Summary
Highlights
The ninhydrin method is used for the quantitative estimation of amino acids. Alpha-amino acids react with excess ninhydrin to produce a purple product called Ruhemann's purple. This reaction is highly reliable for detecting amino acids, even in microgram concentrations, as the color intensity is proportional to the amino acid concentration.
The necessary reagents for this experiment include standard amino acid stock solution (150 µg/ml), ninhydrin reagent, 50% ethanol, and distilled water. The apparatus and glassware required are test/boiling tubes, pipettes (glass/micropipette), and a water bath.
First, pipette varying volumes (0.2ml-1ml) of standard amino acid solution into labeled test tubes and 0.8ml into an 'unknown' tube. Add distilled water to all tubes to a final volume of 4ml. A blank tube receives 4ml of distilled water. Then, add 1ml of ninhydrin reagent to all tubes, mix, and add marble chips. Cover the tubes with aluminum foil and heat them in a boiling water bath for 15 minutes. Cool the tubes and add 1ml of ethanol to each, mixing well.
To set up the colorimeter, switch it on and set the wavelength to 570nm. Rinse a cuvette with the blank solution, drain it, and fill it with the blank solution to the appropriate mark. Insert the cuvette into the colorimeter slot, ensure proper placement, and tare the instrument to zero. Finally, measure the optical density (OD) values of the solutions from the lowest to the highest concentration.