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Author Topic: AMINO ACID ASSAY:Quantitative estimation of amino acids using Ninhydrin Reaction  (Read 18461 times)
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Francis Umeoguaju
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« on: April 10, 2010, 08:51:26 am »



Quantitative estimation of amino acids using the Ninhydrin Reaction

Ninhydrin (triketohydrindene hydrate), a powerful oxidizing agent reacts with all amino acid between Ph 4 and 8 to give purple coloured compound. The reaction is also given by primary amino and ammonia, but without the liberation of CO2.

Heating in the presence of Ninhydrin causes oxidative deamination of the alpha amino group amino acids and peptides with the concomitant reduction of Ninhydrin molecule. The reduced Ninhydrin react with ammonia and with a molecule of the oxidized Ninhydrin to yield coloured compound (Ruhemann’s violet-blue complex).

REAGENTS AND PREPARATION

   Amino acid (0.2 mmol/L of Aspartic acid, arginine, lysine and proline)
   
   Acetate buffer (4mol/L, pH 5.5)
   
   Methyl cellosolve (ethylene  glycol Monomethyl ether)
   
   Ethanol (50% V/V)
   
   Ninhydrin reagent (this is prepared by dissolving 0.8g of Ninhydrin and 0.12g of hydridantin in 30ml of Methyl cellosolve. 10ml of acetate buffer is added to this. The reagent is prepared fresh each time it is required.
   
PROCEDURE

   Varying concentration of the standard amino acid solution was pipette into labeled test tubes; the volume was made up to 1ml  by the addition of IN acetate buffer. For the unknown sample, 0.1ml of it was added to 0.9ml of the buffer. 1ml of Ninhydrin reagent was then added to all the test tubes. This was made to boil for 15 minutes. When the solutions were cooled to room temperature, 2ml of the diluent (50% ethanol) was added  to each test tube.
   
   The absorbance  of the solutions were read at 570nm. The unknown sample were then extrapolate from the calibration curve.
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jesus
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« Reply #1 on: April 28, 2011, 01:18:36 am »


what to know the method and results of similar or same project
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jesus
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« Reply #2 on: April 28, 2011, 02:09:04 am »


more information on results and discussion
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Francis Umeoguaju
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« Reply #3 on: May 24, 2011, 09:38:42 am »


The results and discussion of the result actually depends on your work. You can get samples publications that uses this method to determine protein content by Googling it. Still any of our bioscience member with such results can still post it here. Regards
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