The HPLC system consisted of a lowpressure
pump and a photodiode array detector (SPDM20A;
Shimadzu). Chromatographic separation was
performed with a C18 column (YMC-Pack ODSAM,
5 μm, 4.6 mm ID × 250 mm; YMC, Japan)
which was maintained at 20°C using a thermostatic
compartment. The mobile phases were a mixture of
A: 75% acetonitrile, 20% methanol, 4.935% hexane,
0.05% butylated hydroxytoluene (BHT) and 0.015%
triethylamine (TEA) and B: 50% acetonitrile, 25%
THF, 24.985% hexane and 0.015% TEA with a flow
rate of 1 ml/min. The gradient system of the mobile
phase commenced from 0 min (100% A) to 30 min
(50% A), 32 min (0% A) and 34 min (100% A), and
then maintained this state to 45 min prior to the next
injection. Chlorophylls a and b from a 20-μl injection
were detected at 665 and 652 nm, respectively.
Standards of chlorophylls a and b (Sigma-Aldrich)
were dissolved in methanol to obtain concentrations
of 25-250 and 10-200 μg/ml respectively for the
calibration curves.