Solvents and Purification Methods. Methanol, n-hexane, and acetone were Carlo Erba HPLC solvents (Milan, Italy).
β-Carotene (>97% purity) and triolein were purchased by Fluka(Milan, Italy).
β-Carotene was used as such, while triolein was purified by washing cycles with pure MeOH and freezing/partial crystallization of the mixture at T < −20 °C, followed by a phase separation, until the measured absorbance of triolein was equal to that of the previous washing cycle (minimum four cycles).
24 Lutein and zeaxanthin were obtained through HPLC semipreparative isolation/purification of methanol extracts of maize flour.
The mobile phase was methanol.
The column was a Waters Spherisorb S5-ODS2 10 × 250 mm RP-C18, the flux was 2.4 mL/min at T = 25 °C, and the spectrophotometric detector was at 450 nm.
Retention times (Rt) were 15 and 16 min, respectively, for lutein and zeaxanthin.
Pheophytin-a and pheophytin-b were obtained through HPLC semipreparative isolation, by Waters Spherisorb S5-ODS2 10 × 250 mm RP-C18 column, detector at 415 nm, of acetone extracts of leaves of Acer Negundo L.
(Aceraceae) acidified with 1 N HCl, after complete pheophytinization of chorophylls present in these extracts.
Pheophytinization was controlled through UV spectrophotometry by observing the sharp diminution of absorbance at 434 nm, where the maximum of chlorophyll-a is located, and the corresponding increase at 410 nm near the absorbance maximum of pheophytin-a (65% of spectral change in 12 min). R
etention times were Rt (pheo-b) = 25.9 min; Rt (pheo-a) = 32.5 min; flux 4.5 mL/min; T = 45 °C; mobile phase: methanol.