Milli q 50 system

Potassium phosphate was provided by Sigma-Aldrich Chemical Co. (Madrid, Spain). HPLC LC/MS grade solvents were supplied by Panreac (Barcelona, Spain). The deionized water used was obtained from a Milli-Q 50 system (Millipore Corp., Milford, MA, USA). Na (NaCOOH) (10 mM NaOH in 300 μL of formic acid) was used for calibration.

Ammonium acetate was supplied by Fluka (Zwijndrecht, The Netherlands). Solvents used for chromatography were HPLC grade (Prolabo, VWR International Eurolab, Barcelona, Spain). Analysis grade solvents were supplied by Scharlau (Microdur, Sevilla, Spain). The deionized water was obtained from a Milli-Q^® 50 system (Millipore Corporation, Milford, MA, USA). For all purposes, analytical grade (American Chemical Society) reagents were used (Merck, Madrid, Spain). Standards of chlorophylls a and b were supplied by Sigma Chemical Co. (St. Louis, MO, USA), and standards of pheophytin a and pyropheophytin a were provided by Wako chemicals Gmbh (Neuss, Germany). Standards of pheophorbide a, pyropheophorbide a, and chlorine e₆ and rhodin g₇ sodium salts, were purchased from Frontier Scientific Europe Ltd. (Carnforth, Lancashire, UK). The C-13 epimers (chlorophylls a’ and b’) were prepared by treatment of the respective chlorophyll with chloroform, and 13²-OH-chlorophylls (a or b) were obtained by selenium dioxide oxidation of the corresponding chlorophyll at reflux-heating for 4 h in pyridine solution under argon [42 (link)]. Pheophytin b was prepared from a solution of chlorophyll b in ethyl ether by acidification with 13% HCl (v/v), and shaking the mixture for 5 min [43 (link)]. Standards purity, evaluated by HPLC, was ≥95% in all cases with the exception of rhodin g₇ sodium salt (~90%).

All chemical reagents were of analytical grade. The standard of chlorophyll a was purchased at Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Sodium hydroxide pellets, phenolphthalein, and diethyl ether, were from Panreac Química, S.A.U. (Castellar del Valles, Barcelona, Spain). Acetone and methanol were from Romil Chemicals Ltd. (Waterbeach, Cambridge, GB, UK). The deionized water used was obtained from a Milli-Q 50 system (Millipore Corp., Burlington, MA, USA).