Total free amino acids were determined using Hamilaton & Slyke (1943) method. 0.5g of plant material was ground in 10ml of phosphate buffer using pestle and mortar that was placed on an ice tub. The phosphate buffer used was made of two salts K2HPO4 and KH2PO4 and the buffer pH was maintained at 7.8. The ground was then centrifuged at 4C for 10 minutes at 10000rpm. The reaction mixture contained 0.5ml extract in a test tube and added with 0.5ml of 4% ninhydrin and 0.5ml of 2% pyridine. The test tubes were shaken well for homogenous mixing. The test tubes were then cotton plugged and placed at 100C for 30miutes in the water bath. After the test tubes were cooled down the distilled water was added to the test tubes to make the volume up to 7.5ml. The absorbance was taken at 570nm and as we were following the dilution procedure in reaction the formula would be:
Total amino acid (mg/g fresh weight) =
Graph reading of sample ×volume of the sample ×dilute factor Weight of the tissue ×1000