After each centrifugation, the fractions were collected and
transferred to the same 50 mL volumetric flask and its volume
was topped up with purified water. An aliquot of the final solution
(1 mL) was filtered through 0.45 lm membrane filters from
Millipore (Brazil) using a plastic syringe with a capacity of 10 mL
of Plascalpand and transferred to a vial with capacity of 1.5 mL of
Uniglas and then injected into the chromatograph. The concentration
of each sample was determined by HPLC–PAD.