Principles and methods for the determination of total amino acids in plant root wound sap.
Operation method
Experiments for the determination of total amino acids in plant root wound sap
Principle
Amino acids and hydrated ninhydrin action, can produce diketoindene - diketoindene amine substitution salt and other blue-purple compounds, the color of its shade and the content of amino acids proportional to the proportion. Determination of the reaction product at 520nm absorbance value , you can calculate the content of amino acids in the sample.
Materials and Instruments
Root injury solution Move I. Material Instrumentation and Reagents For more product details, please visit Aladdin Scientific website.
95 % ethanol Glutaric acid nitrogen standard master batch Ninhydrin hydrate
Spectrophotometer Heating equipment Test tubes Test tube racks Pipettes Earwash balls
1. Materials: root wounding fluid.
2. Instrumentation: spectrophotometer, heating equipment, test tubes, test tube racks, pipettes, ear-washing balls.
3. Reagents and preparation: 95% ethanol.
50μg- ml-1 glutamic acid nitrogen standard master batch: 52.52 mg of glutamic acid was accurately weighed and dissolved in 100 ml of 10% isopropanol solution.
3% hydrated ninhydrin: weigh 3g of hydrated ninhydrin and dissolve in 100 ml of 95% ethanol.
Experimental Procedure
1. Glutamic acid nitrogen standard curve
(1) Take 6 test tubes, number them, and prepare the glutamate nitrogen standard solution with the content of 0-25μg in each tube according to the table below. 
Add the reagents in the table, shake well, placed in a boiling water bath and accurately heated for 10min, the solution becomes blue, indicating the presence of amino acids, remove the tube immediately add 5 ml of 95% ethanol, shake well, cooled to 0 tube solution as a blank control, at 520nm wavelength to determine the absorbance (A) value.
(2) Standard curve
The standard curve was plotted with the nitrogen content of glutamate in tubes No. 1-5 as the horizontal coordinate and the absorbance (A) value as the vertical coordinate.
2. Determination of amino acid content in wound flow solution
Take three test tubes (three replicates), numbered, each add 0.5 ml of wound flow solution, distilled water 1 ml, 3 ﹪ hydrated ninhydrin solution 0.5 ml, shake well, placed in a boiling water bath accurately heated for 10min, take out the tubes and immediately add 5 ml of 95 ％ ethanol, shake well, cool down and take the solution of the standard curve No. 0 tube as a blank control, and then measure the absorbance (A) value at the wavelength of 520nm.
Calculation of results
According to the absorbance value of the sample tube, find out the corresponding glutamic acid nitrogen content from the standard curve, and calculate the amino acid content in the wounded fluid by substituting the average value of three repetitions into the following formula. 