Reagents ninhydrin

Ninhydrin reagent. Most peptides and amino acids can be detected on papers by dipping the paper in a solution containing ninhydrin (or spraying the paper with this solution) and heating in an oven at 60°C for 15-60 min. Further color development will sometimes occur overnight. Various colors ranging from yellow to violet are often observed and can be used to help identify certain peptides or amino acids. 

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Fig. 1 Comparative recordings of the reflectance scans of a mixture of phenylethylamine (1), tyramine (2), serotonin (3) and histamine (4) A. ninhydrin reagent (q.v.), B. ninhydrin — collidine reagent.

Moore, S., and W. H. Stein A Modified Ninhydrin Reagent for the Photo-... 

Penicillamine (50 mg) was mixed with 50 mg of formaldehyde, 50 pL of concentrated HC1, and 2.5 mL of propan-2-ol, and the solution heated at 60 °C for 2 h. An aliquot of the reaction mixture was subjected to TLC on a Chiralplate (Macherey-Nagel) with a mobile phase of methanol-H20-aeetonitrile (1 1 4). Detection was made using 0.1% ninhydrin reagent, and the limit of detection was approximately 0.5%. Good separation of (d)- and (L)-penicillamine was achieved. 

Rao et al. [46] reported the use of a spectrophotometric method for the determination of primaquine phosphate with ninhydrin. Standard solution of 0.01% primaquine phosphate solution (3 mL) or solution prepared from the drug or its tablets was mixed with 2 mL of water, 5 mL of 2-methoxyethanol and then with 4 mL of ninhydrin reagent. The mixture was boiled for 35 min, and, after cooling and dilution to 25 mL with water, the absorbance was measured at 570 nm versus a reagent blank. Beer s law was obeyed from 4 to 20 pg/mL with recoveries of 99.4— 100.2% for 25 mg of primaquine phosphate. 

Example Mixture of amino acids obtained from protein hydrolysates are separated by this method and spots located by using Ninhydrin Reagent that forms a pink to purple product with amino acids. 

Figure 10.18 Schematic diagram of an amino acid analyser using the ninhydrin reagent for quantitation.

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. 

Ninhydrin assay. Our method to determine reactivity towards ninhydrin was a modification of a method described previously (Moore and Stein, 1954 Moore, 1968). Briefly, dried samples were dissolved in 0.10 ml 0.1 M acetic acid and mixed with an equal volume of ninhydrin reagent (Sigma). After 15 minutes in a boiling water bath, samples were diluted with 0.80 ml ethanol/water (1 1 by vol.) and measured for absorbance at 550 nm on a flow-through spectrophotometer (Vitatron). Standards containing 0-0.6 mM leucine in 0.1 M HAc were included. Values were therefore calculated as leucine-equivalents. 

Moore S and Stein WH (1954) A modified ninhydrin reagent for the photometric determination of amino acids and related compounds. J Biol Chem 211, 907-913. 

Silufol Five mobile phases, and combination for two dimensional TLC. Dragendorff, iodoplatinate and ninhydrin reagents. In eye lotions and infusion solutions. 109... 

Amino acids can be separated without prior derivatization on a cation-exchange resin column. The elution buffers are classically lithium citrate buffers with different pH values and salt concentrations, which are applied stepwise. There is usually a programmed increase in column temperature. Consequently, there are numerous variables affecting the separation of the individual amino acids [6]. For the detection of the amino acids, the column effluent is mixed with the ninhydrin reagent. Nowadays there are only very few manufacturers of AAAs left. The considerable cost of purchase and the operation costs are a potential threat to the widespread application of this technique, although it is still considered to be the definitive method for diagnosing disorders of amino acid metabolism. 

As described above, the amino acids are separated at a constant flow on a high-resolution cation-exchange column using buffer and temperature gradients. The postcolumn reaction with the ninhydrin reagent is carried out at 135°C and the absorbances of the reaction products are read at both 570 and 440 nm. Amino acids are identified by comparing their retention time and 570/440 ratio with that of authentic reference substances (see Fig. 2 .2). 

Ninhydrin (2,2-dihydroxy-l,3-indandione) Amax = 570 nm, Amax = 440 nm. This reagent has already been described earlier in this chapter (Sec. III.A. Ion Exchange). Additionally, it should be noted that ninhydrin reagent solutions are susceptible to photodegradation and air oxidation. Older solutions will yield progressively poorer response as the ninhydrin oxidizes. It is an arbitrary decision when to swap a new solution for the old. Representative references include 172-174. 

Remove the chromatogram, dry it at 100 °C for 10 minutes and spray with ninhydrin reagent [0.2% solution in butan-l-ol, (1)] heat at 110°C for 5-10 minutes in order to develop the colour. Mark the centre of each spot with the metal scriber and evaluate and record the RF values. 

Clegg, K. M., Lee, Y. K., and McGilligan, J. F. (1982). Trinitrobenzene sulfonic acid and ninhydrin reagents for the assessment of protein degradation in cheese samples. /. Food Technol. 17, 517-520. 

Write the equation for the reaction of a given amino acid with ninhydrin reagent. 

To prepare the ninhydrin reagent, ninhydrin (2 g) and hydrindantin (300 mg) are dissolved in dimethyl sulfoxide (DMSO) (75 ml) in a foil-covered 400 ml flask (to exclude light) and are adjusted to 100 ml with 4 M lithium acetate buffer. The jar is stoppered with a rubber bung with inlet and outlet tubes, and the reagent is flushed with 02-free N2 for 30 minutes. The operations must be done in a fume hood. The final solution is then stored sealed at room temperature. Ninhydrin is toxic and stains the skin, so gloves should be worn at all times. 

Triplicate aliquots (0.75 ml) of standard solutions, blanks, and soil extracts are pipetted into labeled 20 ml thick-wall test tubes (15 mm diameter). Citric acid buffer (1.75 ml 0.2 M) is pipetted into each tube. Ninhydrin reagent (1.25 ml) is then pipetted slowly into each tube and vortex-mixed for two seconds. The top of each test tube is loosely covered with aluminum foil and placed in a vigorously boiling water bath for 25 minutes. There should be sufficient water in the bath so that the water level is always above the level of the solutions in the test tubes. The water should return to boiling within 2 minutes of the tubes being placed in the bath. After 25 minutes, the test tube... 

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