Amino acid analyzer

The procedure claimed formerly was used to ascertain amino acid content [138 (link)]. Fifty milligrams of freeze dried powder samples was hydrolyzed with 1 mL hydrochloric acid (6N HCl, 24 h, 110 °C). The derivation was then evaporated and condensed under vacuum (80 °C, 24 h). The condensed remnant was then dissolved with 1 mL hydrochloric acid (0.02N HCl) and passed through a filter membrane (0.45-μm). At the end, the solution was loaded in Amino Acid analyzer (Hitachi High-Technologies Corporation, Tokyo, Japan).

The contents of chlorophyll a (Chl a), chlorophyll b (Chl b), total chlorophyll (Total Chl), and carotenoid were determined according to Hosseini et al. (2017 (link)). Freshly harvested leaves (0.5 g) were immersed and homogenized in 80% acetone solution (20 ml). The absorbance of the extract was then recorded at the selected wavelength using a spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
Waqas et al. (2015 (link)) proposed a method for determining the amino acid content. Powdered freeze-dried leaves (50 mg) were hydrolyzed with 1 ml of hydrochloric acid (6 N HCl, 24 h, 110°C), followed by evaporation and condensation under vacuum (80°C, 24 h). Then, hydrochloric acid (1 ml of 0.02 N HCl) was added to dissolve the condensed remnant. The extract was filtered (0.45-μm membrane) before loading into Amino Acid Analyzer (Hitachi High-Technologies Corporation, Tokyo, Japan).

Then, 50 mg of freeze-dried sample were macerated with an acidic mixture comprising 2 mL hydrochloric acid (6 M HCl), 1 mL anhydrous trifluoroacetic acid (TFA), and 0.5% 2-mercaptoethanol (2-ME). Hydrolysis was done for 40 min at 170 °C under N₂ in sealed ampules [41 (link)]. The obtained hydrolysate was dried out with an evaporator, dissolved in distilled water (1 mL), and centrifuged (12,000 rpm, 2 min, 4 °C). The amino acid composition was evaluated with an Amino Acid analyzer (Hitachi High-Technologies Corporation, Tokyo, Japan).