FIG. 1.

HPLC and TLC profiles of purified Chlide. Purity of Chlide prepared from spinach was checked with HPLC with a C₁₈ reverse-phase column and TLC. (A) In HPLC, the sample was eluted with a linear gradient made by solvents A (methanol-28 mM thiobarbituric acid [70:30]) and B (99% methanol). The elution profile was monitored by absorbance at 664 nm. (B) In TLC, the sample was applied onto Silica Gel 60 plates (Merck) and developed with a solvent system (ethyl acetate-ethanol-methanol at 80:20:10, vol/vol/vol). In panel B, commercial Chlide from Sigma Aldrich (1) and indigenously prepared Chlide from commercially available spinach (2) were detected with UV at 360 nm. For experimental details, see “Preparation of Chlide” in Materials and Methods. AU, arbitrary units.