furosine. 

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. 

The fractions may be analyzed spectrophotometrically using the ninhydrin reaction or spectrofluorometrically using fluorescamine (Chapter 6). If radioactive glutamate or aspartate is used, the radioactivity in an appropriate aliquot should be determined with a scintillation counter (Chapter 3). 

The ninhydrin reaction for resin bound peptides gives ambiguous results probably due to inaccessibility of the N-terminal amino group. Therefore a small fraction of the peptide was cleaved from the resin. After concentration by an N2 stream the ninhydrin-reaction or monitoring by TLC or HPLC were performed. 

K2. Kakei, M., Studies on the ninhydrine reaction of gastric juice. Arch. Japan. Chirurgie 28, 2868-2871 (1959). 

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