SIMPLE AND RAPID PREPARATION METHOD OF ST ANDARD SAMPLES OF PHEOPHYTINS

A rapid and simple method for preparation of pheophytins from spinach was established. Spinach leaves (500 g) were dipped into boiling water for 3 min. The pigments were extracted from the leaves with acetone ( 1000 cm(3)) by ultrasonic vibration for 10 min. Chlorophylls were prec ipitated with addition of dioxane (150 cm(3)) and water (100 cm(3)), f ollowed by their pheophytinization with 0.1 M hydrochloric acid. Pheop hytin a and b were separated by preparative reversed-phase HPLC with a n ODS column (24 mm i.d. X 360 mm length) and a mobile phase of aceton e-acetonitrile (50/50, v/v). The purities of the fractionated pheophyt ins a and b were checked by analytical reversed-phase HPLC with an ODS column (4.6 mm i.d. X 250 mm length) and a mobile phase of acetone-ac etonitrile (50/50, v/v). The purity was 98.1% for pheophytin a, with i ts epimer, i.e. pheophytin a', being the sole impurity. No impurity wa s detected for pheophytin b. From 500 g of leaves, more than 400 mg an d 120 mg of pheophytins a and b were obtained respectively. These yiel ds were 1.5 similar to 3 times as high as those of the conventional me thods. Effect of the storage of pheophytins at -5 degrees C was also e xamined by the analytical reversed-phase HPLC. After 60 days, no purit ies were changed. However after 180 days, the purities of both pheophy tin a and b were down to 91.6%. Hence, it is confirmed that these pheo phytins are stable for at least 60 days.