HPLC Analysis for Chlorophyll Composition

Chlorophyll used for HPLC analysis was extracted from fresh leaf samples of 6-week-old pem1, pem2, and “FT” plants, using 100% acetone. After centrifuged at 12,000 × g for 15 min, the supernatant was dried under N₂ gas, redissolved in acetone, and filtered with 0.22 μm organic filter membrane. HPLC analysis was performed, using an Alliance e2695 instrument with a C8 column (4.6 mm i.d. × 250 mm long; 5 μm; Agilent) according to the method of Zapata et al. (2000), using an isocratic system, consisting of methanol: acetonitrile: acetone at 1:3:1 at 40°C and a flow rate of 1 m min⁻¹. Eluted samples were monitored, using a 2,489 UV/Vis detector at 660 nm. Chl a and b standards (Sigma, St. Louis, USA) were used as controls. Three biological repeats were determined for each material, and three times the technical repeats were performed in each biological repeat.