In this article we will discuss about the colorimetric method for estimation of amino acids in plants.
Principle:
Amino acid content can be estimated quantitatively by Colorimetric method using ninhydrin reagent. The reaction of ninhydrin (triketohydrindene hydrate) with amino acids is of particular importance in the regard.
Ninhydrin is a powerful oxidizing agent and elicits the oxidative deamination of the L-amino group, liberating ammonia, CO2, the corresponding aldehyde and a reduced form of the ninhydrin. The ammonia then reacts with an additional mole of ninhydrin and the reduced ninhydrin yields a purple substance. The coloured soln. thus formed is estimated colorimetrically at 570 nm.
The reaction steps are:
Requirements:
(a) Reagents:
1. Ninhydrin soln. (3% in ethylene glycol mono-methyl ether).
2. Acetate buffer (0.1 M, pH 5.3).
(b) Standard glycine solution:
(1 ml of soln. contains 100 µgm.)
(c) Glassware’s and instruments:
1. Test tubes, pipettes, beakers etc.
2. Colorimeter.
Test Procedure:
1. The following sets are made in the tubes by taking standard sample solution:
Set I — 1 ml of sample solution + 0.5 ml of buffer + 0.5 ml of Ninhydrin soln.
Set II — 2 ml of sample solution + 0.5 ml of buffer + 0.5 ml of Ninhydrin soln.
Set III — 3 ml of sample solution + 0.5 ml of buffer + 0.5 ml of Ninhydrin soln.
Set IV — 4 ml of sample solution + 0.5 ml of buffer + 0.5 ml of Ninhydrin soln.
Set V — 5 ml of sample solution + 0.5 ml of buffer + 0.5 ml of Ninhydrin soln.
A replica of five for each set is prepared.
2. All these sets are heated in a water bath for 20-30 mins. until a purple blue colour is developed. Then the mixture is transferred to a 50 ml volumetric flask and diluted up to 50 ml.
3. Finally, the colour intensity of different solutions is determined by colorimeter at 570 mm. One control set is also prepared for blank.
4. The results are plotted on a graph paper and a standard curve is prepared.
5. 2 ml of the unknown sample is also used in the same colour reaction and, finally, Colorimetric readings are taken. The corresponding unknown amino acid concentration is determined by comparing with the standard curve.