Quantifying Plant ADP/ATP Levels by HPLC

The contents of ADP/ATP were measured by High Performance Liquid Chromatography (HPLC) with 20 mg of fresh weight plant materials (Liang et al., 2015 (link)). Briefly, 0.2 ml of 0.1 M HCL was mixed with the plant materials on ice. 15 µl extract/standard (different concentration) was mixed with 77 µl CP Buffer (62 mM, citric acid monohydrate and 76 mM (Na)₂HPO₄ × 2H₂O) and 8 µl 45% chloroacetaldehyde and incubated at 80°C for 10 min. The mixture was then centrifuged at 16.000 xg at 20°C for 30 min. 90 µl of the supernatant was then measured by the HPLC (Hyperclone C18 (ODS) column (Phenomenex)). The result was calculated by the standard ADP and ATP gradient.